biological function of rna interference (rnai) pathways in

Biological function of RNA interference (RNAi) pathways in the moss Physcomitrella patens

(Hedw.) Bruch & Schimp.

Inaugural-Dissertation

zur Erlangung der Doktorwürde

der Fakultät für Biologie

der Albert-Ludwigs-Universität

Freiburg im Breisgau

von

Basel Khraiwesh

aus Jinin Camp - Palästina

Freiburg im Breisgau, 2009

Dekan: Prof. Dr. Ad Aertsen

Promotionsvorsitzender: Prof. Dr. Eberhard Schäfer

Betreuer: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank

Referent: Prof. Dr. Ralf Reski, PD Dr. Wolfgang Frank

Koreferent: Prof. Dr. Wolfgang R. Hess

Tag der Verkündigung des Ergebnisses: 24. April 2009

This work has been created in the

Department of Plant Biotechnology

Institute of Biology II

Faculty of Biology

Albert-Ludwigs University of Freiburg

under the guidance of Prof. Dr. Ralf Reski and PD Dr. Wolfgang Frank

To my marvelous mother and dear family

To my wife and my lovely boys,

For your support, understanding and

always being there for me…

Index

I

Index List of contents I Publications and manuscripts related to this work II

1 Chapter Ι: Introduction and Overview……………………………….. 1 1.1 Background………………………………………………………………………… 1

1.1.1 RNA Interference: function and technology…………………………………………… 1 1.1.2 Small RNAs and gene silencing………………………………………………………… 2

1.1.2.1 MicroRNAs (miRNAs)…………………………………………………………………3 1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA)…………………………………… 5 1.1.2.3 Repeat-associated RNAs (ra-siRNA)………………………………………………. 6 1.1.2.4 Natural antisense transcript-derived small interfering RNAs (nat-siRNA)……… 6 1.1.2.5 Piwi-associated RNAs (piRNAs)……………………………………………………. 7 1.1.2.6 Secondary transitive siRNA…………………………………………………………. 7

1.1.3 Dicer proteins……………………………………………………………………………... 9 1.1.4 Physcomitrella patens as a model system…………………………………………… 11

1.2 Results and Discussion………………………………………………………… 14 1.2.1 DICER-LIKE genes in Physcomitrella patens………………………………………...14

1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout mutants…………. 16 1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders…………………….. 17 1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNA-

targets is abolished in ΔPpDCL1b mutant lines………………………………..17 1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines…………………...18 1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild type………….19 1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants…………………….20 1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing amiR-GNT1…………………………………………………………………………21

1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the phytohormone abscisic acid (ABA)………………………………………….. 21

1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b mutant lines…………………………………………………………………………………22

1.2.2 Highly specific gene silencing by artificial miRNAs in Physcomitrella patens……. 24 1.3 Conclusion………………………………………………………………………… 27 1.4 References………………………………………………………………………… 29 2 Chapter II: Manuscript 1……………………………………………..34

Transcriptional control of gene expression by microRNAs………………35 3 Chapter III: Publication 1…………………………………………..121

Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockout……………………….122

4 Chapter IV: Appendices……………………………………………. 136 4.1 Flow cytometric measurements (FCM)……………………………………...136 4.2 Physcomitrella patens DCL1b (PpDCL1b) mRNA……………………….. 137 4.3 DNA vectors……………………………………………………………………... 140 4.4 Genes downregulated in ΔPpDCL1b mutants…………………………….. 141 4.5 Genes upregulated in ΔPpDCL1b mutants………………………………… 146 4.6 Acknowledgments……………………………………………………………... 152 4.7 Erklärung………………………………………………………………………....153

Publications

II

Publications and manuscripts related to this Work: Manuscript #1 - Khraiwesh, B., M. A. Arif, G. I. Seumel, S. Ossowski, D. Weigel, R. Reski, W. Frank.

(2009): Transcriptional control of gene expression by microRNAs. Submitted. Publication #1 - Khraiwesh, B., S. Ossowski, D. Weigel, R. Reski, W. Frank (2008): Specific gene

silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiology, 148: 684–693.

This work has been presented at the following conferences: Talks (presented by W. Frank) − Frank, W., Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R. (2007): Specific

epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. Botanical Congress, September 3-7, 2007, University of Hamburg, Germany.

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Specific

epigenetic control of microRNA target genes to compensate for RNAi dysfunctions in a Physcomitrella patens DICER-LIKE mutant. The Annual International Conference for Moss Experimental Research, August 2-5, 2007, Korea University, Seoul, Korea.

Posters − Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., Frank, W. (2008): Specific gene

silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Annual Meeting of the RNA Society, July 28-August 3, 2008, Free University Berlin, Germany.

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a

DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. 5th Colmar Symposium: The New RNA Frontiers, November 8-9, 2007, Colmar, France.

− Khraiwesh, B., Seumel, G. I., Baar, K. M., Reski, R., Frank, W. (2007): Knockout of a

DICER-LIKE gene causes silencing of microRNA targets in Physcomitrella patens. The Annual International Conference for Moss Experimental Research, August 2-5, 2007, Korea University, Seoul, Korea.

Chapter I Background

1

1 Chapter Ι: Introduction and Overview

1.1 Background

1.1.1 RNA Interference: function and technology

RNA interference (RNAi) is a mechanism regulating gene transcript levels by either

transcriptional gene silencing (TGS) or by posttranscriptional gene silencing (PTGS), which

acts in genome maintenance and the regulation of development (Hannon, 2002; Agrawal et

al., 2003). Since the discovery of RNAi in Caenorhabditis elegans (Lee et al., 1993; Fire et

al., 1998) extensive studies have been performed focusing on the different aspects of RNAi.

In particular, the elucidation of the essential components of RNAi pathways has advanced

extensively (Tomari and Zamore, 2005). RNAi has been discovered in a wide range of

organisms from plants and fungi to insects and mammals suggesting that it arose early in the

evolution of multicellular organisms (Sharp, 2001; Hannon, 2002).

The RNAi pathway is typically initiated by ribonuclease III-like nuclease enzymes, called

Dicer, that cleave double stranded RNA molecules (dsRNAs; typically >200 nt) into small

fragments bearing a 3’ overhang of two nucleotides. One of these two strands is coupled to a

second endonuclease enzyme called Argonaute (AGO) and then integrated into a large

complex (RNA-induced silencing complex, RISC). Subsequently, it has been shown that

RISC contains at least one member of the AGO protein family, which is likely to act as an

endonuclease and cuts the mRNA. In Drosophila and humans, AGO2 has been identified as

being responsible for this cleavage and the catalytic component of the RISC complex. It was

proposed that small interfering RNA (siRNA) guide the cleavage of mRNA. SiRNAs are key

to the RNAi process and they have complementary nucleotide sequences to the targeted RNA

strand. In certain systems, in particular plants, worms and fungi, an RNA dependent RNA

polymerase (RdRP) plays an important role in generating siRNA (Cogoni and Macino, 1999).

Another outcome are epigenetic changes such as histone modification and DNA methylation

(Matzke and Matzke, 2004; Schramke and Allshire, 2004) (Figure1).

In medical research, RNAi is on the way to becoming an important tool to treat HIV,

hepatitis C, and cancer (Hannon and Rossi, 2004) and in plants RNAi technology has been

used to improve their nutritional value (Tang and Galili, 2004). For science in general it is

already a tool of large scale reverse genetic approaches and aids in unravelling gene

functions in many species.


2

Figure 1: Overview of RNA interference (adapted from Matzke and Matzke, 2004). The Dicer enzymes produce siRNA from dsRNA and mature miRNA from hairpin-like miRNA precursor transcripts. MiRNA or siRNA is bound to an AGO enzyme and an effector complex is formed, either a RISC or RITS (RNA-induced transcriptional silencing) complex. RITS affects the rate of transcription by histone and DNA modifications whereas RISC cleaves mRNA or inhibits its translation.

1.1.2 Small RNAs and gene silencing Small non-coding RNAs (20-24 nucleotides in size) have been increasingly investigated and

they are important regulators of PTGS in eukaryotes (Hamilton and Baulcombe, 1999; Mello

and Conte, 2004; Baulcombe, 2005). They were first discovered in the nematode

Caenorhabditis elegans (Lee et al., 1993) and are responsible for phenomena described as

RNAi, co-suppression, gene silencing or quelling (Napoli et al., 1990; de Carvalho et al.,

1992; Romano and Macino, 1992). Shortly after these reports were published, it was shown

that PTGS in plants is correlated to small RNAs (Hamilton and Baulcombe, 1999). These

small RNAs regulate various biological processes, often by interfering with mRNA

translation. Based on their biogenesis and function small RNAs are classified as repeated-

associated small interfering RNAs (ra-siRNAs), trans-acting siRNAs (ta-siRNAs), natural-

antisense transcript-derived siRNAs (nat-siRNAs) and microRNAs (miRNAs) (Vazquez,

2006) (Table 1).


3

Table 1: Classes of small RNAs identified in eukaryotes (Chapman and Carrington, 2007)

1.1.2.1 MicroRNAs (miRNAs)

MiRNAs are ~21nt small RNAs which are encoded by endogenous MIR genes. Their primary

transcripts form precursor RNAs exhibiting a partially double-stranded stem-loop structure

which are processed by DICER-LIKE proteins to release mature miRNAs (Bartel, 2004). In

animals, the primary miRNAs (pri-miRNAs) are cleaved in the nucleus by an enzyme named

Drosha to form the pre-miRNAs (Lee et al., 2003). The pre-miRNAs are then transported

into the cytoplasm where they are processed into the mature double stranded miRNAs,

through cleavage by a second enzyme, Dicer (Bartel, 2004). The first enzyme, Drosha,

required for processing of pri-miRNAs in animals, does not exist in plants, so the precursor

miRNA is directly cleaved within the nucleus to generate the mature miRNA (Baulcombe,

2004) (Figure 2a).

Computational analysis of miRNAs and their potential target mRNAs revealed that many of

the miRNA targets belong to the group of transcription factors (Palatnik et al., 2003; Wang

et al., 2004). In addition to the control of targets at the post-transcriptional level miRNAs

regulate gene expression by epigenetic changes such as DNA and histone methylation (Bao et

al., 2004; Lippman and Martienssen, 2004). Overexpression or knockdown of miRNA genes

can lead to abnormalities during development (Palatnik et al., 2003; Chen, 2004). For

example, plants expressing MIR159, which targets members of MYB transcription factors,

exhibit delayed flowering time and male sterility (Achard et al., 2004; Schwab et al., 2005).

Plants expressing MIR160, which targets members of the ARF transcription factor family,

exhibits agravitropic roots with disorganized root caps and increased lateral rooting (Wang

Class Description Biogenesis and genomic origin Function miRNA

MicroRNA Processing of foldback miRNA gene transcripts by members of the Dicer and RNaseIII-like families

Posttranscriptional regulation of transcripts from a wide range of genes

Primary siRNA

Small interfering RNA

Processing of dsRNA or foldback RNA by members of the Dicer family

Binding to complementary target RNA; guide for initiation of RdRP dependent secondary siRNA synthesis

Secondary siRNA

Small interfering RNA

RdRP activity at silenced loci (Caenorhabditis elegans) processing of RdRP derived long dsRNA or long foldback RNA by members of the Dicer family (Arabidopsis thaliana)

Posttranscriptional regulation of transcripts; formation and maintenance of heterochromatin

tasiRNA

Trans-acting siRNA

miRNA-dependent cleavage and RdRP dependent conversion of TAS gene transcripts to dsRNA, followed by Dicer processing

Posttranscriptional regulation of transcripts

natsiRNA

Natural antisense transcript-

derived siRNA

Dicer processing of dsRNA arising from sense and antisense transcript pairs

Posttranscriptional regulation of genes involved in pathogen defense and stress responses in plants

piRNA

Piwi-interacting RNA

A biogenesis mechanism is emerging which is Argonaute dependent but Dicer-independent

Suppression of transposons and retroelements in the germ lines of flies and mammals


4

(g) Piwi-interacting RNA (piRNA)

et al., 2005). Plants expressing MIR166, which targets members of HD-ZIP transcription

factors, are arrested in seedling development, and show fasciated apical meristems and femal

sterility (Williams et al., 2005).

Figure 2: Small RNA pathways (modified after Vazquez, 2006; Chapman and Carrington, 2007). (a) The miRNA (b) trans-acting siRNA precursors are non-coding RNAs (c) nat-siRNA precursors derive from cis-antisense overlapping coding transcripts. All three precursors are transcribed by RNA polymerase Pol II. (e) Two miRNAs can guide AGO1-mediated cleavage of TAS precursors. The resultant 5’ fragment (TAS1a, 1b, 1c and 2) or 3’ fragment (TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA , which is then cleaved in a phased fashion every 21-nt by DCL4. (f) ta-siRNAs are then 2’-O-methylated at their 3’ ends by HEN1 and guide a slicer-competent AGO protein (AGO7 for TAS3 siRNAs or an unidentified AGO protein for other ta-siRNAs) to their targets for cleavage. (d) A self-amplifying loop believed to depend on RNA Pol IVa is involved in maintaining ra-siRNA-guided methylation of certain DNA repeats. (g) Piwi-interacting RNA (piRNA) biogenesis. Black bars represent genes, with their transcription initiation sites indicated by arrows. Thin black strands represent transcripts of genes encoding small RNAs, and thin blue strands represent target mRNAs. Boxes with broken lines indicate parts of the ta-siRNA and nat-siRNA pathways for which the cellular location is not well established. In small RNA duplexes, the red strands correspond to the guide strand and the black strands correspond to the passenger strand to show that, in the case of siRNAs, the guide strand can originate from either the original sense strand or the newly RDR-synthesized complementary strand.

Target sites in plant miRNAs normally share perfect or nearly perfect complementarity with

their target sequence and are often in coding regions (Schwab et al., 2005), whereas in


5

animals, target sites are often only partially complementary to their miRNAs and are mostly

located in the 3'UTR of target genes (Filipowicz, 2005). Currently, hundreds of miRNAs have

been identified in plant species and deposited in the miRBase database

(http://microrna.sanger.ac.uk/sequences/index.shtml) (Table 2).

Table 2: Plant species and number of miRNAs deposited in miRBase database

(Version 12.0, 2008)

Species Group Number of miRNAs

Arabidopsis thaliana Eudicots 184

Medicago truncatula Eudicots 30

Populus trichocarpa Eudicots 215

Oryza sativa Monocots 243

Zea mays Monocots 96

Physcomitrella patens Mosses 220

1.1.2.2 Trans-acting short interfering RNAs (ta-siRNA) MiRNAs are required for the biogenesis of ta-siRNAs, and both miRNAs and ta-siRNAs

regulate mRNA stability and translation (Baulcombe, 2004). Ta-siRNAs arise in plants from

specific TAS loci (Figure 2b). TAS transcripts are RNA polymeraseII-dependent and function

as highly specialized precursors that feed into an RdRP-dependent siRNA biogenesis

pathway. They are targets for cleavage by miRNA-guided mechanisms and yield siRNAs that

are in a 21-nt register with the cleavage site (Allen et al., 2005; Rajagopalan et al., 2006;

Chapman and Carrington, 2007).

Arabidopsis contains different characterized TAS gene families. TAS1a-c and TAS2 ta-siRNA

biogenesis is initiated by miR173-guided cleavage on the 5′ side of the ta-siRNA generating

region, while TAS3 ta-siRNAs form by miR390-guided cleavage on the 3′ side. MiR390 also

interacts in a non cleavage mode with a second site near the 5′ end (Axtell et al., 2006;

Montgomery et al., 2008). The resultant 5’ fragment (TAS1a-c and TAS2) or 3’ fragment

(TAS3) is used by RDR6 and SGS3 as a template for the production of a long dsRNA, which

is then cleaved in a phased fashion every 21-nt by DCL4. This processing involves an

interaction between DCL4 with DRB4 for TAS3. Ta-siRNAs are later incorporated into the

RISC-like complex and guide cleavage of the complementary mRNAs. However, in

Physcomitrella patens, miR173 is absent and therefore miR390 is responsible for the

generation of TAS precursors (Axtell et al., 2006). TAS3 ta-siRNAs, but not those from

TAS1a-c or TAS2, are dependent on a specialized AGO7 (also called ZIP) (Montgomery et al.,

2008). The mechanisms for recognition and routing of transcripts through the ta-siRNA or

RDR6/DCL4-dependent pathway are not well understood. Axtell et al. (2006) proposed a


6

two-hit trigger mechanism, in which transcripts with two or more small RNA target sites are

preferentially routed into the RDR6/DCL4 pathway.

1.1.2.3 Repeat-associated RNAs (ra-siRNA)

The mechanism of small interfering RNAs (siRNA) and ra-siRNA production is quite similar.

They originate from transgenes, viruses and transposons and may require RdRP for dsRNA

formation (Waterhouse et al., 2001; Aravin et al., 2003). Unlike miRNAs, the diced siRNA

products derived from the long complementary precursors are not uniform in sequence, but

correspond to different regions of their precursor. Because these small RNAs are often single

stranded, in fact this means that siRNAs work through the same mechanism, but they are not

evolutionary conserved (Bartel and Bartel, 2003; Allen et al., 2004; Jones-Rhoades et al.,

2006; Axtell et al., 2007). In plants, the cleavage of siRNA occurs by different DICER-LIKE

enzymes than the miRNA processing (Xie et al., 2004). They were first described in plants,

where it was shown that the silencing of three transgenes involved a small antisense RNA

complementary to each targeted mRNA (Hamilton and Baulcombe, 1999; Hamilton et al.,

2002; Bonnet et al., 2006). In plants, siRNA have different functions that can be divided into

two broad categories: those that are involved in formation and maintenance of

heterochromatin and those that derive from and defend against viruses or sense transgene

transcripts (Baulcombe, 2004; Bonnet et al., 2006).

Ra-siRNAs (24-nt) are derived from repetitive elements and control the maintenance of DNA

and histone modifications (Hamilton et al., 2002; Xie et al., 2004) (Figure 2d). Previous

studies on Arabidopsis reported that the transcripts of the two canonical repeats, the

retrotransposable element AtSN1 and the 5S ribosomal DNA are converted by RDR2 into

long dsRNA as a template for DCL3, which processes 24-nt siRNAs that are O-methylated by

HEN1 (Xie et al., 2004; Yang et al., 2006). 24-nt ra-siRNAs guide methylation of AtSN1 and

5S rDNA repeat loci by the action of the AGO4 and they have been implicated in chromatin

modifications (Zilberman et al., 2003). The ra-siRNA pathway is a positive feedback loop

because methylation of these loci is essential for ra-siRNA accumulation (Xie et al., 2004).

1.1.2.4 Natural antisense transcript-derived small interfering RNAs (nat-siRNA)

Borsani et al. (2005) identified a new class of small RNAs derived from a natural-antisense

overlapping transcript pair. The transcripts of P5CDH, a stress-related gene, and SRO5, a

gene of unknown function, partially overlap (Figure 2c). In the proposed nat-siRNA

pathway, a 760-nt double-stranded region resulting from pairing of cis-antisense transcripts

is thought to be processed by DCL2 to generate a unique 24-nt nat-siRNA that guides

cleavage of P5CDH transcript through an unidentified AGO protein. In principle, RDR6 and


7

SGS3 could synthesize a strand complementary to cleaved fragments of P5CDH mRNA

leading to a dsRNA that is processed like TAS duplexes in a phased fashion by DCL1. Each

resulting 21-nt nat-siRNA reinforces cleavage of P5CDH mRNAs. NRPD1a could be involved

in a reinforcing amplification loop using the dsRNA generated by RDR6 and SGS3 as a

template. Accumulation of the 24-nt nat-siRNA is correlated with salt stress-induced

transcription of the SRO5 gene and is abolished in dcl2, rdr6, sgs3 and nrpd1a mutants. The

finding that 4–20% of the genes in many eukaryotes show cis-antisense overlapping

organization raises the possibility that the nat-siRNA could be a major mechanism for gene

expression regulation (Borsani et al., 2005).

1.1.2.5 Piwi-associated RNAs (piRNAs) Recent studies have revealed a new class of 24-30-nt RNAs that are generated by a Dicer-

independent mechanism and that interact with members of the Piwi subfamily of AGO

proteins (Chapman and Carrington, 2007; Klattenhoff and Theurkauf, 2008). The proposed

piRNA-biogenesis model involves initial targeting of transcripts from transposons and

retroelements by a Piwi-like protein that is programmed with a small RNA (Figure 2g).

PiRNAs are important for spermatogenesis in mammals and insects. Plants appear to lack

these piRNAs (Faehnle and Joshua-Tor, 2007). In Drosophila, the previously identified ra-

siRNAs have been shown to bind the Piwi family members Piwi and Aubergine (Aub) and

thus represent a subset of Drosophila piRNAs. Piwi-associated ra-siRNAs (now referred to

as piRNAs) are 24−29nt long than AGO-bound siRNAs/miRNAs and are derived

predominately from repetitive genomic loci like transposons or satellite repeats (Aravin et

al., 2003; Chapman and Carrington, 2007).

1.1.2.6 Secondary transitive siRNA Previous studies on A. thaliana and C. elegans reported the amplification of silencing related

RNA via transitivity, and explain how strong, persistent silencing can be initiated with small

amounts of initiator dsRNA (Axtell et al., 2006; Pak and Fire, 2007; Sijen et al., 2007). The

emergence of transitivity has an important role for RNAi in controlling gene expression and

for understanding the effects of silencing RNAs on cell function and organism development.

The initiator of transitivity is a dsRNA which is produced by RdRP activity and then

processed by DICER-LIKE family into secondary siRNA or a related type of RNA referred to

as miRNA, these 21-25 nucleotide single stranded RNAs are the primary silencing RNAs in

the transitive process (Baulcombe, 2007; Moissiard et al., 2007). Upon binding to target

transcripts, siRNAs can not only trigger their cleavage and subsequent destruction, but also

serve as primers for RdRP. These extend the local RNA double strands and generate

templates for production of secondary siRNAs by Dicer action. These secondary siRNAs are


8

unrelated in sequence to the initial trigger (Moissiard et al., 2007; Mlotshwa et al., 2008). In

plants, the transitivity occurs in both directions of the initial dsRNA trigger whereas in

animals spreading of the initial signal occurs only upstream of the trigger (Figure 3).

Figure 3: Models for amplification of silencing signals in C. elegans and A. thaliana (adapted from Chapman and Carrington, 2007). (a) Processing of trigger dsRNA in C. elegans by Dicer 1 (DCR-1) releases primary siRNAs.The primary siRNA associates with an AGO protein, such as RDE-1, and the complexes bind to complementary target RNA. The bound complexes might then recruit RdRP, which uses the target transcript as template for synthesis of the secondary siRNA. Abundant secondary siRNAs are formed by independent initiation events (rather than by Dicer-mediated processing), are complementary to the target RNA and accumulate in phased pools. (b) Processing of trigger dsRNA in A. thaliana by one or more Dicer-like enzymes releases primary siRNAs, which associate with an AGO protein, such as AGO1, and guide cleavage of the target RNA. This event is proposed to recruit an RdRP, such as RDR6, which uses the target transcript as template for synthesis of a long dsRNA. This dsRNA precursor is processed by DCL enzymes to release abundant secondary siRNAs.


9

1.1.3 Dicer proteins Dicer and DICER-LIKE (DCL) proteins are RNAaseIII-type enzymes that cleave RNA

molecules with dsRNA features into small fragments bearing a 3’ overhang of two

nucleotides during PTGS (Elbashir et al., 2001). Dicer is a large multidomain protein

conserved in most eukaryotes, consisting of DExD-helicase, helicase-C, Duf283, PAZ (Piwi-

Argonaute-Zwille), dual RNAase III , and double stranded RNA-binding (dsRB) domains

(Bartel and Bartel, 2003) (Figure 4).

Figure 4: Crystal structure of Dicer (adapted from Macrae et al., 2006). The linear arrangement of domains typically found in DCL or DCR proteins is depicted above the figure. (a) Front and side view ribbon representations of Dicer showing the N-terminal platform domain (blue), the PAZ domain (orange), the connector helix (red), the RNase IIIa domain (yellow), the RNase III b domain (green) and the RNase-bridging domain (gray). Disordered loops are drawn as dotted lines. (b) Close-up view of the Dicer catalytic sites; conserved acidic residues (sticks); erbium metal ions (purple); and erbium anomalous difference electron density map, contoured at 20s (blue wire mesh).

The DExD and helicase-C domains are found towards the N-terminal and C-terminal

regions, respectively. There are always two RNase III domains (termed A and B) in a Dicer

protein, and the Duf283 is a domain of unknown function but which is strongly conserved

among Dicer proteins. The role of the dsRB domain in human Dicer is generally thought to

mediate unspecific reactions with dsRNA, with the PAZ, RNase III A and RNase III B

domains being crucial for the recognition and spatial cleavage of dsRNAs into siRNA or

miRNA (Zhang et al., 2004). In organisms with only one Dicer, this enzyme, with its

associated proteins, is presumably the only generator of siRNAs and miRNAs. In organisms

with two or more Dicers, there is probably a division of labour. Plants have at least four main

types of DCLs (Margis et al., 2006). In A. thaliana, the four different DICER-LIKE proteins

(DCL1-DCL4) exhibit predominant functions in particular small RNAs pathways, even


10

though functional redundancies among these proteins were identified (Henderson et al.,

2006). DCL1 produces mature miRNAs which direct cleavage of transcripts containing

sequence elements in reverse complementary orientation (Kurihara and Watanabe, 2004).

DCL2 mediates the generation of siRNA from RNA of exogenous sources (Xie et al., 2004).

DCL3 is required for the formation of heterochromatin-associated endogenous siRNA (Xie et

al., 2004) and DCL4 is needed for the formation of ta-siRNA involved in systemic cell-to-cell

transmission of silencing signals (Xie et al., 2005). Also all DCLs have been shown to be

involved in generation of viral-derived RNAs in coordinated hierarchical actions (Moissiard

and Voinnet, 2006). Human, mice and nematodes each contain one Dicer gene, involved in

miRNA biogenesis and the generation of siRNAs. Insects and fungi possess two Dicer genes,

the two Dicers have related but different roles, one processes miRNAs and the other is

necessary for siRNA-mediated RNAi (Margis et al., 2006). Knockout and knockdown

experiments indicate that Dicer is essential for vertebrate development (Jaskiewicz and

Filipowicz, 2008). Disruption of Dicer in mice arrests embryogenesis (Bernstein et al.,

2003). Dicer function was also found to be essential for Zebrafish development and many

processes in C. elegans. In Drosophila, Dicer involved in miRNA biogenesis is likewise an

essential gene (Jaskiewicz and Filipowicz, 2008). In mammals, Dicer is important for

protection against influenza A virus infection (Matskevich and Moelling, 2007). Dicer is also

required for the maintenance of epigenetic silencing in human to protect against cancer

(Ting et al., 2008).

Loss-of-function mutants of DCL1 in Arabidopsis revealed its role in a number of

developmental processes including embryogenesis and flower morphogenesis (Golden et al.,

2002; Park et al., 2002). The pleiotropic effects observed in these mutants were ascribed to

the lack of miRNA which was caused by the loss of miRNA biogenesis. However, other

mechanisms controlled by Dicer and related to RNAi, such as DNA methylation, chromatin

structure and centromeric silencing, may also contribute to developmental or cellular

defects.

Although recombinant Dicer is active as a dsRNA endonuclease in vitro, in cells it generally

functions in association with other proteins as a component of multiprotein complexes. In

animals, Dicer proteins were shown to be associated with other proteins forming complexes

which act as RISC or RISC loading complexes (RLC) (Tomari et al., 2004). Thus, miRNA

processing and target-RNA cleavage could be coupled. In Drosophila, it was shown that Dcr-

2, which produces siRNA, also acts in the RISC assembly together with its partner R2D2 by

loading one of the two siRNA strands into RISC (Tomari et al., 2004). The C. elegans

homologue of this protein, RDE-4 was also found to interact with Dicer (Tabara et al., 2002).

Similarly, human Dicer may function in loading siRNA into RISC, as siRNA does not cause

PTGS in human cells lacking Dicer (Doi et al., 2003). However, this is dependent on


11

particular cell types as siRNA triggers gene silencing in Dicer knockout embryonic stem cells

(Kanellopoulou et al., 2005). Moreover, human Dicer, TRBP and AGO2 were present in a

protein complex which is able to perform siRNA or miRNA directed target RNA cleavage

(Gregory et al., 2005). In plants, members of the HYL1/DRB family proteins were identified

as DCL-interacting dsRBD (dsRNA-binding domain) partners and implicated in small RNA

pathways in Arabidopsis (Hiraguri et al., 2005). Another group of well characterized Dicer

partners in animals is represented by PPD or AGO proteins. Members of the PPB protein

family contain two signature domains: a PAZ domain in the center and a PIWI domain at the

carboxyl terminus (Carmell et al., 2002; Tolia and Joshua-Tor, 2007). Several other proteins

have been found to interact with Dicer. RNA-helicase-related protein, which is required for

RNAi, was found to interact with RDE-4 and Dicer in C. elegans (Tabara et al., 2002).

FMRP, an mRNA-binding protein involved in the pathogenesis of fragile X syndrome, has

been shown to interact with Dicer and AGO-1 in mammalian cells (Jin et al., 2004).

Biochemical analysis of fission yeast Dicer (Dcr1) revealed its physical and functional

association with RNA-directed RNA polymerase complex (RDRC) in transcriptional

silencing (Shiekhattar, 2007). Identification of so many Dicer-interacting proteins indicates

that Dicer participates in many cellular processes (Jaskiewicz and Filipowicz, 2008).

1.1.4 Physcomitrella patens as a model system

The moss Physcomitrella patens is a member of the bryophytes. Physcomitrella patens

occupies an important phylogenetic position for the elucidation of the development of higher

plants (Figure 5), including other model organisms, such as Arabidopsis, and plants of

commercial importance, such as poplar, corn, soybean, sorghum, and rice. In terms of

evolutionary distance, Physcomitrella is to the flowering plants what fish is to humans.

Figure 5: Land plant evolution (adapted from Rensing et al., 2008). Bryophytes comprise three separate lineages which, together with the vascular plants (including the flowering plants), make up the embryophytes (land plants). These four lineages, remnants of the initial radiation of land plants in the Silurian, began to diverge from each other about 450 million years ago.


12

The gametophytic phase is divided into the protonema and the gametophore stages, which

produce the sporophyte upon particular conditions. Typical for mosses is the heteromorphic-

heterophasic alteration of generations, which is responsible for the predominant haploid

phase of the gametophyte (Figure 6), and a diploid phase that produces haploid spores.

Physcomitrella patens is a monoecious, self-fertile species, i.e. one plant carries both the

male (antheridia) and the female (archegonia) sex organs.

Figure 6: Life cycle of Physcomitrella patens (adapted from http://www.plant-biotech.net). A haploid spore germinates and grows into the filamentous protonema cells. Starting with a three-faced apical cell bud formation is initiated which gives rise to the leafy adult gametophyte. In monoecious moss species both sex organs (antheridia and archegonia) are present on one and the same plant. Fertilization of the egg cell takes place in the presence of water. From the fertilized egg the sporophyte grows out of the archegonia. Within the spore capsule the cells undergo meiosis and new spores are formed.

These features, among others, make Physcomitrella advantagous for scientific use. Because

protonema grows quickly and simultaneously, it can be cultivated in a bioreactor as a

genetically stable cell suspension. Another advantage is that Physcomitrella can be easily

manipulated using molecular biology methods (Reski, 1998a).

A unique feature is the high efficiency of homologous recombination, therefore targeted

disruption and manipulation of single genes can be performed easily (strepp et al., 1998;

Schaefer and Zryd, 1997). The rate of homologous recombination in Physcomitrella is found

to be several orders of magnitudes higher than in any other characterized plant species


13

(Reski, 1998b). The high rate of homologous recombination together with the predominant

haploid phase make Physcomitrella a highly suitable system to initiate forward and reverse

genetics approaches, enabling the study of gene functions related to almost all aspects of

plant biology. A considerable collection of mutants (Egener et al., 2002) and around 210.000

expressed sequence tag (EST) sequences are available (Rensing et al., 2002). Analyses have

shown that around 95% of Physcomitrella’s transcriptome is covered by these data.

The moss Physcomitrella patens genome comprises about 511 Mbp which are dispersed on

27 chromosomes. The sequence contains approximately 30,000 protein coding genes. Most

predicted genes are supported by multiple types of evidence, and 84% of the predicted

proteins appear to be complete. About 20% of the analyzed genes show alternative splicing, a

frequency similar to that of A. thaliana and O. sativa (Rensing et al., 2008). Despite its low

evolutionary position at the basis of land plants Physcomitrella shares more features with

the seed plant A. thaliana, than Arabidopsis as dicotyledonous plant with the

monocotyledon O. sativa (Reski, 1998b). Recently, a small RNA database has been

established in Physcomitrella (Arazi et al., 2005; Axtell et al., 2006; Axtell et al., 2007;

Fattash et al., 2007) and they are highly conserved in plants. Recent reports have shown that

the RNAi machinery is present and working correctly in Physcomitrella. However, in

contrast to A. thaliana and other plant species the biological function of the RNAi pathways

in Physcomitrella were not studied.

The objectives of this study are:

1. To study the biological function of RNAi pathways in the moss Physcomitrella patens,

focussing on the function of the key protein of RNAi, Dicer, by the generation of targeted

knockout plants and analyzing the pathways of small RNAs and miRNA target genes in Dicer

mutants.

2. To study gene silencing using artificial miRNAs (amiRNAs) in the moss Physcomitrella

patens as an alternative tool to targeted gene knockouts.

Chapter I Results and Discussion

14

1.2 Results and Discussion

1.2.1 DICER-LIKE genes in Physcomitrella patens To identify genes encoding DCL proteins BLAST searches of a Physcomitrella EST database

(Rensing et al., 2002) were performed using the four Arabidopsis DCL proteins as query.

The corresponding cDNA clones of the identified ESTs were sequenced. Analysis of these

partial cDNA sequences suggested the existence of four DCL genes in Physcomitrella (Table

3).

Table 3: Identification of DICER-LIKE genes in Physcomitrella patens. Closest A. thaliana homologue obtained by reverse BLAST searches using the deduced amino acid sequences of the four P. patens genes in the GenPept/nr database. The PpDCL1b sequence used to generate ΔPpDCL1b mutant lines are underlined.

These DCL genes have recently been deduced from the Physcomitrella patens genome

sequence independently (Axtell et al., 2007). Two of the Physcomitrella patens DCL proteins

(PpDCL1a and PpDCL1b) group together with AtDCL1 (Figure 7), the only A. thaliana DCL

involved in miRNA processing (Kurihara and Watanabe, 2004). Prediction of protein

domains in the Pfam database (Bateman et al., 2004) revealed the existence of all functional

domains in the two Physcomitrella patens DCL1 proteins present in the AtDCL1 protein

(Figure S2 and S3, Manuscript 1). However, compared to AtDCL1 the PpDCL1b protein

lacks approximately 240 amino acids at the N terminus. The other two PpDCL proteins are

homologs of AtDCL3 and AtDCL4, whereas an AtDCL2 homolog is lacking.

Using the homologous recombination system, M. Asif Arif generated and analyzed (in his

ongoing Ph.D. work) two targeted PpDCL1a knockout mutants (ΔPpDCL1a) (Figure S2,

Manuscript 1). The ΔPpDCL1a mutant lines show severe developmental abnormalities.

A.th. homologues (Acc. No.) P.p. DCL cDNA (Acc. No.)

AtDCL1 (Q9SP32)

AtDCL2 (NP_566199)

AtDCL3 (NP_189978)

AtDCL4 (P84634)

PpDCL1a (EF670436) 69% identity

81% similarity

PpDCL1b (DQ675601) 65% identity

78% similarity

PpDCL3 (EF670437) 32% identity

48% similarity

PpDCL4 (EF670438) 35% identity

53% similarity


15

Most drastically the ΔPpDCL1a mutant lines are not able to develop leafy gametophores and

are developmentally arrested at the protonema stage (Figure 1A and 1B, Manuscript 1). The

results show that PpDCL1a is the functional equivalent of the A. thaliana DCL1 protein

required for the biogenesis of miRNAs and ta-siRNAs. Compared to wild type the expression

levels of miRNA and ta-siRNA target genes were upregulated in ΔPpDCL1a mutant lines

(Figure 1C-E, Manuscript 1). However, in Physcomitrella patens miRNAs might be

processed by additional DCLs as the detection particular miRNAs albeit at significantly

reduced expression levels (Figure 1, Manuscript 1).

The additional presence of a second AtDCL1 homolog in Physcomitrella patens suggested

potential differences in endogenous RNAi pathways in comparison to the seed plant A.

thaliana.

Figure 7: Neighbour-joining tree showing the phylogenetic relationships between DICER-LIKE proteins. DICER-LIKE proteins from animals and plants are indicated by vertical lines. The four groups of DICER-LIKE proteins in plants are marked by coloured boxes. Species abbreviations are At (Arabidopsis thaliana), Ce (Caenorhabditis elegans), Cr (Chlamydomonas reinhardtii), Dm (Drosophila melanogaster), Hs (Homo sapiens), Mm (Mus musculus), Mt (Medicago truncatula), Nc (Neurospora crassa), Os (Oryza sativa), Pp (Physcomitrella patens), Pt (Populus trichocarpa), Sp (Schizosaccharomyces pombe). Pp DCL proteins are highlighted in bold. * The sequence of DCL from Chlamydomonas reinhardtii can be retrieved at: http://genome.jgi-psf.org/chlre2. (Figure S1, Manuscript 1).


16

1.2.1.1 Generation and molecular analysis of ΔPpDCL1b knockout mutants

To generate PpDCL1b knockout lines a PpDCL1b gene disruption construct was prepared by

inserting an nptII selection marker cassette into a 560 bp fragment of the PpDCL1b cDNA

which encompasses the coding region of the second RNAseIII domain present in the DCL1b

protein (Seumel, 2004; Figure S3, Manuscript 1). The resulting construct was used for

transfection of Physcomitrella protoplasts. After selection of regenerating plants they were

analyzed by PCR to identify mutant lines which had integrated the disruption construct at

the DCL1b genomic locus. Out of a total of 520 analyzed transgenic lines 8 lines (1.54%)

unable to produce PpDCLb1 transcripts were identified. Four lines were used for further

studies (ΔPpDCL1b 1-4). The full-length cDNA sequence of PpDCL1b was obtained (Figure 8,

Appendix 4.2), the cDNA was termed DCL1b encoding a protein of 1695 amino acids.

Furthermore, the haploidy of all ΔPpDCL1b mutant lines was verified by flow cytometry to

exclude the possible generation of diploid lines by protoplast fusion during the

transformation process (Appendix 4.1).

Figure 8: Cloning and sequencing of the PpDCL1b cDNA. The PpDCL1b cDNA is indicated by a black line. The colored arrows above depict cDNA fragments obtained by different cloning steps. The numbers in the arrows refer to the corresponding nucleotide positions in the DCL1 cDNA. First, a cDNA clone comprising the 3’ end of PpDCL1b was sequenced. Subsequently, three 5’ RACE-PCRs using the BD Smart RACE cDNA Amplification Kit (Clontech) and one RT-PCR was performed. The primers for the RT-PCR were derived from available Physcomitrella genomic trace files. All PCR and 5’ RACE primers were selected to give rise to overlapping PCR fragments of already known sequence stretches to confirm that the amplicons were derived from the same cDNA. The following primers were used: 5‘RACE-PCR 1: 5’- GAACTCCCAACGATGGTCGAGACGC-3’ 5’RACE-PCR 2: 5’- CCAGCT CATCGTGATCAGTAAAGTCGGG -3’ 5‘RACE-PCR 3: 5’-TCCCAGCGCCCGTGTCTAGAAATGCAAC -3’ RT-PCR: 5’-GAGAGGCGGTCTGTGTCGAGGTCTAG -3’ and 5’-TTGTAGCCACCAGCAACGTCACCCGT -3’


17

1.2.1.1.1 Knockout of PpDCL1b causes developmental disorders The ΔPpDCL1b mutant lines showed developmental disorders throughout all stages of

protoplast regeneration including abnormalities in cell division, growth polarity, cell size,

cell shape and growth of tissues (Figure 2A, Manuscript 1). Moreover, these mutants

developed only a small number of gametophores, which in addition were malformed (Figure

9). The observed developmental effects are consistent with previous studies of Dicer mutants

in animals and plants. The pleiotropic effects observed in these mutants were ascribed to the

lack of miRNA which was caused by the loss of miRNA biogenesis.

Figure 9: Phenotypic analysis of the ΔPpDCL1b mutants. Electron micrographs of gametophores from wild type plants and ΔPpDCL1b mutant 1 (Figure 2B, Manuscript 1).

1.2.1.1.2 MiRNA biogenesis is not affected and miRNA-directed cleavage of mRNA-targets is abolished in ΔPpDCL1b mutant lines

The isolated PpDCL1b gene from Physcomitrella shows 65% identitiy and 78% similarity to

to the DCL1 gene from Arabidopsis (Table 3), encoding the essential enzyme required for the

generation of miRNAs from pre-miRNA precursors. If the high sequence conservation causes

considerable overlap in function one would expect the absence of miRNAs in the ΔPpDCL1b

mutant lines. Interestingly, the accumulation of miRNAs in ΔPpDCL1b mutant lines

compared to the wild-type was present in almost equal amounts (Figure 2C, Manuscript 1),

indicating that PpDCL1b is not required for processing of miRNA precursors in

Physcomitrella. In contrast, miRNA-triggered cleavage of miRNA target genes, encoding

different transcription factors, was abolished in the ΔPpDCL1b mutant lines (Figure 3A,

Manuscript 1). The abolished miRNA-directed cleavage of target mRNAs in the ΔPpDCL1b

mutant lines suggests a direct involvement of PpDCL1b in this step of miRNA action. The

requirement of DCL proteins for target cleavage was not shown in plants. It is unlikely that

PpDCL1b directly cleaves mRNA targets as this function is commonly associated with AGO

proteins present in the RISC (Liu et al., 2003). Studies in animals have shown Dicer in


18

association with protein complexes (Tabara et al., 2002; Liu et al., 2003; Lee et al., 2004;

Pham et al., 2004). Some of these complexes, like the Dcr-2/R2D2 heterodimer from

Drosophila act in loading siRNA into RISC (Liu et al., 2003). It is possible that

Physcomitrella patens DCL1b functions in RISC loading like Dcr-1 and Dcr-2 from

Drosophila. Until now, only the Arabidopsis protein HYL1 was shown to interact with

Arabidopsis DCL1 in vitro (Hiraguri et al., 2005). However, even though HYL1 shows high

similarity to the Drosophila R2D2, a function in RISC loading is unlikely as dsRNA triggered

gene silencing is not affected in hyl1 mutants. Analysis of miRNA expression indicated that

HYL1 plays a role in miRNA biogenesis as miRNA levels were reduced in the hyl1 mutant

(Han et al., 2004).

1.2.1.1.3 Generation of transitive siRNA in ΔPpDCL1b mutant lines In Physcomitrella wild type, 5’RACE-PCRs performed from the miRNA targets yielded

additional fragments besides the expected cleavage products, indicating additional cleavage

of the mRNAs at sites other than the miRNA binding site (Figure 3A, Manuscript 1). The

mRNA cleavage products may serve as templates for synthesizing cRNA by RdRP (Vaistij et

al., 2002) leading to the formation of dsRNA. Subsequently, the dsRNA may be processed

into secondary siRNAs resulting in spreading of the initial miRNA signal (Figure 3B,

Manuscript 1). In plants, this mechanism, known as transitivity, is initiated by dsRNA

triggers (e. g. viruses and transgene transcripts) and transcripts that are targeted by more

than one small RNA (Moissiard et al., 2007). In seed plants, the generation of transitive

siRNAs from miRNA cleavage products is the exception. To prove the occurrence of

transitivity in Physcomitrella, sense and antisense oligonucleotides derived from PpARF and

PpC3HDZIP1 mRNA regions upstream and downstream of the miRNA binding sites were

used. Sense and antisense siRNAs were only detected in wild type whereas siRNAs derived

from miRNA targets were lacking in the ΔPpDCL1b mutants (Figure 10). In Physcomitrella

the generation of siRNAs depends on PpDCL1b function and is specific for miRNA-directed

cleavage of target RNAs.

Figure 10: Detection of transitive siRNAs derived from miRNA target genes. Detection of sense and antisense siRNAs derived from PpARF and PpC3HDZIP1 with oligonucleotides derived from regions upstream and downstream the miRNA binding sites. Hybridisation with an

antisense probe for U6snRNA served as control to indicate equal loading (Figure 3C, Manuscript 1).


19

1.2.1.1.4 Analysis of DNA methylation in ΔPpDCL1b mutants and wild type

When miRNA targets are not cleaved, the respective mRNAs are likely to accumulate to

higher levels in the ΔPpDCL1b mutant lines. Conversely, in ΔPpDCL1b mutants all miRNA

targets analyzed had reduced transcript levels when compared to wild type (Figure 11),

although these mRNAs were not cleaved in these mutants. It is tempting to speculate that

other RNAi components may sense the defective target cleavage as some of them were shown

to direct heterochromatin formation and gene silencing. One probable explanation for these

unexpected findings is a yet undiscovered epigenetic control of genes encoding miRNA

targets in Physcomitrella. Since methylation of cytosine residues is the most prominent

mechanism for transcriptional silencing in eukaryotes (Bender, 2004), this possibility was

tested by methylation-specific PCR from the miRNA target genes and the control gene

(PpGNT1) which is not regulated by a miRNA.

Figure 11: Expression levels of miRNA target genes in ΔPpDCL1b mutants and wild type. RNA blots analysis of miRNA target genes PpARF, PpC3HDZIP1, PpHB10 and PpSBP3 and two control genes, PpGNT1 and PpEF1α. (Figure 4B, Manuscript 1).

In some cases, endogenous siRNAs have an influence

on epigenetic control, DNA methylation and

chromatin structure at target loci and are associated with RNA-directed DNA methylation

(RdDM) and chromatin remodeling (Hamilton et al., 2002; Zilberman et al., 2003; Xie et al.,

2004). In plants, dsRNAs which contain sequences that are homologous promoter regions

can trigger promoter methylation and transcriptional gene silencing (Melquist and Bender,

2003; Matzke and Birchler, 2005). A function of miRNA 165/166 in directing DNA

methylation was shown in the regulation of the homeodomain-leucine zipper (HD-ZIP)

transcription factor genes PHABULOSA (PHB) and PHAVOLUTA (PHV) in Arabidopsis

(Bao et al., 2004).

Promoter regions of the miRNA target genes and the control gene were unmethylated in wild

type, whereas in the ΔPpDCL1b mutants the promoters of the genes encoding miRNA targets

were methylated (Figure 4D, Manuscript 1). In the latter, methylation occurred specifically

at CpG residues (Figure S7, Manuscript 1). In contrast, the promoter of the control gene

PpGNT1 remained unaffected in the mutants. Taken together, this reveals a specific

epigenetic control of genes encoding miRNA targets upon PpDCL1b dysfunction and

subsequent impeded miRNA-directed mRNA cleavage.


20

In Physcomitrella the pC3HDZIP1 and PpHB10 harbor an intron within their miRNA

binding site (Figure S8, Manuscript 1). Therefore, it is unlikely that DNA methlyation is

initiated by the formation of an miRNA:DNA hybrid. The miRNA:mRNA duplex may be

required to control the DNA methlyation. In Arabidopsis, the composition of a nucleolar

complex involved in the siRNA-directed silencing of endogenous repeat regions has been

recently identified (Bao et al., 2004). This complex combines several proteins which have

been linked to RdDM including RDR2, DCL3 and AGO4. In the yeast Schizosaccharomyces

pombe, the RITS complex containing AGO1, a chromodomain protein (Chp1) and other

proteins, was shown to bind siRNAs to direct DNA heterochromatin formation (Verdel et al.,

2004). In Physcomitrella, the detection of the miRNA:mRNA duplexes in the ΔPpDCL1b

mutant lines (Figure 4G, Manuscript 1) suggests that the miRNAs are not incorporated

into the RISC but may form a free duplex. Subsequently, this duplex guides the RITS

complex to the corresponding genomic region resulting in the initiation of DNA methylation.

1.2.1.1.5 Analysis of the ta-siRNA pathway in ΔPpDCL1b mutants To challenge the findings obtained from the transitivity and miRNA-dependent DNA

methylation the ta-siRNA pathway was analysed. In Physcomitrella, all four ta-siRNA

precursors (TAS1-4 RNAs) analyzed to date are cleaved within two distinct miRNA390

binding sites resulting in the production of ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et

al., 2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of the

ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). The abolished

miRNA390-directed cleavage of TAS4 precursor resulted the lack of ta-siRNAs in ΔPpDCL1b

mutant lines (Figure 5A and B, Manuscript 1) revealing that PpDCL1b is required to

initiate the ta-siRNA pathway. According to the findings obtained from miRNA target genes,

the lack of ta-siRNAs in the ΔPpDCL1b mutants should abolish cleavage of the EREBP/AP2

mRNA and subsequently transitive siRNAs derived from it should be missing. In agreement

with findings for miRNA target genes the level of target TAS4-RNA was down-regulated in

the ΔPpDCL1b mutants (Figure 12), and the TAS4 genomic locus was methylated in the

ΔPpDCL1b mutants but not in wild type (Figure 5D, Manuscript 1). Indeed, the mRNA

level of EREBP/AP2 was increased in the ΔPpDCL1b mutants (Figure 12) and the cognate

genomic locus was unmethylated in wild type but methylated in the ΔPpDCL1b mutants

(Figure 5D, Manuscript 1).

Figure 12: Analysis of expression levels of PpTAS4 and PpEREBP/AP2 in ΔPpDCL1b mutants and wild type. RNA blots analysis of PpTAS4 and PpEREBP/AP2. Ethidium bromide staining shown as loading control below. (Figure 5C, Manuscript 1).


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1.2.1.1.6 Analysis of ΔPpDCL1b mutants and wild type lines expressing amiR-GNT1

To check whether the mechanism of epigenetic silencing occurred in Physcomitrella wild

type, an amiRNA targeting the control gene PpGNT1 in Physcomitrella wild type as well as in

the ΔPpDCL1b mutants was used. PpGNT1-amiRNA was expressed from the Arabidopsis

thaliana miR319a precursor fused to a constitutive promoter. Transgenic Physcomitrella

lines harboring the overexpression construct showed precise processing of the PpGNT1-

amiRNA (Figure 6B, Manuscript 1). However, normalization of the PpGNT1-amiRNA

hybridization signal to the U6snRNA control revealed amiRNA expression levels which

differed between the individual lines (Figure 6B, Manuscript 1). In agreement with the

results obtained from miRNA target genes, the cleavage product of PpGNT1 in the

ΔPpDCL1b mutant background was not detect (Figure 6C, Manuscript 1), and an efficient

knock-down of the PpGNT1 gene in the plants expressing the PpGNT1-amiRNA and the

transcript level of PpGNT1 even lower in the ΔPpDCL1b mutant background (Figure 6D,

Manuscript 1). The PpGNT1 promoter was methylated in the ΔPpDCL1b mutant

background (Figure 6E and Figure S9, Manuscript 1). Also DNA methylation at the

PpGNT1 promoter in the wild type background which showed a strong expression of the

PpGNT1-amiRNA was detected whereas the PpGNT1 promoter was unmethylated in the wild

type background expressing the PpGNT1-amiRNA at a low level (Figure 6E and Figure S9,

Manuscript 1). This finding suggests that the ratio of the miRNA and its target mRNA is

crucial for the induction of DNA methylation at the target locus. At low concentrations the

miRNA might be effectively loaded into a cleavage competent RISC to direct target cleavage.

If the miRNA concentration reaches a certain threshold the RISC loading capacity for the

miRNA might be limited and excessive miRNAs form a duplex with their targets, the excess

miRNA might be loaded immediately into an effector complex such as RITS triggering

duplex formation that directs DNA methylation (Figure 7, Manuscript 1).

1.2.1.1.6.1 Specific methylation of a miRNA1026 target gene in response to the phytohormone abscisic acid (ABA)

The results obtained from the analysis of PpGNT1-amiRNA expressing lines indicated that in

Physcomitrella patens miRNAs control the expression of target RNAs at the post-

transcriptional and transcriptional level (Figure 7, Manuscript 1). Expression profiling

experiments using a Physcomitrella microarray (unpublished data) revealed an ABA-

mediated repression of a gene encoding a basic helix-loop-helix transcription factor

(PpbHLH) in wild type and was down-regulated in ΔPpDCL1b mutants (Figure 13). This


22

gene has been predicted to be targeted by the Physcomitrella miRNA1026 (Axtell et al.,

2007).

Figure 13: Expression profile of PpbHLH. Expression level of PpbHLH down-regulated in response to 10 µM ABA and in ΔPpDCL1b mutants.

RNA gel blots confirmed the down-regulation of

PpbHLH in response to ABA and corresponding

ABA-induced increase of miRNA1026 expression

levels (Figure 6G and H, Manuscript 1). In

agreement with hypothesis that miRNAs control the expression of their targets at the post-

transcriptional and transcriptional level, the PpbHLH promoter as well as intragenic regions

were found to be methylated in the plants treated with ABA (Figure 6J, Manuscript 1).

From these results, epigenetic silencing of miRNA target loci contributes to the control of

target gene expression in Physcomitrella was concluded. Although this phenomenon in

ΔPpDCL1b mutants was initially discovered, subsequent analyses of the miR1026/PpbHLH

regulon confirmed that this type of miRNA-dependent control operates also in wild type.

1.2.1.1.7 Expression profiling of transcription factor genes in ΔPpDCL1b mutant lines

Approximately 6% of the protein coding genes are considered to encode transcription factors

in Arabidopsis (Riechmann et al., 2000). In addition, more than 50 % of the predicted

miRNA target genes belong to the class of transcription factor encoding mRNAs (Rhoades et

al., 2002). In Physcomitrella patens, the comparison of the expression pattern of

transcription factor encoding genes between wild type and ΔPpDCL1b mutants will identify

putative candidate genes, which are regulated by RNAi. It is likely that more genes which are

miss-regulated in the ΔPpDCL1b mutants and direct miRNA and ta-siRNA targets were able

to be identified.

RNA from wild type and two ΔPpDCL1b mutants was hybridized on a custom Combimatrix

12K oligonucleotide microarray representing 1,427 Physcomitrella patens assembled

transcript sequences encoding more than 400 Transcription Associated Proteins (TAPs).

Corresponding gene models assigned for 1,200 assembled transcripts (Richardt, 2009). In

ΔPpDCL1b mutants, all previously analyzed miRNA targets (PpARF, PpC3HDZIP1, PpHB10

and PpSBP3) were downregulated and the ta-siRNA target gene (PpEREBP/AP2) was

upregulated when compared to wild type. I hypothesized that the downregulated genes of

transcription factors in Physcomitrella ΔPpDCL1b mutants could be putative miRNA target

genes and the upregulated ones could be ta-siRNA target genes. Clustering of expression


23

profiles showed different gene expression between ΔPpDCL1b mutant lines and wild-type

plants (Figure 14).

Figure 14: Expression profiling of genes in ΔPpDCL1b mutant lines and Wild type. Differential gene expression in ΔPpDCL1b mutant lines and wild-type plants, 213 genes which were downregulated and 273 genes upregulated in ΔPpDCL1b mutant lines (Appendix 4 and 5). MiRNA and ta-siRNA target genes supposed to be downregulated and upregulated in ΔPpDCL1b mutant lines, respectively.

46 miRNA target genes are present on the Combimatrix 12K oligonucleotide microarray.

Normalization and statistical analysis identified 20 miRNA target genes differentially

expressed within Physcomitrella ΔPpDCL1b mutant lines; the analysis revealed 13 miRNA

target genes downregulated (Table 4) and 7 miRNA target genes upregulated in ΔPpDCL1b

mutant lines. By analyzing all upregulated genes in ΔPpDCL1b mutants, the ta-siRNA target

genes were predicted using the RNA hybrid program (I. Fattash, personal communication).

The parameters used in this program are adopted from Schwab et al. (2005). In agreement

with findings for ta-siRNA target genes, the analysis revealed 19 ta-siRNA target genes

upregulated in ΔPpDCL1b mutant lines (Table 5).


24

Table 4: MiRNA target genes downregulated in ΔPpDCL1b mutant lines

MiRNAs

Target accession (Gene model)

Sequence ID (EST) Target description (Annotation)

Fold change

miR1026ab Phypa1_132150 † PP015054317R 12-oxophytodienoate reductase (OPR1) -1.5 miR1026ab Phypa1_209063 ‡ PP_12500_C1 basic helix-loop-helix (bHLH) family protein -3.2 miR166 Phypa1_116038 ‡ PP015020123R class III HD-Zip protein HB12 -1.5 miR166 Phypa1_182184 ‡ PP020016117R class III HD-Zip protein HB11 -2.0 miR166 Phypa1_184087 ‡ PP_SD_92_C1 class III HD-Zip protein HB10 -2.0 miR166 Phypa1_192868 ‡ BJ580674 class III HD-Zip protein HB14 -2.0 miR414 Phypa1_167487 ‡ PP_9369_C1 Helix-loop-helix DNA-binding -1.6 miR414 Phypa1_145753 ‡ PP_4238_C1 translation initiation factor 3 subunit 3 / eIF-3 -1.6 miR477a Phypa1_130477 ‡ PP_323_C1 Photosystem subunit V, chloroplast precursor -1.6 miR538abc Phypa1_109598 ‡ PP020062195R MADS-domain protein PPM2 -2.0 miR538abc Phypa1_94754 ‡ PP_SD_0_C1 agamous-like MADS box protein AGL1 -2.0 miR902f Phypa1_199042 † PP030015063R polyubiquitin (UBQ4), identical to GI:17677 -1.5 miR904 Phypa1_141045 ‡ PP015071162R AGO1-1 (Nicotiana benthamiana) -1.4

Table 5: Ta-siRNA target genes upregulated in ΔPpDCL1b mutant lines

Ta-

siRNAs

Target accession (Gene model)

Sequence ID (EST) Target description (Annotation)

Fold change

PpTAS2 Phypa1_160018 † PP_10130_C2 Q8H9A2 Dehydratiion responsive element binding protein 1 like protein

1.8

PpTAS1 Phypa1_188484 † PP_10320_C1 Putative nuclear DNA-binding protein G2p 1.8 PpTAS1 Phypa1_170836 † PP_13554_C1 Q9LKG4 Putative DNA binding protein. 1.3 PpTAS2 Phypa1_53217 † PP_12145_C1 Homolog of hypothetical protein sativa 1.8 PpTAS1 Phypa1_184404 † PP_13985_C1 Arabidopsis thaliana genomic DNA, 1.4 PpTAS1 Phypa1_123311 † PP_15546_C1 Q9LW84 Gb|AAF26996.1. 1.7 PpTAS2 Phypa1_61310 † PP_15997_C1 Q9SI75 F23N19.11 Hypothetical protein 3.0 PpTAS1 Phypa1_168363 † PP_17900_C1 Homolog of (AJ131113) VP1/ABI3-like protein 1.4 PpTAS1 Phypa1_175333 † PP_18393_C1 not annotated Physcomitrella patens 2.0 PpTAS3 Phypa1_203982 † PP_10621_C1 Q9FPV8 Putative methionine aminopeptidase 1.3 PpTAS1 Phypa1_13874 † PP_584_C1 scarecrow-like transcription factor 3 (SCL3) 2.1 PpTAS1 Phypa1_216494 † PP_12254_C1 Homolog of lateral suppressor protein 1.7 PpTAS1 Phypa1_165365 † PP_8332_C1 Homolog of AP2 domain, 7.8 PpTAS1 Phypa1_142162 † PP_8343_C1 Putative 2-isopropylmalate synthase 3.0 PpTAS1 Phypa1_15899† PP004007192R Q9FJ91 Dbj|BAA78737.1 AT5g52010 2.0 PpTAS1 Phypa1_138749 † PP004043210R Q9SGT9 T6H22.8.2 protein. 4.1 PpTAS1 Phypa1_167719 † PP004103024R O99018 Chloroplast protease precursor. 1.7 PpTAS3 Phypa1_79139 ‡ PP015028003R Homolog of zinc finger B-box type family 3.6 PpTAS3 Phypa1_161831 † PP020043294R Mitochondrial transcription termination factor 3.3

‡ Target validated, † Target predicted

1.2.2 Highly specific gene silencing by artificial miRNAs in Physcomitrella patens

Artificial miRNA (amiRNA) are single-stranded 21-nt small RNAs, which have been used to

downregulate single or multiple protein coding genes by guiding their cleavage based on

sequence complementarity. Their sequences are designed according to known determinants

of target selection for natural miRNAs (Schwab et al., 2005; Schwab et al., 2006). Previous

reports have shown that DNA sequences encoding Arabidopsis pre-miRNAs can be

expressed from the constitutive CaMV35S promoter in transgenic plants to produce mature


25

miRNAs. Moreover, alterations of several nucleotides within a miRNAs 21-nt sequence do

not affect its biogenesis and maturation (Vaucheret et al., 2004). These findings raise the

possibility of modifying miRNA sequences according to the determinant miRNA target

selection, such that the 21-nt specifically silence their intended target gene(s). In humans

miR30 precursor has been modified to generate an amiRNA to downregulate gene

expression by translation inhibition (Boden et al., 2004; Dickins et al., 2005). Arabidopsis

miRNA precursors have been modified to silence endogenous and exogenous target genes in

the dicotyledonous plants Arabidopsis, tomato and tobacco (Parizotto et al., 2004; Alvarez et

al., 2006; Niu et al., 2006; Schwab et al., 2006; Qu et al., 2007). Gene silencing in

monocotyledon species by amiRNAs has been reported (Warthmann et al., 2008). Previous

results have shown that artificial ta-siRNAs (ata-siRNAs) confer consistent and effective

gene silencing in Arabidopsis by engineering the TAS1c (ta-siRNAs1c) locus to silence the

FAD2 gene (de la Luz Gutierrez-Nava et al., 2008). So amiRNAs and ata-siRNAs make an

effective tool for specific gene silencing in plants.

In the moss Physcomitrella patens analysis of gene function can be carried out by the

generation of targeted gene knockout lines. However, the development of an amiRNA

expression system will be a valuable alternative to speed up such analyses. As a proof of

concept two amiRNAs, targeting the gene PpFtsZ2-1, which is indispensable for chloroplast

division (Strepp et al., 1998), and the gene PpGNT1 encoding an N-

acetylglucosaminyltransferase (Koprivova et al., 2003) were designed.

Both amiRNAs were expressed from the Arabidopsis thaliana miR319a precursor fused to a

constitutive promoter (Figure 1A, Publication 1). Based on the conservation of the miR319

family among land plants and similar secondary structures of miR319 precursor transcripts

from Arabidopsis and Physcomitrella (Figure 1B, Publication 1), the PpFtsZ2-1-amiRNA

and PpGNT1-amiRNA were correctly processed from the Arabidopsis miR319a precursor.

Transgenic Physcomitrella lines harboring the overexpression constructs showed precise

processing of the amiRNAs and an efficient knockdown of the cognate target mRNAs (Figure

1D and 2A, Publication 1). Furthermore, chloroplast division was impeded in PpFtsZ2-1-

amiRNA lines which phenocopied PpFtsZ2-1 knockout mutants (Figure 15). The formation of

macrochloroplasts in the PpFtsZ2-1-amiRNA lines was observed in all tissues and cells

analyzed indicating an efficient production of mature amiRNAs from constitutively

expressed precursor transcripts. To investigate the possibility of transitivity, sense and

antisense oligonucleotides derived from a PpFtsZ2-1 and PpGNT1 mRNA regions

downstream the amiRNA recognition site were used for RNA gel blot analysis.

Sense and antisense siRNAs were only detected in PpFtsZ2-1-amiRNA and PpGNT1-

amiRNA lines, but not in wild type (Figure 2B and C, Publication 1). Additionally, these


26

siRNAs do not seem to have a major effect on sequence-related mRNAs, confirming

specificity of the amiRNA approach.

Figure 15: Impeded plastid divison and formation of macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors. A, Light microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (Size bars: 100 µm). B, Confocal laser scanning microscopy from protonema and leaves of wild type (WT) and one PpFtsZ2-1-amiRNA overexpression line (Size bars: 50 µm). Red: chlorophyll autofluorescence in plastids. (Figure 3, Publication 1).

Chapter I Conclusion

27

1.3 Conclusion

These findings reveal the existence of novel RNAi pathways in Physcomitrella patens. As

opposed to Arabidopsis thaliana, the miRNA-directed posttranscriptional control of target

mRNAs in Physcomitrella patens is amplified by transitive siRNAs. Furthermore, we

identified a pathway that depends on miRNA:targetRNA duplexes and triggers the silencing

of genes encoding miRNA targets. Consequently, ΔPpDCL1b mutants deficient in miRNA

target cleavage are not viable in some plants and animals. In contrast, Physcomitrella

ΔPpDCL1b mutant lines are viable, although severely affected in several cellular features and

in development. From that a model for gene-specific sensing of the levels of specific miRNAs

and their target-RNAs (by miRNA:mRNA or miRNA:TAS-RNA duplex formation) was

proposed, effective (or ineffective) target cleavage, and subsequent epigenetic control of

target-RNA accumulation (Figure 16).

In summary, the conclusions are:

1- PpDCL1a is the functional equivalent of Arabidopsis AtDCL1 (miRNA biogenesis).

2- PpDCL1b is essential for miRNA target cleavage (including the ta-siRNA pathway).

3- In Physcomitrella, amplification of miRNA and ta-siRNA signals by transitive siRNAs is a

common mechanism.

4- The accumulation of miRNAs and their cognate RNA targets in the ΔPpDCL1b mutants

causes a specific hypermethylation of the corresponding genomic loci.

5- MiRNAs induce a specific epigenetic silenicng of miRNA target genes which depends on

the miRNA:target ratio and is mediated by the formation of stable miRNA:RNA duplexes.

6- The expression of amiRNAs in Physcomitrella leads to an efficient silencing of their target

mRNAs comparable to the effects of targeted gene knockouts.

7- The amplification of the initial amiRNA signal by secondary transitive siRNAs, these

siRNAs do not have a major effect on highly conserved gene families, confirming specificity

of the amiRNA approach in Physcomitrella.

Chapter I Conclusion

28

Figure 16: Model for the post-transcriptional and epigenetic control of miRNA target genes in Physcomitrella patens. (A) Pathways leading to miRNA target cleavage. The maturation of miRNAs from stem-loop precursors is catalyzed by PpDCL1a. PpDCL1b is required for loading miRNAs into cleavage competent RISC. After loading of miRNAs into RISC (consisting of PpDCL1b, AGO and unknown proteins) transient miRNA:target-RNA duplexes form based on sequence complementarity. Subsequently, target-RNAs are cleaved. From the cleavage products dsRNA is produced by the action of RdRP. Subsequently, the dsRNA is processed to generate transitive siRNAs (from mRNA cleavage products) or ta-siRNAs (from TAS-RNAs).Transitive siRNAs lead to an amplification of the miRNA trigger; ta-siRNAs are directed to their mRNA targets guiding their cleavage. (B) Epigenetic control of miRNA target genes. In the ΔPpDCL1b mutant lines miRNAs are not loaded into cleavage competent RISC and target cleavage is abolished. Also in Physcomitrella patens wild type miRNAs can accumulate which cannot be loaded efficiently into RISC. In both cases miRNAs may be loaded into alternative complexes such as the RITS complex and targeted to cognate target-RNAs. These miRNA:RNA duplexes bound by RITS enter the nucleus and initiate DNA methylation at complementary genomic loci. The RITS complex expands into adjacent regions (e. g. promoters) and completes CpG methylation of the entire genomic locus. In consequence, genomic loci are silenced and accumulation of mRNAs is inhibite

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Zhang, H., F. A. Kolb, et al. (2004). "Single processing center models for human Dicer and bacterial RNase III." Cell 118(1): 57-68.

Zilberman, D., X. Cao, et al. (2003). "ARGONAUTE4 control of locus-specific siRNA accumulation and DNA and histone methylation." Science 299(5607): 716-9.

Chapter II Transcriptional control of gene expression by microRNAs

34

2 Chapter II: Manuscript 1

Transcriptional control of gene expression by microRNAs

Own contribution:

Carried out all molecular and phenotypic analyes of the ΔPpDCL1b mutants, generated and

analyzed the amiR-GNT1 overexpression lines, and performed the complete analysis of the

miR1026 and its PpbHLH target. Writing parts of the manuscript and preparing the figures.

The work was supervised by W. Frank


Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef

Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*

1Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-

79104 Freiburg, Germany

2Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-

72076 Tübingen, Germany

3Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of

Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany

4Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,

D-79104 Freiburg, Germany

* Corresponding author

Phone: +49 (0)761-203-2820

Fax: +49 (0)761-203-6945

Email: [email protected]

1

Summary

MicroRNAs (miRNAs) control gene expression in animals and plants. They share with

another class of small RNAs, siRNAs, the ability to post-transcriptionally affect target

mRNAs. In contrast to siRNAs, however, the role of miRNAs in transcriptional

regulation has been less clear. Here we reveal dual transcriptional and post-

transcriptional activities of miRNAs in Physcomitrella patens. In plants lacking activity

of one DICER-LIKE gene (PpDCL1b), miRNA target genes are silenced. The specific

function of PpDCL1b in miRNA-mediated target cleavage suggests that changes in the

ratio of the miRNA and its targets cause miRNA:target-RNA duplex formation, which in

turn triggers DNA methylation. We propose that miRNA-mediated transcriptional

silencing, which also occurs in wild type plants, provides a mechanism critical for

homeostasis of miRNA-dependent gene expression.

Introduction

Small RNAs (sRNAs) are important regulators of post-transcriptional and transcriptional

gene expression (Meister and Tuschl, 2004). In plants, microRNAs (miRNAs), which

are produced from hairpin-like precursor transcripts, are also required for the

biogenesis of trans-acting small interfering RNAs (ta-siRNAs). Both miRNAs and ta-

siRNAs regulate mRNA stability and translation, siRNAs, which originate from perfectly

double-stranded RNA (dsRNA) precursors post-transcriptionally silence transposons,

viruses and transgenes and are important for the establishment and maintenance of

cytosine DNA methylation (Baulcombe, 2004). Even though the role of plant siRNAs in

the methylation of cognate genomic loci is well understood (Matzke et al., 2007),

evidence for a similar function of miRNAs in directing DNA methylation is limited. The

biogenesis of sRNAs from dsRNA is catalyzed by Dicer proteins and the size of the

Dicer gene family varies between organisms, reflecting different degrees of

specialization of Dicer proteins. For example, in D. melanogaster Dcr1 produces

2

miRNAs from hairpin precursors, whereas Dcr2 generates siRNAs from dsRNA

molecules (Tomari and Zamore, 2005). By contrast, in C. elegans the single Dicer

protein DCR-1 is directed by accessory proteins such as PIR-1, ER-1 and RRF-3 to

produce sRNAs from different dsRNA triggers (Duchaine et al., 2006). Besides their

function in dicing dsRNA, animal Dicers are associated with accessory proteins in

complexes which act as RISC (RNA-induced silencing complex) or RISC loading

complexes (Doi et al., 2003; Pham et al., 2004). Thus, Dicer proteins are also essential

components in the executive phase of RNAi, indicating that miRNA/siRNA processing

and target RNA cleavage are coupled. Dcr-2 from Drosophila, which produces siRNA,

acts together with its partner R2D2 to load one of the two siRNA strands into RISC(Liu

et al., 2003; Tomari et al., 2004). Similarly, human Dicer associated with Ago2, TRBP

and RHA acts in RISC assembly (MacRae et al., 2008) which is further supported by

the observation that siRNAs cannot cause post-transcriptional gene silencing in human

cells lacking Dicer (Doi et al., 2003).

In the plant A. thaliana, the four DCL proteins (AtDCL1-4) act in specific sRNA

pathways, with some functional redundancies of the four isoforms (Gasciolli et al.,

2005; Henderson et al., 2006). The maturation of miRNAs from imperfect RNA

foldbacks relies on AtDCL1 activity. In consequence, A. thaliana dcl1 mutants have

significantly reduced miRNA levels and a corresponding increase in target mRNA

levels, which causes a multitude of developmental defects (Golden et al., 2002; Park et

al., 2002). AtDCL2 mediates the generation of siRNAs from exogenous RNA sources

(Xie et al., 2004), AtDCL3 is required for the formation of heterochromatin-associated

endogenous siRNAs (Herr et al., 2005; Xie et al., 2004) and AtDCL4 is needed for the

formation of ta-siRNAs involved in systemic cell-to-cell transmission of silencing signals

(Dunoyer et al., 2005; Xie et al., 2005).

The genome of the moss Physcomitrella patens encodes four DCL proteins

(Axtell et al., 2007). PpDCL1a and PpDCL1b are very similar to AtDCL1 (Figure S1).

3

PpDCL3 and PpDCL4 proteins are orthologs of AtDCL3 and AtDCL4, whereas an

AtDCL2 ortholog is lacking. The primary PpDCL1a transcript harbors a miRNA

precursor within one intron, which is reminiscent of AtDCL1, and suggests a conserved

autoregulatory control of mRNA maturation (Axtell et al., 2007). Together with the

slightly greater sequence similarity, this led us to hypothesize that PpDCL1a as the

functional equivalent of AtDCL1 is required for miRNA biogenesis, while the additional

presence of PpDCL1b suggested also potential differences in sRNA pathways between

P. patens and A. thaliana. Here, we present an analysis of P. patens ΔPpDCL1a and

ΔPpDCL1b knockout mutants, which supports differences in sRNA pathways such as

the formation of transitive siRNAs. Moreover, we propose a mechanism for the miRNA-

mediated transcriptional silencing of miRNA target genes that relies on miRNA

abundance, formation of miRNA:target-RNA duplexes and DNA methylation.

Results

Requirement of PpDCL1a for miRNA biogenesis

Taking advantage of the efficient homologous recombination system in P. patens

(Strepp et al., 1998) we generated two PpDCL1a knockout mutants (ΔPpDCL1a)

(Figure S2). Complete loss of PpDCL1a function resulted in retarded growth and

developmental disorders including abnormalities in cell size, and cell shape. The

mutants were arrested at the filamentous protonema stage and did not form

gametophores (Figure 1A and 1B).

To test whether miRNA biogenesis is affected in the ΔPpDCL1a mutants, we

analyzed the accumulation of miR156, 160, 166, and 390 (Arazi et al., 2005; Fattash et

al., 2007). Due to the limited amount of plant material of the ΔPpDCL1a mutant lines,

miRNA expression was investigated by RT-PCR (Varkonyi-Gasic et al., 2007). PCR

products were sequenced to rule out unspecific amplification. Compared to wild type,

4

miR156, 160 and 166 had drastically reduced levels, while miR390 was undetectable in

ΔPpDCL1a mutants (Figure 1C). In P. patens, all known trans-acting siRNA (ta-siRNA)

precursors (PpTAS1-4 RNAs) are initially cleaved at two distinct miR390 target sites.

Subsequently, dsRNAs are generated from the cleavage products and processed in a

phased manner to generate ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al., 2006).

Two ta-siRNAs (pptA079444 processed from PpTAS1, and pptA013298 processed

from PpTAS3) (Axtell et al., 2006) were detected in wild type, but were absent in the

ΔPpDCL1a mutants, indicating that the lack of miR390 abolishes ta-siRNA production

(Figure 1D). To test whether reduced levels of miRNAs result in elevated transcript

levels of miRNA targets, as observed in A. thaliana dcl1 mutants, we analyzed the

expression of the miRNA targets PpSPB3 for miR156 (Arazi et al., 2005),

PpC3HDZIP1 and PpHB10 for miR166 (Axtell et al., 2007; Floyd and Bowman, 2004),

PpARF for miR160 (Fattash et al., 2007), and PpTAS1 for miR390(Axtell et al., 2006).

RT-PCR analysis revealed increased transcript levels of all analyzed miRNA targets in

the ΔPpDCL1a mutants (Figure 1E). From these results we conclude that PpDCL1a is

the major P. patens DCL protein required for the processing of miRNAs and thus the

functional equivalent of A. thaliana DCL1.

Requirement of PpDCL1b for miRNA guided target cleavage

The presence of PpDCL1b as a second P. patens AtDCL1 homolog raised the question

whether PpDCL1b acts redundantly with PpDCL1a, given that some miRNAs were not

completely abolished in ΔPpDCL1a mutants. We generated four targeted PpDCL1b

knockout mutants (ΔPpDCL1b) (Figure S3). The PpDCL1b mutants were strongly

affected in cell division, growth polarity, cell size, cell shape and tissue differentiation

(Figure 2A and Figure S4A). The developmentally arrested mutants produced only a

small number of malformed gametophores (Figure 2B and Figure S4B). Thus, while

5

both ΔPpDCL1a and ΔPpDCL1b mutants suffer from severe developmental defects,

the exact mutant phenotypes differed.

Analysis of six different miRNAs, miR156, 160, 166, 390, 535, and 538(Arazi et

al., 2005; Fattash et al., 2007), revealed that their levels were unchanged in ΔPpDCL1b

mutants (Figure 2C-2E). Thus, PpDCL1b is not essential for miRNA maturation from

precursor RNAs. The severe developmental defects of the mutants prompted us

nevertheless to examine miRNA targets PpSPB3, PpC3HDZIP1, PpHB10, and PpARF.

We verified cleavage of these miRNA targets in P. patens wild type based on 5’ RACE,

cDNA cloning and sequencing (Figure 3A). An unrelated mRNA (PpGNT1) (Koprivova

et al., 2003), which is not miRNA regulated, was examined as control (Figure 3A).

Although the ΔPpDCL1b mutants produced apparently normal levels of miRNAs, the

miRNA target transcripts were not cleaved (Figure 3A), indicating a surprising

requirement of PpDCL1b for miRNA-guided mRNA cleavage. We propose that

PpDCL1b may act in loading miRNAs into an RNA-cleavage competent RISC, in

analogy to what has been reported for animal Dicer proteins (Doi et al., 2003; Liu et al.,

2003; MacRae et al., 2008; Pham et al., 2004; Tomari et al., 2004).

Generation of transitive siRNA triggered by miRNA-guided transcript cleavage

In P. patens wild type we had not only detected 5’ RACE products resulting from

miRNA-guided cleavage of the target mRNA, but also a variety of shorter and longer

products (Figure 3A). In analogy with other plant systems, where targeting of a

transcript with dsRNA-derived siRNAs or multiple miRNAs (Axtell et al., 2006; Howell et

al., 2007; Vaistij et al., 2002) causes the production of secondary siRNAs, the miRNA

cleavage products may serve as templates for synthesizing cRNA by RNA-dependent

RNA polymerase (RdRP). Subsequently, the resulting dsRNA may be processed into

secondary siRNAs resulting in spreading of the initial trigger signal (Figure 3B). In

6

flowering plants, this phenomenon, known as transitivity, is, however, rarely observed

after targeting of an mRNA with a single miRNA (Axtell et al., 2006; Howell et al.,

2007).

To investigate the possibility of transitivity in P. patens, we asked whether one

could synthesize cDNA from both the sense and antisense strand of miRNA target

mRNAs (PpARF and PpC3HDZIP1). Indeed, this was the case in wild type, indicating

the presence of dsRNA. Such dsRNA molecules were lacking in ΔPpDCL1b mutants

(Figure 3C). To determine whether transitive siRNAs were generated from these

dsRNAs, we performed small RNA blots using probes for both upstream and

downstream sequences relative to the miRNA targeting site. Such small RNAs

corresponding to sense and antisense strands of PpARF and PpC3HDZIP1 mRNAs

were detected in wild type, but not in ΔPpDCL1b mutants (Figure 3D). Thus, in P.

patens transitive siRNAs arise from regions upstream as well as downstream of the

miRNA targeting motif after miRNA-directed cleavage of mRNAs. These siRNAs most

likely cause cleavage of the cognate mRNAs at additional sites, which explains why we

observed additional mRNA fragments in the 5’ RACE analyses. We did not detect such

siRNAs in the ΔPpDCL1b mutants, nor for the control mRNA (PpGNT1) in wild type

(Figure 3C and 3D), indicating that the generation of transitive siRNAs depends on

PpDCL1b and is specific for mRNAs subject to miRNA-directed cleavage.

DNA methylation of miRNA target loci in ΔPpDCL1b mutants

A. thaliana ago1 and dcl1 mutants are defective in miRNA-directed target cleavage or

miRNA biogenesis, respectively. Consequently, transcript levels of miRNA targets are

elevated in both mutants (Ronemus et al., 2006). Conversely, all miRNA targets

analyzed had reduced transcript levels in ΔPpDCL1b mutants (Figure 4A and 4B),

even though they were not cleaved. As explanation for these unexpected findings we

7

considered the possibility that miRNA target loci are under epigenetic control in P.

patens. Since methylation of cytosine residues is the most prominent mechanism for

transcriptional silencing in plants and other eukaryotes (Bender, 2004), we evaluated

this scenario by methylation-specific PCR of four miRNA target loci, along with an

unrelated locus, PpGNT1 (Figure 4C-4F and Figure S5 and Figure S6). Promoters of

all five genes were unmethylated in wild type, but in ΔPpDCL1b mutants the four

miRNA target promoters were methylated (Figure 4D). These findings were confirmed

by sequencing the PCR products of the PpARF promoter from wild type and

ΔPpDCL1b mutants. In the latter, methylation occurred specifically at CpG residues

(Figure S7). Taken together, we conclude that disruption of PpDCL1b causes specific

epigenetic changes in genes encoding miRNA targets, and that this is accompanied by

a loss of miRNA-directed mRNA cleavage.

It is well-known that siRNA pathways govern DNA methylation in A. thaliana,

e.g. at repeat-associated loci (Herr et al., 2005). However, only one study has

suggested a function of miRNAs in the silencing of cognate target genes, at the PHB

and PHV loci, which are targeted by miR165/166. Normally methylated DNA

sequences downstream of the miRNA complementary motif became hypomethylated in

plants with dominant alleles of PHB and PHV, while the promoters remained

unmethylated (Bao et al., 2004). The dominant alleles carry mutations in the miRNA

targeting motif, such that the encoded mRNAs are no longer susceptible to miRNA-

guided cleavage.

Like PHB and PHV, the P. patens HD-ZIP homologs PpC3HDZIP1 and PpHB10

harbor an intron within their miRNA binding site (Figure S8), whereas the miRNA

targeting motif in PpARF is not disrupted by an intron. Similar to the promoters,

PpC3HDZIP1 and PpARF sequences flanking the miRNA targeting motif as well as the

8

intron disrupting the miR166 binding site of PpC3HDZIP1 were CpG methylated in

ΔPpDCL1b mutants, but not in P. patens wild type (Figure 4E and 4F and Figure S7).

One scenario that can account for the findings presented so far is that

PpDCL1b normally acts in loading miRNAs into an RNA-cleavage competent RISC. In

the absence of PpDCL1b, miRNAs might be loaded instead into an RNA-induced

transcriptional silencing complex (RITS) directing DNA methylation of miRNA target

loci. As the sequence of the miR166 binding site is disrupted by introns in two genes

(PpC3HDZIP1 and PpHB10), it is unlikely that their methylation in ΔPpDCL1b mutants

is initiated by the formation of miRNA:DNA hybrids. Instead, the miRNA-loaded RITS

complex might interact with the target mRNA, resulting in the formation of a stable

miRNA:mRNA duplex. Subsequently, this duplex could guide the RITS complex to the

corresponding genomic region, resulting in the initiation and spreading of DNA

methylation.

If stable miRNA:mRNA duplexes are present in ΔPpDCL1b mutants, it should

be possible to synthesize cDNA without added exogenous primers, which can

subsequently be detected by conventional PCR. In support of such a scenario we

obtained RT-PCR products for all miRNA targets examined, but not for a control locus

in ΔPpDCL1b mutants. No such products were obtained with RNA from wild type plants

(Figure 4G). In addition, in the ΔPpDCL1b mutants no PCR products were obtained

when using PCR primers located downstream of the miRNA targeting site. As a further

control, we heated the RNA samples prior to cDNA synthesis. This should lead to

denaturation of a miRNA:mRNA complex and hence eliminate priming; indeed, this

procedure prevented the amplification of PCR products in the ΔPpDCL1b mutants

(Figure 4H). These results are compatible with base-paired miRNA:mRNA duplexes

being present specifically in RNA samples from ΔPpDCL1b mutants.

9

To further scrutinize our hypotheses of transitivity and miRNA-dependent DNA

methylation, we analyzed the ta-siRNA pathway in ΔPpDCL1b mutants. After miR390-

mediated cleavage of TAS precursors, the RNA cleavage products are converted into

dsRNA and further processed into ta-siRNAs (Axtell et al., 2006; Talmor-Neiman et al.,

2006). The mRNA encoding an EREBP/AP2 transcription factor is targeted by one of

the ta-siRNAs derived from the TAS4 precursor (Talmor-Neiman et al., 2006). Hence,

the production of ta-siRNAs presents an intermediate step in the miRNA-dependent

control of mRNAs. TAS4 RNA cleavage products resulting from miRNA390-directed

cleavage were detected by 5’ RACE-PCR in P. patens wild type, but not in the

ΔPpDCL1b mutants (Figure 5A), even though miR390 was present in equal amounts in

ΔPpDCL1b mutants and wild type (Figure 2C). Furthermore, ta-siRNAs of both sense

and antisense orientation were present in wild type, but were undetectable in

ΔPpDCL1b mutants (Figure 5B), confirming that PpDCL1b is required to initiate the ta-

siRNA pathway.

In agreement with our findings for other miRNA targets, TAS4 transcript levels

were reduced in ΔPpDCL1b mutants (Figure 5C). Likewise, the TAS4 genomic locus

was methylated only in ΔPpDCL1b mutants (Figure 5D). If, similar to miRNAs, ta-

siRNA-mediated cleavage of target mRNAs also initiates the generation of transitive

secondary siRNAs, the lack of ta-siRNAs in ΔPpDCL1b mutants should abolish both

cleavage of EREBP/AP2 mRNA and transitive siRNAs. Consistent with this scenario,

the EREBP/AP2 mRNA was cleaved in wild type, but not in ΔPpDCL1b mutants (Figure

5A), and only wild type produced EREBP/AP2 mRNA-derived siRNAs in sense and

antisense orientation (Figure 5B). This observation indicates that the siRNA-dependent

amplification of target RNA degradation that was initially triggered by ta-siRNA/miRNA-

guided cleavage is a common mechanism in P. patens. In addition, we expected that,

in contrast to direct miRNA targets, EREBP/AP2 mRNA levels should be elevated in

10

ΔPpDCL1b mutants, as stable ta-siRNA:mRNA duplexes that could guide DNA

methylation at the corresponding genomic locus should be absent. Indeed, the

EREBP/AP2 RNA levels were increased in ΔPpDCL1b mutants (Figure 5C), and the

genomic locus was methylated neither in wild type nor in ΔPpDCL1b mutants (Figure

5D).

Dependence of DNA methylation at miRNA target loci on miRNA expression

levels

We propose that the formation of stable miRNA:target RNA duplexes leads to

methylation of the corresponding genomic regions in ΔPpDCL1b mutants. Is this

mechanism of epigenetic silencing also relevant in wild type P. patens? To investigate

this question, we generated wild type and ΔPpDCL1b mutant plants expressing

different levels of an artificial miRNA targeting our control gene PpGNT1.

Artificial miRNAs (amiRNAs) can be generated by exchanging the

miRNA/miRNA* sequence of endogenous miRNA precursor genes, while maintaining

the general pattern of matches and mismatches in the foldback. We engineered an

amiRNA against PpGNT1 into the A. thaliana miR319a precursor (Khraiwesh et al.,

2008) and expressed the hybrid construct in P. patens wild type and ΔPpDCL1b

mutants (Figure 6A). RNA blots confirmed precise maturation of amiR-GNT1 in

transformed lines, independent of expression level (Figure 6B). The expected PpGNT1

mRNA cleavage products were present in wild type, but not in ΔPpDCL1b mutants

(Figure 6C). Consequently, compared to P. patens wild type the PpGNT1 transcript

levels were reduced in amiR-GNT1 plants (Figure 6D). Despite abolished amiRNA-

directed cleavage of PpGNT1 mRNA, transcript levels were even lower in the

ΔPpDCL1b mutant background (Figure 6D).

11

Consistent with our model of miRNA-dependent epigenetic silencing, the

PpGNT1 promoter was methylated in the ΔPpDCL1b mutant background (Figure 6E

and Figure S9). Importantly, the PpGNT1 promoter was also methylated in wild type

lines, with strong expression of the amiR-GNT1, while it was unmethylated in lines with

low levels of amiR-GNT1 (Figure 6E and Figure S9). Thus, specific methylation of

miRNA target loci is not limited to ΔPpDCL1b mutants. Based on the observation that

methylation in wild type is miRNA-dosage dependent, we hypothesized that the ratio of

the miRNA and its target mRNA is crucial for the induction of DNA methylation at the

target locus. If the miRNA concentration exceeds a certain threshold, the miRNA may

either interact directly with its target and the duplex might then be recruited into a DNA

methylation silencing complex, or the excess miRNA might be loaded immediately into

an effector complex such as RITS triggering duplex formation that directs DNA

methylation. We obtained supporting evidence for the expected amiR-GNT1:PpGNT1-

mRNA duplexes by cDNA synthesis without exogenous primers and subsequent PCR

in ΔPpDCL1b mutants. Importantly, we could amplify such products also in a wild type

line with high levels of amiRNA expression, but not in a wild type line expressing only

moderate amounts of amiR-GNT1 (Figure 6F).

Hormone-dependent DNA methylation of a miR1026 target locus

The analysis of amiRNA-GNT1 lines had shown that miRNA-directed epigenetic

silencing occurs also wild type, but that it is dependent on miRNA levels. We therefore

sought to identify endogenous miRNAs that might be induced to high levels in

response to specific stimuli, which in turn should be reflected by downregulation of

target mRNAs. In separate experiments we had found that treatment with the hormone

abscisic acid (ABA) strongly represses expression of a basic helix-loop-helix (bHLH)

transcription factor gene, PpbHLH, which has been predicted to be targeted by

12

miR1026 (Axtell et al., 2007). ABA is a well known signaling molecule in abiotic stress

signaling pathways in plants including mosses (Frank et al., 2005b). RNA gel blots

confirmed downregulation of PpbHLH in response to ABA (Figure 6G). This effect

correlated well with an ABA-induced increase of miR1026 levels (Figure 6H),

suggesting direct regulation of PpbHLH by miR1026. We confirmed miR1026-mediated

cleavage of PpbHLH transcript by 5’ RACE (Figure 6I).

To evaluate transcriptional effects of miR1026, we analyzed DNA methylation of

the PpbHLH gene, including the promoter and transcribed sequences. Upon ABA

treatment, PpbHLH became methylated at specific CpG sites (Figure 6J and Figure

S10), consistent with the methylation patterns we had found before in ΔPpDCL1b

mutants. The promoter of an unrelated gene, PpGNT1, was unmethylated regardless

of ABA treatment (Figure 6J).

Our model posits that DNA methylation will be initiated if the miRNA target ratio

exceeds a certain threshold. That DNA methylation of the PpbHLH locus is not

quantitative, as deduced from the observation that unmethylation-specific primers

allowed albeit inefficient PCR amplification in ABA-treated samples, may reflect cell

type-specific differences in miRNA or target expression levels. Finally, we tried to

obtain evidence for stable miR1026:PpbHLH-mRNA duplexes by unprimed RT-PCR.

Consistent with the DNA methylation status, such duplexes were only found in the

ABA-treated samples (Figure 6K).

We conclude that in P. patens, epigenetic silencing of miRNA target loci

contributes to the control of target gene expression. Although we initially discovered

this phenomenon in ΔPpDCL1b mutants, subsequent analyses of the

miR1026/PpbHLH regulon confirmed that this type of miRNA-dependent control

operates also in wild type.

13

Discussion

Our studies suggest that PpDCL1a is the functional ortholog of AtDCL1 required for

miRNA and ta-siRNA biogenesis. Even though PpDCL1b shares a similar level of

sequence identity with AtDCL1, we propose that it has a distinct function, since its

inactivation does not affect miRNA biogenesis, but abolishes miRNA-directed target

cleavage. It is unlikely that PpDCL1b directly cleaves target RNAs, as AGO proteins in

RISC are the catalytic enzymes in sRNA-dependent target cleavage (MacRae et al.,

2008). Biochemical analysis of AGO1 complexes immunoprecipitated from Arabidopsis

dcl1-7, dcl2-1 and dcl3-1 mutants provided evidence for distinct functional properties.

An AGO1 complex extracted from dcl1-7 mutants was not able to cleave RNA targets

due to the lack of ~21 nt small RNA accumulation in this mutant. In contrast, cleavage

of RNA targets was not affected in AGO1 complexes from dcl2-1 and dcl3-1 mutants

(Qi et al., 2005). Furthermore, purification of Arabidopsis AGO1 revealed a ~160 kDa

complex, most likely only consisting of AGO1 and associated sRNA (Baumberger and

Baulcombe, 2005). Thus, there is so far no evidence to support a function of plant DCL

proteins in sRNA-mediated target cleavage. In contrast, studies in animals have shown

that Dicer proteins are part of the RNA loading complex (RLC), which loads sRNAs into

RISC. Human RLC comprises the proteins Ago2, Dicer and TRBP and the purified

protein components assemble spontaneously in vitro without requirement of any

cofactors. The reconstituted RLC is fully functional and once Ago2 is loaded with a

miRNA it tends to dissociate from the rest of the complex (MacRae et al., 2008).

Similarly, Dcr-2 from D. melanogaster, which produces siRNA, acts in the RISC

assembly together with its partner R2D2 by loading one of the two siRNA strands into

RISC (Liu et al., 2003; Tomari et al., 2004). The C. elegans homolog of this protein,

RDE-4, was also found to interact with Dicer (Tabara et al., 2002). Given this particular

function of animal Dicer proteins, we hypothesize that P. patens PpDCL1b may exhibit

14

an equivalent function in loading miRNAs into RISC, making it indispensable for

miRNA-directed target cleavage.

Arabidopsis dcl1 and ago1 mutants, which are affected in miRNA biogenesis or

miRNA-directed target cleavage, respectively, exhibit elevated transcript levels of

miRNA targets (Ronemus et al., 2006). Likewise, miRNA target transcripts are

increased in ΔPpDCL1a mutants due to the lack of miRNAs. In contrast, levels of

miRNA target mRNAs are drastically reduced in ΔPpDCL1b mutants, in spite of

abolished target RNA cleavage. We have shown that cytosine residues within the

corresponding genomic loci are methylated in ΔPpDCL1b mutants, suggesting

epigenetic control at the transcriptional level. Small RNAs initiate transcriptional

silencing of homologous sequences by methylation of cytosine residues at CpG,

CpNpG, and CpHpH sequence motifs or by histone modifications (Bender, 2004; Cao

and Jacobsen, 2002). In all genomic regions analyzed in the ΔPpDCL1b mutants, we

only detected methylation at CpG dinucleotides, but cannot exclude that cytosine

methylation may also occur at different sequence contexts in other regions. Moreover,

we detected CpG methylation in large regions of the genomic loci encoding miRNA

targets including introns, exons and promoter regions pointing to methylation that is

able to spread over considerably long distances.

Although spreading of siRNA-directed DNA methylation into adjacent non

repeated sequences is not common in A. thaliana, siRNA-mediated spreading of DNA

methylation has been observed for the SUPPRESSOR OF drm1 drm2 cmt3 (SDC)

locus, where methylation spreads beyond siRNA generating repeat regions present in

the SDC promoter (Henderson and Jacobsen, 2008). In Arabidopsis, cytosine

methylation can also spread in the PHV and PHB genes, which are targets of

miR165/166 (Bao et al., 2004). In both genes, the miR165/166 complementary motif is

disrupted by an intron and the coding sequence was found to be heavily methylated

15

downstream of the miRNA complementary site in differentiated, but not undifferentiated

cells of wild type plants. Furthermore, methylation was reduced in phv-1d and phb-1d

mutants, which have an altered miRNA recognition motif or a mutation in the intron

splice donor sequence, suggesting that miR165/166 needs to bind to nascent PHV and

PHB transcripts to trigger gene silencing (Bao et al., 2004).

Similarly, the P. patens HD-Zip genes PpC3HDZip1 and PpHB10 are targeted

by miR166 and the miR166 binding sites are only reconstituted after splicing of an

intron from the primary transcripts. These loci are hypermethylated in ∆PpDCL1b

mutants, but not in wild type, suggesting that the initiation of CpG methylation upon

defective target cleavage cannot be mediated by miR166, but involves binding of

miR166 to the cognate target mRNAs. We have obtained evidence for the presence of

stable duplexes of a miRNA and its target RNA in ∆PpDCL1b mutants. We propose

that such duplexes guide a DNA modification complex.

In Arabidopsis, RNA-directed DNA methylation (RdRM) by siRNAs requires

RDR2, DCL3 and RNA PolIVa, which are all involved in siRNA biogenesis (Herr et al.,

2005; Kanno et al., 2005; Onodera et al., 2005; Pontier et al., 2005; Xie et al., 2004),

whereas AGO4, DRM2, DRD1 and RNA PolIVb are indispensable for DNA methylation

(Cao and Jacobsen, 2002; Kanno et al., 2005; Zilberman et al., 2004). In fission yeast,

RNA-directed heterochromatic gene silencing at centromeres relies on two different

complexes, the RITS complex comprising Ago1, Chp1 and TAS3, and the argonaute

siRNA chaperone complex (ARC) comprising Ago1, Arb1 and Arb2. However, these

complexes are required to direct histone H3 Lys9 methylation, but do not direct

cytosine methylation. Nevertheless, it has been proposed that their action involves the

recognition of nascent transcripts by RITS-bound siRNAs to promote recruitment of

chromatin-modifying enzymes that implement silencing (Buker et al., 2007).

We also detected the specific silencing of miRNA target genes in P. patens wild

type, where the expression of amiR-GNT1 caused methylation of the PpGNT1 genomic

16

locus. Moreover, we found that methylation of the locus is dependent on amiR-GNT1

abundance and only obtained evidence for amiR-GNT1:PpGNT1-mRNA duplexes in

lines with high amiRNA levels, supporting the hypothesis that miRNA:target-RNA

duplexes are required for DNA methylation. Finally, we have been able to show that the

genomic region of the miR1026 target PpbHLH becomes methylated in response to

ABA, which upregulates miR1026 expression. Also in this case, DNA methylation was

miR1026 dosage-dependent and appeared to correlate with the formation of stable

miR1026:PpbHLH-mRNA duplexes. As ABA acts as a mediator of abiotic stress

signaling, we assume that the miR1026-regulated silencing of PpbHLH is part of stress

adaptation in P. patens.

In plants, epigenetic changes as a response to stress conditions have been

previously shown to include DNA methylation, histone modifications and chromatin

remodeling (Boyko and Kovalchuk, 2008; Dyachenko et al., 2006; Henderson and

Dean, 2004). Our analysis of the miR1026:PpbHLH regulon suggests that miRNAs

may act in the epigenetic control of stress-responsive genes in plants.

Taken together, we propose that silencing of genomic loci can be triggered by

stable duplexes of a miRNA and its target RNA, which can be either an mRNA or a

primary TAS transcript. The epigenetic control of genes encoding miRNA target RNAs

discovered in P. patens presents a new mechanism that affects the homeostasis of

miRNA-regulated RNAs (Figure 7). The specific equilibrium of a cleavage-competent

RISC and a DNA-modifying RITS loaded with the same miRNA may determine the

relative contribution of both pathways to miRNA-mediated downregulation of gene

expression. In addition, siRNA-mediated transitivity as a major factor in amplifying the

original miRNA- and ta-siRNA-directed cleavage signal appears to be more prevalent

than in the flowering plant A. thaliana. It seems not unlikely that similar modifications

and specializations of RNAi pathways will be common, which indicates that care needs

17

to be exercised when interpolating the results from single model organisms, in either

plants or animals.

Experimental Procedures

Plant material

Culture of P. patens, protoplast transformation, and molecular analyses of transgenic

plants were performed according to standard procedures (Frank et al., 2005a). Abscisic

acid treatment was carried out by application of 10 µM (±)-cis-trans ABA to P. patens

liquid cultures.

Generation of ΔPpDCL1a and ΔPpDCL1b mutant lines

An nptII selection marker cassette was cloned into single restriction sites present in

PpDCL1a and PpDCL1b, respectively. The gene disruption constructs were transfected

into P. patens protoplasts and G418-resistant lines were analyzed by PCR to confirm

precise integration events at the corresponding genomic loci. Loss of PpDCL1a and

PpDCL1b transcript, respectively, was confirmed by RT-PCR.

P. patens lines expressing amiR-GNT1

The generation of an amiRNA targeting PpGNT1 was described previously (Khraiwesh

et al., 2008). The amiRNA expression construct was transfected into P. patens wild

type and ΔPpDCL1b mutant lines.

RT-PCR of small RNAs

RT-PCR analyses of miRNAs and ta-siRNAs was carried out as described (Varkonyi-

Gasic et al., 2007). Oligonucleotides used for the cDNA synthesis and subsequent

PCR reactions are listed in Table S1.

18

DNA methylation analysis

DNA sequences were analyzed with the MethPrimer program (Li and Dahiya, 2002) to

deduce methylation-specific (MSP) and unmethylation-specific primers (USP) (Figure

S6) for PCR analysis of bisulfite-treated DNA. Two µg of genomic DNA were used for

sodium bisulfite treatment with the EpiTect Bisulfite Kit (Qiagen).

Detection of miRNA:mRNA duplexes by RT-PCR

cDNA was synthesized from 4 µg total RNA with Superscript III (Invitrogen) without the

addition of primers, with the exception of a primer specific for the PpEF1α transcript to

monitor the efficiency of cDNA synthesis. RT-PCRs were carried out with gene-specific

primers located upstream of miRNA binding sites (Table S1). Control experiments were

performed by heating RNA samples to 95°C for 5 min prior to cDNA synthesis. PpEF1α

control primers were added after cooling of the samples.

Supplemental Data

Supplemental Data include Figure S1-S10, Table S1 and Supplemental Experimental

Procedures and can be found with this article online

Acknowledgements

This work was supported by the Landesstiftung Baden-Württemberg (P-LS-RNS/40 to

D.W., W.F. and R.R.), the German Federal Ministry of Education and Research

(FRISYS: 0313921 to W.F. and R.R.), the Excellence Initiative of the German Federal

and State Governments (EXC 294 to R.R.), the European Community FP6 IP

SIROCCO (contract LSHG-CT-2006-037900; D.W.), and the German Academic

Exchange Service (M.A.A.). We thank G. Gierga for assisting us in the small RNA blot

19

technique and T. Laux, W.R. Hess, R. Baumeister, and P. Beyer for comments on the

manuscript.

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Figure Legends

Figure 1. Analysis of ΔPpDCL1a mutants

(A) Protonema filaments of identical density from wild type (WT) and two ΔPpDCL1a

mutants grown for 28 days on solid medium. (B) Protonema filaments of plants grown

in liquid cultures. (C) RT-PCR expression analysis of miR156, 160, 166, and 390. (D)

RT-PCR expression analysis of ta-siRNAs pptA013298 (processed from PpTAS3) and

pptA079444 (processed from PpTAS1). (E) RT-PCR expression analysis of miRNA

target genes in wild type and ΔPpDCL1a mutants. Error bars indicate standard errors

with n=3.

Figure 2. Analysis of ΔPpDCL1b mutants (1-4)

(A) Regeneration of protoplasts from wild type (WT) and ΔPpDCL1b mutant 1 over

indicated time points. (B) Scanning electron micrographs of gametophores. See

Supplementary Fig. 5 for phenotypes of other ΔPpDCL1b mutants. (C) Small RNA blots

with 30 µg total RNA from protonema, probed for miR156, miR390, miR535, and

miR538. An antisense probe for U6snRNA served as loading control. (D) Small RNA

25

blot with 80 µg total RNA from protonema treated with 5 µM auxin (NAA) for 8 hours,

probed for miR160. (E) Small RNA blot with 80 µg total RNA from gametophores,

probed for miR166. Ethidium bromide staining shown as loading control at the bottom

for d and e. Size bars correspond to 100 µm in a, except for the 18 d and 8 week old

plants, 500 µm.

Figure 3. RNA cleavage products, antisense transcripts, and transitive siRNAs of

miRNA target genes

(A) 5’ RACE products of miRNA targets and a control transcript, PpGNT1, from wild

type and ΔPpDCL1b mutants. Arrows indicate PCR fragments of the expected size for

cleavage products. Numbers above miRNA:target alignments indicate sequenced

RACE products with the corresponding 5’ end. (B) Scheme for the generation of

transitive siRNAs. Double stranded RNA is synthesized from cleaved miRNA targets by

an RNA-dependent RNA polymerase (RdRP), processed into transitive siRNAs, which

subsequently mediate cleavage of the miRNA target mRNA upstream and downstream

of the miRNA recognition motif. Black line: mRNA; grey box: miRNA binding site;

curved line: miRNA; arrows indicate oligonucleotide primers for RT-PCR, with grey

indicating primers for cDNA synthesis from antisense strand, and black for sense

strand. (C) RT-PCR products derived from antisense or sense-specific cDNAs from

wild type and two ΔPpDCL1b mutants (KO1, KO2). (D) Detection of sense and

antisense transitive siRNAs derived from PpARF and PpC3HDZIP1 RNAs, using

hybridization probes targeting regions upstream and downstream of the miRNA binding

sites. U6snRNA was used as control.

Figure 4. Expression of miRNA target genes, DNA methylation, and detection of

miRNA:mRNA duplexes

26

(A) RT-PCR expression analysis of miRNA target genes and the control gene PpGNT1

in wild type and ΔPpDCL1b mutants (KO1-KO4). Bars indicate standard error (n=3). (B)

RNA blot analysis of miRNA target genes PpARF and PpC3HDZIP1 and two control

genes, PpGNT1 and PpEF1α. (C) Specificity analysis of bisulfite PCR, using primers

specific for unmodified sequences. PCR was performed with untreated and bisulfite-

treated genomic DNA of wild type and two ΔPpDCL1b mutants (KO1, KO2). (D-F) PCR

reactions with bisulfite-treated genomic DNA using methylation (MSP) and

unmethylation specific primers (USP). (D) Bisulfite PCR for promoters of miRNA target

genes and the PpGNT1 control. (E) Bisulfite PCR analysis of PpARF sequences

surrounding the miR160 targeting motif. (F) Bisulfite PCR analysis of PpC3HDZIP1

sequences upstream of, the intron disrupting, and sequences downstream of the

miR166 targeting motif. Arrows in d-f mark primer bands. (G) PCR products of miRNA

target genes using cDNA synthesized from wild type and two ΔPpDCL1b mutants (1

and 2) without addition of exogenous primers. For the PpGNT1 control, no PCR

products were detected in either wild type or ΔPpDCL1b mutants (not shown). A

PpEF1α primer specific for cDNA synthesis from the sense transcript was added as an

internal control to all reactions, to monitor the efficiency of cDNA synthesis. (H) The

same experiment performed with RNA samples that had been heated for 5 min to 95°C

prior to cDNA synthesis. The control PpEF1α primer was added after cooling of the

RNA samples.

Figure 5. The ta-siRNA pathway in wild type and ΔPpDCL1b mutants

(A) 5’ RACE-PCRs from wild type and ΔPpDCL1b mutants (1-4) for the miR390 target

PpTAS4 and the ta-siRNA target PpEREBP/AP2. Arrows indicate products of the size

expected for cleavage products. The number of sequenced RACE-PCR products with

the corresponding 5’ end is indicated above the alignment. (B) ta-siRNAs derived from

27

PpTAS4 and transitive siRNAs derived from PpEREBP/AP2. U6snRNA served as

control. (C) RNA blots for PpTAS4 and PpEREBP/AP2 transcripts. Ethidium bromide

staining shown as loading control below. (D) Bisulfite PCR with methylation specific

(MSP) and unmethylation specific primers (USP) for PpTAS4 and PpEREBP/AP2. The

arrow marks primer dimers.

Figure 6. Lines expressing amiR-GNT1 and analysis of miR1026 target PpbHLH

(A) PCR-based identification of two transgenic lines each harboring the PpGNT1-

amiRNA expression construct in wild type (lines #1, #2), ΔPpDCL1b mutant 1 (lines #3,

#4), and ΔPpDCL1b mutant 2 (lines #5, #6) backgrounds. PpEF1α served as control.

(B) Detection of amiR-GNT1 on a small RNA blot loaded with 50 µg of total RNA.

U6snRNA served as control. (C) Cleavage mapping of PpGNT1 in amiR-GNT1 lines by

5’ RACE-PCR. The number of sequenced RACE-PCR products with the corresponding

5’ end is indicated above the alignment. (D) RNA blot of wild type and amiR-GNT1

lines, probed for PpGNT1. Hybridization signals were normalized to rRNA. Levels

relative to wild type are indicated (E) Bisulfite PCR on genomic DNA from amiR-GNT1

lines using methylation (MSP) and unmethylation specific primers (USP) derived from

the PpGNT1 promoter. (F) RT-PCR to detect amiR-GNT1:PpGNT1-mRNA duplexes,

using cDNA synthesized without the addition of exogenous primers. PCR was carried

out with a primer pair upstream of the amiR-GNT1 target motif. Amplification controls

were as in Figure 4G and 4H. Arrows mark primer dimers. (G) RNA blots with 20 µg

total RNA from untreated (Untr.) and ABA-treated wild type plants using probes for

PpbHLH, the loading control PpEF1α, and PpCOR47, a known ABA-induced gene.

PpbHLH levels were normalized to PpEF1α. Relative PpbHLH mRNA levels compared

to wild type are given. (H) Small RNA blot with 50 µg total RNA from untreated (Untr.)

and ABA-treated wild type. MiR1026 levels were normalized to the U6snRNA control.

28

Numbers indicate miR1026 levels relative to wild type. (I) 5’ RACE-PCR for PpbHLH

using RNA from untreated (Untr.) and wild type treated for 4 h with ABA. Arrows

indicate PCR fragments of the expected size for cleavage products. Numbers above

miRNA:target alignments indicate sequenced RACE-PCR products with the

corresponding 5’ end. (J) Bisulfite PCR reactions on DNA from untreated (Untr.) and

ABA-treated wild type using methylation (MSP) and unmethylation specific primers

(USP) targeting PpbHLH genomic sequences. PpGNT1 promoter served as control.

Arrows mark primer dimers. (K) RT-PCR to detect miR1026:PpbHLH-mRNA duplexes,

using cDNA synthesized without the addition of exogenous primers. PCR was carried

out with a primer pair upstream of the miR1026 binding site. Amplification controls were

as in Figure 4G and 4H. Arrows mark primer dimers.

Figure 7. MiRNA expression levels determine post-transcriptional and

transcriptional silencing of miRNA target genes in P. patens

At low miRNA:target-RNA ratios, miRNA targets are regulated primarily at the post-

transcriptional level. The maturation of miRNAs from stem-loop precursors is catalyzed

by PpDCL1a. PpDCL1b is required for loading miRNAs into cleavage competent RISC.

After loading of miRNAs into RISC, transient miRNA:target-RNA duplexes form based

on sequence complementarity resulting in target RNA cleavage. In P. patens, the

amplification of the miRNA signal by the generation of transitive siRNAs appears to be

widespread. Elevated miRNA expression levels cause an increase in the miRNA:target

RNA ratio. In addition to the loading of miRNA into RISC (dotted arrow), miRNAs form

stable duplexes with their cognate target RNAs. MiRNAs are either loaded into a RITS

complex and subsequently interact with their target to form a duplex, or these duplexes

are formed at first and then loaded into RITS. The miRNA:RNA duplexes bound by

RITS initiate DNA methylation at complementary genomic loci. The RITS complex is

29

able to act on adjacent regions (e. g., promoters) to complete CpG methylation of the

entire genomic locus.

30

Supplemental Data


Basel Khraiwesh1, M. Asif Arif1, Gotelinde I. Seumel1, Stephan Ossowski2, Detlef

Weigel2, Ralf Reski1,3,4, Wolfgang Frank1,3*

1Plant Biotechnology, Faculty of Biology, University of Freiburg, Schänzlestraße 1, D-

79104 Freiburg, Germany

2Department of Molecular Biology, Max Planck Institute for Developmental Biology, D-

72076 Tübingen, Germany

3Freiburg Initiative for Systems Biology (FRISYS), Faculty of Biology, University of

Freiburg, Schänzlestr. 1, D-79104 Freiburg, Germany

4Centre for Biological Signalling Studies (bioss), University of Freiburg, Schänzlestr. 1,

D-79104 Freiburg, Germany

Supplementary Figures

Figure S1. Neighbor-joining tree of DICER-LIKE proteins

Figure S2. Generation of ΔPpDCL1a mutants

Figure S3. Generation of ΔPpDCL1b mutants

Figure S4. Phenotypic analysis of ΔPpDCL1b mutants

Figure S5. Gene models of PpARF, PpC3HDZIP1, PpHB10, PpSBP3 and PpGNT1

Figure S6. Primer design for bisulfite PCR analyses

Figure S7. DNA methylation analysis of promoter and intragenic regions of the PpARF

gene in P. patens wild type and two ΔPpDCL1b mutants

Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted by

introns

Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines

expressing the amiR-GNT1

Figure S10. DNA methylation analysis of promoter and intragenic regions of PpbHLH

in untreated and ABA-treated P. patens wild type

Supplementary Tables

Table S1. Primers used in this study

Supplemental Experimental Procedures

Figure S6

Promoter region of PpC3HDZIP1

1 CCTGCCTGCGCTGTCCTATCCTCCTCCTCTTCCTACTTCCCCTCACCTCCTCCGCCTCTG ::||::||++:|||::|||::|::|::|:||::||:||::::|:|::|::|:++::|:|| 1 TTTGTTTGCGTTGTTTTATTTTTTTTTTTTTTTTATTTTTTTTTATTTTTTTCGTTTTTG 61 CGCTCTGTGCACTGTCCCTTCCATGTCGTGCCAGGCTCTGCGGAGGGTGCGGCCAGGCAG ++:|:||||:|:|||:::||::||||++||::|||:|:||++|||||||++|::|||:|| 61 CGTTTTGTGTATTGTTTTTTTTATGTCGTGTTAGGTTTTGCGGAGGGTGCGGTTAGGTAG WTfwd >>>>>>>>>>>>>>>>>>>>> 121 GCAGCTGTGATGGCGGTGTTGTACTGCCGCATGATTCTGGACCAACCGGGCCAGGGCGGG |:||:||||||||++||||||||:||:++:||||||:||||::||:++||::||||++|| 121 GTAGTTGTGATGGCGGTGTTGTATTGTCGTATGATTTTGGATTAATCGGGTTAGGGCGGG MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>> 181 CGACTGTTACTGCAGACTGTTACTGACTCCTGAGGGCGCAGCAGGCTGGTGAGAGGGACG ++|:|||||:||:|||:|||||:|||:|::||||||++:||:|||:||||||||||||++ 181 CGATTGTTATTGTAGATTGTTATTGATTTTTGAGGGCGTAGTAGGTTGGTGAGAGGGACG <<<< 241 GCCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGCTTT |:++++|||||||++|||++|||||||||||||||||||||||++++|||||++||:||| 241 GTCGCGGGTGGGGCGGGGCGAAGAAGAGAAAAAGGTGTGTAGGCGCGGGAGGCGTGTTTT <<<<<<<< <<<<<<<<<< <<<<<<<<<<<<<<<<< WTrev 301 GTATGCGCACTACATGCCTTGAGCCTGGTGGTTGTTCATGACACTGTTCATCGCAGTATT |||||++:|:||:|||::|||||::|||||||||||:||||:|:||||:||++:|||||| 301 GTATGCGTATTATATGTTTTGAGTTTGGTGGTTGTTTATGATATTGTTTATCGTAGTATT <<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<< USPrev 361 CTACAACGACATCGTCCCCAGCTGGTAGTAGTATTGCAGTTGTATAAGTTGTCGCTGCAG :||:||++|:||++|::::||:||||||||||||||:|||||||||||||||++:||:|| 361 TTATAACGATATCGTTTTTAGTTGGTAGTAGTATTGTAGTTGTATAAGTTGTCGTTGTAG 421 CCAGGCGCCGCCAGTCAGCATCTTCTCGTTAGTGTTCAGTTAGTAGTTGAAGCGAGGGAG ::|||++:++::|||:||:||:||:|++||||||||:|||||||||||||||++|||||| 421 TTAGGCGTCGTTAGTTAGTATTTTTTCGTTAGTGTTTAGTTAGTAGTTGAAGCGAGGGAG 481 TATATTCCGCCTTCGATTTTTTGTTTCTCAGGGAGTCACAGCGCTGCGAATCAGAAGCCT ||||||:++::||++|||||||||||:|:|||||||:|:||++:||++|||:|||||::| 481 TATATTTCGTTTTCGATTTTTTGTTTTTTAGGGAGTTATAGCGTTGCGAATTAGAAGTTT 541 GTGAGAGCTTTGGGAACTGGTTTTCGTGTTTTAGAAAGCGAGGCCAACGAGAGAGCGAGA |||||||:||||||||:|||||||++||||||||||||++|||::||++||||||++||| 541 GTGAGAGTTTTGGGAATTGGTTTTCGTGTTTTAGAAAGCGAGGTTAACGAGAGAGCGAGA

601 TCGAGAGAGAGAGAGAGCGCGAGCGACAGCATGTCACGCATGAGAGGAGAGAAGAACAGA |++||||||||||||||++++||++|:||:||||:|++:|||||||||||||||||:||| 601 TCGAGAGAGAGAGAGAGCGCGAGCGATAGTATGTTACGTATGAGAGGAGAGAAGAATAGA 661 GGACGGAGCAGGGCTGGCCTATTGGTGTTACAGGAAGGGGGTTGCAGGAATTTGTAGGCG |||++|||:||||:|||::|||||||||||:|||||||||||||:|||||||||||||++ 661 GGACGGAGTAGGGTTGGTTTATTGGTGTTATAGGAAGGGGGTTGTAGGAATTTGTAGGCG 721 TGGCCGTCACTGTTTGGTTTTTGAAAGCTAGTGCTGCGACAAGAGATGCGGGTGGTCCTA |||:++|:|:|||||||||||||||||:|||||:||++|:||||||||++||||||::|| 721 TGGTCGTTATTGTTTGGTTTTTGAAAGTTAGTGTTGCGATAAGAGATGCGGGTGGTTTTA 781 GCTTGAGTACTTGTGCTAGGCGTCTGAGGCGTGAAGTTTCGGCTAGCTGATTGCAAATTC |:|||||||:|||||:||||++|:|||||++||||||||++|:|||:||||||:|||||: 781 GTTTGAGTATTTGTGTTAGGCGTTTGAGGCGTGAAGTTTCGGTTAGTTGATTGTAAATTT 841 AGTAAGATTGGAGAGGGCAATGGCTGACGGTCCGCATCCATTCGTACAAGAATGCCTTCT |||||||||||||||||:|||||:|||++||:++:||::|||++||:|||||||::||:| 841 AGTAAGATTGGAGAGGGTAATGGTTGACGGTTCGTATTTATTCGTATAAGAATGTTTTTT 901 TCTTGAAAAGCTGGTTGATCCTCGTCGTTGTAATCCGACGGTGCGGCTACGGAGCTAAAG |:||||||||:||||||||::|++|++|||||||:++|++|||++|:||++|||:||||| 901 TTTTGAAAAGTTGGTTGATTTTCGTCGTTGTAATTCGACGGTGCGGTTACGGAGTTAAAG 961 TTCAAACGCTTAGTCTCTTCTTTTCTGGTGTGAAGTAGGT ||:|||++:|||||:|:||:||||:||||||||||||||| 961 TTTAAACGTTTAGTTTTTTTTTTTTTGGTGTGAAGTAGGT Used primers: Forward MSP: 5’-ATTGTCGTATGATTTTGGATTAATC-3’ Reverse MSP: 5’-ACATATAATACGCATACAAAACACG-3’ Forward USP: 5’-GTTGTATGATTTTGGATTAATTGG-3’ Reverse USP: 5’-CATATAATACACATACAAAACACACC-3’ Forward WT: 5’-ACTGCCGCATGATTCTGGACC-3’ Reverse WT: 5’-GCATGTAGTGCGCATACAAAG-3’

Promoter region of PpHB10 1 AGGAGGTGGAGGAGGTGGAGGGTTCCAAGGTGAGGGAGCAAGCTGTCATACCGGTAGGAG ||||||||||||||||||||||||::||||||||||||:|||:|||:|||:++||||||| 1 AGGAGGTGGAGGAGGTGGAGGGTTTTAAGGTGAGGGAGTAAGTTGTTATATCGGTAGGAG 61 TCCGTAGAGGGAAATAGAGAGGAAGCAAGTCAGGAAGTGTTGGTGAAGGGGGAGAGAAAG |:++|||||||||||||||||||||:||||:||||||||||||||||||||||||||||| 61 TTCGTAGAGGGAAATAGAGAGGAAGTAAGTTAGGAAGTGTTGGTGAAGGGGGAGAGAAAG 121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG ||||++|||||||||||||||||||||||||||||++|||++|||||||||||||||||| 121 AGAGCGAGAGGAGGAGGAGGAGTAGTAGAGGTGGTCGTGTCGATGATGGAAGAGATGATG 181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGCCATGAAGGAGGGGTTGTTTTTACGGGTAAT |||||||||||||||||||||||||||||::|||||||||||||||||||||++|||||| 181 GTGTAGTTTTTGGTTGTATGTAGTAGTAGTTATGAAGGAGGGGTTGTTTTTACGGGTAAT

241 GGTTGTTGTTCGGAAGGTATGTACAAATGGAGAGGGCTATGTCGGGGATCAGCTGGAGTG ||||||||||++|||||||||||:||||||||||||:|||||++|||||:||:||||||| 241 GGTTGTTGTTCGGAAGGTATGTATAAATGGAGAGGGTTATGTCGGGGATTAGTTGGAGTG 301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC |||||||||||||||||||||||||||++||||||||||||||||||||||||||||||+ 301 ATGATTGATTGAGTGGAGAGGGAGTGGCGGTAGATATGGGGATGGAGTGGAATGGGGTTC 361 GTATGTCATCATTAGAATCCAAGAGTGGAGAGTAGTTTACCTGGAGCAGCAGCGTTGTGC +|||||:||:||||||||::|||||||||||||||||||::|||||:||:||++|||||: 361 GTATGTTATTATTAGAATTTAAGAGTGGAGAGTAGTTTATTTGGAGTAGTAGCGTTGTGT 421 TCTTGCGCATCCTGGCGATGGACATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGCTGT |:|||++:||::|||++|||||:||||||||||||||||||||||||||||++|||:||| 421 TTTTGCGTATTTTGGCGATGGATATTTGTGTTTGAGTAGTAGAGGTGGAGGCGTTGTTGT 481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGACTCTGTAGTGGCTATGGT |||||||||++|||||||||||||||||||||||||||||||:|:||||||||:|||||| 481 TGTTGTTGTCGTGGTTGTTGTGTGGTAGTGGTAGTAGTGTGATTTTGTAGTGGTTATGGT 541 GGGTCTATTGCTGATGGTTTTTGTGTTGCGTCAGGCCGGCGTCACGGTCGTGTAGCATCG ||||:|||||:|||||||||||||||||++|:|||:++|++|:|++||++|||||:||++ 541 GGGTTTATTGTTGATGGTTTTTGTGTTGCGTTAGGTCGGCGTTACGGTCGTGTAGTATCG 601 AGGGCGACGAAAGGTGAATGAACAAAGGGTGTGATTGTGTATAGGCATCCACATATTCTC ||||++|++|||||||||||||:||||||||||||||||||||||:||::|:|||||:|+ 601 AGGGCGACGAAAGGTGAATGAATAAAGGGTGTGATTGTGTATAGGTATTTATATATTTTC WTfwd >>>>>>>>>>>>>>>>>>>>>>>> 661 GGCTGTGGAAGTTGGGAACAGGGATGCCTTGTGTGCGATTCAACTCGTGGTATAGAAGAA +|:|||||||||||||||:|||||||::|||||||++|||:||:|++||||||||||||| 661 GGTTGTGGAAGTTGGGAATAGGGATGTTTTGTGTGCGATTTAATTCGTGGTATAGAAGAA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 721 GAAGAAGAGGAGCTTGAAGGTTGTCAAGAAAAGGGTAGGGTGTTGCTGCAGCAGCAGTAG ||||||||||||:|||||||||||:||||||||||||||||||||:||:||:||:||||| 721 GAAGAAGAGGAGTTTGAAGGTTGTTAAGAAAAGGGTAGGGTGTTGTTGTAGTAGTAGTAG <<<<<<<<<<<<<<<<<<<<<<<<< WTrev 781 CAGCAGGAGCATCAGTAGCAGCTTGAGAGGACGAGGACCTAGGAGGAACAGAAGCTCTTG :||:|||||:||:|||||:||:|||||||||++||||::|||||||||:|||||:|:||| 781 TAGTAGGAGTATTAGTAGTAGTTTGAGAGGACGAGGATTTAGGAGGAATAGAAGTTTTTG <<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev 841 CGTGGTCTGTGAGGAGAATTCTTTGTTAGGGGTTGGAAGCTTCTAGGTTGGGCACGTAGT ++||||:|||||||||||||:||||||||||||||||||:||:|||||||||:|++|||| 841 CGTGGTTTGTGAGGAGAATTTTTTGTTAGGGGTTGGAAGTTTTTAGGTTGGGTACGTAGT 901 AGTGCGTTCTTTGTGTCTTGTCAACTGGGGTTTCAGTCGTATGAGTTGAACACGGGCTGT ||||++||:|||||||:||||:||:||||||||:|||++|||||||||||:|++||:||| 901 AGTGCGTTTTTTGTGTTTTGTTAATTGGGGTTTTAGTCGTATGAGTTGAATACGGGTTGT 961 CGTCACCAACCAGCAATTCGCAACCGGGCCTGCTCACGA ++|:|::||::||:||||++:||:++||::||:|:|++| 961 CGTTATTAATTAGTAATTCGTAATCGGGTTTGTTTACGA

Used primers: Forward MSP: 5’-GATGTTTTGTGTGCGATTTAATTC-3’ Reverse MSP: 5’-ACTTCTATTCCTCCTAAATCCTCGT-3’ Forward USP: 5’-ATGTTTTGTGTGTGATTTAATTTGT-3’ Reverse USP: 5’-ACTTCTATTCCTCCTAAATCCTCATC-3’ Forward WT: 5’-GATGCCTTGTGTGCGATTCAACTC-3’ Reverse WT: 5’-GCTTCTGTTCCTCCTAGGTCCTCGT-3’

Promoter region of PpARF 1 AGGAGTGGTTTGTGATGCGAAGCTGGGAGGGTGACAGAAAGGACATCAGTGGATCTATGC |||||||||||||||||++|||:|||||||||||:||||||||:||:|||||||:||||: 1 AGGAGTGGTTTGTGATGCGAAGTTGGGAGGGTGATAGAAAGGATATTAGTGGATTTATGT 61 TCTTATTAGTCCTAGTATGGATTAGTATTCATTGATTATAGAGGCTGCGCGGGAGAAAAT |:||||||||::|||||||||||||||||:||||||||||||||:||++++||||||||| 61 TTTTATTAGTTTTAGTATGGATTAGTATTTATTGATTATAGAGGTTGCGCGGGAGAAAAT 121 GGAGAGACTAAGAAGATGAATTCTTCGTAGTTGTGACGAGATGGAAGGTTATTCAATTTA |||||||:||||||||||||||:||++|||||||||++|||||||||||||||:|||||| 121 GGAGAGATTAAGAAGATGAATTTTTCGTAGTTGTGACGAGATGGAAGGTTATTTAATTTA 181 TATTAGGGTACAATGGAAGGAATGCACTTAATTTTTGAAAGTTTTTCGCACGCCAGGATG ||||||||||:|||||||||||||:|:|||||||||||||||||||++:|++::|||||| 181 TATTAGGGTATAATGGAAGGAATGTATTTAATTTTTGAAAGTTTTTCGTACGTTAGGATG 241 GAACTCTTGATAATTGCGTATTATCTACGTATTGTTGAGTTTTCAATTTTCCCATACTGT |||:|:||||||||||++||||||:||++||||||||||||||:||||||:::|||:||| 241 GAATTTTTGATAATTGCGTATTATTTACGTATTGTTGAGTTTTTAATTTTTTTATATTGT 301 CTGTCTGGATTTGCTTCTCATGATACAGGAGTTGTCTGTGAATCTCATTGGATATTTCCG :|||:||||||||:||:|:||||||:|||||||||:|||||||:|:|||||||||||:++ 301 TTGTTTGGATTTGTTTTTTATGATATAGGAGTTGTTTGTGAATTTTATTGGATATTTTCG 361 GATGGTTATTAACCGGGTCCAGTTGATCGTCCAAGCTCCCTTGGCATTTGTGAGGGGTTC ||||||||||||:++|||::|||||||++|::|||:|:::||||:||||||||||||||: 361 GATGGTTATTAATCGGGTTTAGTTGATCGTTTAAGTTTTTTTGGTATTTGTGAGGGGTTT 421 TACTAGTTGTGTGAACTCAGCACACGTAAATTTATAGATTTCCTCTCAAGGCTCAAAGTA ||:||||||||||||:|:||:|:|++|||||||||||||||::|:|:||||:|:|||||| 421 TATTAGTTGTGTGAATTTAGTATACGTAAATTTATAGATTTTTTTTTAAGGTTTAAAGTA WTfwd >>>>>>>>>>>>>>>>>>>>>> 481 CCAGACTTTTTATGCTAGGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGT ::|||:||||||||:|||||:|||:||||||||||||::||++|||:|||||||++|||| 481 TTAGATTTTTTATGTTAGGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATCGTGGT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 541 TCTTAAGGGTCGAGTGCTTAGCTCCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAG |:||||||||++||||:||||:|::|:||::|:|||:||||||:|||||||||||||||| 541 TTTTAAGGGTCGAGTGTTTAGTTTTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAG

<<<<<<<<<<<<<<<<<<<<< WTrev 601 GGGACGTAATGACAACACGAAGCTTATAAAAACTCAAAGCTATATGATCATAGGGCTTTC ||||++||||||:||:|++|||:|||||||||:|:||||:||||||||:||||||:|||: 601 GGGACGTAATGATAATACGAAGTTTATAAAAATTTAAAGTTATATGATTATAGGGTTTTT <<<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev 661 ACGATGAGCGAGATATTTTCTCTCAAGCCTGTGAAGCATTTTGAACGTCTTTATTCTAGG |++|||||++|||||||||:|:|:|||::|||||||:||||||||++|:||||||:|||| 661 ACGATGAGCGAGATATTTTTTTTTAAGTTTGTGAAGTATTTTGAACGTTTTTATTTTAGG 721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGCTCTTTCAGAAGTATTTTCAGAAGT ||||++|||||||||||||||||||||||||||++:|:|||:|||||||||||:|||||| 721 AAGACGAGTTTGATGTTTATTGGTATTGAGTTTCGTTTTTTTAGAAGTATTTTTAGAAGT 781 TAGCAACGATTTCCTATGTTAGGTTCTGTTATTGGTTTTTTGCGTATGATTCGTGTCCTT |||:||++||||::|||||||||||:||||||||||||||||++|||||||++|||::|| 781 TAGTAACGATTTTTTATGTTAGGTTTTGTTATTGGTTTTTTGCGTATGATTCGTGTTTTT 841 CTGGTTGTAACCAAGCTGTACAAAAAAACGTGCAATTGATATCATTTGGTGGCGATTAGA :|||||||||::|||:||||:|||||||++||:|||||||||:|||||||||++|||||| 841 TTGGTTGTAATTAAGTTGTATAAAAAAACGTGTAATTGATATTATTTGGTGGCGATTAGA 901 CATTTTGGTGTCATTGACAAGTTCCAATGTACACTTCTCTTTAAGGTTTTTATTTAATTC :||||||||||:|||||:|||||::||||||:|:||:|:||||||||||||||||||||: 901 TATTTTGGTGTTATTGATAAGTTTTAATGTATATTTTTTTTTAAGGTTTTTATTTAATTT 961 CTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTCA :||||||||||||||||||||||||||||||||||||:| 961 TTAAGTATTGATATTTTATTTATTTATTTGTTGTGGTTA Used primers: Forward MSP: 5’-GGATAATTTTTGGATATGTGTTAGC-3’ Reverse MSP: 5’-AACTTTAAATTTTTATAAACTTCGTA-3’ Forward USP: 5’-ATAATTTTTGGATATGTGTTAGTGT-3’ Reverse USP: 5’-AACTTTAAATTTTTATAAACTTCATA-3’ Forward WT: 5’-GGACAATCTTTGGATATGTGCC-3’ Reverse WT: 5’-AAGCTTCGTGTTGTCATTACG-3’

Promoter region of PpSBP3 1 ATAAAAGTCGTAAGGATCTCACTGGGTCCCTCTCACATTTCTCCCTGAAAAATGACGACG ||||||||++|||||||:|:|:|||||:::|:|:|:||||:|:::||||||||||++|++ 1 ATAAAAGTCGTAAGGATTTTATTGGGTTTTTTTTATATTTTTTTTTGAAAAATGACGACG 61 TCGTTTTCATGACGGTGATTCTCGGTTGTCCATTTGTGGCCTTGACGGAAATGTGTGGGC |++||||:||||++||||||:|++|||||::||||||||::||||++||||||||||||+ 61 TCGTTTTTATGACGGTGATTTTCGGTTGTTTATTTGTGGTTTTGACGGAAATGTGTGGGC 121 GATCTTTGATGGCCACTCTTTTTGTTTTGTTGCCAATCCTCCTCCTATATTTAGTGACTG +||:||||||||::|:|:||||||||||||||::|||::|::|::||||||||||||:|| 121 GATTTTTGATGGTTATTTTTTTTGTTTTGTTGTTAATTTTTTTTTTATATTTAGTGATTG

181 GAGGATCTTTGCTGTTGCTGATTTCCTGGCTTATCCTGGGCGCTGCTATAAGTTAGGCTT ||||||:||||:|||||:||||||::|||:||||::||||++:||:|||||||||||:|| 181 GAGGATTTTTGTTGTTGTTGATTTTTTGGTTTATTTTGGGCGTTGTTATAAGTTAGGTTT 241 TTCTTCATCCATTTTGAGGTGTCACAATATATTTATGGTCGTCGTAATTGTTTTTAATTT ||:||:||::||||||||||||:|:||||||||||||||++|++|||||||||||||||| 241 TTTTTTATTTATTTTGAGGTGTTATAATATATTTATGGTCGTCGTAATTGTTTTTAATTT 301 TACCTCCGTCGGGGTCTGCGCCACCATATGCTTGATAAATTGCAGATTTCAAAGCAGAAC ||::|:++|++||||:||++::|::|||||:|||||||||||:||||||:||||:||||+ 301 TATTTTCGTCGGGGTTTGCGTTATTATATGTTTGATAAATTGTAGATTTTAAAGTAGAAC 361 GTTTCGGTGATGCATGGTCACTTGTGCAGGTTTCTAGTTACCTGGTTGGTTATTTCTTTT +|||++||||||:|||||:|:|||||:||||||:||||||::|||||||||||||:|||| 361 GTTTCGGTGATGTATGGTTATTTGTGTAGGTTTTTAGTTATTTGGTTGGTTATTTTTTTT 421 TTGTTTATTTCTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGCTTAGAACGCTGCAAA ||||||||||:|++||||||++||||||||||||||||||||||:||||||++:||:||| 421 TTGTTTATTTTTCGAGTTTGCGGGTAGTGGTGGAGTTATGGATGTTTAGAACGTTGTAAA 481 TAGGCCAGTTTGGTGTTGGTGATGAGGATTGCGCTCCTTCCAGTCACGATTGTGTGCCTG ||||::|||||||||||||||||||||||||++:|::||::|||:|++||||||||::|| 481 TAGGTTAGTTTGGTGTTGGTGATGAGGATTGCGTTTTTTTTAGTTACGATTGTGTGTTTG 541 CATTCTGTGGAGTCTGTAATCCGCAGTTCAGTTTTTGTGTTTTAGCAAATTAGCGCATGC :|||:||||||||:||||||:++:||||:||||||||||||||||:|||||||++:|||: 541 TATTTTGTGGAGTTTGTAATTCGTAGTTTAGTTTTTGTGTTTTAGTAAATTAGCGTATGT 601 TTCGCAGTCTTACGTGCTTATGACGTTCCTATGGACGTCCTTCTATCGTTGCCCGAATTT ||++:|||:|||++||:||||||++||::||||||++|::||:|||++|||::++||||| 601 TTCGTAGTTTTACGTGTTTATGACGTTTTTATGGACGTTTTTTTATCGTTGTTCGAATTT 661 TCTGTGCTTCTTTCAAAGTCGCTGGCAATTGCAGACCTGGAAATTGGGTATTGTTTCCTC |:||||:||:|||:|||||++:|||:|||||:|||::|||||||||||||||||||::|: 661 TTTGTGTTTTTTTTAAAGTCGTTGGTAATTGTAGATTTGGAAATTGGGTATTGTTTTTTT 721 AGTTGCTTACTCTAAGTGCGAATACTACTTAGACGTGCTGTTGAGGGTAAACTTGCTTCT |||||:|||:|:||||||++||||:||:|||||++||:|||||||||||||:|||:||:| 721 AGTTGTTTATTTTAAGTGCGAATATTATTTAGACGTGTTGTTGAGGGTAAATTTGTTTTT WTfwd >>>>>>>> 781 GAGGCTCTCCACAGTTTTAGAAGTTTGATTAATAAGATATAGAGGCTTTTCTCTGATCAC ||||:|:|::|:|||||||||||||||||||||||||||||||||:||||:|:||||:|: 781 GAGGTTTTTTATAGTTTTAGAAGTTTGATTAATAAGATATAGAGGTTTTTTTTTGATTAT MSPfwd >>>>>>>> USPfwd >>>>>>>> >>>>>>>>>>>>>>>>>> 841 TTCAAATGGATGGTGATCGTGTTCTTTGATACTGCTGAAGCTTGGCGAGTTTTTTTGGTT ||:||||||||||||||++||||:|||||||:||:|||||:||||++||||||||||||| 841 TTTAAATGGATGGTGATCGTGTTTTTTGATATTGTTGAAGTTTGGCGAGTTTTTTTGGTT >>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>> <<<<<<<<<<< 901 CAAATCTCCGAAGCCTATGGACCATTCAGCAGCCTGAGCTTCCAATTTGGCCGTCAGTGT :||||:|:++|||::||||||::|||:||:||::||||:||::|||||||:++|:||||| 901 TAAATTTTCGAAGTTTATGGATTATTTAGTAGTTTGAGTTTTTAATTTGGTCGTTAGTGT <<<<<<<<<<< <<<<<<<<<<<

<<<<<<<<<<<<<<< WTrev 961 CGTATGTTACTCCTATGTTGAAGCTTGTGGGCTGGATCGC ++|||||||:|::||||||||||:|||||||:|||||++: 961 CGTATGTTATTTTTATGTTGAAGTTTGTGGGTTGGATCGT <<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<< USPrev Used primers: Forward MSP: 5’-TTGATTATTTTAAATGGATGGTGATC-3’ Reverse MSP: 5’-AAAAATAACATACGACACTAACGAC-3’ Forward USP: 5’-TTGATTATTTTAAATGGATGGTGATT-3’ Reverse USP: 5’-AAAAATAACATACAACACTAACAAC-3’ Forward WT: 5’-CTGATCACTTCAAATGGATGGTGATC-3’ Reverse WT: 5’-TAGGAGTAACATACGACACTGACGGC-3’

Promoter region of PpGNT1 1 CTGTTGATGTATCCTAGATATTGTTGCATAGTTCTTGTCTAGTTTATTAAAATAAGAATA :|||||||||||::||||||||||||:||||||:||||:||||||||||||||||||||| 1 TTGTTGATGTATTTTAGATATTGTTGTATAGTTTTTGTTTAGTTTATTAAAATAAGAATA 61 ATAATAATAAATGTTTATATATTTAATATTAAAATAACCAATGTACAAAATATGTTAGAC |||||||||||||||||||||||||||||||||||||::||||||:|||||||||||||: 61 ATAATAATAAATGTTTATATATTTAATATTAAAATAATTAATGTATAAAATATGTTAGAT 121 ATTTTTGTATCAAATTCAAAAATATATTAAAAAAAGTACACAACATAGGTTACAATGGAT ||||||||||:|||||:|||||||||||||||||||||:|:||:||||||||:||||||| 121 ATTTTTGTATTAAATTTAAAAATATATTAAAAAAAGTATATAATATAGGTTATAATGGAT 181 CATAAATCATTAATTATTCTTGATATTATGTTAAAAAAGTTGAGAAACATCTACAATTAG :||||||:||||||||||:||||||||||||||||||||||||||||:||:||:|||||| 181 TATAAATTATTAATTATTTTTGATATTATGTTAAAAAAGTTGAGAAATATTTATAATTAG 241 TTAGAAACTTTCATATTGTTTAAAATCATTTTGTTATAAAAACAATACCATTTAATAAAG |||||||:|||:||||||||||||||:|||||||||||||||:||||::||||||||||| 241 TTAGAAATTTTTATATTGTTTAAAATTATTTTGTTATAAAAATAATATTATTTAATAAAG 301 ATGAATCTTATTAATAGGTAATTCTGTTGATATATTTCCTTGACACAGCAATATGGATAG ||||||:||||||||||||||||:|||||||||||||::||||:|:||:||||||||||| 301 ATGAATTTTATTAATAGGTAATTTTGTTGATATATTTTTTTGATATAGTAATATGGATAG 361 GAATCATAGTCTTAGATATAGTAGTTTTAAGGTGATTAATGTCAAAAGAACATAACTAGC ||||:|||||:|||||||||||||||||||||||||||||||:|||||||:||||:|||: 361 GAATTATAGTTTTAGATATAGTAGTTTTAAGGTGATTAATGTTAAAAGAATATAATTAGT 421 AAAAGAATTAAAGCATAGTCCACCAAACATATATTTTGAATAGCAAGATAATATAAATTA |||||||||||||:|||||::|::|||:|||||||||||||||:|||||||||||||||| 421 AAAAGAATTAAAGTATAGTTTATTAAATATATATTTTGAATAGTAAGATAATATAAATTA 481 CTTTAAAACAGAATATAATATAATATTAAATTTACTTTTATATTATTTTTAGATTAATGA :|||||||:|||||||||||||||||||||||||:||||||||||||||||||||||||| 481 TTTTAAAATAGAATATAATATAATATTAAATTTATTTTTATATTATTTTTAGATTAATGA

541 AACTTCACAATAATACATGAAAGAAATTTTTGTGACTTTGGCACCTTTTATTAGCAATGT ||:||:|:|||||||:|||||||||||||||||||:|||||:|::|||||||||:||||| 541 AATTTTATAATAATATATGAAAGAAATTTTTGTGATTTTGGTATTTTTTATTAGTAATGT 601 ATTACTCTTACTATGTAAAAGTATCAAATTTAACAAAAATTGAAAAAATATACATCCACT ||||:|:|||:|||||||||||||:||||||||:||||||||||||||||||:||::|:| 601 ATTATTTTTATTATGTAAAAGTATTAAATTTAATAAAAATTGAAAAAATATATATTTATT 661 TATACTATCAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGACTTAAAA ||||:|||:||||||||||||||||||||||||||||||||||||||||||||:|||||| 661 TATATTATTAATTAATTAATTAAAATTTTATTTATTTTTTAATTTTTTGTTGATTTAAAA 721 TATATCTATATATATATATATATATATATATATGTATATTCACATTCTTGAACAAGAATT |||||:||||||||||||||||||||||||||||||||||:|:|||:|||||:||||||| 721 TATATTTATATATATATATATATATATATATATGTATATTTATATTTTTGAATAAGAATT WTfwd >>>>>>>>>>>>>>>>>>>>>>> 781 TTGGATTCAAGGAGGTGAATGCTTTGCACAAAAAAAAGTTTTATCTCTAAATTCTTAGAC |||||||:|||||||||||||:||||:|:|||||||||||||||:|:||||||:|||||: 781 TTGGATTTAAGGAGGTGAATGTTTTGTATAAAAAAAAGTTTTATTTTTAAATTTTTAGAT MSPfwd >>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>> 841 AACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATACGTATTTACACACT ||++|:|||:||||||||||||||||:||++|:||||:||||||||++||||||:|:|:| 841 AACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATACGTATTTATATATT >>>> >>>>> 901 TGTATATGATGTACCATAGACGGTAATCGTACATATTTGCCGACACCCTGCAATTAATAG |||||||||||||::|||||++|||||++||:|||||||:++|:|:::||:||||||||| 901 TGTATATGATGTATTATAGACGGTAATCGTATATATTTGTCGATATTTTGTAATTAATAG <<<<<<<<<<<<<<<<<<<<<<< WTrev 961 AGTTCGAATATCCCCGCCGCGTTCAAGTCGCCTCGTGCAA ||||++|||||:::++:++++||:||||++::|++||:|| 961 AGTTCGAATATTTTCGTCGCGTTTAAGTCGTTTCGTGTAA <<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<< USPrev Used primers: Forward MSP: 5’-TTTATTTTTAAATTTTTAGATAACG-3’ Reverse MSP: 5’-AACGACTTAAACGCGACGA-3’ Forward USP: 5’-TTATTTTTAAATTTTTAGATAATGT-3’ Reverse USP: 5’-AAACAACTTAAACACAACAAA-3’ Forward WT: 5’-GTTTTATCTCTAAATTCTTAGAC-3’ Reverse WT: 5’-GGCGACTTGAACGCGGCGGGGAT-3’

Intron 4 within the miRNA166 binding site of PpC3HDZIP1 1 GGTATGAAGGTATGGATGCCATGCCTTCCTACGGCACGTTCTACAGTGTATTGTGGAGTA ||||||||||||||||||::|||::||::||++|:|++||:||:|||||||||||||||| 1 GGTATGAAGGTATGGATGTTATGTTTTTTTACGGTACGTTTTATAGTGTATTGTGGAGTA MSPfwd >>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>> 61 GCGAGCCTCACCTGTAACTCTTGATCTATAGATTCCATTATCAGAGATATGATCGCACGA |++||::|:|::|||||:|:|||||:||||||||::|||||:|||||||||||++:|++| 61 GCGAGTTTTATTTGTAATTTTTGATTTATAGATTTTATTATTAGAGATATGATCGTACGA <<<<<<< <<<<<<< 121 AATAACTCTTTGTTCCAACCTTTTGTAAAATAAGTATTAGCGGAGTCATGGTACTGGAGC |||||:|:||||||::||::||||||||||||||||||||++||||:||||||:|||||: 121 AATAATTTTTTGTTTTAATTTTTTGTAAAATAAGTATTAGCGGAGTTATGGTATTGGAGT <<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<< USPrev 181 AAAGTCAAACAAATTAATTTGACTCAAAACACGACTTCGAATTAATTTAGGAGCTAACAA |||||:|||:||||||||||||:|:||||:|++|:||++||||||||||||||:|||:|| 181 AAAGTTAAATAAATTAATTTGATTTAAAATACGATTTCGAATTAATTTAGGAGTTAATAA 241 GGTAATGATATTGATTCTTTAATTCAAATTAAAGTGGTTGATTGCAAATGCCATTGCTGA ||||||||||||||||:|||||||:|||||||||||||||||||:|||||::||||:||| 241 GGTAATGATATTGATTTTTTAATTTAAATTAAAGTGGTTGATTGTAAATGTTATTGTTGA 301 TACGTCACTAGT ||++|:|:|||| 301 TACGTTATTAGT Used primers: Forward MSP: 5’-GTTATGTTTTTTTACGGTACGT-3’ Reverse MSP: 5’-AAACAAAAAATTATTTCGTACG-3’ Forward USP: 5’-TGTTATGTTTTTTTATGGTATGT-3’ Reverse USP: 5’-TTAAAACAAAAAATTATTTCATACA-3’

Intron 1 upstream of the miRNA166 binding site of PpC3HDZIP1 1 GGTATGTATCCGTGTCCTTCGCCAGATTCTAGGAGAGGAAAATTGTTTGGGCATAAACCT |||||||||:++|||::||++::|||||:||||||||||||||||||||||:|||||::| 1 GGTATGTATTCGTGTTTTTCGTTAGATTTTAGGAGAGGAAAATTGTTTGGGTATAAATTT 61 CGTATAGAAGTTCGTTCGATCTTGAAATCTTTCTCAAACGGAGATTCTGGATGACATGCA ++||||||||||++||++||:|||||||:|||:|:|||++||||||:|||||||:|||:| 61 CGTATAGAAGTTCGTTCGATTTTGAAATTTTTTTTAAACGGAGATTTTGGATGATATGTA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 121 CGTGAAGTTGATTCACTATATTATGTCGCCTCGAATTTCACTTCGTATTGAGCCATCTCA ++|||||||||||:|:||||||||||++::|++|||||:|:||++|||||||::||:|:| 121 CGTGAAGTTGATTTATTATATTATGTCGTTTCGAATTTTATTTCGTATTGAGTTATTTTA

181 TGTTTTATTCACTTCTGTCTTGAGATTGTCATACGTGCCGCTCCGTGATGTGCGGATAGG |||||||||:|:||:|||:||||||||||:|||++||:++:|:++|||||||++|||||| 181 TGTTTTATTTATTTTTGTTTTGAGATTGTTATACGTGTCGTTTCGTGATGTGCGGATAGG <<<<<<<< <<<<<<<<<< 241 TGGACAGGTGCACAACATGATAATCCATGTTGTGGTCGAGGGGTAGGGGGGTGGTACACG ||||:|||||:|:||:||||||||::||||||||||++||||||||||||||||||:|++ 241 TGGATAGGTGTATAATATGATAATTTATGTTGTGGTCGAGGGGTAGGGGGGTGGTATACG <<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<< USPrev 301 ACACAATTAATTGAAATGAGTGGAGAGTGTATTGCAG |:|:||||||||||||||||||||||||||||||:|| 301 ATATAATTAATTGAAATGAGTGGAGAGTGTATTGTAG Used primers: Forward MSP: 5’-TTCGATTTTGAAATTTTTTTTAAAC-3’ Reverse MSP: 5’-TATTATACACCTATCCACCTATCCG-3’ Forward USP: 5’-TGATTTTGAAATTTTTTTTAAATGG-3’ Reverse USP: 5’-ATTATACACCTATCCACCTATCCACA-3’

Exon 14 downstream of the miRNA166 binding site of PpC3HDZIP1 1 GGACGGAATCGGGCTGAATCGTACACTTGATCTGGCTTCCACACTTGAAGATCACGAGGC |||++||||++||:|||||++||:|:|||||:|||:||::|:|:||||||||:|++|||: 1 GGACGGAATCGGGTTGAATCGTATATTTGATTTGGTTTTTATATTTGAAGATTACGAGGT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 61 AGGATTGAATGGAGAGAGCAAGTCTAATGGCAGCTCTAGCCAAGTGCGATCCGTTCTGAC ||||||||||||||||||:||||:||||||:||:|:|||::|||||++||:++||:|||: 61 AGGATTGAATGGAGAGAGTAAGTTTAATGGTAGTTTTAGTTAAGTGCGATTCGTTTTGAT 121 AATAGCTTTTCAGTTTGCGTATGAAGTTCATACACGCGAAACATGCGCAGTGATGGCCCG |||||:||||:||||||++|||||||||:|||:|++++|||:|||++:||||||||::++ 121 AATAGTTTTTTAGTTTGCGTATGAAGTTTATATACGCGAAATATGCGTAGTGATGGTTCG <<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<< 181 CCAGTATGTTCGCACAGTGGTTGCATCCGTGCAGCGGGTTGCCATGGCTTTGGCACCGTC ::||||||||++:|:||||||||:||:++||:||++|||||::||||:|||||:|:++|: 181 TTAGTATGTTCGTATAGTGGTTGTATTCGTGTAGCGGGTTGTTATGGTTTTGGTATCGTT <<<<<<<<<< MSPrev <<<<<<<< USPrev 241 CCGTGGTCAGCCCCGTCCAGCACTGGGCAACTCAGATGCCATCAGTTTGGCGCGTCACAT :++||||:||:::++|::||:|:||||:||:|:|||||::||:|||||||++++|:|:|| 241 TCGTGGTTAGTTTCGTTTAGTATTGGGTAATTTAGATGTTATTAGTTTGGCGCGTTATAT 301 CCTGAGCAGCTACAG ::||||:||:||:|| 301 TTTGAGTAGTTATAG

Used primers: Forward MSP: 5’-TGGTTTTTATATTTGAAGATTACGA-3’ Reverse MSP: 5’-AACATACTAACGAACCATCACTACG-3’ Forward USP: 5’-TGGTTTTTATATTTGAAGATTATGA-3’ Reverse USP: 5’-CATACTAACAAACCATCACTACACA-3’

Exon 1 upstream of the miRNA160 binding site of PpARF 1 GGTATCGATCTGGAGCCCGTTGCAAACTCAATGGTGTATTTTATAGGGCAAAAGTCTGAT |||||++||:|||||::++|||:|||:|:|||||||||||||||||||:||||||:|||| 1 GGTATCGATTTGGAGTTCGTTGTAAATTTAATGGTGTATTTTATAGGGTAAAAGTTTGAT 61 CTATATGGAATGCATCCTCTCAGAGTTGCAAATCATGGACTGCATGTCACTCTGGGTTAT :|||||||||||:||::|:|:|||||||:||||:|||||:||:||||:|:|:|||||||| 61 TTATATGGAATGTATTTTTTTAGAGTTGTAAATTATGGATTGTATGTTATTTTGGGTTAT 121 TCTCGATCACCTAGCTTTGCTGGAGTTCAAATTGGTGAGTACGAGTATTATGAGTGATCT |:|++||:|::|||:||||:|||||||:|||||||||||||++|||||||||||||||:| 121 TTTCGATTATTTAGTTTTGTTGGAGTTTAAATTGGTGAGTACGAGTATTATGAGTGATTT 181 CGAGTTTATGGTCCCCTTCTTTCATGATCAAGGGTAATTTATATCAAGGGTGTATATGAG ++||||||||||::::||:|||:|||||:|||||||||||||||:||||||||||||||| 181 CGAGTTTATGGTTTTTTTTTTTTATGATTAAGGGTAATTTATATTAAGGGTGTATATGAG 241 AGATACGCACTTATTGAGTGGACCTTTTCTCATACTGCATTTACACCCCTGTCAGTTGCA |||||++:|:||||||||||||::||||:|:|||:||:|||||:|::::|||:|||||:| 241 AGATACGTATTTATTGAGTGGATTTTTTTTTATATTGTATTTATATTTTTGTTAGTTGTA 301 GCATCCTGGTTTGGAATGCCGGGTCCAGTCCCTCTATTATCCATGAGTGTAAAATCGGAG |:||::||||||||||||:++|||::|||:::|:||||||::|||||||||||||++||| 301 GTATTTTGGTTTGGAATGTCGGGTTTAGTTTTTTTATTATTTATGAGTGTAAAATCGGAG 361 AGTCTCGATGACATTGGAGGTCACGAGAAAAAATCTGTAACTGGGTCGGAAGTGGGTGGC |||:|++||||:|||||||||:|++|||||||||:|||||:|||||++|||||||||||: 361 AGTTTCGATGATATTGGAGGTTACGAGAAAAAATTTGTAATTGGGTCGGAAGTGGGTGGT 421 CTCGATGCTCAGCTGTGGCATGCCTGTGCTGGGGGTATGGTTCAACTGCCTCATGTGGGT :|++|||:|:||:|||||:|||::||||:|||||||||||||:||:||::|:|||||||| 421 TTCGATGTTTAGTTGTGGTATGTTTGTGTTGGGGGTATGGTTTAATTGTTTTATGTGGGT 481 GCTAAGGTTGTCTATTTTCCCCAAGGCCATGGCGAACAAGCTGCTTCAACTCCCGAGTTC |:|||||||||:||||||::::||||::||||++||:|||:||:||:||:|::++||||: 481 GTTAAGGTTGTTTATTTTTTTTAAGGTTATGGCGAATAAGTTGTTTTAATTTTCGAGTTT 541 CCCCGCACTTTGGTTCCAAATGGAAGTGTTCCCTGCCGAGTTGTGTCAGTTAACTTTCTG :::++:|:|||||||::|||||||||||||:::||:++||||||||:||||||:|||:|| 541 TTTCGTATTTTGGTTTTAAATGGAAGTGTTTTTTGTCGAGTTGTGTTAGTTAATTTTTTG MSPfwd >>>>> USPfwd >>>>> 601 GCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC |:|||||:|||||:|||++|||||||||:|++||||||::||:||::|||||||||:|:: 601 GTTGATATAGAAATAGACGAGGTATTTGTTCGTATTTGTTTGTAGTTTGAGATTGGTTTT >>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>

661 TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCA |:++:|:|||||||||:||||||||:|:|||++|:|:++::|:||||||||::||:||:| 661 TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTCGTTTTTAGAGAAATTAGTTTTA 721 TTTGCCAAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTCGT ||||::||||++:|:|:|:|||||||||:|||:||++|||||||:||||:||||::|++| 721 TTTGTTAAAACGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTCGT <<<<<<<<<<<<<<<<<<<<<<<<<MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<USPrev 781 TATTGTGCTGAAACTATTTTCCCACCTCTCGATTACTGTATCGATCCTCCTGTTCAAACT |||||||:|||||:||||||:::|::|:|++||||:|||||++||::|::||||:|||:| 781 TATTGTGTTGAAATTATTTTTTTATTTTTCGATTATTGTATCGATTTTTTTGTTTAAATT 841 GTTCTTGCAAAAGATGTCCATGGAGAGGTGTGGAAATTTCGTCACATTTACAGG |||:|||:|||||||||::||||||||||||||||||||++|:|:|||||:||| 841 GTTTTTGTAAAAGATGTTTATGGAGAGGTGTGGAAATTTCGTTATATTTATAGG Used primers: Forward MSP: 5’-TTTTGGTTGATATAGAAATAGACGA-3’ Reverse MSP: 5’-GAAATATTAAAAAACCTCCACCGTT-3’ Forward USP: 5’-TTTTGGTTGATATAGAAATAGATGA-3’ Reverse USP: 5’-AAAATATTAAAAAACCTCCACCATT-3’

Exon 4 downstream of the miRNA160 binding site of PpARF 1 AGGAATTCCATGGAGACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCT |||||||::|||||||:|||:|||++::|:|||:|::||:||:||||||::|||||||:| 1 AGGAATTTTATGGAGATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTT 61 AAAGCTTGATCATAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAA ||||:|||||:||||:|:||||:::|:|:|:||||++||||||||||||:||:|||:||| 61 AAAGTTTGATTATAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>> 121 CAGAATTGCACGGTAAAGGAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTT :|||||||:|++|||||||||||:||||:||||:||||||||||||||||++|||++:|| 121 TAGAATTGTACGGTAAAGGAAAATTGTATTAGGTATGTTATATGGGAATTCGGATCGTTT 181 CTTGCAATTAAACACGCTAGCGCCGTTTGGTGCCAATGTTATTCTGGCATTTGTTTTGTT :|||:|||||||:|++:|||++:++|||||||::|||||||||:|||:|||||||||||| 181 TTTGTAATTAAATACGTTAGCGTCGTTTGGTGTTAATGTTATTTTGGTATTTGTTTTGTT <<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<<< USPrev 241 TCCTTTGGAAACAAATTGCTATATTTCAAAGTCCTTTGGAGGAGCTCGC |::||||||||:||||||:|||||||:|||||::||||||||||:|++: 241 TTTTTTGGAAATAAATTGTTATATTTTAAAGTTTTTTGGAGGAGTTCGT Used primers: Forward MSP: 5’-AGTTTATAATTTTTTTATAGGACGT-3’ Reverse MSP: 5’-AAAATAACATTAACACCAAACGAC-3’

Forward USP: 5’-TAGTTTATAATTTTTTTATAGGATGT-3’ Reverse USP: 5’-CCAAAATAACATTAACACCAAACAAC-3’

Intron 2 upstream of the miRNA160 binding site of PpARF 1 AGTTCTCCATGGCGGGTTCTGCAGGTTAGCTTTTTGTTTGTCTAATCAAAGCAATCAATG ||||:|::||||++||||:||:|||||||:|||||||||||:||||:||||:|||:|||| 1 AGTTTTTTATGGCGGGTTTTGTAGGTTAGTTTTTTGTTTGTTTAATTAAAGTAATTAATG MSPfwd >>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>> 61 TGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAGTCAGTGGAA ||:|||||||++||:||::||:|||||:||:|||||:||||||:||++||||:||||||| 61 TGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAGTTAGTGGAA >>>>>>>>>>>>> >>>>>>>>>>>>> 121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCTATT |||||||||||||||||||||||||||||||:||||:|:||||||||||:++||::|||| 121 ATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTTATT 181 GCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCA |:|||||||||||++||||||:::||||:||||||||:||||||++|:|:||::||||:| 181 GTAGGGAGTGAATCGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTA <<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<< 241 GCTGCCCCCTGTCTCTCTTCCCAAAAAGAAACTGCG |:||:::::|||:|:|:||:::|||||||||:||++ 241 GTTGTTTTTTGTTTTTTTTTTTAAAAAGAAATTGCG <<<<<<< MSPrev <<<<<<< USPrev Used primers: Forward MSP: 5’-TAAAGTAATTAATGTGTTAATGAACGT-3’ Reverse MSP: 5’-AAACAACTACATAAAAAATATCGCC-3’ Forward USP: 5’-TTAAAGTAATTAATGTGTTAATGAATGT-3’ Reverse USP: 5’-AAACAACTACATAAAAAATATCACC-3’

Intron 3 downstream of the miRNA160 binding site of PpARF 1 AGGATCAGGTTTGTATCAACAAATCTCTAGTTCGTTTTGGCATGGAGTTCACAATGTGCT |||||:||||||||||:||:||||:|:|||||++||||||:||||||||:|:||||||:| 1 AGGATTAGGTTTGTATTAATAAATTTTTAGTTCGTTTTGGTATGGAGTTTATAATGTGTT MSPfwd >>>>>>>>>>>>> USPfwd >>>>>>>>>>>>> 61 CTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCCTTTTCTTT :||:|||:||++|||||||:||||:|:||:||:||||:||:||++|++||::||||:||| 61 TTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTTTTTTTTTT >>>>>>>>>>>>> >>>>>>>>>>>>>

121 TTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATGGAG |||++||||||||||||||||::|||||||:||||||||||++|:|||||||::|||||| 121 TTTCGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATGGAG <<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<< 181 ACAGTCAGACGCCTCATACTCCTGCATCTGGTAGCCAATGAGGCTAAAGCTTGATC |:|||:|||++::|:|||:|::||:||:||||||::|||||||:|||||:|||||: 181 ATAGTTAGACGTTTTATATTTTTGTATTTGGTAGTTAATGAGGTTAAAGTTTGATT <<<< MSPrev <<<< USPrev Used primers: Forward MSP: 5’-TTTATAATGTGTTTTTTAAGTTTCGT-3’ Reverse MSP: 5’-CTATCTCCATAAAATTCCTATCGTA-3’ Forward USP: 5’-TTTATAATGTGTTTTTTAAGTTTTGT-3’ Reverse USP: 5’-CTATCTCCATAAAATTCCTATCATA-3’

Sequence of PpTAS4 1 ACCAAAGTAGATTGAATCAATCCGTGCATCGATTCACAGGAGCACTGACCTGATCTATGC |::||||||||||||||:|||:++||:||++|||:|:|||||:|:|||::||||:||||+ 1 ATTAAAGTAGATTGAATTAATTCGTGTATCGATTTATAGGAGTATTGATTTGATTTATGC 61 GACGGTGCGAGAAAAATCATCCCAGCGTGGTGCTACGCTAGTCACCTAGTCATCAGCATC +|++|||++||||||||:||:::||++|||||:||++:||||:|::||||:||:||:||+ 61 GACGGTGCGAGAAAAATTATTTTAGCGTGGTGTTACGTTAGTTATTTAGTTATTAGTATC MSPfwd >>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>> 121 GGTGCTACGTAAACTTATGGCAATGGTGCGTTCCTATCAGTGCGTCACTTCAAGGAAGCC +|||:||++||||:||||||:|||||||++||::|||:||||++|:|:||:|||||||:: 121 GGTGTTACGTAAATTTATGGTAATGGTGCGTTTTTATTAGTGCGTTATTTTAAGGAAGTT <<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<< 181 CTTCCCAAGTCTTCATCCGGCCCGCTACCTTTGCGTAGCTGCTTCACTGGAGGCCTGGGT :||:::||||:||:||:++|::++:||::||||++|||:||:||:|:||||||::||||| 181 TTTTTTAAGTTTTTATTCGGTTCGTTATTTTTGCGTAGTTGTTTTATTGGAGGTTTGGGT <<<<< MSPrev <<<<< USPrev 241 GGAGACTGGCGGACAGCTTTGCGATGTTACGGTTGTAGCCAATTCTTGTTGCACTTAGAT |||||:|||++||:||:||||++||||||++|||||||::||||:||||||:|:|||||| 241 GGAGATTGGCGGATAGTTTTGCGATGTTACGGTTGTAGTTAATTTTTGTTGTATTTAGAT 301 TTCCACTGGGCGTTATCCCTCTTGAGCTGAGAAGACAAGGGCTCCCTCCTAGGGGGCGAA ||::|:||||++||||:::|:|||||:||||||||:|||||:|:::|::|||||||++|| 301 TTTTATTGGGCGTTATTTTTTTTGAGTTGAGAAGATAAGGGTTTTTTTTTAGGGGGCGAA 361 AATAGGTGAGCTGGGGTCACCTTGTTAGCGGGGTGTTAAGCATTTGAATGCAACACTCCT ||||||||||:||||||:|::|||||||++||||||||||:|||||||||:||:|:|::| 361 AATAGGTGAGTTGGGGTTATTTTGTTAGCGGGGTGTTAAGTATTTGAATGTAATATTTTT

421 ACGCAAGACCCTAGCTATGGCTCCATAGGGTGTGATGAGTGCTTCATCCGGTGCTCTTCT |++:||||:::|||:|||||:|::|||||||||||||||||:||:||:++|||:|:||:| 421 ACGTAAGATTTTAGTTATGGTTTTATAGGGTGTGATGAGTGTTTTATTCGGTGTTTTTTT 481 ACTGCCTTGCCCACCTACCCTTGTGATATGGGCCGCGCGTGTCTGCGTGTCTCCTGTATC |:||::|||:::|::||:::||||||||||||:++++++|||:||++|||:|::|||||+ 481 ATTGTTTTGTTTATTTATTTTTGTGATATGGGTCGCGCGTGTTTGCGTGTTTTTTGTATC 541 GGTTGTATATCACTCCTGAGCTACGGGTGTGCAATTCCCATGTCTTTTGGGAATAGGCGT +|||||||||:|:|::||||:||++||||||:||||:::||||:|||||||||||||++| 541 GGTTGTATATTATTTTTGAGTTACGGGTGTGTAATTTTTATGTTTTTTGGGAATAGGCGT 601 CAAGACTAGAGGTAGTTTTGTTGTCTTAGCCGGCCACAGGCGGCGGTGATAAAACCTGCA :||||:||||||||||||||||||:||||:++|::|:|||++|++|||||||||::||:| 601 TAAGATTAGAGGTAGTTTTGTTGTTTTAGTCGGTTATAGGCGGCGGTGATAAAATTTGTA 661 GTTGATGTAATGGAGTCACATACTGAATCCACTTGACTGGCTGTGGCTGAAATAAAAACA ||||||||||||||||:|:|||:|||||::|:||||:|||:|||||:|||||||||||:| 661 GTTGATGTAATGGAGTTATATATTGAATTTATTTGATTGGTTGTGGTTGAAATAAAAATA 721 TTTTCCAC ||||::|: 721 TTTTTTAT Used primers:Forward MSP: 5’-GGTGCGAGAAAAATTATTTTAGC-3’ Reverse MSP: 5’-AAAAAAACTTCCTTAAAATAACGCA-3’ Forward USP: 5’-GTGTGAGAAAAATTATTTTAGTGT-3’ Reverse USP: 5’-AAAAAAACTTCCTTAAAATAACACA-3’

Coding Sequence of PpEREBP/AP2

1 NATGTCTGGTAGCGGAAGCATAGGCACTTCCGGAGTGGACTCATGGGTTGAGCAGAGCTA |||||:||||||++||||:|||||:|:||:++|||||||:|:||||||||||:||||:|| 1 NATGTTTGGTAGCGGAAGTATAGGTATTTTCGGAGTGGATTTATGGGTTGAGTAGAGTTA MSPfwd >>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 61 ACTGACATGCTTTGCGGGGGAATTACTCACTGATTCAGCAGCACTGGCAGATCTGTGTGA |:|||:|||:||||++|||||||||:|:|:|||||:||:||:|:|||:||||:||||||| 61 ATTGATATGTTTTGCGGGGGAATTATTTATTGATTTAGTAGTATTGGTAGATTTGTGTGA 121 GGCGTTGAAGGTTTTCTACGAGTCGCTGCTTCCCCAGCTAATCCAAAGAGATGCGGATAG ||++|||||||||||:||++|||++:||:||::::||:||||::|||||||||++||||| 121 GGCGTTGAAGGTTTTTTACGAGTCGTTGTTTTTTTAGTTAATTTAAAGAGATGCGGATAG <<<<<<<<<<<<<<<<<<<<<<<<< MSPrev <<<<<<<<<<<<<<<<<<<<<<<<< USPrev 181 AGATTCCGCAACATCATGCTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC |||||:++:||:||:|||:||||||||||||||||||||++||||||||||||||||||+ 181 AGATTTCGTAATATTATGTTGAAGTGGAAGAAAGTGTTGCGAGTGTGGGTTTATAGATTC 241 GCGCTTTATTGACGATTGAGGTGAAGCAGAGGGTGGGGTTTTCGGAAAGCTGCAGCGTCA +++:||||||||++||||||||||||:|||||||||||||||++|||||:||:||++|:| 241 GCGTTTTATTGACGATTGAGGTGAAGTAGAGGGTGGGGTTTTCGGAAAGTTGTAGCGTTA

301 CTCGAGTGCAGAGGGTGTGCCAGGTTGTAGTGGTTTTCGAATTTCAGCGGACGAGGTAAG :|++||||:||||||||||::||||||||||||||||++|||||:||++||++||||||| 301 TTCGAGTGTAGAGGGTGTGTTAGGTTGTAGTGGTTTTCGAATTTTAGCGGACGAGGTAAG 361 GAAGCCTAGCAGACGAGAACGTGAGTAAGTCAGCGCAAGATGGTAGGCAAAGTGCAGGCG ||||::|||:|||++||||++|||||||||:||++:|||||||||||:||||||:|||++ 361 GAAGTTTAGTAGACGAGAACGTGAGTAAGTTAGCGTAAGATGGTAGGTAAAGTGTAGGCG 421 TCCCTAGCTGGTGCCCATGGCAAGCAGTCTACTCATCATGCCATGGTTCGAAGCAGTCAT |:::|||:|||||:::||||:|||:|||:||:|:||:|||::||||||++|||:|||:|| 421 TTTTTAGTTGGTGTTTATGGTAAGTAGTTTATTTATTATGTTATGGTTCGAAGTAGTTAT 481 CACACCCTAGTTCCGGAGATCATGGGTCGCTCGCGACCTGTTTCACAAAAGCTGAAAGCC :|:|:::|||||:++|||||:||||||++:|++++|::|||||:|:|||||:||||||:+ 481 TATATTTTAGTTTCGGAGATTATGGGTCGTTCGCGATTTGTTTTATAAAAGTTGAAAGTC 541 GCCAGCATCAAGAGGGCCAAAAAGGTTCAAGAGGGGAGGTACAGAGGGGTGCGGCAGCGG +::||:||:|||||||::|||||||||:|||||||||||||:|||||||||++|:||++| 541 GTTAGTATTAAGAGGGTTAAAAAGGTTTAAGAGGGGAGGTATAGAGGGGTGCGGTAGCGG 601 CCGTGGGGGCGATTTGCGGCGGAGATTAGAGACCCCAATACTAAGGAACGGAAGTGGCTA :++||||||++|||||++|++|||||||||||::::||||:|||||||++|||||||:|| 601 TCGTGGGGGCGATTTGCGGCGGAGATTAGAGATTTTAATATTAAGGAACGGAAGTGGTTA 661 GGCACTTTTGACACTGCTGAGGATGCAGCTCTCGCTTACGACACTGGTAAGAATATCAAC ||:|:||||||:|:||:||||||||:||:|:|++:|||++|:|:||||||||||||:||: 661 GGTATTTTTGATATTGTTGAGGATGTAGTTTTCGTTTACGATATTGGTAAGAATATTAAT 721 TTCTCCATTGCGCATTTGGTTACAAGGTGGCGATGACGATCACGTATCTCTATCCCTGAT ||:|::||||++:|||||||||:|||||||++||||++||:|++|||:|:|||:::|||| 721 TTTTTTATTGCGTATTTGGTTATAAGGTGGCGATGACGATTACGTATTTTTATTTTTGAT 781 AGAGCTAACTCTATCCGCCTTCCGTTTCTTGTAGCGGCAAGATCTATGAGAGGACCTAAG ||||:|||:|:|||:++::||:++|||:||||||++|:|||||:||||||||||::|||| 781 AGAGTTAATTTTATTCGTTTTTCGTTTTTTGTAGCGGTAAGATTTATGAGAGGATTTAAG 841 GCACGTACCAACTTTGTGTACCCTACGCATGAGACCTGTCTTCTTTCCGCTGCAGCGGCA |:|++||::||:||||||||:::||++:||||||::|||:||:|||:++:||:||++|:| 841 GTACGTATTAATTTTGTGTATTTTACGTATGAGATTTGTTTTTTTTTCGTTGTAGCGGTA 901 CTGGCGGCGCCAAATGGTAATTCGCAGCATCACCAGGTGGGTCTAATCGCTCAGAAGACC :|||++|++::|||||||||||++:||:||:|::||||||||:||||++:|:||||||:: 901 TTGGCGGCGTTAAATGGTAATTCGTAGTATTATTAGGTGGGTTTAATCGTTTAGAAGATT 961 TTGGGAAGTGCTGCTGCTCTCAGCAGCAGTACCGGCTTATTGCACNNAACCCTNNNNNNN ||||||||||:||:||:|:|:||:||:||||:++|:||||||:|:||||:::|||||||| 961 TTGGGAAGTGTTGTTGTTTTTAGTAGTAGTATCGGTTTATTGTATNNAATTTTNNNNNNN 1021 GGGGA ||||| 1021 GGGGA Used primers: Forward MSP: 5’-GGTAGCGGAAGTATAGGTATTTTC-3’ Reverse MSP: 5’-TTAACTAAAAAAACAACGACTCGTA-3’ Forward USP: 5’-GTAGTGGAAGTATAGGTATTTTTGG-3’ Reverse USP: 5’-TTAACTAAAAAAACAACAACTCATA-3’

Promoter region of PpEREBP/AP2 1 GCGTGACATCGTAGATATTGAGGATGAAGATTCGTCTGAGAATGGAACTTGCGTGGATAG |++|||:||++|||||||||||||||||||||++|:|||||||||||:|||++||||||| 1 GCGTGATATCGTAGATATTGAGGATGAAGATTCGTTTGAGAATGGAATTTGCGTGGATAG 61 CAGACATTTTCTGGGCTCGATGACTCCAAGCTCTGAACCTGTGTCTTCGAAATTTACGAT :|||:|||||:||||:|++||||:|::|||:|:||||::|||||:||++|||||||++|| 61 TAGATATTTTTTGGGTTCGATGATTTTAAGTTTTGAATTTGTGTTTTCGAAATTTACGAT 121 CACGCTGGAGTCCACGTACTTCGACACTACTTGGTCCAGTGTGCCACTTATAATTTTAGA :|++:||||||::|++||:||++|:|:||:|||||::||||||::|:||||||||||||| 121 TACGTTGGAGTTTACGTATTTCGATATTATTTGGTTTAGTGTGTTATTTATAATTTTAGA 181 CTCAAACTGCCACTGCATTAGTGCTTGCGAAGGAGAAGTTACTGTAGAAGCTTTTTTGTA :|:|||:||::|:||:|||||||:|||++||||||||||||:||||||||:||||||||| 181 TTTAAATTGTTATTGTATTAGTGTTTGCGAAGGAGAAGTTATTGTAGAAGTTTTTTTGTA 241 AAGCATAATCGAATCGCTTGGTGGTGAATCGTTTTCGGAGGAAACTGAGATATCATCGCT |||:|||||++|||++:||||||||||||++||||++|||||||:||||||||:||++:| 241 AAGTATAATCGAATCGTTTGGTGGTGAATCGTTTTCGGAGGAAATTGAGATATTATCGTT 301 GTCAAAGACCTGCCACAAGGATTTGAAAAAGTGGTGAACTCCAGTAAGGTGGGATATCTC ||:|||||::||::|:||||||||||||||||||||||:|::|||||||||||||||:|: 301 GTTAAAGATTTGTTATAAGGATTTGAAAAAGTGGTGAATTTTAGTAAGGTGGGATATTTT 361 CAAGGATGAAATGCATTTAGCAGAGCTTATTCCAATAACAACTGCACCATCACTACGTAA :||||||||||||:||||||:||||:|||||::|||||:||:||:|::||:|:||++||| 361 TAAGGATGAAATGTATTTAGTAGAGTTTATTTTAATAATAATTGTATTATTATTACGTAA 421 TGACCAAATAAACTAGTTATAAACAAAATGCTACGAGCATCTTCATAATGCGAAACATAA |||::|||||||:||||||||||:||||||:||++||:||:||:||||||++|||:|||| 421 TGATTAAATAAATTAGTTATAAATAAAATGTTACGAGTATTTTTATAATGCGAAATATAA 481 AGCCTGCTTCAAACACACCTTGAAACAGGTGTGAGTATTCACGTTGCTGGTTCACAAGAC ||::||:||:|||:|:|::||||||:|||||||||||||:|++|||:|||||:|:||||: 481 AGTTTGTTTTAAATATATTTTGAAATAGGTGTGAGTATTTACGTTGTTGGTTTATAAGAT 541 TCCGGAAAAAGTAATAAGTTTCTGGATCGAGTAGTGAAAGAGAATTACCTGAAATGGTTG |:++|||||||||||||||||:|||||++||||||||||||||||||::||||||||||| 541 TTCGGAAAAAGTAATAAGTTTTTGGATCGAGTAGTGAAAGAGAATTATTTGAAATGGTTG 601 GGATCCTGCTCCGCAATAGCGTGACACAAACAGCTCGGAACTGACAGTTGGACTCCGTTT ||||::||:|:++:|||||++|||:|:|||:||:|++|||:|||:|||||||:|:++||| 601 GGATTTTGTTTCGTAATAGCGTGATATAAATAGTTCGGAATTGATAGTTGGATTTCGTTT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>>> 661 GAATTGGGTCAGACGAAATTGTTGGTCTTCGTCAGAGACGCAATGCCATTGCTCTTGCTT |||||||||:|||++|||||||||||:||++|:|||||++:||||::||||:|:|||:|| 661 GAATTGGGTTAGACGAAATTGTTGGTTTTCGTTAGAGACGTAATGTTATTGTTTTTGTTT 721 CCCCGGACTTACTTTAAAACCTTCTACCTCAACCTTGTTGATCGAGGGACTTTTCGGCAT :::++||:|||:|||||||::||:||::|:||::||||||||++|||||:||||++|:|| 721 TTTCGGATTTATTTTAAAATTTTTTATTTTAATTTTGTTGATCGAGGGATTTTTCGGTAT <<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<< 781 CACCAATCTTTCATCCTGCGAAGGGCTGTGGAGAGCGTTGGGAGGAGGCATCTGTTGAGA :|::|||:|||:||::||++|||||:|||||||||++|||||||||||:||:|||||||| 781 TATTAATTTTTTATTTTGCGAAGGGTTGTGGAGAGCGTTGGGAGGAGGTATTTGTTGAGA

<<<<<<< MSPrev <<<<< USPrev 841 AGCTGGTTGGATCTTTCTGTCTACGTACCTACACCCATACCCAGGCAACCTGTATCCTTC ||:|||||||||:|||:|||:||++||::||:|:::|||:::|||:||::|||||::||: 841 AGTTGGTTGGATTTTTTTGTTTACGTATTTATATTTATATTTAGGTAATTTGTATTTTTT 901 TACCAGTTGCTCCTGTACTGCTGGTGCATTGAACATATTGTTGGAATGTTCATCATAATT ||::|||||:|::||||:||:|||||:||||||:||||||||||||||||:||:|||||| 901 TATTAGTTGTTTTTGTATTGTTGGTGTATTGAATATATTGTTGGAATGTTTATTATAATT 961 CAGCTGAGTATTTTTCCAGTTGCAGTTGCGAGCATTGTTT :||:|||||||||||::|||||:|||||++||:||||||| 961 TAGTTGAGTATTTTTTTAGTTGTAGTTGCGAGTATTGTTT Used primers: Forward MSP: 5’-TAATAGCGTGATATAAATAGTTCGG-3’ Reverse MSP: 5’-ATTAATAATACCGAAAAATCCCTCG-3’ Forward USP: 5’-GTAATAGTGTGATATAAATAGTTTGG-3’ Reverse USP: 5’-TAATAATACCAAAAAATCCCTCAAT-3’

Promoter region of PpbHLH 1 GAACAAGGGTTTAAAGCATTGCAGGCAGGTGATTGCATTTGTATTAACCGAGTAGTACAA |||:||||||||||||:||||:|||:|||||||||:|||||||||||:++|||||||:|| 1 GAATAAGGGTTTAAAGTATTGTAGGTAGGTGATTGTATTTGTATTAATCGAGTAGTATAA 61 TTCGAGTTTGTGTGTCATTTCGCAGAATATTGGTGGTTGGGGTTCCATGATATTGTTCAC ||++|||||||||||:||||++:|||||||||||||||||||||::|||||||||||:|: 61 TTCGAGTTTGTGTGTTATTTCGTAGAATATTGGTGGTTGGGGTTTTATGATATTGTTTAT 121 TGCTTTGATGTTTTTATTTGTGTGATTGTGATTTTATCATGATCAAACGCAAACAAAAGT ||:||||||||||||||||||||||||||||||||||:|||||:|||++:|||:|||||| 121 TGTTTTGATGTTTTTATTTGTGTGATTGTGATTTTATTATGATTAAACGTAAATAAAAGT 181 ATTCTTCTGTTGCTGCTGTATCACGTTTTACTGTGGGTTGAAGAATGTTGCAGTCTAACA |||:||:|||||:||:|||||:|++|||||:|||||||||||||||||||:|||:|||:| 181 ATTTTTTTGTTGTTGTTGTATTACGTTTTATTGTGGGTTGAAGAATGTTGTAGTTTAATA 241 ATGTGGTTCTCTAGAAGGACTGTCTAAGGCGACGGAATATTTCAGGCCTCTGTTGGGCTG ||||||||:|:||||||||:|||:|||||++|++||||||||:|||::|:|||||||:|| 241 ATGTGGTTTTTTAGAAGGATTGTTTAAGGCGACGGAATATTTTAGGTTTTTGTTGGGTTG 301 TGTTTATTATTTCTTTTTTGTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTTTACCTCATT ||||||||||||:||||||||||:||:||:||:||:||:||:||:||:|||||::|:||| 301 TGTTTATTATTTTTTTTTTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTATTTTATT 361 CATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT :||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| 361 TATATATATATATATATATATATATATATATATGTGTATGGATTTGTGAATGATATGAAT 421 TCACAGTTTTAATCATAACCTTGGATGCTGGGGGATACTCTGAATGAAATAGTTTTCCCC |:|:|||||||||:||||::|||||||:|||||||||:|:||||||||||||||||:::: 421 TTATAGTTTTAATTATAATTTTGGATGTTGGGGGATATTTTGAATGAAATAGTTTTTTTT

481 TACAGCAGTTATTCACGAAGTTGCTTTGAGCAATACCCGATATTACCATGGCTCAAGCTT ||:||:|||||||:|++||||||:||||||:||||::++||||||::||||:|:|||:|| 481 TATAGTAGTTATTTACGAAGTTGTTTTGAGTAATATTCGATATTATTATGGTTTAAGTTT 541 ATTGAATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAAC |||||||:|||:|||||:++:||:::|++:|:||||:|||||:|||:|||||||:||||: 541 ATTGAATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAAT MSPfwd >>>>>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 601 TGCTGATTTTGTCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAG ||:|||||||||:::||++||||||:||||||||:||++|:|++|:||:|||||:||:|| 601 TGTTGATTTTGTTTTATCGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAG 661 TTTCGAAATCCCAGCTTGGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGG |||++||||:::||:|||||:||||:||++||||||:||||++|||||:|::||||:||| 661 TTTCGAAATTTTAGTTTGGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGG <<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<< 721 TAACACGTCAAGAATTGGCATCGGCTCTTCAATTAGTACTATTGAACTTTCCGAGGACGT |||:|++|:|||||||||:||++|:|:||:||||||||:|||||||:|||:++||||++| 721 TAATACGTTAAGAATTGGTATCGGTTTTTTAATTAGTATTATTGAATTTTTCGAGGACGT <<<< MSPrev <<<< USPrev 781 GCTATGGCGTCGCAATCCAATTCCGACATGTGGATGGGCGCTGCATCATCAGAAATCAGG |:|||||++|++:|||::||||:++|:|||||||||||++:||:||:||:||||||:||| 781 GTTATGGCGTCGTAATTTAATTTCGATATGTGGATGGGCGTTGTATTATTAGAAATTAGG 841 TTCAGTTCATGATCACCCCTGCATTTCCTTCCAACAACCATTCTCATTACGAACAGGAGT ||:||||:|||||:|::::||:||||::||::||:||::|||:|:||||++||:|||||| 841 TTTAGTTTATGATTATTTTTGTATTTTTTTTTAATAATTATTTTTATTACGAATAGGAGT 901 TCTGCTGGTTTCTGTGATTGATAAAGACTCGGTTCGGAGTTAAATCACTTGGAATAAATC |:||:||||||:|||||||||||||||:|++|||++|||||||||:|:|||||||||||: 901 TTTGTTGGTTTTTGTGATTGATAAAGATTCGGTTCGGAGTTAAATTATTTGGAATAAATT 961 CATGAATGTGTTTTTTTTATTTTTTATTTTATTTTCCCATAGTTTGCCT :||||||||||||||||||||||||||||||||||:::||||||||::| 961 TATGAATGTGTTTTTTTTATTTTTTATTTTATTTTTTTATAGTTTGTTT Used primers: Forward MSP: 5’- ATTTTTTAAATTTCGTTTTTTTCGT-3’ Reverse MSP: 5’- ATTACCAATCCTAATAACCTCCGTC-3’ Forward USP: 5’- ATTTTTTAAATTTTGTTTTTTTTGT-3’ Reverse USP: 5’- ATTACCAATCCTAATAACCTCCATC-3’

Gene model of PpbHLH (Phypa1_209063) 1 ATTACCAAATCAAGTTGATCCATCGATTGCTAATTTGCAGACTGGAGTGCAGGAAAATGT ||||::||||:||||||||::||++||||:|||||||:|||:|||||||:|||||||||| 1 ATTATTAAATTAAGTTGATTTATCGATTGTTAATTTGTAGATTGGAGTGTAGGAAAATGT 61 AGGAACACCAAGTTTCAGCAAGGGCGTGCTGGACGAGGAGTGGTACACGCCCGAGACCTC |||||:|::||||||:||:|||||++||:||||++||||||||||:|++::++|||::|: 61 AGGAATATTAAGTTTTAGTAAGGGCGTGTTGGACGAGGAGTGGTATACGTTCGAGATTTT

121 CTTAATGGAGCTCTCTTACTCATTACCATATGGGATATCCGATACTCGCACAGGCTTTGG :|||||||||:|:|:|||:|:||||::|||||||||||:++|||:|++:|:|||:||||| 121 TTTAATGGAGTTTTTTTATTTATTATTATATGGGATATTCGATATTCGTATAGGTTTTGG 181 AATGCTCGAGTCGTCGCTGAATTTTGACAGCAGCAGCAACCTCATGTCTAGCTTCCGCCC ||||:|++|||++|++:||||||||||:||:||:||:||::|:||||:|||:||:++::: 181 AATGTTCGAGTCGTCGTTGAATTTTGATAGTAGTAGTAATTTTATGTTTAGTTTTCGTTT 241 TGCTCCCTCAGCCTTGAGCATGGGCCTTGAGAGCAACCGCAGTCTGGAGGATCTCGTTTG ||:|:::|:||::|||||:|||||::|||||||:||:++:|||:||||||||:|++|||| 241 TGTTTTTTTAGTTTTGAGTATGGGTTTTGAGAGTAATCGTAGTTTGGAGGATTTCGTTTG 301 CACTGGTCAGGGCTCGAGCAACGTTGGCCTCCTCTCAAGTCTTTCTCCAGGTCTTGTGGT :|:||||:||||:|++||:||++||||::|::|:|:||||:|||:|::||||:||||||| 301 TATTGGTTAGGGTTCGAGTAACGTTGGTTTTTTTTTAAGTTTTTTTTTAGGTTTTGTGGT 361 CTTGTCCCATTTTTCAGCAATTGTACACTTGTGATATCCGTTTCTCAACACATACACCGC :||||:::||||||:||:|||||||:|:|||||||||:++|||:|:||:|:|||:|:++: 361 TTTGTTTTATTTTTTAGTAATTGTATATTTGTGATATTCGTTTTTTAATATATATATCGT 421 AGCATTTTATAAAATTCATCTCAACAATGGATGAGAACCATGGTACCTGCTCAACTTACA ||:|||||||||||||:||:|:||:||||||||||||::||||||::||:|:||:|||:| 421 AGTATTTTATAAAATTTATTTTAATAATGGATGAGAATTATGGTATTTGTTTAATTTATA 481 AGGTCCTCAAGTAGGGAATTGAAGGATATCCTGTTGGTTGTGATTGCAGGCGGTCAACTC ||||::|:|||||||||||||||||||||::|||||||||||||||:|||++||:||:|+ 481 AGGTTTTTAAGTAGGGAATTGAAGGATATTTTGTTGGTTGTGATTGTAGGCGGTTAATTC 541 GGCCGGAGCACCGTAATGGAAAGCTTCAGCTCAGGTCTGCCAACAAGCTTCAACCAAGGA +|:++|||:|:++||||||||||:||:||:|:||||:||::||:|||:||:||::||||| 541 GGTCGGAGTATCGTAATGGAAAGTTTTAGTTTAGGTTTGTTAATAAGTTTTAATTAAGGA 601 ATCATCAACGCTGGTGGAAGCATAACCAACATCACCAGTAGCAACATAAATAACGTCCGC ||:||:||++:|||||||||:||||::||:||:|::|||||:||:||||||||++|:++: 601 ATTATTAACGTTGGTGGAAGTATAATTAATATTATTAGTAGTAATATAAATAACGTTCGT 661 TCTAACTTCCCCCTCATGGCCTCACCTTCGAACTTTTCCGATGCGTACCGCGCTCGATCA |:|||:||:::::|:||||::|:|::||++||:||||:++|||++||:++++:|++||:| 661 TTTAATTTTTTTTTTATGGTTTTATTTTCGAATTTTTTCGATGCGTATCGCGTTCGATTA 721 GTGAGCGAAGACAAGTCTGGGAAAGTTGTTGGCTCTGGCGGCCCACGGAATGAACTTGTG |||||++||||:||||:|||||||||||||||:|:|||++|:::|++|||||||:||||| 721 GTGAGCGAAGATAAGTTTGGGAAAGTTGTTGGTTTTGGCGGTTTACGGAATGAATTTGTG 781 CCATATCACAGAAACAAGGGGGCGGAAACTCGGAGCCATGGTCAGGGTCAGCAAACTCTA ::||||:|:|||||:|||||||++||||:|++|||::|||||:|||||:||:|||:|:|| 781 TTATATTATAGAAATAAGGGGGCGGAAATTCGGAGTTATGGTTAGGGTTAGTAAATTTTA 841 TTCTTGAAGCGCGCAGCTTCTCGCCGTTGTGCGGGGTCTAGTGGGACTGTCTCTCCAGTG ||:||||||++++:||:||:|++:++|||||++||||:||||||||:|||:|:|::|||| 841 TTTTTGAAGCGCGTAGTTTTTCGTCGTTGTGCGGGGTTTAGTGGGATTGTTTTTTTAGTG 901 AGCAAGTCACCCCCACGCGTGGTCACTAGTGCCTCCAACGATTCTTCTGTGGACACGCCG ||:||||:|:::::|++++||||:|:|||||::|::||++|||:||:||||||:|++:++ 901 AGTAAGTTATTTTTACGCGTGGTTATTAGTGTTTTTAACGATTTTTTTGTGGATACGTCG 961 GATAAAGACAGCCCTCATCCGCGTAATGCTCACTTGCAGAGCGCATCTGGAAGATTAAAT ||||||||:||:::|:||:++++|||||:|:|:|||:||||++:||:||||||||||||| 961 GATAAAGATAGTTTTTATTCGCGTAATGTTTATTTGTAGAGCGTATTTGGAAGATTAAAT

1021 ATCAACTCCGGATCGGATGATCCCAACGACATGGGATTAGATGGTGACGACTACGATGCC ||:||:|:++|||++||||||:::||++|:|||||||||||||||||++|:||++|||:: 1021 ATTAATTTCGGATCGGATGATTTTAACGATATGGGATTAGATGGTGACGATTACGATGTT 1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGCTCAGGGGGGCCCTACGAGGTGGAA |||||++|++||||||||||||||||||||||++|:|:|||||||:::||++|||||||| 1081 AAAGACGACGATGATTTGGATGAGAGTGGTGACGGTTTAGGGGGGTTTTACGAGGTGGAA 1141 GAAGGCGCAGGCAATGGAGCAGATCAAAGCATTGGAAAGGGAAACGGCAAAGGGAAACGA |||||++:|||:|||||||:||||:||||:||||||||||||||++|:|||||||||++| 1141 GAAGGCGTAGGTAATGGAGTAGATTAAAGTATTGGAAAGGGAAACGGTAAAGGGAAACGA 1201 GGACTTCCTGCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGC |||:||::||++|||||::|:||||:||||++:|||++:++:||||||:|:||++||++: 1201 GGATTTTTTGCGAAAAATTTTATGGTTGAGCGTAGGCGTCGTAAAAAATTTAACGATCGT MSPfwd >>>>>>>>>>>>>>>>>>>>>> USPfwd >>>>>>>>>>>>>>>>>>>>>>>>> 1261 CTGTACACGCTACGGTCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTT :||||:|++:||++||:|||||||::||||||||:|||||||:||::|||:|:|||:||| 1261 TTGTATACGTTACGGTTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTT 1321 GAACATGTTGCCCGCCTCGATTGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTA |||:||||||::++::|++||||:|||||||:|:||:||||||||||||||||::|:||| 1321 GAATATGTTGTTCGTTTCGATTGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTA 1381 CGAATCAGTGGGGTGTGGAGTACAGATGGATAGAGCCTCCATATTGGGGGATGCGATTGA ++|||:||||||||||||||||:||||||||||||::|::|||||||||||||++||||| 1381 CGAATTAGTGGGGTGTGGAGTATAGATGGATAGAGTTTTTATATTGGGGGATGCGATTGA 1441 GTACCTAAAGGAGCTCCTGCAACGCATCAATGAAATCCATAACGAACTGGAAGCAGCAAA |||::||||||||:|::||:||++:||:||||||||::||||++||:||||||:||:||| 1441 GTATTTAAAGGAGTTTTTGTAACGTATTAATGAAATTTATAACGAATTGGAAGTAGTAAA <<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<< 1501 GCTGGAGCAGTCGCGGTCGATGCCGTCTAGCCCCACTCCACGATCCACCCAAGGTTATCC |:|||||:|||++++||++|||:++|:|||::::|:|::|++||::|:::||||||||:: 1501 GTTGGAGTAGTCGCGGTCGATGTCGTTTAGTTTTATTTTACGATTTATTTAAGGTTATTT <<<<<< MSPrev <<<<< USPrev 1561 AGCTACAGTTAAAGAAGAATGCCCCGTCTTGCCGAATCCTGAATCCCAGCCTCCTCGAGT ||:||:|||||||||||||||:::++|:|||:++|||::|||||:::||::|::|++||| 1561 AGTTATAGTTAAAGAAGAATGTTTCGTTTTGTCGAATTTTGAATTTTAGTTTTTTCGAGT 1621 ATGTTGTTTATAATTTCTCACCTTCTTGGAATTGCATCTCAGTACTTATTTCGCAATGCC ||||||||||||||||:|:|::||:|||||||||:||:|:||||:||||||++:||||:: 1621 ATGTTGTTTATAATTTTTTATTTTTTTGGAATTGTATTTTAGTATTTATTTCGTAATGTT 1681 AACGACGTTCTGAAATGTCTACACTTTGCACTGTTCTGAAGTTCTGGAATGCTGAACATA ||++|++||:||||||||:||:|:||||:|:||||:|||||||:|||||||:||||:||| 1681 AACGACGTTTTGAAATGTTTATATTTTGTATTGTTTTGAAGTTTTGGAATGTTGAATATA 1741 GTTTACTTTGCACATTGTTTCATAGGTGGAAGTGAGGAAAAGAGAAGGTCAGGCCCTCAA |||||:||||:|:|||||||:||||||||||||||||||||||||||||:|||:::|:|| 1741 GTTTATTTTGTATATTGTTTTATAGGTGGAAGTGAGGAAAAGAGAAGGTTAGGTTTTTAA 1801 CATTCATATGTTCTGTGCCCGCCGGCCTGGACTCCTCCTCTCTACTGTGAAGGCGCTGGA :|||:|||||||:||||::++:++|::||||:|::|::|:|:||:||||||||++:|||| 1801 TATTTATATGTTTTGTGTTCGTCGGTTTGGATTTTTTTTTTTTATTGTGAAGGCGTTGGA

1861 CGCCCTTGGCTTGGATGTACAACAGGCTGTCATCAGCTGCTTCAATGGTTTCGCCCTTGA ++:::||||:|||||||||:||:|||:|||:||:||:||:||:||||||||++:::|||| 1861 CGTTTTTGGTTTGGATGTATAATAGGTTGTTATTAGTTGTTTTAATGGTTTCGTTTTTGA 1921 CCTCTTCCGTGCAGAGGTAAGAGTCTTTCGCCTCAAGAATTCGATGTGATTGCAACTAAT ::|:||:++||:||||||||||||:|||++::|:|||||||++|||||||||:||:|||| 1921 TTTTTTTCGTGTAGAGGTAAGAGTTTTTCGTTTTAAGAATTCGATGTGATTGTAATTAAT 1981 AGAGTTTGTGCGTTGACATGGGCGGGAAATGTACATCGGTTGTCTTATTTAGCTGTGTTA ||||||||||++||||:|||||++|||||||||:||++|||||:||||||||:||||||| 1981 AGAGTTTGTGCGTTGATATGGGCGGGAAATGTATATCGGTTGTTTTATTTAGTTGTGTTA 2041 GGCCTCAAGCTGAAGATCACCTTGGGTGTGGGTTATTGCTGTGGGCAGGCCAAAGATGTG ||::|:|||:|||||||:|::|||||||||||||||||:||||||:|||::||||||||| 2041 GGTTTTAAGTTGAAGATTATTTTGGGTGTGGGTTATTGTTGTGGGTAGGTTAAAGATGTG 2101 GACGTTGGACCAGAAGAAATAAAGGCCGTTCTGCTGCTCACTGCGGGATGTGATTTGCAC ||++|||||::||||||||||||||:++||:||:||:|:|:||++||||||||||||:|: 2101 GACGTTGGATTAGAAGAAATAAAGGTCGTTTTGTTGTTTATTGCGGGATGTGATTTGTAT 2161 TCTTTGCAGTAGATCCCACAATGCAGACGGACAAGTTGAATGAATTCTCTTCTTTTCTGC |:||||:|||||||:::|:||||:|||++||:||||||||||||||:|:||:||||:||: 2161 TTTTTGTAGTAGATTTTATAATGTAGACGGATAAGTTGAATGAATTTTTTTTTTTTTTGT 2221 ATGGGAAACAAAACACAAATTGATACGAGTGTGGTTTCAAAGTCTCTCCTCACCAGGCAG ||||||||:||||:|:|||||||||++||||||||||:|||||:|:|::|:|::|||:|| 2221 ATGGGAAATAAAATATAAATTGATACGAGTGTGGTTTTAAAGTTTTTTTTTATTAGGTAG 2281 TGTTCTTCAATTTTCACCATACAAGCTAAAAAATTTGACGCTACCTAAATTCAGTGGTTT ||||:||:||||||:|::|||:|||:||||||||||||++:||::||||||:|||||||| 2281 TGTTTTTTAATTTTTATTATATAAGTTAAAAAATTTGACGTTATTTAAATTTAGTGGTTT 2341 GAACCTGACATTGTTGTAGGCTGTACTCAGCTTTTTCTGTTTTTGTATAACTTAGTATAC |||::|||:|||||||||||:||||:|:||:|||||:|||||||||||||:||||||||+ 2341 GAATTTGATATTGTTGTAGGTTGTATTTAGTTTTTTTTGTTTTTGTATAATTTAGTATAC 2401 GGTAAAGGCCCACAGAGCAGAGAACGGTGGGACCTTCACGGTACTGCCACTAGACCAAGC +|||||||:::|:||||:||||||++||||||::||:|++|||:||::|:||||::|||: 2401 GGTAAAGGTTTATAGAGTAGAGAACGGTGGGATTTTTACGGTATTGTTATTAGATTAAGT 2461 CTTGAAAACGTTATGCAGGTGAAATGTTTCTGTACATCATCTTCAAGACTTGTGTGCTGT :|||||||++|||||:|||||||||||||:||||:||:||:||:||||:|||||||:||| 2461 TTTGAAAACGTTATGTAGGTGAAATGTTTTTGTATATTATTTTTAAGATTTGTGTGTTGT 2521 GTGTGCCTCTATGTGTTTGCCTATGTAACCAAGTTGTCCGTTGCTATCCAACACCTGCCA |||||::|:||||||||||::|||||||::|||||||:++|||:|||::||:|::||::| 2521 GTGTGTTTTTATGTGTTTGTTTATGTAATTAAGTTGTTCGTTGTTATTTAATATTTGTTA

Used primers: Forward MSP: 5’-GCGAAAAATTTTATGGTTGAGC-3’ Reverse MSP: 5’-TCCAACTTTACTACTTCCAATTCGT-3’ Forward USP: 5’-TGTGAAAAATTTTATGGTTGAGTGT-3’ Reverse USP: 5’-CCAACTTTACTACTTCCAATTCATT-3’

Figure S6. Primer design for bisulfite PCR analyses

Primer design for promoter, exon and intron regions of miRNA target genes

(PpC3HDZIP1, PpHB10, PpSBP3, PpARF, PpbHLH, PpTAS4), the ta-siRNA target

gene PpEREBP/AP2, and the PpGNT1 control gene as described in the manuscript. In

the gene model sequence of PpbHLH start and stop codons are highlighted in grey and

intron sequences are marked in blue. The upper strand of each sequence depicts the

wild type sequence, the lower strand indicates the expected cytosine to thymine

conversions after the bisulfite treatment. Upright lines mark unaltered nucleotides,

double points mark cytosine to thymine conversions. CpG dinucleotides are marked by

plus signs (+).

Figure S7

A WT GGACAATCTTTGGATATGTGCCAGCGTATCTTGTGATCGTGGTTCTTAAGGGTCGAGTGCTTAGCT WT BT GGATAATTTTTGGATATGTGTTAGTGTATTTTGTGATTGTGGTTTTTAAGGGTTGAGTGTTTAGTT KO1 BT GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT KO2 BT GGATAATTTTTGGATATGTGTTAGCGTATTTTGTGATTGTGGTTTTTAAGGGTCGAGTGTTTAGTT WT CCTCATCCTCATGCTTAGGTCTGGAAATATGTAAAAGGGGACGTAATGACAACACGAAGCTTATAA WT BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGATGTAATGATAATATGAAGTTTATAA KO1 BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA KO2 BT TTTTATTTTTATGTTTAGGTTTGGAAATATGTAAAAGGGGACGTAATGATAATACGAAGTTTATAA WT AAACTCAAAGCT WT BT AAATTTAAAGTT KO1 BT AAATTTAAAGTT KO2 BT AAATTTAAAGTT B WT TTTCTGGCTGATACAGAAACAGACGAGGTATTTGCTCGTATTTGCCTGCAGCCTGAGATTGGCTCC WT BT TTTTTGGTTGATATAGAAATAGATGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT KO1 BT TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT KO2 BT TTTTTGGTTGATATAGAAATAGACGAGGTATTTGTTTGTATTTGTTTGTAGTTTGAGATTGGTTTT WT TCCGCTCAGGATTTAACAGATGATTCTCTTGCGTCTCCGCCTCTAGAGAAACCAGCTTCATTTGCC WT BT TTTGTTTAGGATTTAATAGATGATTTTTTTGTGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT KO1 BT TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT KO2 BT TTCGTTTAGGATTTAATAGATGATTTTTTTGCGTTTTTGTTTTTAGAGAAATTAGTTTTATTTGTT WT AAAACGCTCACTCAAAGTGATGCAAACAACGGTGGAGGCTTTTCAATACCTC WT BT AAAATGTTTATTTAAAGTGATGTAAATAATGGTGGAGGTTTTTTAATATTTTKO1 BT AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC KO2 BT AAAATGTTTATTTAAAGTGATGTAAATAACGGTGGAGGTTTTTTAATATTTC

C WT TAGCTCATAACCCTCTCACAGGACGTAATGGGGGTGACAACATGCTAACAGAATTGCACGGTAAAG WT BT TAGTTTATAATTTTTTTATAGGATGTAATGGGGGTGATAATATGTTAATAGAATTGTATGGTAAAG KO1 BT TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG KO2 BT TAGTTTATAATTTTTTTATAGGACGTAATGGGGGTGATAATATGTTAATAGAATTGTACGGTAAAG WT GAAAACTGTACTAGGCATGTTATATGGGAATTCGGATCGCTTCTTGCAATTAAACACGCTAGCGCC WT BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATTGTTTTTTGTAATTAAATATGTTAGTGTTKO1 BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC KO2 BT GAAAATTGTATTAGGTATGTTATATGGGAATTTGGATCGTTTTTTGTAATTAAATACGTTAGTGTC WT GTTTGGTGCCAATGTTATTCTGG WT BT GTTTGGTGTTAATGTTATTTTGG KO1 BT GTTTGGTGTTAATGTTATTTTGG KO2 BT GTTTGGTGTTAATGTTATTTTGG D WT TCAAAGCAATCAATGTGCTAATGAACGTGCTGCCTGCATTGTCTGCAATGACTATGTACTGCGTAG WT BT TTAAAGTAATTAATGTGTTAATGAATGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGTGTAG KO1 BT TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG KO2 BT TTAAAGTAATTAATGTGTTAATGAACGTGTTGTTTGTATTGTTTGTAATGATTATGTATTGCGTAG WT TCAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGCAGGTCACTTGGGATGAGCCGGACCT WT BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTTGGATTT KO1 BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT KO2 BT TTAGTGGAAATAGGTGGTTGATTATGAGATTTTGGTTGTGTAGGTTATTTGGGATGAGTCGGATTT WT ATTGCAGGGAGTGAATCGTGTAAGCCCATGGCAGTTAGAGCTTGTGGCGACACTTCCTATGCAGCT WT BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGTGATATTTTTTATGTAGTT KO1 BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT KO2 BT ATTGTAGGGAGTGAATTGTGTAAGTTTATGGTAGTTAGAGTTTGTGGCGATATTTTTTATGTAGTT WT GCCC WT BT GTTT KO1 BT GTTT KO2 BT GTTT E WT TTCACAATGTGCTCTTCAAGCTTCGTTGTATGCTAAACTCTACTGCAATACTGCTACGGCGTGCC WT BT TTTATAATGTGTTTTTTAAGTTTTGTTGTATGTTAAATTTTATTGTAATATTGTTATGGTGTGTT KO1 BT TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT KO2 BT TTTATAATGTGTTTTTTAAGTTTCGTTGTATGTTAAATTTTATTGTAATATTGTTACGGCGTGTT WT TTTTCTTTTTTCGAAGTATGATATGATGACCTAATGGTCTTTTTGAATACGACAGGAATTCCATG WT BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATATGATAGGAATTTTATG KO1 BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG KO2 BT TTTTTTTTTTTTGAAGTATGATATGATGATTTAATGGTTTTTTTGAATACGATAGGAATTTTATG WT GAGACAG WT BT GAGATAG KO1 BT GAGATAG KO2 BT GAGATAG Figure S7. DNA methylation analysis of promoter and intragenic regions of the

PpARF gene in P. patens wild type and two ΔPpDCL1b mutants

Nucleotide sequences of PCR products obtained from methylation-specific PCRs are

aligned. (A) Promoter region. (B) Exon 1 upstream of the miR160 binding site. (C) Exon

4 downstream of the miR160 binding site. (D) Intron 2 upstream of the miR160 binding

site. (E) Intron 3 downstream of the miR160 binding site. WT: Wild type nucleotide

sequence of the analyzed region; CpG residues are highlighted in green. WT BT:

Sequences of PCR products obtained with USP primers from bisulfite-treated DNA

from wild type. KO1+2 BT: Sequences of PCR products obtained with MSP primers

from bisulfite-treated DNA from two ΔPpDCL1b mutants. Cytosine residues of CpG

dinucleotides which are methlyated in the ΔPpDCL1b mutants are indicated in red.

Cytosine residues of CpG dinucleotides which are not methylated are highlighted in

yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.

Five independent clones from each PCR product were sequenced.

Figure S8

A PpC3HDZIP1 mRNA GACGGATTTCCTGGCGAAGGCAACGGGAACCGCAGTGGATTGGATACAGT |||||||||||||||||||||||||||||||||||||||||||||||||| PpC3HDZIP1 genomic gacggatttcctggcgaaggcaacgggaaccgcagtggattggatacagt PpC3HDZIP1 mRNA TACCTGGTATGAAG------------------------------------ |||||||||||||| PpC3HDZIP1 genomic tacctggtatgaagGTatggatgccatgccttcctacggcacgttctaca PpC3HDZIP1 mRNA -------------------------------------------------- PpC3HDZIP1 genomic gtgtattgtggagtagcgagcctcacctgtaactcttgatctatagattc PpC3HDZIP1 mRNA -------------------------------------------------- PpC3HDZIP1 genomic cattatcagagatatgatcgcacgaaataactctttgttccaaccttttg PpC3HDZIP1 mRNA -------------------------------------------------- PpC3HDZIP1 genomic taaaataagtattagcggagtcatggtactggagcaaagtcaaacaaatt PpC3HDZIP1 mRNA -------------------------------------------------- PpC3HDZIP1 genomic aatttgactcaaaacacgacttcgaattaatttaggagctaacaaggtaa PpC3HDZIP1 mRNA -------------------------------------------------- PpC3HDZIP1 genomic tgatattgattctttaattcaaattaaagtggttgattgcaaatgccatt PpC3HDZIP1 mRNA --------------------------CCTGGTCCGGATGCCATTGGCATC |||||||||||||||||||||||| PpC3HDZIP1 genomic gctgatacgtcactagtgcaatgcAGcctggtccggatgccattggcatc

PpC3HDZIP1 mRNA ATTGCTATATCCCATGGTTGCGTGGGCATAGCAGCTCGAGCGTGCGGCCT |||||||||||||||||||||||||||||||||||||||||||||||||| PpC3HDZIP1 genomic attgctatatcccatggttgcgtgggcatagcagctcgagcgtgcggcct B PpHB10 mRNA AGCTACCGCTGAGGAGACGCTGACAGAATTCCTGGCTAAAGCCACAGGAA |||||||||||||||||||||||||||||||||||||||||||||||||| PpHB10 genomic agctaccgctgaggagacgctgacagaattcctggctaaagccacaggaa PpHB10 mRNA CGGCGGTGGATTGGATTCAGTTACCTGGTATGAAG--------------- ||||||||||||||||||||||||||||||||||| PpHB10 genomic cggcggtggattggattcagttacctggtatgaagGTatgtcatctctcc PpHB10 mRNA -------------------------------------------------- PpHB10 genomic gcgatggtgatgagtgattcaccgcatcacctcttaccgtaatctgagtg PpHB10 mRNA -------------------------------------------------- PpHB10 genomic atttgaagtaattgcaatgctctgaaattgaaatctgaagttctgaaagg PpHB10 mRNA -------------------------------------------------- PpHB10 genomic gcggtttggctgaatttgttaactggtgagactattgctgttgcactaat PpHB10 mRNA --------------------CCTGGTCCGGATGCCATTGGCATTATTGCT |||||||||||||||||||||||||||||| PpHB10 genomic agtaagatgtttatttgcAGcctggtccggatgccattggcattattgct

Figure S8. The miR166 binding sites of PpC3HDZIP1 and PpHB10 are disrupted

by introns

(A) PpC3HDZIP1; (B) PpHB10. The miR166 binding sites are indicated in red and are

underlined. Intron borders (GT and AG) are marked in bold.

Figure S9

WT TTTATCTCTAAATTCTTAGACAACGTCATTCAAAATAAGTTTTAAAACAGCGACTAGTCATAAAATA WT1+amiRNA BT TTTATTTTTAAATTTTTAGATAATGTTATTTAAAATAAGTTTTAAAATAGTGATTAGTTATAAAATA WT2+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA KO1+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA KO2+amiRNA BT TTTATTTTTAAATTTTTAGATAACGTTATTTAAAATAAGTTTTAAAATAGCGATTAGTTATAAAATA WT CGTATTTACACACTTGTATATGATGTACCATAGACGGTAACCGTACATATTTGCCGACACCCTGCAA WT1+amiRNA BT TGTATTTATATATTTGTATATGATGTATTATAGATGGTAATTGTATATATTTGTTGACATTTTGTAA WT2+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA KO1+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTTGACATTTTGTAA KO2+amiRNA BT CGTATTTATATATTTGTATATGATGTATTATAGACGGTAATTGTATATATTTGTCGACATTTTGTAA WT TTAATAGAGTTCGAATATCCCCGCCGCGTTCAAGTCGCCT WT1+amiRNA BT TTAATAGAGTTTGAATATTTTTGTTGTGTTTAAGTTGTTT WT2+amiRNA BT TTAATAGAGTTCGAATATTTTCGTCGCGTTTAAGTCGTTT KO1+amiRNA BT TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT KO2+amiRNA BT TTAATAGAGTTTGAATATTTTCGTCGCGTTTAAGTCGTTT

Figure S9. DNA methylation analysis of the PpGNT1 promoter region in lines

expressing the amiR-GNT1


aligned. WT: Wild type nucleotide sequence of the analyzed region; CpG residues are

highlighted in green. WT1 + amiRNA BT: Sequences of PCR products obtained with

USP primers from bisulfite-treated DNA from wild type line 1 expressing the amiR-

GNT1 at low levels. WT2 + amiRNA BT: Sequences of PCR products obtained with

MSP primers from bisulfite-treated DNA from wild type line 2 expressing the amiR-

GNT1 at high levels. KO1 + amiRNA BT and KO2 + amiRNA BT: Sequences of PCR

products obtained with MSP primers from bisulfite-treated DNA from ΔPpDCL1b

mutants expressing the amiR-GNT1. Cytosine residues of CpG dinucleotides which are

methlyated are indicated in red. Cytosine residues of CpG dinucleotides which are not

methylated are highlighted in yellow. Cytosine to thymine conversions are highlighted

in bold and are underlined. Five independent clones from each PCR product were

sequenced.

Figure S10 A WT ATCTTTCAAATTCCGCTTCCCTCGCACATGACTAAATCTAACATAATTTCTAAACTGCTGATTTTG WT/Con. BT ATTTTTTAAATTTTGTTTTTTTTGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG WT/ABA BT ATTTTTTAAATTTCGTTTTTTTCGTATATGATTAAATTTAATATAATTTTTAAATTGTTGATTTTG WT TCCCATCGGTGTGTCAGGAAAGACTTCGACTCGTCAGCTGTAACTTCAGTTTCGAAATCCCAGCTT WT/Con. BT TTTTATTGGTGTGTTAGGAAAGATTTTGATTTGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT WT/ABA BT TTTTATTGGTGTGTTAGGAAAGATTTCGATTCGTTAGTTGTAATTTTAGTTTTGAAATTTTAGTTT WT GGACAGAACTTCGTTTTATCTAGACGGAGGTCACCAGGACTGGTAAC WT/Con. BT GGATAGAATTTTGTTTTATTTAGATGGAGGTTATTAGGATTGGTAAT WT/ABA BT GGATAGAATTTCGTTTTATTTAGACGGAGGTTATTAGGATTGGTAAT B WT GCGAAAAACCTCATGGCTGAGCGCAGGCGCCGCAAAAAACTCAACGATCGCCTGTACACGCTACGG WT/Con. BT GTGAAAAATTTTATGGTTGAGTGTAGGTGTTGTAAAAAATTTAATGATTGTTTGTATATGTTATGG WT/ABA BT GCGAAAAATTTTATGGTTGAGCGTAGGTGTCGTAAAAAATTTAATGATCGTTTGTATACGTTACGG WT TCTGTAGTTCCTAAGATTACAAAGGTGCTTCCAAACTCTATCTTTGAACATGTTGCCCGCCTCGAT WT/Con. BT TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTTGAT WT/ABA BT TTTGTAGTTTTTAAGATTATAAAGGTGTTTTTAAATTTTATTTTTGAATATGTTGTTTGTTTCGAT WT TGCTGAATTGCACATCATGTATGTTGAGATGTCCACTTACGAATCAGTGGGGTGTGGAGTACAGAT WT/Con. BT TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTATGAATTAGTGGGGTGTGGAGTATAGAT WT/ABA BT TGTTGAATTGTATATTATGTATGTTGAGATGTTTATTTACGAATTAGTGGGGTGTGGAGTATAGAT WT GGATAGAGCCTCCATATTGGGGGATGCGATTGAGTACCTAAAGGAGCTCCTGCAACGCATCAATGA WT/Con. BT GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAATGTATTAATGA WT/ABA BT GGATAGAGTTTTTATATTGGGGGATGTGATTGAGTATTTAAAGGAGTTTTTGTAACGTATTAATGA WT AATCCATAACGAACTGGAAGCAGCAAAGCTGGA WT/Con. BT AATTTATAATGAATTGGAAGTAGTAAAGTTGGA WT/ABA BT AATTTATAACGAATTGGAAGTAGTAAAGTTGGA Figure S10. DNA methylation analysis of promoter and intragenic regions of

PpbHLH in untreated and ABA-treated P. patens wild type


aligned. (A) Promoter region of PpbHLH. (B) Coding Sequence of PpbHLH (intron

sequences are marked in blue). WT: Wild type nucleotide sequence of the analyzed

region; CpG residues are highlighted in green. WT/Con. BT: Sequences of PCR

products obtained with USP primers from bisulfite-treated DNA from untreated wild

type. WT/ABA BT: Sequences of PCR products obtained with MSP primers from

bisulfite-treated DNA from ABA-treated wild type. Cytosine residues of CpG

dinucleotides which are methlyated in the ABA-treated plants are indicated in red.

Cytosine residues of CpG dinucleotides which are not methylated are highlighted in

yellow. Cytosine to thymine conversions are highlighted in bold and are underlined.

Five independent clones from each PCR product were sequenced.

Table S1. Primers used in this study

Primer sequen ) Description of

TGGCATACAG Antisense oligon iR160.

ce (5’ 3’

GGAGCCAGGCA

experiment

ucleotide of m

GGGGAATGAAGCCTGGTCCGA Antisense oligonucleotide of miR166.

GGCGCTATCC Antisense oligon f miR390. CTCCTGAGCTT ucleotide o

GTGCTCACTCT TTCTGTCA Antisense oligonu of miR156. C cleotide

GCGTGCTCTCTCTCGTTGTCA Antisense oligonucleotide of miR535.

TCCAGACATAGACT Antisense oligonuCCATGCAA cleotide of miR538.

TGTCCTCTCAAGTCTTTCTCA Antisense oligon of miR1026. ucleotide

AAGCGTCCTGATTATTTGGAA Antisense oligonucleotide of amiR-GNT1.

GGGGCCATGC Antisense oligon A. TAATCTTCTCTG ucleotide of U6snRN

GGGTGTACAAGAGCTCTATAGTGCCACCG 5’ RACE primer eavage product of

PpC3HDZIP1.

to detect the cl

GCCACCGTTT GAGTTCC 5’ RACE neste the cleavage

product of PpC3

CCTGTCGGGA d primer to detect

HDZIP1.

AACCGCCGCCATCACACGGCCGGATC 5’ RACE primer to detect the cleavage product of

PpHB10.

CACACGGCCGGATCAGGTAACCACTTG 5’ RACE nested p to detect the cleavage

product of PpHB10.

rimer

CGCAGATCGG GTGCTCAC 5’ RACE primer he cleavage product of

PpSBP3.

TGAACCCGCG to detect t

CCCGCGGTGCT CAACTGAGACCGGA 5’ RACE nested p to detect the cleavage

product of PpSB

CAC rimer

P3.

ATGAGGGCTG CGTGT 5’ RACE primer age product of

PpbHLH.

TCTTTATCCGG to detect the cleav

ACTTTGGAGC AGGTGGA 5’ RACE primer he cleavage product of

PpGNT1 in PpG ssing lines.

AAGTTCTTCCC to detect t

NT1-amiRNA expre

CGGTGAGAAATACACGCTTTTGACCCT 5’ RACE primer gene PpGNT1. of the control

Primer sequence (5’ 3’) Description of experiment

GATGCTTACCATCCCCAGCAACGGA 5’ RACE primer to detect the cleavage product of

PpARF.

PpARF revers sense and

antisense trans the miR160

binding site by R

Primer used for e PpARF sense

transcript derived cDNA.

e primer to detect

cript downstream of

T-PCR.

the synthesis of th

CAAGATCATCAAG T PpARF forward prim and

antisense transcript do 0

binding site by R

Primer used f

antisense transcript de

TCTTCCATCC er to detect sense

wnstream of the miR16

T-PCR.

or the synthesis of the PpARF

rived cDNA.

CAAAGAGTGTCCAATCCTGGC PpC3HDZIP1 fo ct sense and

antisense trans he miR166

binding site by R

Primer used for of the PpC3HDZIP1

antisense transcript derived cDNA.

PpC3HDZIP1 forwa he miRNA:

mRNA duplexes by

rward primer to dete

cript upstream of t

T-PCR.

the synthesis

rd primer to detect t

RT-PCR.

TTGAAGCCACACCAGCCTGAC PpC3HDZIP1 re se and

antisense trans tream of the miR166

binding site by RT-PCR.

Primer used for PpC3HDZIP1

sense transcript

PpC3HDZIP1 re ct the miRNA:

mRNA duplexes

verse primer to detect sen

cript ups

the synthesis of the

derived cDNA.

verse primer to dete

by RT-PCR.

CAAAGATGTG PpGNT1 forwa t sense and

antisense transc

Primer used fo PpGNT1

antisense transc

PpGNT1 forwar r expression analysis by

RT-PCR.

PpGNT1 forwa the miRNA:

mRNA duplexes

Forward primer used for the amplification of the

PpGNT1 hybrid

GCGGAGAAAT rd primer to detec

ript by RT-PCR.

r the synthesis of the

ript derived cDNA.

d primer fo

rd primer to detect

by RT-PCR.

isation probe from cDNA.


ATAACCTGGCGACCTTTCCT PpGNT1 reverse primer to detect sense and

antisense transcript by RT-PCR.

Primer used fo the PpGNT1

sense transcript

PpGNT1 revers on analysis by

RT-PCR.

PpGNT1 revers t the miRNA:

mRNA duplexes

Reverse primer used for the amplification of the

PpGNT1 hybrid NA.

r the synthesis of

derived cDNA.

e primer for expressi

e primer to detec

by RT-PCR.

isation probe from cD

CCCCTGTCAG PpARF forward the miRNA:

mRNA duplexes by

TTGCAGCATCC primer to detect

RT-PCR.

CTAGAGGCGGCGACGCAAGAG PpARF reverse the miRNA:

mRNA duplexes

primer to detect

by RT-PCR.

GGAAAGAAGCAACAAGGTTGG PpHB10 forwa the miRNA:

mRNA duplexes .

rd primer to detect

by RT-PCR

ATCCCGCAGGACTGGAATCGC PpHB10 revers r t the miRNA:

mRNA duplexes

e prime to detec

by RT-PCR.

GTGCAGGGTTGTGATGCCGAC PpSBP3 forward p RNA:

mRNA duplexes by

rimer to detect the mi

RT-PCR.

ATGCAAGAAACTGGACTGCTTC PpSBP3 revers he miRNA:

mRNA duplexes

e primer to detect t

by RT-PCR.

TTGATCCATCGATTGCTAATTT PpbHLH forwa the miRNA:

mRNA duplexes R.

rd primer to detect

by RT-PC

AGCCCTGACCAGTGCAAAC PpbHLH revers the miRNA:

mRNA duplexes

e primer to detect

by RT-PCR.

AGCGTGGTATCACAATTGAC PpEF1α forward ion analysis by

RT-PCR and PC A.


PpEF1α hybridisation probe from cDNA.

primer for express

Rs from genomic DN


GATCGCTCGATCATGTTATC PpEF1α reverse primer for for expression analysis

by RT-PCR and PCRs from genomic DNA.

Primer used fo F1α sense

transcript derive

Reverse primer of the

PpEF1α hybridi from cDNA.

r the synthesis of PpE

d cDNA.

used for the amplification

sation probe

CAGGCTTTCGCGTAATTCCCGTTG Forward primer used for the amplification of the

PpC3HDZIP1 h cDNA. ybridisation probe from

AGTGCCTCCAACTTCGGGCCTAAC Reverse primer u

PpC3HDZIP1 hybridisation

sed for the amplification of the

probe from cDNA.

TTCTGCTGTCACTGGTGGACTT PpC3HDZIP1 forward primer for expression

analysis by RT-PCR.

AGAGTTCCAAGAACCTCCATGC PpC3HDZIP1 reverse p or expression

analysis by RT-

rimer f

PCR.

GATTCTGCTGTCACTGGTGGTC PpHB10 forward n analysis by

RT-PCR.

primer for expressio

GTCTTGTAACCAACGTGGACGA PpHB10 reverse primer for expression analysis by

RT-PCR.

GGCTATCACTTCCTGGATGGAC PpSBP3 forward primer for expression analysis by

RT-PCR.

ACAAGGAAGTTGCAGATGGTGA PpSBP3 reverse primer for expression analysis by

RT-PCR.

TGGTTCTCGGTTCAAGATGAAA PpARF forward primer for expression analysis by

RT-PCR.

CAACTTGTTGGACTGCTGAGGA PpARF reverse primer for expression analysis by

RT-PCR.

GGTGAACGTTTTGAGGTTGTG PpARF forward primer for the amplification of the

PpARF hybridisation probe.

CAAAGGAAACAAAACAAATGCC PpARF reverse primer for the amplification of the

PpARF hybridisation probe.

GGACGTTGGACCAGAAGAAA PpbHLH forward primer for the amplification of the

PpbHLH hybridisation probe.


CGCTTTATTCAGCCTCCTCA PpbHLH reverse primer for the amplification of the

PpbHLH hybridisation probe.

GGTTGGTCAT PpCOR47 forw the amplification of

the PpCOR47 h

GGGTTGCG ard primer for

ybridisation probe.

GAGGTCAACT CTCGCC PpCOR47 reve ification of

the PpCOR47 h probe.

GT rse primer for the ampl

ybridisation

CAGCCACAGCCAGTCAAGTGGATTCAGT 5’ RACE primer ge product of

PpTAS4.

to detect the cleava

ATGTGACTCCATTACATCAACTGCAGGT 5’ RACE neste ct the cleavage

product of PpTAS4

d primer to dete

.

GACCCACCTGGTGATGCTGCGAATTACC 5’ RACE primer ge product of

PpEREBP/AP2.

to detect the cleava

ATTTGGCGCCGCCAGTGCCGCTGCAGCG 5’ RACE neste cleavage

product of PpTA

d primer to detect the

P2.

GCACTTAGATTTCCACTGGGCG PpTAS4 forward for expression analysis by

RT-PCR.


PpTAS4 hybridi

primer

sation probe from cDNA.

AAGACATGGGAATTGCACACCC PpTAS4 reverse analysis by

RT-PCR.

Reverse primer for the amplification of the

PpTAS4 hybridisation probe from cDNA.

primer for expression

used

TTTGCGATGTTACGGTTGTAGC PpTAS1 forward primer for expression analysis by

RT-PCR.

ACAGCCAGTCAAGTGGATTCAG PpTAS1 reverse primer for expression analysis by

RT-PCR.

CCGCAACATCATGCTGAAGTGG PpEREBP/AP2 forward primer for expression

analysis by RT-PCR.


PpEREBP/AP2 hybridisation probe from cDNA.

GATGCTGGCGGCTTTCAGCTTT PpEREBP/AP2 reverse primer for expression

analysis by RT-PCR.

Reverse primer used for the amplification of the

PpEREBP/AP2 hybridisation probe from cDNA.


GAAGGAAGCAACGAGGCTGGTGGCGTGAAT

GCTAAGCTGACAGCC

Oligonucleotide to detect PpC3HDZIP1 sense

siRNA upstream of the miR166 binding site.

GTTCTTGGAAC GGAAACGGTG

GCACTATAGA

Oligonucleotide C3HDZIP1 sense

siRNA downstre inding site.

TCTCCCGACA

GCTCTT

to detect Pp

am of the miR166 b

GGCTGTCAGCTTAGCATTCACGCTCACCAGC

CTCGTTGCTTCCTTC

Oligonucleotide antisense

siRNA upstream of the miR166 binding site.

to detect PpC3HDZIP1

AAGAGCTCTATAGTGCCACCGTTTCCTGTCG

GGAGAGTTCCAAGAAC

Oligonucleotide IP1 antisense

siRNA downstre ing site.

to detect PpC3HDZ

am of the miR166 bind

TACACGATTCACTCCCTGCAATAGGTCCGGC

TCACTCCAAGTGAC

Oligonucleotide F sense siRNA

upstream of the miR1

to detect PpAR

60 binding site.

CTCCAGCTGTGAGCTTCCAGAAGCAGACATG

AAGGCTAAGGGCTG

Oligonucleotide sense siRNA

downstream of t .

to detect PpARF

he miR160 binding site

GTCACTTGGGATGAGCCGGACCTATTGCAGG

GAGTGAATCGTGTA

Oligonucleotide ense siRNA

upstream of the site.

to detect PpARF antis

miR160 binding

CAGCCCTTAGCCTTCATGTCTGCTTCTGGAA

GCTCACAGCTGGAG

Oligonucleotide F antisense siRNA

downstream of t ite.

to detect PpAR

he miR160 binding s

TGAAGCACTCATCACACCCTATGGAGCCATA

GCTAGGGTCTTGCG

Oligonucleotide se ta-

siRNA.

to detect PpTAS4 sen

TGGCTCCATAGGGTGTGATGATGTCTTCATC

CGGTGCTCTTCTACTGCCTT

Oligonucleotide to ct PpTAS4 antisense ta-

siRNA.

dete

GGCAAAGTGCAGGCGTCCCTAGCTGGTGCC

CATGGCAAGCAGTCT

Oligonucleotide to detect PpEREBP/AP2 sense

siRNA.

AGACTGCTTGCCATGGGCACCAGCTAGGGA

CGCCTGCACTTTGCC

Oligonucleotide to detect PpEREBP/AP2

antisense siRNA.

GTCGTATCCAGTGCAGGGTCCGAGGTATTCG

CACTGGATACGACGTGCTC

Oligonucleotide for miR156-specific cDNA

synthesis.


CACTGGATACGACTGGCAT


synthesis.


CACTGGATACGACGGGGAA


synthesis.


CACTGGATACGACGGCGCT


synthesis.



CACTGGATACGACGCAGAG

Oligonucleotide for ta-siRNA (pptA013298)-

specific cDNA synthesis.

GTCGTATCCA GAGGTATTCG

CACTGGATAC

Oligonucleotide pptA079444)-

specific cDNA s

GTGCAGGGTCC

GACTTGCCC

for ta-siRNA (

ynthesis.

GCGGCGGTGACAGAAGAGAGT Forward primer for miR156 RT-PCR.

CCTCCCGTGCCTGGCTCCCTGT Forward primer . for miR160 RT-PCR

GCGGCGGTCGGACCAGGCTTCA Forward primer 166 RT-PCR. for miR

GCGGCGGAAGCTCAGGAGGGAT Forward primer for miR390 RT-PCR.

GCGGCGGGTGATTGCACTGCAG Forward primer 013298) RT-

PCR.

for ta-siRNA (pptA

GCGGCGGATCACAAGGGTAGGT Forward primer NA (pptA079444) RT-

PCR.

for ta-siR

GTGCAGGGTCCGAGGTAT Universal reverse primer for miRNA and ta-siRNA

RT-PCR.

Supplemental Experimental Procedures

Isolation of PpDCL full-length cDNAs

Partial cDNA sequences of P. patens DCL genes were initially identified by tblastn

searches in P. patens EST sequences (Rensing et al., 2002) using A. thaliana DCL1-4

protein sequences (accession numbers P84634, Q9SP32, NP_189978, NP_566199) as

queries. Corresponding cDNA clones were sequenced and used to obtain the full-length

sequences. The cloning of full-length cDNA sequences was performed by 5’RACE-

PCRs and RT-PCRs using primers derived from available P. patens genomic sequence

data. To confirm that the amplicons were derived from the same cDNA all PCR and 5’

RACE primers were selected to produce overlapping PCR fragments of already known

sequence stretches.

Generation and molecular analysis of PpDCL1a and PpDCL1b knockout lines

For the generation of PpDCL1a and PpDCL1b knockout constructs we amplified a

PpDCL1a genomic region with the primers 5’-CCAGTTGCGCATAAAGTTGA-3’ and 5’-

TCCAAGGCATCCAGAGAGTC-3’ and a PpDCL1b cDNA region using the primers 5’-

GCATTCCTGTGGAGTTTGATG-3’ and 5’-ACCTTCCACACTTGGTGTGTG-3’. An nptII

selection marker cassette was cloned into a single Eco72I restriction site present in the

PpDCL1b cDNA fragment and into a single EcoRV restriction site of the PpDCL1a

genomic fragment. The complete knockout cassettes were released from the vector prior

to transformation. Primers used to identify ΔPpDCL1a transgenic lines were: 5’-

TTATGTGGATTCAGTGCGCTTC-3’ and 5’-CCATCGACTTAGCCAAACCAGT-3’. To

confirm a precise 5’ and 3’ integration of the PpDCL1a knockout construct we used the

primers 5’-TTTGCAGTTGACTGACCTCAAGA-3’ and 5’-

GCGGCTGAGTGGCTCCTTCA-3’ (5’ integration) and 5’-

CCAAGGATCCCGGAAGAGGA-3’ and 5’-AAATTATCGCGCGCGGTGTC-3’ (3’

integration). To confirm the loss of PpDCL1a transcript by RT-PCR the primers 5’-

TTGGTCCGTTGGAATACACA-3’ and 5’- AATCTTTGTGCGCCTCTCAC-3’ were used.

Primers used for the screening of transgenic ΔPpDCL1b lines were: 5’-

GCATTCCTGTGGAGTTTGATG-3’ and 5’-ACCTTCCACACTTGGTGTGTG-3’. The

same primers were used to confirm the loss of PpDCL1b transcript by RT-PCR. A

second primer pair upstream of the integration site was used for RT-PCR: 5’-

AGGATTGTTACTGCGGTGCA-3’ and 5’-AAGCTCTGCACGCTCATAGC-3’. To confirm

a precise 5’ and 3’ integration of the PpDCL1b knockout construct we used the primers

5'-TGCTACTCACTTCATGAACTG-'3 and 5'-ACGTGACTCCCTTAATTCTCC-'3 (5’

integration) and 5'-CCCGCAATTATACATTTAATACG-'3 and 5'-

GCACCATGGCTGCAACAAAG-'3 (3’ integration). RT-PCR control primers for the

PpEF1α control gene are listed in Table S1.

P. patens lines expressing an artificial miRNA (amiRNA) targeting PpGNT1

The amiR-GNT1 sequence was introduced into the A. thaliana miRNA319a precursor

by overlapping PCR as described (Khraiwesh et al., 2008). The resulting construct was

used for transfections of P. patens wild type and ΔPpDCL1b mutant lines. To identify

transgenic lines harboring the amiR-GNT1 expression construct PCR was performed

using the primers 5’-TGATATCTCCACTGACGAAAGGG-3’ and 5’-

GGATCCCCCCATGGCGATGCCTTAAAT-3’.

Detection of RNA cleavage products

Synthesis of 5’ RACE-ready cDNAs was carried out according to Zhu et al. (Zhu et al.,

2001) with the BD Smart RACE cDNA Amplification Kit (Clontech). PCR reactions were

performed using the UPM Primer-Mix in combination with gene-specific primers derived

from target RNAs (Table S1). Cleavage products were excised from the gel, cloned and

sequenced.

Small RNA Blots

Total RNA was separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea

in TBE buffer. The RNA was electroblotted onto nylon membranes for 1 h at 400 mA.

Radiolabeled probes were generated by end-labeling of DNA oligonucleotides

complementary to miRNA, siRNA and ta-siRNA sequences and the U6snRNA control

(Table S1) with γ32P-ATP using T4 polynucleotide kinase. Blot hybridization was carried

out in 0.05 M sodium phosphate (pH 7.2), 1 mM EDTA, 6 x SSC, 1 x Denhardt’s, 5%

SDS. Blots were washed 2-3 times with 2 x SSC, 0.2% SDS and one time with 1 x SSC,

0.1% SDS. Blots were hybridized and washed at temperatures 5°C below the Tm of the

oligonucleotide. The sequences of the oligonucleotides used for the detection of small

RNAs are listed in Table S1.

Detection of small RNAs by RT-PCR

The RT-PCR analyses of the miR156, 160, 166, 390, and the ta-siRNAs pptA079444

(processed from the PpTAS1 gene) and pptA013298 (processed from the PpTAS3

gene) were carried out as described (Varkonyi-Gasic et al., 2007). The sequences of

oligonucleotides used for the cDNA synthesis and subsequent PCR reactions are listed

in Table S1.

Expression analysis by RT-PCR and RNA gel blots

RT-PCRs were performed for PpEF1α, PpGNT1, PpC3HDZIP1, PpHB10, PpSBP3,

PpARF, and PpTAS1 from three independent biological replicates with gene-specific

primers (Table S1). PCR products were quantified with the Quantity One Software (Bio-

Rad). The relative amounts of the transcripts were normalized to the constitutive control

PpEF1α. 20 µg of total RNA isolated from wild type, ΔPpDCL1b mutants and transgenic

lines expressing the amiR-GNT1, respectively, were separated in denaturing agarose

gels and blotted onto nylon membranes. Hybridization probes for PpARF, PpC3HDZIP1,

PpGNT1, PpEF1α, PpTAS4 PpEREBP/AP2, PpbHLH, and PpCOR47 were amplified

from wild type cDNA (primers listed in Table S1). The ABA-responsive gene PpCOR47

(Frank et al., 2005) was used to control the efficiency of the ABA treatments.

DNA methylation analysis

The cDNA sequences of PpC3HDZIP1 (DQ385516), PpHB10 (AB032182), PpARF

(AR452951), PpSBP3 (AJ968318) and PpGNT1 (AJ429143) were used for BLASTN

searches to identify corresponding genomic sequences from the P. patens whole-

genome-shotgun (WGS) traces (accessible via www.ncbi.nlm.nih.gov/Traces/trace.cgi).

The identified genomic sequences were clustered and assembled using the Paracel

Transcript Assembler to determine the genomic exon/intron structure (Figure S5). The

parameters for clustering threshold, overlap length and overlap identity were 100 nt, 80

nt and 90%, respectively. Primers to analyse the PpTAS4 genomic locus were derived

from the reported PpTAS4 sequence (Talmor-Neiman et al., 2006). Primers for the

analysis of the PpEREBP/AP2 and PpbHLH gene were derived from the corresponding

gene model of the available P. patens genomic sequence (http://genome.jgi-

psf.org/Phypa1_1/Phypa1_1.home.html; gene model accession numbers

Phypa1_129196 [PpEREBP/AP2] and Phypa1_209063 [PpbHLH]). The derived

promoter, exon and intron regions were analyzed with the MethPrimer program (Li and

Dahiya, 2002) to deduce methylation-specific (MSP) and unmethylation-specific primers

(USP) (Figure S6) for PCR analysis of bisulfite-treated DNA.

Detection of sense and antisense transcripts

cDNA from wild type plants and ΔPpDCL1b mutants was synthesized from 4 µg total

RNA with Superscript III (Invitrogen) using primers specific for sense and antisense

transcripts, respectively (Table S1). To monitor the efficiency of cDNA synthesis, primers

specific for the PpEF1α sense transcript were added to each cDNA synthesis reaction.

RT-PCRs were carried out with gene-specific primers (Table S1).

Supplemental Experimental Procedures References

Frank, W., Ratnadewi, D., and Reski, R. (2005). Physcomitrella patens is highly tolerant

against drought, salt and osmotic stress. Planta 220, 384-394.

Khraiwesh, B., Ossowski, S., Weigel, D., Reski, R., and Frank, W. (2008). Specific gene

silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted

gene knockouts. Plant Physiol.

Li, L. C., and Dahiya, R. (2002). MethPrimer: designing primers for methylation PCRs.

Bioinformatics 18, 1427-1431.

Rensing, S. A., Rombauts, S., Van de Peer, Y., and Reski, R. (2002). Moss

transcriptome and beyond. Trends Plant Sci 7, 535-538.

Talmor-Neiman, M., Stav, R., Klipcan, L., Buxdorf, K., Baulcombe, D. C., and Arazi, T.

(2006). Identification of trans-acting siRNAs in moss and an RNA-dependent RNA

polymerase required for their biogenesis. Plant J 48, 511-521.

Varkonyi-Gasic, E., Wu, R., Wood, M., Walton, E. F., and Hellens, R. P. (2007).

Protocol: a highly sensitive RT-PCR method for detection and quantification of

microRNAs. Plant Methods 3, 12.

Zhu, Y. Y., Machleder, E. M., Chenchik, A., Li, R., and Siebert, P. D. (2001). Reverse

transcriptase template switching: a SMART approach for full-length cDNA library

construction. Biotechniques 30, 892-897.

Chapter III Artificial microRNAs in Physcomitrella patens

3 Chapter III: Publication 1

Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockout Plant Physiology, October 2008, Vol. 148, pp. 684–693 Received August 13, 2008 Accepted August 22, 2008 Own contribution:

Carried out all experimental work reported in the publication and contributed to prepare the

manuscript (drafting the manuscript and preparing the figures). The work was supervised by

W. Frank.

121

Breakthrough Technologies

Specific Gene Silencing by Artificial MicroRNAs inPhyscomitrella patens: An Alternative to TargetedGene Knockouts1[C][W][OA]

Basel Khraiwesh, Stephan Ossowski, Detlef Weigel, Ralf Reski, and Wolfgang Frank*

Plant Biotechnology, Faculty of Biology (B.K., R.R., W.F.), Freiburg Initiative for Systems Biology (R.R., W.F.),and Centre for Biological Signaling Studies (R.R.), University of Freiburg, 79104 Freiburg, Germany; andDepartment of Molecular Biology, Max Planck Institute for Developmental Biology, 72076 Tuebingen,Germany (S.O., D.W.)

MicroRNAs (miRNAs) are approximately 21-nucleotide-long RNAs processed from nuclear-encoded transcripts, whichinclude a characteristic hairpin-like structure. MiRNAs control the expression of target transcripts by binding to reversecomplementary sequences directing cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) canbe generated by exchanging the miRNA/miRNA* sequence within miRNA precursor genes, while maintaining the pattern ofmatches and mismatches in the foldback. Thus, for functional gene analysis, amiRNAs can be designed to target any gene ofinterest. The moss Physcomitrella patens exhibits the unique feature of a highly efficient homologous recombination mechanism,which allows for the generation of targeted gene knockout lines. However, the completion of the Physcomitrella genomenecessitates the development of alternative techniques to speed up reverse genetics analyses and to allow for more flexibleinactivation of genes. To prove the adaptability of amiRNA expression in Physcomitrella, we designed two amiRNAs, targetingthe gene PpFtsZ2-1, which is indispensable for chloroplast division, and the gene PpGNT1 encoding an N-acetylglucosami-nyltransferase. Both amiRNAs were expressed from the Arabidopsis (Arabidopsis thaliana) miR319a precursor fused to aconstitutive promoter. Transgenic Physcomitrella lines harboring the overexpression constructs showed precise processing ofthe amiRNAs and an efficient knock down of the cognate target mRNAs. Furthermore, chloroplast division was impeded inPpFtsZ2-1-amiRNA lines that phenocopied PpFtsZ2-1 knockout mutants. We also provide evidence for the amplification of theinitial amiRNA signal by secondary transitive small interfering RNAs, although these small interfering RNAs do not seem tohave a major effect on sequence-related mRNAs, confirming specificity of the amiRNA approach.

During the last decade, small nonprotein-codingRNAs (20–24 nucleotides [nt] in size) have been dem-onstrated to be involved in RNA-mediated phenomenasuch as RNA interference (RNAi), cosuppression, genesilencing, and quelling (Matzke et al., 1989; Napoli et al.,1990; deCarvalhoet al., 1992;RomanoandMacino, 1992;Lee et al., 1993; Hamilton and Baulcombe, 1999). Major

classes of small RNAs include microRNAs (miRNAs)and small interfering RNAs (siRNAs),which differwithrespect to their biogenesis (Bartel, 2004; Chapman andCarrington, 2007). MiRNAs are approximately 21-ntRNAs that are encoded by endogenous MIR genes.Their primary transcripts form precursor RNAs exhib-iting a partially double-stranded stem-loop structurethat is processed by DICER-LIKE proteins to releasemature miRNAs (Bartel, 2004). MiRNAs are recruitedto the RNA-induced silencing complex (RISC), wherethey become activated by unwinding of the doublestrandandsubsequentlybind tocomplementarymRNAsequences resulting in either direct cleavage of themRNA or repression of their translation by RISC(Bartel, 2004; Kurihara and Watanabe, 2004; Brodersenet al., 2008). Recently, miRNAs have been identified asimportant regulators of gene expression in both plantsand animals (Jones-Rhoades et al., 2006), and particularmiRNA families were shown to be highly conserved inevolution (Jones-Rhoades et al., 2006; Axtell et al., 2007;Fahlgren et al., 2007; Fattash et al., 2007; Axtell andBowman, 2008). In contrast, precursors of siRNAsform perfectly complementary double-stranded RNA(dsRNA) molecules (Myers et al., 2003). They originatefrom transgenes, viruses, and transposons and mayrequire RNA-dependent RNApolymerases for dsRNAformation (Waterhouse et al., 2001; Aravin et al., 2003).

1 This work was supported by the Landesstiftung Baden-Wurttemberg (grant no. P–LS–RNS/40 to W.F., R.R., and D.W.), theFederal Ministry of Education and Research (Freiburg Initiative forSystems Biology grant no. 0313921 to R.R. and W.F.), the ExcellenceInitiative of the German Federal State Governments (BiologicalSignaling Studies grant no. EXC294 to R.R.), and European Com-munity FP6 IP SIROCCO (contract no. LSHG–CT–2006–037900 toD.W.).

* Corresponding author; e-mail [email protected].

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Wolfgang Frank ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.128025

684 Plant Physiology, October 2008, Vol. 148, pp. 684–693, www.plantphysiol.org � 2008 American Society of Plant Biologists

UnlikemiRNAs, thediced siRNAproductsderived fromthe long complementary precursors are not uniform insequence, but correspond to different regions of theirprecursor. Whereas miRNAs mainly mediate posttran-scriptional control of endogenous transcripts, siRNAshave been implicated in transcriptional silencing oftransposable elements as well as posttranscriptionalcontrol of endogenous and exogenous RNAs, for exam-ple, viral transcripts (Waterhouse et al., 2001; Aravinet al., 2003; Myers et al., 2003; Ossowski et al., 2008).Previous reports demonstrated that the alteration of

several nucleotides within the miRNA sequence doesnot affect its biogenesis as long as the positions ofmatches and mismatches within the precursor stemloop remain unaffected (Vaucheret et al., 2004). Thisraises the possibility of modifying miRNA sequencesand creating artificial miRNAs (amiRNA) directedagainst any gene of interest resulting in posttran-scriptional silencing of the corresponding transcript(Zeng et al., 2002; Parizotto et al., 2004; Alvarez et al.,2006; Niu et al., 2006; Schwab et al., 2006; Warthmannet al., 2008). In addition, genome-wide expressionanalyses in Arabidopsis (Arabidopsis thaliana) haveshown that plant amiRNAs exhibit high specificitysimilar to natural miRNAs (Schwab et al., 2005, 2006),such that their sequences can easily be optimized toknock down the expression of a single gene or severalhighly conserved genes without affecting the expres-sion of other genes.The moss Physcomitrella patens has become a recog-

nized model system to study diverse processes inplant biology, which was mainly based on the uniqueability to efficiently integrate DNA into its nucleargenome by means of homologous recombination en-abling the generation of targeted gene knockout lines(Schaefer, 2002). Furthermore, based on the predomi-nant haploid phase of Physcomitrella’s life cycle, thefrequency of phenotypic deviations caused by thedisruption of a single gene is higher compared toseed plants (Egener et al., 2002). Nevertheless, thegeneration of targeted knockout mutants in Physcomi-trella has limitations. For example, despite the haploidgenome, homologs might still compensate for eachother and one cannot recover knockouts of genes withessential functions. Furthermore, the targeted knock-out of a single gene requires several cloning steps,repetitive selection of transgenic lines, and detailedmolecular analysis of putative knockout candidates.The recently published Physcomitrella genome (Rensinget al., 2008) now opens the way for medium- to large-scale analysis of gene functions in a postgenomic era(Quatrano et al., 2007) requiring the development ofnew techniques. The posttranscriptional silencing ofgenes by amiRNAs may serve as an appropriate toolto speed up such analyses because they can be de-signed to target several genes (as long they contain atleast one conserved sequence stretch), amiRNAs canbe expressed from inducible promoters, and amiRNAconstructs can easily be generated using a standard-ized cloning procedure (Schwab et al., 2006).

Alternative approaches to analyze gene functionsin Physcomitrella were recently reported, which werebased on the expression of classical inverted repeatsequences resulting in the formation of dsRNA mol-ecules, which give rise to siRNAs and consequentlysilence the target transcript (Bezanilla et al., 2003, 2005;Vidali et al., 2007). One drawback to applying classicalRNAi constructs is the production of a diverse setof siRNAs from the complete dsRNA, which may af-fect off-target transcripts. Furthermore, in some cases,gene silencing triggered by the expression of invertedrepeat sequences was found to be unstable in Phys-comitrella (Bezanilla et al., 2005). Additional differ-ences in the action of siRNAs and amiRNAs mayresult from their varying mobility within the plant.Recent studies have shown that transgene-derived orviral-induced siRNAs are able to move from cell tocell, whereas miRNAs are not mobile and act cellautonomously (Tretter et al., 2008).

Independent studies on small RNAs in Physcomitrellarevealed the existence of a diverse miRNA repertoire,including highly conserved miRNA families (Araziet al., 2005; Axtell et al., 2006, 2007; Talmor-Neimanet al., 2006; Fattash et al., 2007). Furthermore, theircorresponding precursor transcripts share the charac-teristic hairpin-like structure known from seed plants.Thus, the design and expression of amiRNAs for thespecific knock down of genes in Physcomitrella shouldbe feasible. To test amiRNA function in Physcomitrella,we targeted the gene PpFtsZ2-1, which is requiredfor chloroplast division (Strepp et al., 1998), and thePpGNT1 gene encoding an N-acetylglucosaminyltrans-ferase (Koprivova et al., 2003). PpFtsZ2-1 null mutantsform macrochloroplasts, presenting an obvious pheno-type, which enables direct evaluation of the efficiency ofthe intended amiRNA approach.

RESULTS

Expression and Detection of PpFtsZ2-1-amiRNA and

PpGNT1-amiRNA in Physcomitrella

The use of amiRNAs for efficient gene silencing hasbeen reported in various seed plants (Alvarez et al.,2006; Niu et al., 2006; Schwab et al., 2006; Warthmannet al., 2008), but it has not been tested in nonseedplants such as the bryophyte P. patens. This is an issuebecause functional studies of essential members of theRNAi machinery in Physcomitrella, such as Dicer andArgonaute proteins, are still missing (Axtell et al.,2007). Furthermore, the complement of essential RNAi-related proteins in Physcomitrella differs from that inseed plants (Axtell et al., 2007; Rensing et al., 2008).

We designed two amiRNAs that were predicted totarget the genes PpFtsZ2-1 and PpGNT1, respectively,using the amiRNA designer interface WMD (Schwabet al., 2006; Ossowski et al., 2008). The designedamiRNAs contain a uridine residue at position 1 andan adenine residue at position 10, both of which are

Artificial MicroRNAs in Physcomitrella patens

Plant Physiol. Vol. 148, 2008 685

overrepresented among natural plant miRNAs andincrease the efficiency ofmiRNA-mediated target cleav-age (Schwab et al., 2005). Furthermore, we also pre-ferred that the amiRNAs exhibit 5# instability relativeto the miRNA*, which positively affects separation ofboth strands during RISC loading (Fig. 1A; Malloryet al., 2004; Schwab et al., 2005). In previous studies, theArabidopsis miR319a precursor was used to introducespecific nucleotide changeswithin themiRNA/miRNA*stem-loop region. Based on the conservation of themiR319 family among land plants (Jones-Rhoades et al.,2006; Fattash et al., 2007; Axtell and Bowman, 2008;Warthmann et al., 2008) and similar secondary struc-tures of miR319 precursor transcripts from Arabidopsisand Physcomitrella (Fig. 1B; Supplemental Fig. S1),we hypothesized that the PpFtsZ2-1-amiRNA andPpGNT1-amiRNAwill be correctly processed from theArabidopsismiR319a precursor. ThePpFtsZ2-1-amiRNAand PpGNT1-amiRNA and the corresponding miRNA*sequences were introduced into the miR319a precur-sor by overlapping PCR using primers harboring therespective amiRNA and miRNA* sequences, clonedinto the plant expression vector pPCV downstreamof a double cauliflower mosaic virus 35S promoter(Fig. 1A) and used for transfection of Physcomitrellaprotoplasts. After selection of regenerating plants,genomic DNA of individual lines was analyzed byPCR with primers flanking the amiRNA sequencepresent in the expression constructs to identify trans-genic lines that had integrated the PpFtsZ2-1-amiRNAand PpGNT1-amiRNA constructs, respectively. Eightof 12 regenerated lines derived from the transforma-tion with the PpFtsZ2-1-amiRNA construct and sevenof 12 regenerated lines derived from the transforma-tion with the PpGNT1-amiRNA construct producedthe expected PCR amplicon. Thus, we cannot ex-clude that some lines survived the antibiotic selectionwithout the integration of the DNA constructs. Fromthe lines harboring an overexpression construct, threePpFtsZ2-1-amiRNA lines and two PpGNT1-amiRNAlines were selected for further analysis (Fig. 1C). As thegenerated amiRNA overexpression constructs do notcontain homologous sequences of the Physcomitrella ge-nome, the constructs are expected to integrate into thePhyscomitrella genome by an illegitimate recombina-tion event. To prove the correct maturation of thePpFtsZ2-1-amiRNA and PpGNT1-amiRNA from theArabidopsis miR319a precursor and its accumulationin the transgenic lines, we performed small RNA gel-blot analyses with antisense probes for both amiRNAs.Accumulation of the mature PpFtsZ2-1-amiRNA andPpGNT1-amiRNA was detected in all lines analyzed,demonstrating that the amiRNAs are efficiently pro-cessed from the Arabidopsis miR319a precursor inPhyscomitrella (Fig. 1D). However, normalization ofthe PpFtsZ2-1-amiRNA and PpGNT1-amiRNA hybri-dization signals to the U6snRNA controls revealedamiRNA expression levels that differed up to 8-foldfor the PpFtsZ2-1-amiRNA and up to 5-fold for thePpGNT1-amiRNAbetweenthe individual lines (Fig.1D).

AmiRNA-Directed Cleavage of PpFtsZ2-1 and

PpGNT1 mRNAs

The expression of the PpFtsZ2-1-amiRNA andPpGNT1-amiRNA should cause cleavage of the cog-nate mRNAs within the region complementary to theamiRNA sequences. To prove this, we performed 5#RACE-PCRs to detect specific PpFtsZ2-1 and PpGNT1mRNA cleavage products. Using 5# RACE-readycDNA prepared from one PpFtsZ2-1-amiRNA, onePpGNT1-amiRNA overexpression line, and wild type,cleavage products of the expected size were onlyamplified from the amiRNA lines. Conversely, inwild type, the 5# RACE-PCRs yielded exclusivelyfragments derived from the full-length transcripts(Fig. 1E). The PCR products corresponding to theexpected size of the PpFtsZ2-1 and PpGNT1 mRNAcleavage products in the amiRNA lines were clonedand sequenced to determine the precise mRNA cleav-age sites. In 12 of 18 clones analyzed, PpFtsZ2-1mRNAcleavage occurred between nucleotide positions 11and 12 with respect to the PpFtsZ2-1-amiRNA se-quence, whereas the remaining six clones resultedfrom cleavage of the PpFtsZ2-1 mRNA between nu-cleotides 12 and 13 (Fig. 1E). Normally, in plants,cleavage within a target transcript that is mediated bya 21-nt miRNA occurs between positions 10 and 11with respect to the miRNA sequence (Llave et al.,2002), suggesting that the actual amiRNAs producedfrom the PpFtsZ2-1-amiRNA construct were shifted by1 or 2 nt. However, the sequencing of six independentclones of PpGNT1 mRNA cleavage products revealedthat cleavage occurred between positions 10 and 11with respect to the PpGNT1-amiRNA sequence (Fig.1E), indicating precise processing of the PpGNT1-amiRNA from the precursor construct.

Target sites in plant mRNAs normally share highsequence complementarity to the respective miRNA(Schwab et al., 2005). To prove the specificity of theexpressed PpFtsZ2-1-amiRNA, we analyzed whetherthe mRNA of PpFtsZ2-2, the closest homolog ofPpFtsZ2-1, is targeted by the PpFtsZ2-1-amiRNA.Compared to the PpFtsZ2-1-amiRNA recognition sitein PpFtsZ2-1, the corresponding region within thePpFtsZ2-2 sequence contains two mismatches at posi-tions 12 and 16. 5# RACE-PCRswere performed using aPpFtsZ2-2 gene-specific primer. PCR products indicat-ing amiRNA-guided cleavage products were not ob-tained. Instead, the 5# RACE-PCR yielded exclusivelyfragments corresponding to the PpFtsZ2-2 full-lengthtranscript (Fig. 1E). Thus, the PpFtsZ2-1-amiRNA ex-hibits high specificity, comparable to natural miRNAs.

AmiRNAs Efficiently Down-Regulate PpFtsZ2-1 and

PpGNT1 mRNA Levels

As we detected amiRNA-directed cleavage of thePpFtsZ2-1 and PpGNT1 target mRNAs, we next ana-lyzed the target transcript levels by RNA gel blots.Compared to wild type, we detected strongly reduced

Khraiwesh et al.

686 Plant Physiol. Vol. 148, 2008

Figure 1. Analysis of Physcomitrella lines expressing PpFtsZ2-1-amiRNA and PpGNT1-amiRNA. A, Scheme illustrating thePpFtsZ2-1-amiRNA and PpGNT1-amiRNA overexpression constructs. The modified ath-miRNA319a precursor DNA fragmentswere cloned into the SmaI and BamHI sites of the pPCV plant expression vector containing a double 35S promoter, nosterminator, and hpt selection marker cassette. Primers that were used for molecular analyses of the transgenic lines are indicated



steady-state levels of PpFtsZ2-1 and PpGNT1mRNAs inthe respective amiRNA overexpression lines (Fig. 2A).However, PpFtsZ2-1 transcript levels were reducedto 1% to 2% in PpFtsZ2-1-amiRNA lines, whereasPpGNT1 mRNA levels dropped to 10% to 20% inPpGNT1-amiRNA lines when compared to wild-typeplants. Furthermore, the efficiency of posttranscrip-tional silencing of PpFtsZ2-1 was similar in all threeamiRNA overexpression lines, even though they dif-fered with respect to the PpFtsZ2-1-amiRNA accumu-lation (Fig. 1D), whereas the reduction of PpGNT1transcript levels correlated with the PpGNT1-amiRNAexpression levels. From these results, we conclude thatamiRNAs confer efficient down-regulation of theirtarget mRNAs in Physcomitrella. As a control, we alsoanalyzed the steady-state levels of the sequence-related PpFtsZ2-2 mRNA in PpFtsZ2-1-amiRNA over-expression lines. In agreement with the absence ofamiRNA-induced mRNA cleavage products, PpFtsZ2-2transcript levels were similar in wild-type and the threePpFtsZ2-1-amiRNA lines (Fig. 2A).

The 5# RACE-PCR experiments performed withone of the PpFtsZ2-1-amiRNA lines yielded additionalfragments that differed substantially in size from theexpected cleavage products (Fig. 1E). After amiRNA-mediated cleavage of the mRNA, the cleavage prod-ucts may serve as templates for synthesizing cRNAby RNA-dependent RNA polymerase (Vaistij et al.,2002) leading to the formation of dsRNA. Subse-quently, the dsRNA may be processed into secondarysiRNAs, resulting in spreading of the initial amiRNAsignal (Fig. 2B). This mechanism, known as transitiv-ity, usually is initiated by dsRNA triggers. In plants,the transitivity occurs in both directions of the initialdsRNA trigger (Moissiard et al., 2007), whereas inanimals, spreading of the initial signal occurs onlyupstream of the trigger (Pak and Fire, 2007). However,the onset of transitivity is a rare event after miRNA-mediated target cleavage (Howell et al., 2007; Moissiardet al., 2007) and is normally not observed afteramiRNA-mediated target cleavage (Schwab et al.,2006). To investigate the possibility of transitivity, weused sense and antisense oligonucleotides derived

from PpFtsZ2-1 and PpGNT1 mRNA regions down-stream of the amiRNA recognition site for RNA gel-blot analysis. Sense and antisense siRNAs were onlydetected in PpFtsZ2-1-amiRNA and PpGNT1-amiRNAlines, respectively, but not in wild type (Fig. 2C). Weconclude that amiRNAs allow for efficient down-regulation of mRNAs in Physcomitrella and the genera-tion of transitive siRNAs frommRNAcleavage productsmay amplify the initial amiRNA trigger. However,the transitive effects are apparently not sufficient tohave a major impact on sequence-related genes, as thePpFtsZ2-2 steady-state RNA levels were unaffected inPpFtsZ2-1-amiRNA overexpression lines (Fig. 2A).

PpFtsZ2-1-amiRNA Overexpressors Phenocopy PpFtsZ2-1Null Mutants

In this study, we have chosen two genes to evaluatethe use of an amiRNA expression system in Physco-mitrella. The targeted deletion of PpGNT1 that is in-volved in theN-glycosylation of proteins did not causeany phenotypic deviations (Koprivova et al., 2003). Inagreement with this previous study, the two charac-terized PpGNT1-amiRNA lines were indistinguish-able from Physcomitrella wild-type plants. In contrast,PpFtsZ2-1 null mutants, which were generated by tar-geted gene disruption and lack expression of PpFtsZ2-1mRNA, are impeded in chloroplast division leadingto the formation of macrochloroplasts (Strepp et al.,1998). In our study, the expression of PpFtsZ2-1-amiRNA led to strongly reduced PpFtsZ2-1 mRNAlevels. To compare knockout and amiRNA lines, weinvestigated the phenotypes of the three PpFtsZ2-1-amiRNA lines. In all lines, the accumulation of theamiRNA targeting PpFtsZ2-1 resulted in impairedchloroplast division and the formation of macrochlo-roplasts that phenocopied the PpFtsZ2-1 null mutants(Fig. 3; Supplemental Fig. S2). The formation of macro-chloroplasts in the PpFtsZ2-1-amiRNA lines was ob-served in all tissues and cells analyzed indicatingan efficient production of mature amiRNAs from con-stitutively expressed precursor transcripts. Further-more, we did not observe any particular phenotypic

Figure 1. (Continued .)by arrows. B, Secondary structures of foldbacks of the P. patens miR319d precursor (ppt-MIR319d) and Arabidopsis miR319aprecursor (ath-MIR319a). The mature miRNA is highlighted in green with uppercase letters. C, PCR screen to identify transgeniclines harboring the PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression constructs. WT, Wild type; amiRNA lines, 1, 2, and 3for PpFtsZ2-1-amiRNA; 1 and 2 for PpGNT1-amiRNA; PpEF1a, control PCRs. D, Expression analysis of PpFtsZ2-1-amiRNA andPpGNT1-amiRNA in Physcomitrella wild type (WT), and lines harboring the PpFtsZ2-1-amiRNA or PpGNT1-amiRNAexpression constructs. Fifty micrograms of each RNA was blotted and hybridized with a PpFtsZ2-1-amiRNA and PpGNT1-amiRNA antisense probe, respectively. Hybridization with an antisense probe for U6snRNA served as control. PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels were normalized to the U6snRNA control hybridization. Numbers indicate therelative PpFtsZ2-1-amiRNA and PpGNT1-amiRNA expression levels. E, Top, 5# RACE-PCRs for the genes PpFtsZ2-1 andPpFtsZ2-2 from wild type (WT) and line 1 expressing the PpFtsZ2-1-amiRNA; bottom, 5# RACE-PCR for the gene PpGNT fromwild type (WT) and line 1 expressing the PpGNT1-amiRNA. The arrows mark PCR fragments corresponding to the expected sizeof the cleavage products that were isolated, cloned, and sequenced. The right images show the sequence complementarity ofPpFtsZ2-1, PpFtsZ2-2, and PpGNT1 to the amiRNA sequences. The determined cleavage sites within the PpFtsZ2-1 and PpGNT1mRNAs are marked by vertical arrows and numbers above indicate the number of sequenced products cleaved at this site.[See online article for color version of this figure.]

Khraiwesh et al.


differences among the transgenic lines expressing thePpFtsZ2-1-amiRNA, which is consistent with the sim-ilar degree of PpFtsZ2-1 mRNA reduction. Our resultsdemonstrate that the expression of amiRNAs in Phys-comitrella leads to efficient silencing of their targetmRNAs comparable to the effects of targeted geneknockouts.

DISCUSSION

The successful use of amiRNAs for the specificdown-regulation of genes was shown for the dicoty-ledonous plants Arabidopsis, tomato (Solanum lyco-persicum), and tobacco (Nicotiana tabacum), and for themonocot rice (Oryza sativa; Parizotto et al., 2004;Alvarez et al., 2006; Niu et al., 2006; Schwab et al.,2006; Qu et al., 2007; Ossowski et al., 2008; Warthmannet al., 2008). In most cases, the amiRNAwas expressedfrom endogenous miRNA precursors. However, highexpression rates of amiRNAswere achieved in tobaccoand tomato using the Arabidopsis miR164b precursorsequence indicating correct processing of conserved

pre-miRNAs within seed plants (Alvarez et al., 2006).In our study, we tested the application of amiRNAs forthe specific silencing of genes in the bryophyte Phys-comitrella making use of an amiRNA expression sys-tem, where the Arabidopsis miR319a precursor servesas the backbone for amiRNA expression and subse-quent maturation and was developed to control geneexpression in Arabidopsis (Schwab et al., 2006). ThemiR319 family belongs to the highly conserved amiRNAfamilies, even over large evolutionary distances, andwas also found in Physcomitrella (Arazi et al., 2005;Jones-Rhoades et al., 2006; Fattash et al., 2007). Nota-bly, miR319 stands out in that there is also consider-able sequence conservation in the foldback, not onlyin the miRNA itself. Our comparison of the Arabidop-sis miR319a precursor and the Physcomitrella miR319precursor sequences confirmed nucleotide sequenceconservation outside the miRNA/miRNA* region,implying similar foldback structures of the Arabidop-sis and Physcomitrella miR319 pre-miRNAs. Indeed,we detected the correct processing of a mature 21-ntPpFtsZ2-1-amiRNA and PpGNT1-amiRNA, respec-tively, from the Arabidopsis miR319a precursor in

Figure 2. Expression analysis ofPpFtsZ2-1,PpFtsZ2-2,and PpGNT1, and detection of transitive siRNAs. A,Left, RNA gel blots (20 mg each) from wild type (WT)and PpFtsZ2-1-amiRNA overexpression lines (1–3)hybridized with PpFtsZ2-1 and PpFtsZ2-2 probes;right, RNA gel blots (20 mg each) from wild type (WT)and PpGNT1-amiRNA overexpression lines (1 and 2)hybridized with a PpGNT1 probe. The ethidiumbromide-stained gels below indicate equal loading.The hybridization signalswere normalized to the rRNAbands, and the PpFtsZ2-1, PpFtsZ2-2, and PpGNT1expression levels in wild type were set to 1. Numbersindicate the relative PpFtsZ2-1, PpFtsZ2-2, andPpGNT1 mRNA levels. B, Scheme illustrating thegeneration of transitive siRNAs from amiRNA targetcleavage products requiring an RNA-dependent RNApolymerase (RdRP) to generate dsRNA, which issubsequently processed into siRNAs. Black line,mRNA; gray box, amiRNA binding site; curved line,amiRNA.C,Detection of sense and antisense transitivesiRNAs produced from PpFtsZ2-1 (left) and PpGNT1(right) mRNA cleavage products by RNA gel blotshybridizedwith oligonucleotides derived from regionsdownstream of the amiRNA binding sites. Hybridiza-tion with an antisense probe for U6snRNA servedas control.



transgenic Physcomitrella lines, indicating that the re-constructed miR319a pre-miRNA contains the essen-tial recognition and processing information to enterthe Physcomitrella miRNA biogenesis pathway. ThePpFtsZ2-1 mRNA cleavage products were, however,offset by 1 and 2 nt relative to the expected products(Llave et al., 2002), suggesting that the PpFtsZ2-1-amiRNAwas shifted by 1 or 2 nt, respectively, relativeto the intended amiRNA. A similar effect has beenobserved for some Arabidopsis amiRNAs (Schwabet al., 2006). Because the originally designed amiRNAswere perfectly complementary, the shifted amiRNAsshould still adhere to the targeting rules for miRNAs.The observation of shifted cleavage products suggestedthat release of the PpFtsZ2-1-amiRNA/miRNA* du-plex from the precursor was not always precise,consistent with observations on endogenous miRNAs(Rajagopalan et al., 2006). Nevertheless, the Arabidop-sis miR319a precursor can be used routinely for theexpression of amiRNAs in Physcomitrella as the appar-ent shift by 1 nt during the maturation of the amiRNAmay result in a mismatch at the 3# end of the miRNA,which is not affecting target mRNA cleavage (Schwab

et al., 2005). Furthermore, cleavage of the PpFtsZ2-1mRNA within the amiRNA recognition site indicatescorrect amiRNA/amiRNA* duplex recognition andamiRNA loading into the RISC complex.

Previous studies have shown that the transcriptlevels of amiRNA targets are in most cases anticorre-lated with corresponding amiRNA levels (Schwabet al., 2006). Among PpFtsZ2-1-amiRNA and PpGNT1-amiRNA lines analyzed, the amiRNA expression levelsvaried 8-fold and 5-fold, respectively. Nevertheless, theamiRNA expression caused a similar reduction ofPpFtsZ2-1 and PpGNT1 mRNA levels to 1% to 2% and10% to 20%, respectively, compared to transcript levelsin wild type. This suggests that the amount of amiRNAsis not limiting in any of the lines. Instead, it islikely that the competition of natural miRNAs andamiRNAs in RISC loading determines the efficiencyof posttranscriptional silencing of the PpFtsZ2-1 andPpGNT1 transcripts.

The formationofmacrochloroplasts in thePpFtsZ2-1-amiRNA lines indicated impeded plastid divisionand resembled the phenotype of PpFtsZ2-1 knockoutlines, which completely lack a functional transcript

Figure 3. Impeded plastid division andformation of macrochloroplasts inPpFtsZ2-1-amiRNA overexpressors. A,Light microscopy from protonema andleaves of wild type (WT) and onePpFtsZ2-1-amiRNA overexpression line(size bars, 100 mm). B, Confocal laser-scanning microscopy from protonemaand leaves of wild type (WT) and onePpFtsZ2-1-amiRNA overexpression line(size bars, 50 mm). Red, Chlorophyll auto-fluorescence in plastids. See SupplementalFigure S2 for phenotypes of the othertwo PpFtsZ2-1-amiRNA lines.

Khraiwesh et al.


(Strepp et al., 1998). We therefore conclude that theremaining PpFtsZ2-1 transcripts in the amiRNA linesare not able to generate sufficient PpFtsZ2-1 protein tosupport proper plastid division. In addition, amiRNAexpression in the transgenic lines seems to be stableover long time periods as we did not observe anyphenotypic reversion to wild-type plastids in thePpFtsZ2-1-amiRNA overexpression lines after 1 year ofsubculture. We anticipate that the described amiRNAexpression system will result in similar silencingefficiencies of any target gene and thus can be rou-tinely used as an alternative to the generation ofknockout mutants in Physcomitrella.The efficient silencing of PpFtsZ2-1 and PpGNT1 by

amiRNAs might be enhanced by the generation oftransitive siRNAs, as we detected such siRNAs fromthe 3# cleavage products of the PpFtsZ2-1 and PpGNT1mRNAs. Usually, transitive siRNAs are producedfrom exogenous RNA sequences such as viruses orsense transgene transcripts (Baulcombe, 2004), but theformation of transitive siRNAs from miRNA-guidedcleavage products appears to be the exception (Howellet al., 2007; Moissiard et al., 2007). Furthermore, tran-sitivity was suggested not to be a major factor con-tributing to amiRNA efficacy in previous studies,although this was inferred only indirectly from thelack of effects on sequence-related transcripts (Schwabet al., 2006; Warthmann et al., 2008). Although wecannot exclude that siRNAs, which are produced fromamiRNA-mediated mRNA cleavage products, can af-fect other genes not targeted by the original amiRNA,the PpFtsZ2-1 homolog PpFtsZ2-2, which shares highidentity in sequence stretches of the coding region(Supplemental Fig. S3), seemedunaffected inPpFtsZ2-1-amiRNA lines. We detected neither cleavage productsby 5# RACE-PCR, indicating siRNA-mediated cleav-age of PpFtsZ2-2 transcripts, nor reduced PpFtsZ2-2steady-state mRNA levels, pointing to a posttran-scriptional silencing of this gene. Thus, even thoughtransitivity might be more common in Physcomitrella,the specificity of posttranscriptional silencing is ap-parently sufficient to silence single members of highlyconserved gene families. Moreover, it might be pref-erable to design amiRNAs lacking perfect sequencecomplementarity at the 3# end, as this reduces tran-sitivity (Moissiard et al., 2007).

CONCLUSION

Compared to the conventional targeted gene knock-out approach in Physcomitrella, the expression ofamiRNA provides several advantages. (1) The gener-ation and molecular analysis of amiRNA overexpres-sion lines is sped up as each regenerated transgenicline harboring an amiRNA expression construct andshould produce the desired mature amiRNA. (2)Instead of the generation of multigene knockout lines,which is experimentally difficult, but feasible (Hoheet al., 2004), amiRNAs are likely to be particularly

useful for targeting groups of closely related genes(Alvarez et al., 2006; Schwab et al., 2006). (3) AmiRNAscan be expressed from inducible or tissue-specificpromoters (Schwab et al., 2006) enabling the analysisof genes with essential functions that cannot be ana-lyzed by targeted gene disruption. Provided that otheramiRNAs have a similar effect on the knock down oftheir cognate target genes in Physcomitrella as observedin this study, they can be considered as an efficientalternative tool to the targeted gene knockout approachfor reverse genetics studies in Physcomitrella.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Physcomitrella patens plants were cultured in modified liquid Knop me-

dium containing 250 mg L21 KH2PO4, 250 mg L21 KCl, 250 mg L21

MgSO4·7H2O, 1,000 mg L21 Ca(NO3)2, and 12.5 mg L21 FeSO4·7H2O (pH 5.8)

or on solid Knop plates. Erlenmeyer flasks containing 400 mL of suspension

culture were agitated on a rotary shaker at 120 rpm at 25�C under a 16-h-light/

8-h-dark regime (Philips TLD 25; 50 mM m22 s21). Liquid cultures were

mechanically disrupted every week to maintain the plants in the protonema

stage. Gametophore development was induced by transferring protonema

tissue to solidified Knop medium.

Transformation of Physcomitrella Protoplasts

Polyethylene glycol-mediated transformation of Physcomitrella protoplasts

was performed according to standard procedures (Frank et al., 2005). Briefly,

transformation was carried out using 25 mg of linearized plasmid DNA.

Transformed protoplasts were cultivated for 24 h under standard conditions

in the dark and were then transferred to light. After 10 d, the protoplasts were

transferred to solid Knop medium. Three days later, regenerating plants were

transferred to Knop medium supplemented with hygromycin (Promega). The

selection lasted 2 weeks and was followed by a 2-week release period on Knop

medium without antibiotic followed by another round of selection and

release. Plants surviving the second round of selection were screened by

PCR to confirm integration of the DNA construct.

Generation of Physcomitrella Lines ExpressingAmiRNAs Targeting PpFtsZ2-1 and PpGNT1

AmiRNAs targeting PpFtsZ2-1 (accession no. AJ001586; amiRNA,

5#-TTCGTAATTAACGTGTCCGCG-3#) and PpGNT1 (accession no. AJ429143;

amiRNA, 5#-TTCCAAATAATCAGGACGCTT-3#) were designed using the

amiRNA designer interface WMD (Schwab et al., 2006; Ossowski et al., 2008).

The PpFtsZ2-1-amiRNA and PpGNT1-amiRNA sequences were introduced into

theArabidopsis (Arabidopsis thaliana) miR319a precursor by overlapping PCR us-

ing the following primers. PpFtsZ2-1-amiRNA, miRNA-sense, 5#-GATTCGTA-

ATTAACGTGTCCGCGTCTCTCTTTTGTATTCC-3#; miRNA-antisense, 5#-GAC-

GCGGACACGTTAATTACGAATCAAAGAGAATCAATGA-3#; miRNA*-

sense, 5#-GACGAGGACACGTTATTTACGATTCACAGGTCGTGATATG-3#;miRNA*-antisense, 5#-GAATCGTAAATAACGTGTCCTCGTCTACATATATAT-

TCCT-3#; primer A, 5#-CCCGGGTGCAGCCCCAAACACACGCTC-3#; primer

B, 5#-GGATCCCCCCATGGCGATGCCTTAAAT-3#. PpGNT1-amiRNA, miRNA-

sense, 5#-GATTCCAAATAATCAGGACGCTTTCTCTCTTTTGTATTCC-3#;miRNA-antisense, 5#-GAAAGCGTCCTGATTATTTGGAATCAAAGAGAATC-

AATGA-3#; miRNA*-sense, 5#-GAAAACGTCCTGATTTTTTGGATTCACAGG-

TCGTGATATG-3#; miRNA*-antisense, 5#-GAATCCAAAAAATCAGGACGT-

TTTCTACATATATATTCCT-3#; same primers A and B as described above. The

plasmid pRS300 harboring the Arabidopsis miR319a precursor was used as

PCR template (Schwab et al., 2006). The resulting precursor fragments were

cloned into the pJET1.2 cloning vector (Fermentas) and sequenced. The modified

ath-miRNA319a precursor DNA fragments were cloned into SmaI and BamHI

sites of the plant expression vector pPCV (Koncz et al., 1989) containing the

cauliflower mosaic virus 35S promoter, nos terminator, and hpt selection marker

cassette. Transgenic lines were analyzed by PCR to identify lines that had



integrated the amiRNA overexpression constructs using the primers 5#-TGA-

TATCTCCACTGACGAAAGGG-3# and 5#-GGATCCCCCCATGGCGATGCCT-

TAAAT-3#. PCR primers for the amplification of the Physcomitrella control gene

EF1a were 5#-AGCGTGGTATCACAATTGAC-3# and 5#-GATCGCTCGAT-

CATGTTATC-3#. The one-step isolation of genomic DNA was performed ac-

cording to the method of Schween et al. (2002).

Small RNA Blots

Total RNAwas isolated from protonema using TRIzol reagent (Invitrogen)

and separated in a 12% denaturing polyacrylamide gel containing 8.3 M urea

in Tris-borate/EDTA buffer. The RNA was electroblotted onto nylon mem-

branes for 1 h at 400 mA. Radiolabeled probes were generated by end labeling

of DNA oligonucleotides with [g-32P]ATP using T4 polynucleotide kinase.

The following probes were used. Antisense probe for PpFtsZ2-1-amiRNA,

5#-CGCGGACACGTTAATTACGAA-3#; antisense probe for PpGNT1-amiRNA,

5#-AAGCGTCCTGATTATTTGGAA-3#; detection of sense transitive PpFtsZ2-1

siRNAs, 5#-CCCCAGTGACGGAAGCGTTCAATCTTGCAGACGACATCCTT-

CGGC-3#; detection of antisense transitive PpFtsZ2-1 siRNAs, 5#-GCCGAAG-

GATGTCGTCTGCAAGATTGAACGCTTCCGTCACTGGGG-3#; detection of

sense transitive PpGNT1 siRNAs, 5#-GTGAATTTCCTGCAGCATTTAG-

ATGAAAATCCTCCCAAGACAAGG-3#; detection of antisense transitive

PpGNT1 siRNAs, 5#-CCTTGTCTTGGGAGGATTTTCATCTAAATGCTGCAG-

GAAATTCAC-3#; detection of the U6snRNA control, 5#-GGGGCCATGC-

TAATCTTCTCTG-3#. Blot hybridization was carried out in 0.05 M sodium

phosphate (pH 7.2), 1 mM EDTA, 63 SSC, 13 Denhardt’s, 5% SDS. Blots were

washed three timeswith 23 SSC, 0.2%SDS, and one timewith 13 SSC, 0.1%SDS.

Blots were hybridized and washed at temperatures 5�C below the melting

temperature of the oligonucleotide.

Detection of mRNA Cleavage Products

Synthesis of 5# RACE-ready cDNAs was carried out according to Zhu et al.

(2001) using the BD Smart RACE cDNA amplification kit (CLONTECH).

Subsequent PCR reactions were performed using the UPM Primer-Mix

supplied with the kit in combination with gene-specific primers derived

from the target gene PpFtsZ2-1 (5#-GACTATCCCTGTGGCTCGCTCAA-

TACCC-3#), a PpFtsZ2-1 nested primer (5#-CCAATAGAGGAGATTGGA-

TTGCGCTCA-3#), a gene-specific primer derived from the gene PpFtsZ2-2

(5#-CCAATACGCGACTTGCATACTGCATAC-3#), and a gene-specific primer

derived from the gene PpGNT1 (5#-ACTTTGGAGCAAGTTCTTCCCAGGTG-

GA-3#). Amplification products corresponding to the size of the expected

cleavage products were excised from the gel, cloned and sequenced.

Total RNA Gel Blots

Twenty micrograms of total RNA were mixed with an equal volume of

RNA denaturing buffer and incubated for 10 min at 65�C. The RNA gel was

blotted to a Hybond-N+ nylon membrane (GE Healthcare) using a Turbo

blotter (Schleicher & Schuell) with 203 SSC. RNA was fixed by UV cross-

linking. Hybridization was carried out with an [a-32P]dCTP-labeled DNA

probe derived from PpFtsZ2-1 amplified with primers 5#-AGACACGTCAT-

TAAAGGT-3# and 5#-TAAGTGTGCAAGAAGATA-3#, a probe derived from

PpFtsZ2-2 amplified with primers 5#-AAGGTAGTACAAATGGGATGGC-3#and 5#-TCATTAAGTCTGCCACTCCAC-3#, and a probe derived from

PpGNT1 amplified with primers 5#-GCACTCTCGATCGGATTCTC-3# and

5#-TCGGGAGAGATTTCCATGTC-3#. DNA labeling was carried out with the

Rediprime II random prime labeling kit (GE Healthcare). Prehybridization

was carried out at 67�C for 4 h, subsequent hybridization at 67�C overnight.

Blots were washed three times with 0.53 SSC, 0.1% SDS, and one time with

13 SSC, 0.1% SDS at 67�C. Signals were detected using the Molecular Imager

FX (Bio-Rad).

Microscopy

For microscopic analyses, we used the Axioplan 2 epifluorescence micro-

scope equipped with an AttoArc HBO 100-W bulb and the stereomicroscope

Stemi 2000-C (Carl Zeiss). Image acquisition was achieved using the Canon

digital camera EOS D300 (Canon), and confocal laser-scanning microscopy

(TCS 4D; Leica).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers AJ001586 (PpFtsZ2-1), XM_001766723

(PpFtsZ2-2), and AJ429143 (PpGNT1).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Analysis of Physcomitrella and Arabidopsis

miR319 precursors.

Supplemental Figure S2. Impeded plastid division and formation of

macrochloroplasts in PpFtsZ2-1-amiRNA overexpressors.

Supplemental Figure S3.Nucleotide sequence alignment of PpFtsZ2-1 and

PpFtsZ2-2 coding regions.

ACKNOWLEDGMENTS

We thank Andras Viczian for providing the pPCV expression vector, Enas

Qudeimat for assisting with confocal laser-scanning microscopy, and Bjorn

Voß and Isam Fattash for advice on miR319 precursor sequence analysis.

Received August 13, 2008; accepted August 22, 2008; published August

27, 2008.

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Supplemental Figure S3 FtsZ2-1 1 ATGGCGTTGTTTAGTGGGTGCTCGGGATGGGCGGGGCTCAAGGTGTCATC 50 |||||| |||| | | ||||||| | || ||| |||| |||| || FtsZ2-2 1 ATGGCGCTGTTAGGCAGTCGCTCGGGCTTGGTGGGCCTCAGGGTGAGCTC 50 FtsZ2-1 51 GCGAGTGGGTGGGGAGGCTTGCAGAA-----CCCCCCCCGTT-GTTCACT 94 ||||||||| |||||| | |||| ||| | || | || || FtsZ2-2 51 GCGAGTGGGCGGGGAGAGCAGTAGAATAGTGCCCGCGACGAGAGATCGCT 100 FtsZ2-1 95 GCAGCATGCATTCTAGGTCAAGCGTTCGAGCTCTACGCCGAATCGACCGA 144 | || |||| | ||| | ||| ||| || | || || | || | FtsZ2-2 101 TCTGCGTGCACTTGAGGCCGAGCACTCGGGCGCATCGTCGTCTGGATAGG 150 FtsZ2-1 145 GCTTTGAGTAATGGGGGTCTTTGCAATTTTGGAGAGAGGGACTTGTTGGC 194 || | | |||| | ||||||||| | | |||||||||| |||| FtsZ2-2 151 ACTGTAGGGAATGAGAGTCTTTGCACTCCCCGGGAGAGGGACT---TGGC 197 FtsZ2-1 195 TTTGGAAGCAAAATC---GCCTTTGCGATGTGAACCCCCCTCGA------ 235 | ||| | |||| || | |||| || | | || FtsZ2-2 198 TGCGGAGCCTAAATTCTTGCACACGGGATGGGAGTCTTCTTCTTCTTCTT 247 FtsZ2-1 236 ------------GCGTGATGCGGAATCCTGTCATGGCATTTGAAGGAAGC 273 ||| || ||| || |||| |||||||| ||| | FtsZ2-2 248 CTTCTTCTTCTTGCGAGACTGGGATACCCGTCACGGCATTTGGAGGTAAT 297 FtsZ2-1 274 GGAGACGACACTGGAAGTTATAACGAAGCGAAAATTAAAGTAATAGGGGT 323 |||||||| || |||| || || |||||||||||||| ||||| || FtsZ2-2 298 GGAGACGAATATGAGAGTTCCAATGAGGCGAAAATTAAAGTGATAGGCGT 347 FtsZ2-1 324 CGGAGGTGGGGGTTCCAACGCCGTAAACCGAATGCTTGAGAGCGAGATGC 373 || || ||||||||||||||||| |||||||||||||||||||| |||| FtsZ2-2 348 GGGGGGCGGGGGTTCCAACGCCGTCAACCGAATGCTTGAGAGCGAAATGC 397 FtsZ2-1 374 AAGGGGTAGAATTTTGGATCGTGAATACTGATGCGCAGGCTATGGCCTTG 423 |||| || ||||| ||||| |||||||| ||||| ||||| ||||| ||| FtsZ2- 398 AAGGTGTGGAATTCTGGATTGTGAATACGGATGCTCAGGCAATGGCGTTG 447 FtsZ2-1 424 TCCCCTGTTCCGGCTCAGAATCGTCTGCAGATTGGGCAAAAATTGACGAG 473 || || ||||||||||||||||||||||||||||| || ||||||||| | FtsZ2-2 448 TCTCCGGTTCCGGCTCAGAATCGTCTGCAGATTGGTCAGAAATTGACGCG 497 FtsZ2-1 474 AGGTCTGGGGGCGGGCGGGAATCCAGAAATAGGGTGTAGTGCTGCGGAAG 523 ||||||||||||||| || ||||| ||||||||||||||||| ||||||| FtsZ2-2 498 AGGTCTGGGGGCGGGTGGTAATCCGGAAATAGGGTGTAGTGCCGCGGAAG 547 FtsZ2-1 524 AGAGCAAAGCTATGGTGGAAGAAGCCCTACGCGGAGCTGACATGGTTTTC 573 |||||||||||||||||||||||||| ||||||||||||||||||||||| FtsZ2-2 548 AGAGCAAAGCTATGGTGGAAGAAGCCTTACGCGGAGCTGACATGGTTTTC 597 FtsZ2-1 574 GTAACGGCGGGTATGGGTGGCGGCACTGGCAGCGGTGCAGCACCAATAAT 623 || || ||||| |||||||| ||||||||||||||||| |||||||| || FtsZ2-2 598 GTTACAGCGGGCATGGGTGGTGGCACTGGCAGCGGTGCTGCACCAATCAT 647 FtsZ2-1 624 TGCGGGTGTGGCGAAGCAGTTGGGAATTCTTACTGTAGGAATAGTTACTA 673 ||| ||||| |||||||| |||||||||||||| || |||||||| |||| FtsZ2-2 648 TGCTGGTGTAGCGAAGCAATTGGGAATTCTTACCGTGGGAATAGTAACTA 697 FtsZ2-1 674 CTCCTTTCGCCTTTGAAGGGCGGAGACGAGCTGTCCAAGCCCACGAGGGT 723 | ||||| ||||||||||||||||||||| | || ||||| ||||| || FtsZ2-2 698 CGCCTTTTGCCTTTGAAGGGCGGAGACGATCCGTTCAAGCTCACGAAGGC 747

FtsZ2-1 724 ATTGCAGCTCTCAAAAATAACGTGGACACGTTAATTACGATTCCAAACAA 773 || || |||||||||||||| || ||||| ||||||||||| |||||||| FtsZ2-2 748 ATCGCGGCTCTCAAAAATAATGTTGACACTTTAATTACGATACCAAACAA 797 FtsZ2-1 824 CAAACTTTTGACTGCAGTTGCGCAGTCTACCCCAGTGACGGAAGCGTTCA 823 ||| ||||||||||||||||||||||||||||| ||||||||||| |||| FtsZ2-2 798 CAAGCTTTTGACTGCAGTTGCGCAGTCTACCCCCGTGACGGAAGCATTCA 847 FtsZ2-1 824 ATCTTGCAGACGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT 873 ||||||| || ||||||||||||||||||||||||||||||||||||||| FtsZ2-2 848 ATCTTGCCGATGACATCCTTCGGCAGGGAGTGCGGGGTATTTCAGATATT 897 FtsZ2-1 874 ATCACGGTCCCTGGGCTGGTTAACGTAGATTTTGCCGACGTGCGGGCGAT 923 ||||| || ||||| || |||||||| || ||||| || ||||||||||| FtsZ2-2 898 ATCACTGTTCCTGGTCTCGTTAACGTGGACTTTGCGGATGTGCGGGCGAT 947 FtsZ2-1 924 CATGGCTAATGCAGGATCATCTTTGATGGGCATAGGGACCGCCACAGGTA 973 |||||| ||||||||||||||||||||||| || || ||||| ||||| | FtsZ2-2 948 CATGGCCAATGCAGGATCATCTTTGATGGGAATTGGAACCGCTACAGGGA 997 FtsZ2-1 974 AGTCAAGAGCTAGAGAAGCAGCATTGAGCGCAATCCAATCTCCTCTATTG 1023 |||||| ||||||||| ||||||||||| || || || ||||| | ||| FtsZ2-2 998 AGTCAAAAGCTAGAGAGGCAGCATTGAGTGCCATTCAGTCTCCATTGTTG 1047 FtsZ2-1 1024 GATGTGGGTATTGAGCGAGCCACAGGGATAGTCTGGAATATCACTGGGGG 1073 ||||||||||||||||||||||||||||| || |||||||| |||||||| FtsZ2-2 1048 GATGTGGGTATTGAGCGAGCCACAGGGATCGTTTGGAATATTACTGGGGG 1097 FtsZ2-1 1074 AAGCGACATGACTCTCTTTGAGGTAAATGCTGCAGCAGAGGTGATTTATG 1123 |||||||||||| |||||||| || ||||||||||||||||| || |||| FtsZ2-2 1098 AAGCGACATGACCCTCTTTGAAGTCAATGCTGCAGCAGAGGTAATCTATG 1147 FtsZ2-1 1124 ATTTGGTCGATCCCAACGCAAATCTTATTTTTGGAGCCGTAGTAGACGAA 1173 ||||||| ||||| ||||||||||||||||| |||||||||||||||||| FtsZ2-2 1148 ATTTGGTGGATCCTAACGCAAATCTTATTTTCGGAGCCGTAGTAGACGAA 1197 FtsZ2-1 1174 GCACTTCATGGCCAAGTTAGTATAACTTTGATAGCAACAGGATTTAGTTC 1223 |||||||||| |||| |||| ||||| || ||||||||||| |||||||| FtsZ2-2 1198 GCACTTCATGACCAAATTAGCATAACCTTAATAGCAACAGGGTTTAGTTC 1247 FtsZ2-1 1224 TCAAGATGAACCTGATGCGCGTAGTATGCAAAATGTGAGTCGTATTTTGG 1273 ||||||||| |||||||| || |||||||| ||| ||||| | |||| FtsZ2-2 1248 TCAAGATGATCCTGATGCACGGAGTATGCAGTATGCAAGTCGCGTATTGG 1297 FtsZ2-1 1274 ATGGACAAGCTGGTCGATCACCGACAGGTTTATCTCAAGGCAGCAATGGC 1323 | || ||||||||||||||| ||| | | ||| | ||| ||||| || FtsZ2-2 1298 AGGGTCAAGCTGGTCGATCATCGATGGCCTCATCCCGAGGTGGCAATAGC 1347 FtsZ2-1 1324 TCTGCGATCAATATACCAAGTTTCTTAAGGAAGCGAGGCCAGACACGTCA 1373 ||| |||| || ||||||| ||||||| | |||||||| || | FtsZ2-2 1348 TCTACGATTAACATACCAAATTTCTTACGAAAGCGAGGGCAAAGG----- 1392 FtsZ2-1 1374 TTAA 1377 || FtsZ2-2 1393 –TAG 1395 Supplemental Figure S3. Nucleotide sequence alignment of PpFtsZ2-1 and PpFtsZ2-2 coding regions. The highly

conserved central region with 89% sequence identity is highlighted in yellow. The PpFtsZ2-1-amiRNA target site is

highlighted in green.

Chapter IV Appendices

136

4 Chapter IV: Appendices

4.1 Flow cytometric measurements (FCM)

For ploidy level determination 10-20 mL of protonema liquid culture were used. The plant

material was harvested four to seven days after the last sub-culturing, resuspended with 2

mL of DAPI buffer, and chopped up with a razor blade in a Petri dish. The solution was

filtered through a sieve of 30 μm pore size prior to measuring the fluorescence intensity with

a PAS cell analyser (Partec, Münster) using a 100 W high-pressure mercury lamp for

detection. The ploidy level was derived from the resulting histograms. For Physcomitrella, a

prominent peak at a fluorescence intensity of about 200 indicates a haploid genotype,

whereas signals at fluorescence intensities of about 400 represent a diploid plant.

Figure 1: Flow cytometric analysis. Flow cytometric histograms of protonema from WT and ΔPpDCL1b mutants (1-4) grown in parallel in Knop medium. The abscissa represents the channel numbers corresponding to the relative fluorescence intensities of analyzed particles (linear mode), while the ordinate indicates the number of events counted.


137

4.2 Physcomitrella patens DCL1b (PpDCL1b) mRNA

LOCUS DQ675601 6052 bp mRNA linear PLN 03-JUL-2008 DEFINITION Physcomitrella patens Dicer-like 1b protein (DCL1b) mRNA, complete cds. ACCESSION DQ675601 VERSION DQ675601.1 GI: 110520366 KEYWORDS SOURCE Physcomitrella patens ORGANISM Physcomitrella patens Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Bryophyta; Moss Superclass V; Bryopsida; Funariidae; Funariales; Funariaceae; Physcomitrella REFERENCE 1 (bases 1 to 6052) AUTHORS UKhraiwesh, BU., Seumel, G.I., Reski, R. and Frank, W. TITLE Direct genetic evidence for the involvement of a Dicer-like gene in microRNA mediated target cleavage in plants. JOURNAL Unpublished REFERENCE 2 (bases 1 to 6052) AUTHORS UKhraiwesh, BU., Seumel, G.I., Reski,R. and Frank,W. TITLE Direct Submission JOURNAL Submitted (06-JUN-2006) Plant Biotechnology, University of Freiburg, Schaenzlestrasse 1, Freiburg 79104, Germany FEATURES Location/Qualifiers Source 1..6052 /organism="Physcomitrella patens" /mol_type="mRNA" /db_xref="taxon: 3218" Gene 1..6052 /gene="DCL1b" CDS 607..5694 /gene="DCL1b" /codon_start=1 /product="Dicer-like 1b protein" /protein_id="ABG74922.1" /db_xref="GI:110520367" /translation="MRRARSVRLENGLNGKDEGEEKARTYQLEVLAQAKV KITVAFLDTGAGKTLIAILLMKHKHQVLREYDKRMLALFLVPKVPLVYQQADVIRNGTKFSVGHYCGEMGSRFWDARGWQREFDTKDVFVMTAQILLNILRHSIVKMEAIHLLILDECHHAVKKHPYSLVMSEFYLMTPKDKRPCVFGMIASPVNLKGVSNQEDCAIEIRNLESKLDSIVCTIRDRKELEKHVPLPSETMILYDKPALLFSLRKERKQMEATVEKAANASVRRSKWKCMGARDAGAKEELQLVYSVAERTESDGAASLSQKLRAITYALDELGQWCAYKVSLGYLTSLHNDERVNHQLDVKFQKLYLKKVCTLLRCSLREGAAGWEVPAEIGESEGDKAQDPMDVEEGSFLTLVSVGEHLDEILGAAVADGKVTPKVQSLIKVLIGYQHTDDFRAIIFVERVWVGVTLCRSLQSCPSLKFVKCASLIGHNNNQDMPTRQMQETISKFRDGRVTLLVATSVAAEGLDIRQCNVVIRFDLAKTVLAYIQSRGRARKPGSDYILMLERGNLQHEAFFRNAKNSEETLRKEAIERTDLGEKRENAILASIDIGEGEIYQVPATGAVVSMNSAVGLIHFYCSQLPSDRYSLLRPEFIMNKIEDQRGAIRYSCRLQLPCHAPFEAVEGPECNSMRGAQQSCVLEGLQKMHEMGAFTDMLLSNKGSREEAAKLEGSEEGESLPGTSRHREYYPEGIADILKGDRIVAEKDSDTKEGSKVLVFMYTVKCENVGFSRDSLLTETSDFTLLVGQQLHDQVLTMTINLFVANPTLLITMSWKKRDLDCSNSQLTELKSFHVRLMSIVLDVNVEPATTRWDPAKAYLFAPVLHKDASDPKDLVDWVVMRRTIETDSWSNPLQRASPDVNLGTDERALGGDRREYGFGKLRCSLAFGQGAHPTYGARGAKAQFDVVKATGLLPTSDMVEETTVQEVPPEGKLLIVDGFVEVEVLVGRIVTAVHSGKRLYVDSVRFDMTADSSFPQKDGYLGPLEYTSYADYYKQKYGVELVCKKQPLLRGRGVSHCKNLLSPRFETSGDSLDALDKTYYVMLPPELCLIHPLPGSLVRGAQRLPSVMRRVESMLLAIQLKHQIDYPIAASKVALTAASGQETSYERAELLSDAYLEWVVSHRLFLKFPSKHEGQLTRMRQKIVSNSVLYQHALEKGLQSYIQADRFAPSRWAAPGVPPAFDEDLRDGDDSDKESKPEVEREVVEIVGEEGEIVKELNTESENMEDGEIEGDSGSYRVLSSKTLADVVEVFMGMYYVEGGGEAATHFMNWVGIPVEDDVETDLATGGCQVPETVMRSIDFSSLQKNVGHEFRERSLLVEAITHASRPSLGVPCYQRLEFVGDAVLDHLITRYLFFKYTNLPPGRLTDLRAAAVNNENFARVAVKHSYHLHLRHGSTALETQIRNFVNDIHSELDKPGVNSFGLGDFKAPKVLGDIFESIAGALFLDARLDTHQVWKVFEPLLQPMVSPETLPIHPVRGLQERCQQEAEGLEYKVSRAESVATVEVYVDGVQIGSTQSAQKKMAQKLGARNALVKLKDKEVIKVKAEAENGDLNAGKSSKNGHTNFTRKTINDLCLKRQWPMPQYKCVLESGPAHAKKFTLSVRVLTTTDGWTEECVGEPMASVKKAKDSAALVLLATLRRSYPLRNNIIDC"


138

ORIGIN 1 aacgcgggga ggtggagaag tggctctttt tctagcacta tccctctcga ggagcggagg 61 tgaattgtca agtaaggaac gattcaatta gagcgccgcg aattgattga attacgagtg 121 gtttgatgga gcaggtgggg agcggagggg tgaaatgcgc aagcaagggg tgtacatgac 181 gaggctcgtg atcgagggaa gaggagccgg gagagcgacg ggagagttgg cgaggctgga 241 atgaagggta gaggtggtgg tggtgtgaga ggattcgtca ggaggagcag ggagaggcgg 301 tctgtgtcga ggtctaggtc agagagaggc ctaaagagag ggcggagatg acgggagaca 361 gcagggaggg gaggaagcga aggtcggctt tcgaatatgc ctttgatgat aggcgcgatg 421 agaagagggg gcggcatttc catgaattgg gagattatcg agattaccac ggttccatag 481 tgacgcgcga cagaccttgg attggcagag gggattgcga ccggcgatca aggatggagg 541 ctcgggagcg atttgttgcg tcgtctcgtg atcgggagag agagtgggag cggtcgcgtg 601 tttgaaatga gaagggcgcg aagcgtgaga ctggagaatg ggctgaatgg gaaggatgag 661 ggtgaggaga aggcgcggac ttatcagctt gaagtgctgg ctcaggcgaa ggtgaagatt 721 acggttgcat ttctagacac gggcgctggg aagaccctaa ttgcgattct gttgatgaag 781 cataagcacc aggtgttgcg ggagtatgac aagcgtatgc tcgctctgtt cctcgtccct 841 aaagtaccgc tcgtctacca gcaagcagat gtgattcgca acggcacaaa gtttagtgtt 901 ggtcactact gcggagagat gggatcaaga ttttgggacg cccgagggtg gcagcgagaa 961 tttgatacca aagatgtttt tgtaatgacc gcacagattc ttttgaacat ccttaggcat 1021 agcattgtaa aaatggaagc cattcatcta cttattctcg atgagtgcca ccatgccgtg 1081 aagaaacatc cctattcttt ggtgatgtct gaattctatc ttatgacacc taaagataag 1141 cgaccgtgtg tctttgggat gatagcatcg cctgtgaacc tcaaaggggt atcaaaccag 1201 gaagattgtg caatagagat tcgaaattta gaaagcaagt tggactcgat agtgtgtaca 1261 atcagggatc ggaaagagct cgaaaagcac gtgcctttgc cgtcagagac aatgattctg 1321 tacgataagc cggccttgct tttctcgttg cggaaagaga gaaaacagat ggaggccact 1381 gtagaaaagg ctgctaatgc aagtgtcaga cgcagcaaat ggaaatgcat gggcgctcgg 1441 gatgcgggtg ctaaagagga actgcaactt gtgtacagtg tcgcggagag aacggaaagc 1501 gatggcgcag ctagtctttc tcaaaagctt agagccatta cctatgcact tgatgaatta 1561 ggtcaatggt gtgcttacaa ggtctcgctg ggatatctga caagtcttca taatgatgaa 1621 agggttaatc atcagttaga cgtgaagttt caaaagttgt acttgaagaa ggtttgtact 1681 cttctgcgat gcagtctacg tgaaggtgct gcagggtggg aggtacctgc tgaaattgga 1741 gagtctgagg gcgataaagc acaagatcca atggatgtgg aagaaggaag cttcctgact 1801 ctcgtatcag tgggtgaaca tttggatgag attcttgggg ccgctgtagc agatggaaaa 1861 gtgactccga aggtgcagtc tttaattaag gttttaatag gttatcagca tacggatgat 1921 ttccgagcta ttatatttgt ggagcgagtc tgggtgggtg ttacgctttg caggtctttg 1981 cagagttgcc cttcattgaa gtttgtgaaa tgtgccagtc tgatagggca caacaataac 2041 caagacatgc cgacacggca gatgcaggag actatttcca agtttcgaga tggacgggtg 2101 acgttgctgg tggctacaag cgtggccgca gagggattag atattcgcca atgtaatgtg 2161 gtcatccgtt ttgatcttgc taaaaccgtg ttagcctaca tccagtctcg tggtcgtgct 2221 cggaagcctg gttcagatta tattttaatg cttgagagag gaaatctgca acatgaggcg 2281 ttttttcgga atgcaaaaaa tagtgaggag actttacgga aggaggctat tgaaagaact 2341 gatctgggtg aaaaacggga gaatgcgatt ctggcctcca ttgacattgg ggaaggggag 2401 atttaccagg tgccagccac tggggcagtc gtgagcatga actcagctgt aggtcttatt 2461 cacttctact gctctcagct tcccagtgac aggtattctc tcttgcgtcc tgagttcatt 2521 atgaacaaaa ttgaggatca aagaggtgct ataagatact cgtgcagact gcagttgcct 2581 tgccatgctc cgtttgaagc tgtggaaggc ccagaatgta attctatgcg aggagcgcag 2641 cagtcctgtg tgcttgaagg cttgcaaaaa atgcacgaaa tgggggcatt cacggacatg 2701 ctattatcta ataaaggaag tagggaagaa gctgctaagt tggagggtag tgaagaggga 2761 gagtctcttc ctggcacatc ccgtcatcga gaatattatc cagaggggat tgcagatatt 2821 ctgaagggcg atcggatagt ggctgagaaa gattcggata caaaggaagg cagcaaggtg 2881 ctcgtattca tgtacacggt gaagtgtgaa aatgttggct tctcgagaga cagccttttg 2941 accgagacat cagactttac cttacttgtc ggccaacagc ttcatgacca ggtgttaacc 3001 atgacaataa atctttttgt cgcaaacccg actttactga tcacgatgag ctggaaaaag 3061 agggatttgg attgctctaa ttcccagttg actgagctca agagttttca tgtgaggctt 3121 atgagcattg ttttagacgt aaatgtcgag ccggcaacaa ctcgttggga tcccgccaag 3181 gcgtatctct ttgctccagt tctgcataag gatgcctccg atcctaaaga cttggtggac 3241 tgggtcgtta tgagaaggac gatcgagact gattcatgga gtaatcccct ccagcgcgca 3301 tcacctgatg tgaacttggg gactgacgaa cgtgctcttg gtggggatcg tagagagtac 3361 gggtttggaa aactgcgatg tagtctggcc tttgggcagg gagcgcatcc aacgtatggt 3421 gctcgtggcg ctaaagctca atttgatgtt gtgaaagcca caggtctact tcctacctca 3481 gacatggtgg aggagacaac tgtgcaggaa gtacctcccg agggtaagct gttgatagtg


139

3541 gatggttttg ttgaagttga agtattggtg ggaaggattg ttactgcggt gcattctggg 3601 aagaggcttt atgtggattc ggtgcgcttt gacatgacag ccgacagctc ttttcctcaa 3661 aaggatggat accttggtcc actggaatac acatcgtatg cggattatta caaacaaaag 3721 tacggtgtcg agttggtttg caagaaacag cctctgttga ggggtcgtgg ggtttctcat 3781 tgcaaaaatt tattgtcgcc acgttttgag acctctggcg actctctgga tgccttggat 3841 aagacgtatt atgtgatgct gccacctgag ctttgcctta tacatcctct tccgggatcc 3901 ttggtgagag gcgcacaaag attgccatcg gtcatgagac gtgtagagag catgttgctt 3961 gccatacaac taaagcacca aatcgattac cctattgctg cttcgaaggt agcgttgacg 4021 gctgcgtctg gtcaagagac attcagctat gagcgtgcag agcttttaag cgacgcgtac 4081 ctcgaatggg ttgttagtca tcgattgttc ctgaagttcc ctagtaaaca tgaggggcag 4141 cttacacgta tgagacagaa aattgtcagc aattccgttc tgtatcaaca tgccctagag 4201 aaaggtcttc agagttacat tcaggccgac cgctttgcac cgtcccggtg ggccgcaccg 4261 ggagtgcctc ctgcattcga tgaggacttg agagatggcg atgattcgga taaggagtcg 4321 aaacctgaag ttgaaagaga agtagtggag attgtcggtg aggaaggtga aattgttaag 4381 gaactaaata cagaaagtga aaatatggaa gacggtgaaa ttgaaggtga ttccggttcc 4441 tatcgagtgc tttcgagtaa aaccttggca gacgtggtag aggtattcat gggaatgtat 4501 tatgtggagg ggggggggga ggctgctact cacttcatga actgggtagg cattcctgtg 4561 gagtttgatg acgtggagac agacttagcc acaggtggct gccaagttcc tgaaaccgtt 4621 atgcggagca tagacttttc atcattacaa aaaaacgttg gccatgaatt tcgtgaacga 4681 agtttattgg tagaggccat cacgcacgcg tctcgaccat cgttgggagt tccttgctac 4741 caaaggctgg agtttgtggg ggatgccgtg ttggaccatc tgattacacg ttatctattc 4801 tttaaatata ctaatttgcc cccaggtagg ttgaccgatt tgcgagctgc tgcagtgaat 4861 aacgaaaatt tcgcacgtgt tgctgtgaag cactcgtatc atcttcattt gcggcatggt 4921 tcaaccgctt tagaaactca gattcgcaat ttcgtgaatg atatacactc ggagttagac 4981 aagcctggag tgaactcttt tggactaggg gattttaagg cccctaaagt gctgggtgat 5041 attttcgaat ccattgcagg cgctctattc ctggacgctc gtcttgacac acaccaagtg 5101 tggaaggttt ttgagccttt gttgcagccc atggtgtccc cagagacatt gccgatccat 5161 ccagtacgag ggttgcagga gcgttgtcaa caagaagctg aaggtctgga gtacaaagtg 5221 tctcgtgcag agagtgttgc gaccgtggag gtgtatgtag acggtgtaca gataggttct 5281 acgcaaagtg ctcagaagaa aatggcccaa aaattaggtg ctcgtaatgc gttggtcaaa 5341 ttgaaggata aggaggtgat caaagtgaaa gctgaggcag agaatggtga cttgaacgct 5401 ggaaaatcga gcaagaacgg tcacactaac ttcactcgca aaacaattaa cgacctttgt 5461 cttaagagac agtggccgat gccacagtac aaatgcgttc tggagagcgg accagcgcat 5521 gctaagaagt ttacgctctc tgtacgggtt ctgaccacca ctgatggatg gaccgaagaa 5581 tgtgttgggg agcctatggc gagtgtgaag aaagctaagg actctgcagc tcttgtactt 5641 ttggctactt tgagacgatc atatcctttg cgtaataata ttatagactg ctaaaatgac 5701 ccaaattgat cagaaaacat acagaactac ataccggcct gtgggtgctt caggttcaac 5761 atatccgtgc cccgtcaaca taaatttgtg aatgcacaaa tcacaaggtt tggatagcac 5821 tagccagcgc cagttcgttc aaggagctgc aggccagctc agccctcgtt ttataccttt 5881 catgatatgt tttgtctttt tggatcaaat tgtgagagac agagcacagg tcagtatacc 5941 gttcaaagaa agtagtgagt ttttgtaccg taagacagct gcgtctctcc ctcttaattt 6001 tgtctttcat ttttcagttt ttcagagaaa aaaaaaaaaa aaaaaaaaaa aa


140

4.3 DNA vectors

Origin of DNA vectors that were used for cloning and transformations

Name Backbone Insert Reference

pCR4-TOPO pCR4-TOPO PpDCL1b cDNA region; nptII Invitrogen,

Karlsruhe

pPCV

(Figure 2)

----

PpFtsZ2-1-amiRNA and PpGNT1-

amiRNA sequences with A.

thaliana miRNA319a Precursor

Koncz, C. et al.,

1989

Figure 2: pPCV plant overexpression vector containing a double 35S promoter,

nos terminator and hpt selection marker cassette.


141

4.4 Genes downregulated in ΔPpDCL1b mutants

Sequence ID

EST

Cosmoss Annotation Fold change

BJ165956 **: Homolog of OSJNBa0003O19.20|putative MYC transcription factor -1.46 BJ172212 ***: Q7XN04 OSJNBb0038F03.7 protein. -1.49 BJ200093 **: Homolog of Similar to phytochrome and flowering time 1 protein -1.59 BJ200754 ***: Q8RYB8 Aldehyde dehydrogenase Aldh21A1. -1.82 BJ579811 **: Homolog of Roc1-related|o_sativa|chr_8|P0020B10|4196 -1.59 BJ580674 **: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens] -1.80 BJ583348 **: Homolog of aldehyde dehydrogenase, putative|o_sativa|chr_11|OSJNBa0052O08|5272 -3.15 BJ583460 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 -2.21 BJ587267 **: Homolog of Helicase conserved C-terminal domain, -1.60 BJ589432 ***: Q84WK0 At4g33880. -1.66 BJ601044 **: Homolog of (68417.m02544 vernalization 2 protein VRN2) -1.43 PP_10059_C1 ***: Q8SA80 Disease-resistent-related protein. -1.34 PP_10115_C2 **: Homolog of 68416.m05958 protein kinase family protein contains eukaryotic protein

kinase domain -1.50

PP_10382_C1 ***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor -3.18 PP_10385_C1 ***: Q6Z5T6 Putative intensifier. -1.58 PP_10479_C1 ***: Q8S1X3 Putative SUVH4. -1.90 PP_10658_C1 **: Homolog of AP2 domain, putative|o_sativa|chr_6|P0021C04|3631 -1.50 PP_1073_C1 **: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946 -1.70 PP_10745_C1 **: Homolog of (AB084898) mitochondrial aldehyde dehydrogenase [Sorghum bicolor] -1.45 PP_10817_C1 not annotated Physcomitrella patens -1.39 PP_11302_C1 ***: Q8X1E7 Histidine kinase. -2.11 PP_11331_C1 ***: Q9M551 Polyubiquitin. -1.86 PP_11112_C1 ***: Q948P1 Peroxisomal ascorbate peroxidase. -2.22 PP_11390_C1 ***: Q41067 Polyubiquitin. -1.66 PP_1146_C3 ***: Q9LV44 Similarity to signal peptidase. -1.60 PP_11513_C1 ***: Q9M077 Putative serine/threonine protein kinase. -1.87 PP_11795_C1 ***: Q93V58 Putative serine threonine-protein kinase. -1.35 PP020018263R **: Homolog of 68418.m05603 YEATS family protein contains Pfam domain -1.50 PP_12005_C1 **: Homolog of Neutral/alkaline nonlysosomal ceramidase|o_sativa|chr_1|P0501G01|2708 -1.58 PP_12101_C1 ***: Q9ARE4 ZF-HD homeobox protein. -1.65 PP_12167_C1 **: Homolog of (AY034888) aldehyde dehydrogenase Aldh21A1 [Tortula ruralis] -4.19 PP_12301_C1 contains: (COIL:coil) -2.40 PP_12301_C1 contains: (COIL:coil) -2.06 PP_12365_C2 ***: Q9FH40 Similarity to unknown protein (TAF14b) (Hypothetical protein At5g45600). -1.35 PP_12367_C1 ***: Q8W314 Putative dehydratase/deaminase. -1.71 PP_125_C1 **: Homolog of (68415.m03211 plectin-related contains -1.25 PP_12576_C1 **: Homolog of SNF2 family N-terminal domain -1.70 PP_12599_C1 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 -2.34 PP_12681_C1 ***: Q7XPK1 OSJNBa0087O24.9 protein. -2.51 PP_12858_C1 **: Homolog of 68417.m05252 expressed protein contains Pfam profile PF04784 -1.74 PP_12940_C1 ***: Q7Y1Z3 Putative small nuclear ribonucleoprotein Prp4p. -1.42 PP_13101_C1 ***: Q43303 Histone H3 (Fragment). -1.43 PP_13149_C1 ***: Q9ZPN6 Transcription factor MYC7E (Fragment). -1.79 PP_13160_C1 ***: RL71_ARATH 60S ribosomal protein L7-1. -1.38 PP_13170_C1 ***: Q9SII9 Putative ubiquitin protein. -1.42 PP_13256_C1 **: Homolog of expressed protein|o_sativa|chr_2|P0506A08|3865 -1.85 PP_13508_C1 contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112) -1.58


142

PP_13734_C1

**: Homolog of 68415.m02009 ARID/BRIGHT DNA-binding domain-containing protein contains

-2.23

PP_13750_C1 **: Homolog of (AB015183) transcription factor Vp1 [Mesembryanthemum crystallinum] -1.91 PP_13846_C1 ***: Q8LB52 Scarecrow-like protein. -1.37 PP_1387_C1 ***: Q7X9V3 Nuclear shuttle interacting protein. -1.51 PP_14366_C1 **: Homolog of (Transcription factor E2F/dimerisation partner TDP) -1.74 PP_14581_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.75 PP_14609_C1 ***: Q39183 Serine/threonine protein kinase (Protein kinase (EC 2.7.1.37) 5)

(AT5g47750/MCA23_7). -1.56 PP_14674_C1 "**: Homolog of (68417.m04184 acid phosphatase class B family protein similar to acid

phosphatase -1.58

PP_1468_C1 not annotated Physcomitrella patens -1.92 PP_15181_C1 ***: O82527 Polyubiquitin (Fragment). -1.46 PP_1665_C2 ***: RL71_ARATH 60S ribosomal protein L7-1. -2.11 PP_17120_C1 **: Homolog of 68417.m02122 myb family transcription factor contains Pfam profile -1.55 PP_1761_C1 contains: Nascent polypeptide-associated complex

NAC(InterPro:IPR002715,PFAM:PF01849) -1.89

PP_18023_C1 **: Homolog of (68415.m03211 plectin-related contains -2.11 PP_18168_C1 ***: SU91_HUMAN Histone-lysine N-methyltransferase, H3 lysine-9 specific 1 -1.63 PP_11285_C4 **: Homolog of (AB112672) auxin response factor 2 [Cucumis sativus] -1.80 PP_18237_C1 ***: ARP_ARATH Apurinic endonuclease-redox protein (DNA-(apurinic or apyrimidinic site) -1.73 PP_18520_C1 "**: Homolog of ((AP004849) putative CCR4-NOT transcription complex -1.56 PP_2007_C1 ***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB). -1.95 PP_2015_C1 ***: Q8LKS8 Early drought induced protein. -1.38 PP_2094_C3 ***: Q8RXD3 ABI3-interacting protein 2. -1.71 PP_2103_C2 ***: Q8GRK2 Somatic embryogenesis receptor kinase 1. -1.43 PP_2104_C1 **: Homolog of Similar to chloroplast DNA-binding protein

PD3|o_sativa|chr_2|P0017C12|3841 -1.74 PP_214_C9 ***: AHM7_ARATH Potential copper-transporting ATPase 3 (EC 3.6.3.4). -2.08 PP_224_C1 **: Homolog of 68417.m02632 lil3 protein identical to Lil3 protein [Arabidopsis thaliana] -1.34 PP_2272_C1 ***: O81077 Putative cytochrome P450. -40.91 PP_2294_C1 **: Homolog of expressed protein|o_sativa|chr_6|OSJNBa0019F11|6897 -1.59 PP_2113_C3 ***: Q9FIX3 Gb|AAD30619.1. -2.07 PP_2320_C1 ***: Q852K5 Putative zinc finger protein (Putative zinc finger transcription factor ZFP38). -1.65 PP_2360_C1 ***: AHM1_ARATH Potential cadmium/zinc-transporting ATPase HMA1 (EC 3.6.3.3) (EC

3.6.3.5). -1.64

PP_233_C4 ***: Q943L1 Putative Ubiquitin carrier protein UBC7. -1.96 PP_2537_C1 ***: Q9ZS93 T4B21.6 protein. -2.16 PP_2633_C1 contains: Transcription factor, MADS-box -1.67 PP_2646_C2 contains: GCN5-related N-acetyltransferase -1.66 PP_2738_C1 ***: O23310 CCAAT-binding transcription factor subunit A(CBF-A) -1.82 PP_276_C1 **: Homolog of (AB106274) SCARECROW-like protein [Lilium longiflorum] -1.50 PP_2920_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 -1.45 PP_2921_C1 ***: T2AG_ARATH Transcription initiation factor IIA gamma chain (TFIIA-gamma). -1.56 PP_285_C2 **: Homolog of AT3g02790/F13E7_27|o_sativa|chr_6|P0621D05|1946 -1.31 PP_2980_C1 ***: Q8H1G0 Putative flowering protein CONSTANS (GATA-type zinc finger protein). -1.37 PP_2817_C1 contains: Protein of unknown function DUF296(InterPro:IPR005175,PFAM:PF03479) -1.56 PP_6215_C1 ***: Q8S9V3 Putative zinc finger protein. -2.49 PP_3091_C1 ***: Q9SNA4 Receptor-like protein kinase homolog. -1.53 PP_323_C1 ***: PSAG_ARATH Photosystem I reaction center subunit V, chloroplast precursor (PSI-G). -1.59 PP_3684_C1 "**: Homolog of (X58577) DNA-binding protein; bZIP type [Petroselinum crispum] -1.70 PP_3846_C1 **: Homolog of aldehyde dehydrogenase, -1.85 PP_3876_C1 **: Homolog of (68414.m05059 Ras-related GTP-binding protein -1.36 PP_3950_C1 **: Homolog of (AB028078) homeobox protein PpHB7 [Physcomitrella patens] -1.58 PP_3864_C1 **: Homolog of (68416.m02515 basic helix-loop-helix bHLH) family protein -2.01 PP_4087_C4 "**: Homolog of (68416.m01203 aspartate/glutamate/uridylate kinase family protein -1.50


143

PP_4109_C1 **: Homolog of (M62985) protein kinase [Zea mays] -1.86 PP_4163_C1 **: Homolog of (AF029984) COP1 homolog [Lycopersicon esculentum] -1.68 PP_4175_C1 **: Homolog of (68414.m07887 basic helix-loop-helix bHLH) family protein -2.79 PP_4238_C1 **: Homolog of (68414.m01246 eukaryotic translation initiation factor 3 subunit 3 / eIF-3 -1.61 PP_4183_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.50 PP_4368_C1 **: Homolog of (68418.m04281 histidine kinase AHK2) identical to histidine kinase -2.10 PP_438_C1 **: Homolog of 68418.m05840 myb family transcription factor contains Pfam profile -1.42 PP_4383_C1 **: Homolog of (68415.m02898 basic helix-loop-helix bHLH) family protein -1.56 PP_4394_C1 **: Homolog of Protein kinase domain, putative|o_sativa|chr_1|OJ1529_G03|4777 -3.02 PP_4227_C2 **: Homolog of (transcription initiation factor iib general transcription factor tfiib). -1.39 PP_4501_C1 **: Homolog of OSJNBa0003O19.1|putative AT-Hook DNA-binding protein -1.43 PP_4570_C3 "**: Homolog of (68418.m08464 F-box family protein similar to unknown protein

(dbj|BAA78736.1) -1.63

PP_4710_C1 **: Homolog of 68416.m05166 Dof-type zinc finger domain-containing protein [Arabidopsis thaliana]

-1.53

PP_4819_C1 **: Homolog of Similar to histidine kinase-like protein|o_sativa|chr_6|P0709F06|1935 -2.13 PP_4820_C1 **: Homolog of (AF378125) GAI-like protein 1 [Vitis vinifera] -4.01 PP_4988_C1 **: Homolog of (68415.m05233 basic helix-loop-helix bHLH) family protein -1.80 PP_5004_C1 "**: Homolog of (68418.m01242 sensory transduction histidine kinase-related -1.70 PP_5046_C1 "**: Homolog of (68415.m04408 zinc finger (C3HC4-type RING finger) family protein -3.59 PP_5138_C1 **: Homolog of (AF439278) ethylene-responsive transciptional coactivator-like protein

[Retama raetam] -3.39

PP_5262_C1 **: Homolog of 68415.m04505 PHD finger transcription factor, putative -1.92 PP_5396_C1 **: Homolog of (AF311224) C2H2 zinc-finger protein [Zea mays] -1.73 PP_5526_C1 **: Homolog of 68418.m04803 Dof-type zinc finger domain-containing protein [Arabidopsis

thaliana] -1.31

PP_5624_C1 **: Homolog of TAZ zinc finger, putative|o_sativa|chr_1|P0696G06|5647 -1.62 PP_5836_C2 **: Homolog of 68416.m05063 myb family transcription factor -27.29 PP_5905_C1 **: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase -1.50 PP_6171_C1 ***: Q84XK6 Peroxisomal targeting signal type 2 receptor. -1.29 PP_6223_C1 contains: Helix-turn-helix, Fis-

type(InterPro:IPR002197,GO:0003700,GO:0006355,PRINTS:PR01590) -3.32

PP_6242_C1 not annotated Physcomitrella patens -2.62 PP_6285_C1 **: Homolog of (Y10685) G/HBF-1 [Glycine max] -1.94 PP_6114_C1 **: Homolog of F-box domain, putative|o_sativa|chr_4|OSJNBa0043A12|8129 -1.91 PP_6587_C1 ***: O82064 Putative beta-subunit of K+ channels. -3.86 PP_646_C2 **: Homolog of Dof domain, zinc finger, putative|o_sativa|chr_1|P0453A06|2679 -2.29 PP_6731_C1 not annotated Physcomitrella patens -1.89 PP_6766_C1 ***: Q8W2B8 Serine acetyltransferase (Hypothetical protein At4g35640). -2.19 PP_683_C1 ***: YPT6_CHLRE Ras-related protein YPTC6. -1.97 PP_6888_C1 contains: Ubiquitin-conjugating enzymes -1.65 PP_6969_C1 **: Homolog of (68415.m03211 plectin-related contains -9.85 PP_6973_C1 "***: Q9C8A0 Serine/arginine-rich protein, putative; 48931-50251 (TAF7) (At1g55300)." -1.39 PP_6875_C1 contains: U2 snRNP auxilliary factor -1.60 PP_7128_C1 **: Homolog of (AF467900) hypothetical transcription factor [Prunus persica] -2.40 PP_7321_C2 ***: Q9FJC9 26S proteasome regulatory particle chain RPT6-like protein

(AT5g53540/MNC6_8). -1.71

PP_11287_C4 ***: RL5_ARATH 60S ribosomal protein L5. -1.55 PP_7371_C1 contains: Tubby(InterPro:IPR000007,PFAM:PF01167) -2.04 PP_7586_C1 ***: DR1D_ARATH Dehydration responsive element binding protein -1.54 PP_7694_C1 ***: Q39031 Protein kinase. -2.02 PP_7708_C1 ***: Q7X976 Putative AT-Hook DNA-binding protein. -1.43 PP_775_C1 ***: Q9SQ79 Helix-loop-helix protein 1A. -1.79 PP_7994_C1 **: Homolog of (AY077758) WRKY transcription factor 1 [Physcomitrella patens] -1.50 PP_8047_C1 **: Homolog of 68417.m01380 KOW domain-containing transcription factor family protein

chromatin -1.45

PP_8107_C1 **: Homolog of Similar to TINY-related|o_sativa|chr_2|OJ1711_D06|4260 -2.55


144

PP_8413_C1 contains: Zn-finger, RING(InterPro:IPR001841,PFAM:PF00097) -1.47 PP_8463_C1 ***: Q86AZ8 Similar to Anabaena sp. (Strain PCC 7120). Hypothetical WD-repeat protein

alr2800. -1.76

PP_8293_C1 contains: (COIL:coil) -2.52 PP_8547_C1 **: Homolog of 68414.m01463 hypothetical protein -1.84 PP_86_C1 **: Homolog of (AY566696) unknown [Xerophyta humilis] -1.83 PP020016117R **: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens] -1.93 PP_723_C1 ***: Q8LST6 Mitochondrial aldehyde dehydrogenase. -1.51 PP_9108_C3 ***: FTH2_ARATH Cell division protein ftsH homolog 2, chloroplast precursor -1.70 PP_12500_C1 **: Homolog of (68414.m01494 basic helix-loop-helix bHLH) family protein / F-box family

protein -3.17

PP_9253_C1 ***: Q9C1Q7 Putative two-component histidine kinase Fos-1. -1.41 PP_9264_C1 "***: Q9SRM4 Putative nucleic acid binding protein (At3g11200/F11B9.12) -1.33 PP_9369_C1 ***: Q9FJ00 Gb|AAF24948.1. -1.59 PP_9394_C1 ***: O94094 Histidine kinase FIK. -1.81 PP_9399_C1 ***: Q8X1E7 Histidine kinase. -1.67 PP_9419_C1 **: Homolog of 68417.m04480 WRKY family transcription factor contains Pfam profile -2.93 PP_9627_C1 ***: Q7WZ30 MmoS. -3.06 PP_9785_C1 ***: Q8LPA5 MADS-box protein PpMADS1. -1.73 PP_9953_C1 ***: Q852U6 At1g49850. -1.59 PP_9960_C1 contains: Nascent polypeptide-associated complex NAC

(InterPro:IPR002715,PFAM:PF01849) -2.92

PP_6995_C2 ***: Q9ZNX9 Sigma-like factor precursor (RNA polymerase sigma subunit SigE). -1.77 PP020064243R ***: Q9LWW0 Oryza sativa (japonica cultivar-group) genomic DNA, chromosome 6,

clone:P0425F02. -1.40

PP_SD_251_C1 **: Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris] -1.33 PP_SD_60_C1 ****: Physcomitrella patens mRNA for homeobox protein PpHB9, complete cds. -1.87 PP_SD_88_C1 not annotated Physcomitrella patens -3.49 PP_SD_92_C1 ****: Physcomitrella patens PpHB10 mRNA for homeobox protein PpHB10, complete cds. -2.00 PP_SD_245_C1 **: Homolog of (68418.m05453 disease resistance protein TIR-NBS-LRR class) -1.66 PP_5766_C1 **: Homolog of (68418.m02952 zinc finger B-box type) family protein similar to

CONSTANS-like protein -1.47

PP_SD_0_C1 ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1, complete cds.

-1.96

PP001001061F **: Homolog of (X98744) chloroplast DNA-binding protein PD3 [Pisum sativum] -2.13 PP001008059F **: PP_CL_6374.Singlet Homolog of (AB046872) PpSIG2 [Physcomitrella patens] -1.52 PP001009093F **: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein -1.35 PP001019006F **: PP_CL_8395.Singlet Homolog of (68416.m00477 RNA recognition motif (RRM) -1.54 PP001030028F **: Homolog of (AC005315) putative non-LTR retroelement reverse transcriptase -1.96 PP001030033F "**: Homolog of 68414.m08061 paired amphipathic helix repeat-containing protein -1.47 PP001068061R **: PP_CL_18208.Singlet Homolog of 68415.m05104 expressed protein -2.18 PP001085009R **: PP_CL_6389.Singlet Homolog of (AY324646) katanin [Gossypium hirsutum] -1.60 BJ165389 ***: Q41067 Polyubiquitin. -1.41

PP004021140R ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1, complete cds.

-1.48

PP004058038R ***: O49977 Ubiquitin (Fragment). -1.31 PP004067310R **: PP_CL_7066.Singlet Homolog of (AB026657) unnamed protein product -1.60 PP004086076R ***: Q41067 Polyubiquitin. -1.58 PP004087236R ****: Physcomitrella patens subsp. patens mRNA for MADS-box protein PpMADS1,

complete cds. -1.52

BI488008 ***: O82527 Polyubiquitin (Fragment). -1.59 PP004095105R ***: O82527 Polyubiquitin (Fragment). -1.59 PP013015004R **: Homolog of OSJNBb0070O09.2|unknown protein -1.51 PP015004308R ***: Q8LNW1 Putative transcription factor. -1.70 PP015011331R **: Homolog of (68414.m08326 GCN5-related N-acetyltransferase GNAT) family protein -1.43 PP015020123R **: Homolog of (AB032182) homeobox protein PpHB10 [Physcomitrella patens] -1.47 PP015029288R "**: PP_CL_5054.Singlet Homolog of (68418.m08510 TAZ zinc finger family protein -1.99


145

PP015033075R **: Homolog of (AB121445) histidine kinase 3 [Zea mays] -4.00 PP015037157R **: Homolog of Helicase conserved C-terminal domain,

putative|o_sativa|chr_8|OJ1034_C08|7210 -1.73

PP015044155R **: PP_CL_1670.Singlet Homolog of (AJ419328) putative MADS-domain transcription factor

-1.50

PP015054317R **: Homolog of Helix-loop-helix DNA-binding domain, putative|o_sativa|chr_1|OSJNBa0093F16|4658

-1.53

PP015071162R ***: AGO1_ARATH Argonaute protein. -1.40

PP020027384R ****: Physcomitrella patens subsp. patens PPLFY1 mRNA for FLORICAULA/LEAFY homolog

-1.40

PP020032226R **: PP_CL_18682.Singlet Homolog of (AJ011828) NDX1 homeobox protein [Lotus corniculatus

-1.59

PP020034124R **: PP_CL_4373.Singlet Homolog of (AB115546) phototropin 2 [Adiantum capillus-veneris] -1.65 PP020036015R **: PP_CL_9604.Singlet Homolog of OJ1365_D05.10|putative response regulator protein -2.19 PP020036307R ***: Q9FT60 Histidine kinase-like protein. -1.44 PP020044335R "**: Homolog of 68416.m03380 oligopeptide transporter OPT family protein -1.94 PP020051260R ***: Q9SXL4 Histidine kinase 1. -2.12 PP020062195R **: Homolog of (AJ419328) putative MADS-domain transcription factor [Physcomitrella

patens] -1.82

PP020065285R **: PP_CL_10589.Singlet Homolog of (AL670011) related to regulatory protein SET1 -1.49 PP020069226R ***: Q9LRI1 Homeobox protein PpHB10. -1.60 PP030015063R ***: O82527 Polyubiquitin (Fragment). -1.55 AY123146 PP_SD_72.Singlet not annotated Physcomitrella patens -1.76


146

4.5 Genes upregulated in ΔPpDCL1b mutants

Sequence ID

EST

Cosmoss Annotation Fold change

AW561368 "**: Homolog of (68417.m01135 F-box family protein (FBL8) FBL24) contains 1.32 BJ160934 **: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii] 2.24 BJ163321 ***: Q8S0L3 Ankyrin-kinase-like protein. 1.94 BJ181458 ***: Q9AV93 Response regulator 8. 1.97 BJ187175 ***: Q84VL6 Putative polyubiquitin (Fragment). 1.81 BJ189887 **: PP_CL_6916.Singlet Homolog of ((AP003273) histone H1-like protein (Oryza sativa ) 1.30 BJ609923 ***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor. 1.43 BJ610672 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 1.70 BU051751 ***: Q7X864 OSJNBa0093F12.4 protein. 1.55 PP_10130_C2 ***: Q8H9A2 Dehydratiion responsive element binding protein 1 like protein. 1.76 PP_10143_C1 ***: Q8YMN1 All4902 protein. 1.87 PP_10308_C1 **: Homolog of OSJNBb0015I11.23|putative ubiquitin

protein|o_sativa|chr_10|OSJNBb0015I11|31 1.62

PP_1034_C1 ***: Q9SEK4 Putative succinic semialdehyde dehydrogenase 1.53 PP_10379_C1 "**: Homolog of (68417.m00335 AAA-type ATPase family protein 1.95 PP_10320_C1 ***: Q96327 Putative nuclear DNA-binding protein G2p (Nuclear DNA-binding protein) 1.75 PP_10567_C1 not annotated Physcomitrella patens 1.57 PP_10875_C1 ***: Q94H06 Putative zinc finger protein. 2.11 PP_10880_C1 ***: Q8GYN7 Putative SCARECROW gene regulator. 1.67 PP_10919_C1 ***: Q8S8P6 Putative salt-inducible protein. 1.69 PP_11080_C1 ***: Q9SA69 F10O3.17. 1.39 PP_1112_C1 ***: Q9ZVG0 Putative ATP-dependent DNA helicase RECG. 1.41 PP_11139_C1 **: Homolog of 68414.m03428 expressed protein 1.38 PP_12499_C1 ***: PRS7_ARATH 26S protease regulatory subunit 7 (26S proteasome subunit 7) 1.43 PP_13554_C1 ***: Q9LKG4 Putative DNA binding protein. 1.30 PP_11359_C1 **: Homolog of 68416.m01881 DNA-binding protein-related 2.03 PP032001093R **: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein 1.58 PP_18394_C1 **: Homolog of RNA polymerase I specific transcription initiation factor 2.11 PP_1179_C1 **: Homolog of Similar to probable zinc finger protein [imported] - Arabidopsis thaliana 2.07 PP_1012_C1 **: Homolog of 68418.m05158 kelch repeat-containing F-box family protein contains Pfam

profiles 2.43

PP_12120_C1 ***: Q7X6J0 RNA binding protein Rp120. 1.73 PP_12140_C1 **: Homolog of (AF470350) WD40 [Tortula ruralis] 1.41 PP_12145_C1 **: Homolog of hypothetical protein|o_sativa|chr_1|P0470A12|2880 1.78 PP001005057F ***: O82527 Polyubiquitin (Fragment). 1.65 PP_12587_C1 **: Homolog of AP2-related transcription factor,

putative|o_sativa|chr_4|OSJNBa0079A21|8302 2.46

PP_12713_C2 ***: Q9GZS3 Homo sapiens cDNA: FLJ21101 fis, clone CAS04682 (G protein beta subunit)

1.56

PP_12802_C1 ***: Q7XXN2 Putative serine/threonine-protein kinase ctr1. 2.19 PP_1303_C1 contains: Dihydrodipicolinate synthetase(InterPro:IPR002220,PFAM:PF00701) 1.86 PP_13105_C1 ***: Q84QC2 Putative AP2 domain transcription factor. 1.72 PP_13136_C1 ***: Q9SGP0 F3M18.14. 1.96 PP_13592_C1 ***: Q94DZ5 Putative helicase-like transcription factor. 2.17 PP_13985_C1 ***: Q9FHJ4 Arabidopsis thaliana genomic DNA, chromosome 5, P1 clone: MFC19. 1.39 PP_14045_C1 ***: O65567 Puative protein. 1.81 PP_14113_C1 ***: IF35_ARATH Eukaryotic translation initiation factor 3 subunit 5 (eIF-3 epsilon) (eIF3

p32 subunit) 1.47

PP_1440_C1 contains: RNA-binding region RNP-1 (RNA recognition motif) 1.69 PP_14547_C1 **: Homolog of (68414.m00475 zinc finger (C3HC4-type RING finger) family protein 1.32


147

PP_14811_C1 **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.40

PP_15007_C1 ***: Q9ZV05 Expressed protein. 1.60 PP_15083_C1 **: Homolog of zinc finger protein, putative|o_sativa|chr_6|P0550B04|1978 1.69 PP_15177_C2 ***: Q9SJR0 Putative AP2 domain transcription factor (Putative AP2/EREBP transcription

factor). 10.73

PP_15255_C1 ***: Q9FHA7 Emb|CAB62312.1 (Putative bHLH transcription factor). 1.41 PP_15299_C1 ***: Q7XJM3 Putative mitochondrial translation elongation factor G. 2.15 PP_15344_C1 ***: O81763 Protein kinase-like protein. 1.84 PP_15382_C1 **: Homolog of 68416.m05464 phototropic-responsive protein 1.59 PP_15384_C1 **: Homolog of 68417.m00399 elongation factor Tu, putative / EF-Tu, putative 1.50 PP_15546_C1 ***: Q9LW84 Gb|AAF26996.1. 1.65 PP_15582_C1 **: Homolog of Similar to DNA helicase-like|o_sativa|chr_2|P0724B10|6621 1.42 PP_15610_C1 ***: MAT1_MOUSE CDK-activating kinase assembly factor MAT1 (RING finger protein

MAT1) 1.44

PP_15633_C1 ***: Q9SJW0 Transfactor-like protein. 2.38 PP_15636_C1 ***: Q8VZG7 AT5g07350/T2I1_60. 2.24 PP_15695_C1 **: Homolog of expressed protein|o_sativa|chr_4|OSJNBa0013K16|5362 24.51 PP_1585_C2 contains: Zn-binding protein, LIM(InterPro:IPR001781,PFAM:PF00412) 1.53 PP_15995_C1 ***: EFTM_ARATH Elongation factor Tu, mitochondrial precursor. 1.96 PP_15997_C1 ***: Q9SI75 F23N19.11 (Hypothetical protein At1g62750). 2.72 PP_16050_C1 ***: Q851S7 Pescadillo-like protein. 4.28 PP_1618_C1 ***: Q9LVF7 Gb|AAD14441.1. 1.44 PP_16284_C1 "**: Homolog of (68414.m09356 coatomer protein complex, subunit beta 2 (beta prime), 3.16 PP_1662_C1 ***: Q8S3E7 Putative bHLH transcription factor. 1.84 PP_1663_C1 ***: Q9LSQ8 Arabidopsis thaliana genomic DNA, chromosome 5, BAC clone: F24B18. 1.98 PP_16437_C1 ***: EFGM_ARATH Probable elongation factor G, mitochondrial precursor (mEF-G). 2.48 PP_1496_C1 contains: Pathogenesis-related transcriptional factor and ERF 3.69 BJ181914 ***: O82527 Polyubiquitin (Fragment). 1.61 PP_16865_C1 **: Homolog of auxin response factor 1 [imported] - Arabidopsis

thaliana|o_sativa|chr_2|P0506A08|3865 2.62

PP_1691_C1 ***: Q9LS31 Homeobox protein Pphb7 short form. 1.91 PP_16923_C1 **: Homolog of OSJNBa0003O19.20|putative MYC transcription factor 1.52 PP_1738_C3 contains: Zn-finger, Dof type(InterPro:IPR003851,GO:0003677,PFAM:PF02701) 2.99 PP_17440_C1 ***: Q8W3M3 AP2 domain containing protein (Putative AP2/EREBP transcription factor). 20.48 PP_17575_C1 ***: Q9SAK5 T8K14.15 protein. 1.34 PP_17900_C1 **: Homolog of (AJ131113) VP1/ABI3-like protein [Chamaecyparis nootkatensis] 1.43 PP_17924_C1 ***: Q7XSB1 OJ991113_30.18 protein. 1.89 PP_1804_C1 ***: Q9SSF9 F25A4.28 protein. 1.76 PP_18357_C1 contains: (COIL:coil) 1.54 PP_18393_C1 not annotated Physcomitrella patens 2.06 PP_18403_C1 **: Homolog of RNA polymerase I specific transcription initiation factor 2.84 PP_1844_C1 ***: O65567 Puative protein. 1.42 PP_18489_C1 "**: Homolog of (68417.m02830 nucleoside phosphatase family protein / GDA1/CD39

family protein 1.67

PP_18663_C1 **: Homolog of Kelch motif, putative|o_sativa|chr_6|OJ1378_E04|3445 1.72 PP_18676_C1 not annotated Physcomitrella patens 2.63 PP_1999_C1 ***: Q8SB10 Putative crp1 protein. 1.70 PP_2158_C1 **: Homolog of EREBP-type transcription factor, putative|o_sativa 2.02

PP_2271_C5 ***: Q9ZVU6 T5A14.12 protein. 1.40 PP_2334_C1 ***: Q8GZ22 Putative ankyrin (At2g03430). 1.52 PP_2362_C1 ***: Q84TU4 Arm repeat-containing protein. 1.50 PP_7120_C2 contains: (SUPERFAMILY:SSF54171) 2.14 PP_2324_C1 ***: Q94ID6 ERF domain protein12 (Ethylene responsive element binding factor, putative). 1.58 PP_2520_C1 ***: Q9M8Z0 T6K12.4 protein. 2.89 PP_2344_C1 ***: Q9FWR5 F14P1.8 protein. 1.35


148

PP_2372_C1 ***: Q9SAI2 F23A5.13 protein (Putative CCR4-associated factor). 1.39

BQ041789 ***: Q9LMP8 F7H2.20 protein (At1g15870/F7H2_19). 1.55 PP_10621_C1 ***: Q9FPV8 Putative methionine aminopeptidase. 1.33 PP_3086_C1 ***: Q949D4 Putative AP2-related transcription factor. 3.09 PP_3132_C1 "**: Homolog of (68415.m02229 expressed protein contains Pfam profiles 1.49 PP_319_C1 ***: Q9LQ28 F14M2.12 protein (Putative AP2/EREBP transcription factor). 1.55 PP_3479_C1 "**: Homolog of 68416.m01095 KOW domain-containing transcription factor family protein 1.51 PP_3728_C1 **: Homolog of 68417.m00223 WRKY family transcription factor 1.79 PP_3738_C1 "**: Homolog of 68418.m07197 protein kinase family protein similar to protein kinase

[Glycine max] 1.67

PP_4015_C1 **: Homolog of DRE-binding protein 1A|o_sativa|chr_8|OJ1323_A06|7300 5.29 PP_4112_C6 "**: Homolog of (AC016529) putative AP2 domain transcription factor 6.24 PP_4166_C1 **: Homolog of OSJNBb0033N16.2|putative RNA 1.31 PP_4300_C1 **: Homolog of (68414.m05749 basic helix-loop-helix bHLH) family protein contains Pfam

profile 1.51

PP_4459_C1 **: Homolog of myb-like DNA-binding domain, SHAQKYF class 1.77 PP_4595_C1 "**: Homolog of (68414.m00292 GCN5-related N-acetyltransferase (GNAT) family protein 1.35 PP_4414_C1 not annotated Physcomitrella patens 2.27 PP_4697_C1 **: Homolog of (AB067689) MADS-box protein PpMADS2 [Physcomitrella patens] 2.22 PP_4719_C1 **: Homolog of 68414.m01735 expressed protein 22.24 PP_4825_C1 **: Homolog of OSJNBa0053C23.4|putative serine/threonine protein kinase 1.53 PP_4986_C1 "**: Homolog of (68415.m03129 transducin family protein / WD-40 repeat family protein 1.45 PP_5002_C1 **: Homolog of (68415.m04914 eukaryotic translation initiation factor 3 subunit 5 / eIF-3

epsilon 1.54

PP_5288_C1 **: Homolog of (AY346455) histone deacetylase [Solanum chacoense] 1.67

PP_5296_C1 **: Homolog of ((AP002092) unnamed protein product [Oryza sativa japonica cultivar group)]

1.46

PP_543_C1 **: Homolog of 68416.m05511 expressed protein 2.02 PP_4563_C1 **: Homolog of (AF184886) LIM domain protein WLIM2 [Nicotiana tabacum] 1.37 PP_5681_C1 **: Homolog of (AB111943) hypothetical protein [Nicotiana benthamiana] 1.51 PP_5719_C1 **: Homolog of expressed protein|o_sativa|chr_5|OSJNBb0015A05|5000 1.70 PP_573_C2 ***: Q852S5 Nucleoside diphosphate kinase. 1.54 PP_5803_C2 **: Homolog of (AB042267) response regulator 5 [Zea mays] 2.33 PP_584_C1 **: Homolog of (68414.m05651 scarecrow-like transcription factor 3 (SCL3) 2.15 PP_560_C1 "**: Homolog of (68418.m07708 no apical meristem (NAM) family protein 1.75 PP_5870_C1 **: Homolog of 68418.m01679 expressed protein 2.76 PP_5912_C2 **: Homolog of (AF506028) CTV.22 [Poncirus trifoliata] 1.37 PP_12254_C1 **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.70 PP_6275_C1 contains: Pathogenesis-related transcriptional factor and ERF 1.43 PP_63_C1 ***: Q7Y1U0 Kinesin-like calmodulin binding protein. 1.81 PP_6340_C1 ***: Q9SR03 Ankyrin-like protein. 1.52 PP_6354_C2 ***: Q9SAU3 CAO. 1.58 PP_6368_C4 ***: Q762A0 BRI1-KD interacting protein 114 (Fragment). 1.33 PP_6455_C1 ***: Q9SZM7 Protein kinase like protein. 2.29 PP_648_C2 **: Homolog of (L76926) putative zinc finger protein [Arabidopsis thaliana] 1.79 PP_18379_C1 not annotated Physcomitrella patens 1.78 PP_6564_C1 "**: Homolog of 68414.m01006 protein kinase 1.64 PP_6651_C1 ***: O22826 Putative splicing factor (At2g43770). 1.64 PP_6682_C1 ***: Q7XMI6 OSJNBb0006N15.13 protein. 1.37 PP_6732_C1 not annotated Physcomitrella patens 1.66 PP_7252_C1 **: Homolog of ((AP005190) putative p53 binding protein [Oryza sativa japonica cultivar-

group)] 1.65

PP_729_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0466H10|5982 1.66 PP_753_C1 ***: Q94D32 P0712E02.24 protein (P0700A11.5 protein). 1.88 PP_7668_C1 ***: PCNA_TOBAC Proliferating cell nuclear antigen (PCNA). 1.40 PP_10278_C1 ***: Q9FP06 P0038C05.18 protein. 2.75


149

PP_793_C4 **: Homolog of (68418.m08455 basic helix-loop-helix bHLH) family protein 1.56 PP_7999_C1 **: Homolog of 68415.m03352 DC1 domain-containing protein contains Pfam profile 1.37 PP_8_C1 contains: Protein synthesis factor, GTP-binding 1.62 PP_8013_C1 ***: RM21_ARATH 50S ribosomal protein L21, mitochondrial precursor. 1.51 PP_14769_C1 **: Homolog of GRAS family transcription factor, putative|o_sativa|chr_1|P0406G08|2927 1.32 PP_8021_C1 **: Homolog of putative AP2 domain transcription

factor|o_sativa|chr_4|OSJNBb0034G17|5454 8.04

PP_8274_C1 not annotated Physcomitrella patens 1.66 PP_8314_C1 **: Homolog of Similar to Lil3 protein|o_sativa|chr_2|P0018H03|4896 1.61 PP_8332_C1 **: Homolog of AP2 domain, putative|o_sativa|chr_6|P0638H11|5506 7.71 PP_8337_C1 ***: Q7XNE0 OSJNBa0088A01.11 protein. 1.79 PP_8348_C1 ***: Q9ZV05 Expressed protein. 1.68 PP_8372_C1 "***: Q9CAN3 Transcription factor SCARECROW, putative; 52594-50618." 2.32 PP_8392_C1 ***: Q8RYF8 P0592G05.19 protein. 2.63 PP_8343_C1 "***: Q9C550 2-isopropylmalate synthase 2.61 PP_8584_C1 ***: Q39216 RNA polymerase subunit (Isoform B). 2.05 PP_8642_C1 **: Homolog of (AY192369) ethylene response factor 3 [Lycopersicon esculentum] 3.41 PP_8479_C1 contains: Basic helix-loop-helix dimerisation region bHLH 1.63 PP_8784_C1 contains: Protein kinase-like(InterPro:IPR011009,SUPERFAMILY:SSF56112) 1.67 PP_8794_C1 contains: Pathogenesis-related transcriptional factor 1.87 PP_8838_C1 ***: Q7XU78 OSJNBa0029H02.4 protein. 2.11 PP_8990_C3 **: Homolog of (GDA1/CD39 nucleoside phosphatase) family, putative 1.59 PP_900_C1 ***: O49591 Putative zinc finger protein. 2.44 PP_8906_C1 ***: Q9M551 Polyubiquitin. 1.42 PP_9142_C1 "**: Homolog of 68418.m03534 bZIP transcription factor family protein 1.55 PP_9252_C1 ***: Q84QD7 Avr9/Cf-9 rapidly elicited protein 276. 1.49 PP_765_C1 ***: O80582 Expressed protein (At2g44130/F6E13.26). 2.39 PP_9420_C1 ***: Q7XU22 OSJNBb0034G17.2 protein (Transcription factor DREB). 3.06 PP_9498_C1 **: Homolog of 68416.m01360 PHD finger family protein contains Pfam domain 1.63 PP_9599_C1 **: Homolog of similar to CH6 and COP9 complex subunit

6|o_sativa|chr_8|OJ1118_A06|3036 1.35

PP_9374_C1 **: Homolog of SelR domain|o_sativa|chr_3|OSJNBa0048D11|3659 1.91 PP_9607_C1 ***: O65639 Glycine-rich protein. 1.33 PP_9750_C1 ***: Q8LBL6 Cell division protein FtsH-like protein. 1.28 PP_976_C1 ***: Q6ZHJ5 Pentatricopeptide (PPR) repeat-containing protein-like. 1.74 PP_17043_C1 **: Homolog of ((AP004068) GCN5-related N-acetyltransferase protein-like (Oryza sativa

japonica ) 1.79

PP_9949_C1 **: Homolog of Myb-like DNA-binding domain, putative|o_sativa|chr_1|P0038F12|2733 1.71 PP_SD_12_C1 ****: Physcomitrella patens mRNA for RNA polymerase alpha subunit, complete cds. 2.09 PP_SD_17_C1 ****: Physcomitrella patens WRKY transcription factor 1 (WRKY1) gene, complete cds. 1.55 PP_SD_252_C1 **: Homolog of 68418.m05899 protein kinase, putative similar to protein kinase G11A

[Oryza sativa] 1.49

PP_SD_46_C1 ****: Physcomitrella patens mRNA for homeobox protein PpHB7, complete cds. 3.51 PP_SD_67_C1 ****: Physcomitrella patens MADS-domain protein PPM1 (ppm1) mRNA, complete cds. 1.37 PP_SD_90_C1 not annotated Physcomitrella patens 2.32 PP_10747_C1 ***: O81763 Protein kinase-like protein. 1.54

PP001063096R **: PP_CL_18202.Singlet Homolog of (AC026238) Hypothetical protein [Arabidopsis thaliana] 1.89

PP001072036R **: PP_CL_15170.Singlet Homolog of 68418.m06538 myb family transcription factor 1.32 PP001077051R ***: O49459 Predicted protein. 1.89 PP001090095R **: PP_CL_15183.Singlet Homolog of Similar to Z97341 apetala2 domain TINY like protein 1.59 PP002015081R **: PP_CL_5597.Singlet Homolog of (AC006072) putative tubby protein [Arabidopsis

thaliana] 1.56 PP002023001R ***: O24460 Calmodulin-like domain protein kinase. 4.72 PP004003286R "**: PP_CL_18294.Singlet Homolog of (68414.m07536 gibberellin regulatory protein

RGL1) 2.15

PP004006023R ****: Physcomitrella patens phytochrome (phy2) gene, complete cds. 1.44


150

PP004007192R ***: Q9FJ91 Dbj|BAA78737.1 (AT5g52010/MSG15_9). 2.04 PP004009367R **: Homolog of (AF004165) 2-isopropylmalate synthase [Lycopersicon pennellii] 1.77 PP004012159R **: Homolog of 68414.m06061 mechanosensitive ion channel domain 1.79 PP004015085R **: Homolog of OSJNBa0010C11.3|putative transcription regulatory protein 1.64 PP004020107R **: PP_CL_11223.Singlet Homolog of 68414.m05053 expressed protein 1.92 PP004024038R ***: Q8VYE7 Putative calcium-dependent protein kinase. 2.59 PP004029122R ***: RPOA_PSINU DNA-directed RNA polymerase alpha chain (EC 2.7.7.6) (PEP) 1.56 PP004030378R **: Homolog of Similar to DRE binding factor 1|o_sativa|chr_6|P0516A04|3745 1.59 PP004032225R **: Homolog of (AB164647) vascular plant one zinc finger protein [Physcomitrella patens] 1.57 PP004040295R **: PP_CL_18294.Singlet Homolog of (AY269087) GAI-like protein [Lycopersicon

esculentum] 2.13

PP004043210R ***: Q9SGT9 T6H22.8.2 protein. 4.14 PP004046136R **: Homolog of VIP2 protein|o_sativa|chr_2|OJ1311_D08|4248 1.43 PP004054012R **: PP_CL_15491.Singlet Homolog of (68418.m00567 zinc finger (C3HC4-type RING

finger) 1.39

PP004054209R PP_SD_13.Singlet not annotated Physcomitrella patens 1.92 PP004057142R ***: Q94C56 Putative FtsH protease (Fragment). 2.33 PP004062128R **: Homolog of 68418.m06038 phototropic-responsive NPH3 family protein 1.34 PP004075103R ***: Q7XPK1 OSJNBa0087O24.9 protein. 1.52 BQ827548 ***: Q8H386 Casein kinase II alpha subunit. 1.47 PP004034373R ****: Physcomitrella patens phytochrome (phy2) gene, complete cds. 1.58 PP004082282R **: Homolog of (AJ579910) NIN-like protein 1 [Lotus corniculatus var. japonicus] 2.23 PP004083344R ***: Q40164 Ubiquitin. 1.67 PP004088245R ***: Q9M1K4 Leucine zipper-containing protein AT103. 1.43 PP004092140R **: PP_CL_18318.Singlet Homolog of (AB028621) unnamed protein product [Arabidopsis

thaliana] 2.09

PP004094219R ***: Q8LST4 Mitochondrial aldehyde dehydrogenase. 1.33 PP004095066R **: Homolog of NF-X1 type zinc finger, putative|o_sativa|chr_1|P0041E11|2738 1.48 PP004095253R **: Homolog of (AF328842) homeodomain protein HB2 [Picea abies] 1.44 PP004096225R ****: PP_SD_29.Singlet Physcomitrella patens mRNA for putative P-type II calcium 2.55

PP004097116R **: Homolog of ((AP005243) VP1/ABI3 family regulatory protein-like [Oryza sativa] 1.60 PP004103024R ***: O99018 Chloroplast protease precursor. 1.72 PP004105269R **: Homolog of 68416.m01747 scarecrow transcription factor family protein 1.60 PP006002067R ***: Q9M378 TATA box binding protein (TBP) associated factor (TAF)-like protein. 2.78 PP010001010R ***: Q8GV68 Phytochrome. 1.60 PP010002038R **: Homolog of (AC068602) F14D16.2 [Arabidopsis thaliana] 1.64 PP010008086R **: Homolog of (AL163912) putative protein [Arabidopsis thaliana] 1.94 PP011003080R "**: PP_CL_17423.Singlet Homolog of (68418.m02981 transducin family protein 1.52 PP011005059R **: Homolog of ATP-dependent metalloprotease FtsH,

putative|o_sativa|chr_5|OJ1362D02|3955 2.28

PP011006015R **: Homolog of (AY514604) gibberelin response modulator dwarf 8 Zea mays 3.63 PP015001075R ****: PP_SD_46.Singlet Physcomitrella patens gene for homeobox protein 3.50 PP015006184R **: PP_CL_2250.Singlet Homolog of Cyclin, N-terminal domain, putative 2.35 PP015015236R ****: PP_SD_46.Singlet Physcomitrella patens mRNA for homeobox protein PpHB7,

complete cds. 3.32

PP015024237R **: PP_CL_3101.Singlet Homolog of (68414.m03658 DNA-directed RNA polymerase family protein

1.58

PP015028003R **: Homolog of (68414.m07827 zinc finger B-box type) family protein 3.56 PP015028194R **: Homolog of 2-isopropylmalate synthase|o_sativa|chr_12|OSJNBb0034E23|7059 2.35 PP015030306R "**: PP_CL_3039.Singlet Homolog of (68417.m04350 translation initiation factor 3 IF-3) 1.66 PP015033189R **: Homolog of (68414.m08377 CCAAT-box-binding transcription factor-related 2.41 PP015040077R **: Homolog of (AF098674) lateral suppressor protein [Lycopersicon esculentum] 1.54 PP015041065R **: Homolog of Similar to RSSG8|o_sativa|chr_12|OJ1396_C02|7247 1.75 PP015041271R **: Homolog of Helix-loop-helix DNA-binding domain,

putative|o_sativa|chr_11|P0410D09|7510 2.00

PP015042038R **: PP_CL_6025.Singlet Homolog of expressed protein|o_sativa|chr_1|B1064G04|2860 1.72 PP015044222R **: PP_CL_18339.Singlet Homolog of (AF328786) EIL3 [Lycopersicon esculentum] 1.32


151

PP015050353R ***: Q9S786 Calcium-dependent protein kinase. 1.50 PP015058275R ***: Q9FGR7 Similarity to salt-inducible protein. 1.66 PP015060167R ***: Q8W3N8 26S proteasome regulatory particle triple-A ATPase subunit4b (Fragment). 1.39 PP020009267R ***: Q9SI82 F23N19.4. 1.42 PP020016269R **: Homolog of OSJNBb0033N16.3|putative protein kinase|o sativa|chr

3|OSJNBb0033N16|761 1.63

PP020019294R **: PP_CL_15.Singlet Homolog of (AF534891) type-B response regulator [Catharanthus roseus]

1.34

PP020024305R ***: O04235 Transcription factor. 1.72 PP020026165R contains: Response regulator receiver 1.90 PP020029315R "**: Homolog of (68418.m00739 transcription factor jumonji (jmjC) domain-containing

protein 1.56

PP020031042R ***: Q9LPC6 F22M8.8 protein. 2.58 PP020031185R **: Homolog of expressed protein|o_sativa|chr_5|P0683B12|6231 1.64

PP020032141R "**: Homolog of (68416.m05316 bacterial transferase hexapeptide repeat-containing protein

1.54

PP020039216R **: Homolog of A67797 unnamed protein product-related|o sativa|chr 8|OSJNBa0054L03|4675 1.41

PP020041086R ***: Q9LTD4 Similarity to unknown protein. 1.96 PP020043294R ***: Q6K7E2 Mitochondrial transcription termination factor-like. 3.27 PP020053142R ***: Q9S729 GlsA. 1.78 PP020054111R "**: PP_CL_10402.Singlet Homolog of (68414.m01147 NF-X1 type zinc finger family

protein 1.85 PP020054231R ***: Q9FP06 P0038C05.18 protein. 1.50 PP020058260R ***: Q9FLM7 Gb|AAC33480.1 (MYB transcription factor). 1.51 PP020060244R **: PP_CL_10622.Singlet Homolog of (AB107691) AG-motif binding protein-3 [Nicotiana

tabacum] 1.77

PP020063226R ***: Q9LV30 Emb|CAB40755.1. 1.48 PP020063256R **: Homolog of Protein kinase domain, putative|o_sativa|chr_1|P0695A04|2742 1.46 PP020070127R PP_SD_242.Singlet not annotated Physcomitrella patens sporophyte 1.81 PP030007003R ***: Q8S9J9 At1g14000/F7A19_9. 1.93 PP030013070R ***: O82527 Polyubiquitin (Fragment). 1.34 PP032009070R ***: Q8GV68 Phytochrome. 2.22

Acknowledgment

152

4.6 Acknowledgments First of all I would like to thank Prof. Dr. Ralf Reski for giving me the opportunity of

doing my PhD in his research group and for his support and encouragement along the way. I

am also indebted to him for his guidance

I would like to thank PD Dr. Wolfgang Frank for his invaluable supervision, great efforts

in guidance, encouragement throughout the research work.

Great appreciation is also due to my family for their encouragement and support.

Special thanks to my wife Enas for valuable help, encouragement and support.

I would like to say thanks to:

Dr. Volker Speth and Dr. Claudia Gack for sample preparation and valuable help at the

scanning electron micrographs

Andras Viczian for providing the pPCV expression vector

Björn Voß for advice on miR319 precursor sequence analysis

Gregor Gierga for assisting in the small RNA blots technique

Richard Haas for practical support in the lab

Anne Katrin Prowse for proofreading my thesis. Special thanks are expressed to my dearest colleagues; Fattash, I., Arif, M. A., Rödel, P.,

Tomek, M.

Finally, I would like to say thanks to all members from the Reski group for the wonderful

working ambience.

Thank you all…

Erklärung

153

4.7 Erklärung

Hiermit erkläre ich, dass ich die vorliegende Arbeit ohne unzulässige Hilfe Dritter und ohne

Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Die aus anderen

Quellen direkt oder indirekt übernommenen Daten und Konzepte sind unter Angabe der

Quellen gekennzeichnet. Insbesondere habe ich hierfür nicht die entgeltliche Hilfe von

Vermittlungs, beziehungsweise Beratungsdiensten (Promotionsberater oder anderer

Personen) in Anspruch genommen. Niemand hat von mir unmittelbar oder mittelbar

geldwerte Leistungen für Arbeiten erhalten, die im Zusammenhang mit dem Inhalt der

vorgelegten Dissertation stehen.

Die Arbeit wurde bisher weder im In noch im Ausland in gleicher oder ähnlicher Form einer

anderen Prüfungsbehörde vorgelegt.

Die Bestimmungen der Promotionsordnung der Fakultät für Biologie der Universität

Freiburg sind mir bekannt; insbesondere weiß ich, dass ich vor Vollzug der Promotion zur

Führung des Doktortitels nicht berechtigt bin.

Basel Khraiwesh

März, 2009

Freiburg,

biological function of rna interference (rnai) pathways in

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