20-mar-2015 accepted date : 22-jun-2015 article...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12922 This article is protected by copyright. All rights reserved.

Received Date : 20-Mar-2015 Accepted Date : 22-Jun-2015 Article type : Original Article The deubiquitinating enzyme AMSH1 is required for

rhizobial infection and nodule organogenesis in

Lotus japonicus

Authors:

Anna Małolepszy1, Dorian Fabian Urbański1, Euan K. James2, Niels Sandal1, Erika Isono3,

Jens Stougaard1, and Stig Uggerhøj Andersen1*

Author affiliations:

1Centre for Carbohydrate Recognition and Signalling (CARB), Department of Molecular

Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, DK-8000 Aarhus C,

Denmark

2EPI Division, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK

3Department of Plant Systems Biology, Technische Universität München, 85354 Freising,

Germany

*Contact:

Stig Uggerhøj Andersen, Department of Molecular Biology and Genetics, Aarhus University,

Gustav Wieds Vej 10, DK-8000 Aarhus C, Denmark. Phone: +45 87154937. Mobile: +45

20747136. E-mail: [email protected]

mailto:[email protected]

This is the pre-peer reviewed version of the following article: Malolepszy, A, Urbański, DF, James, EK, Sandal, N, Isono, E, Stougaard, J & Andersen, SU 2015, 'The deubiquitinating enzyme AMSH1 is required for rhizobial infection and nodule organogenesis in Lotus japonicus' Plant Journal, vol 83, no. 4, pp. 719-731., 10.1111/tpj.12922, which has been published in final form at http://dx.doi.org/10.1111/tpj.12922 . This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

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This article is protected by copyright. All rights reserved.

Key words: Lotus japonicus, AMSH1, deubiquitination, symbiosis, LORE1

Running title

AMSH1 promotes infection and organogenesis

Abstract

The legume-rhizobium symbiosis contributes large quantities of fixed nitrogen to both

agricultural and natural ecosystems. This global impact and the selective interaction between

rhizobia and legumes culminating in development of functional root nodules have motivated

detailed studies of the underlying mechanisms. We conducted a screen for aberrant

nodulation phenotypes using the Lotus japonicus LORE1 insertion mutant collection. Here,

we describe the identification of amsh1 mutants that only develop small nodule primordia

and display stunted shoot growth, and show that the aberrant nodulation phenotype caused by

LORE1 insertions in the Amsh1 gene can be separated from the shoot phenotype. In amsh1

mutants, rhizobia initially became entrapped in infection threads with thickened cells walls.

Much delayed, some rhizobia were released into plant cells, however no typical symbiosome

structures were formed. Furthermore, cytokinin treatment only very weakly induced nodule

organogenesis in amsh1 mutants, suggesting that AMSH1 function is required downstream of

cytokinin signaling. Biochemical analysis showed that AMSH1 is an active deubiquitinating

enzyme and that AMSH1 specifically cleaves K63-linked ubiquitin chains. Post-translational

ubiquitination and deubiquitination processes comprising the AMSH1 deubiquitinating

enzyme are thus involved in both infection and organogenesis in Lotus japonicus.

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Introduction

Many legumes are rich sources of protein due to their symbiosis with nitrogen fixing rhizobia

accommodated in root nodules. The symbiotic signaling process is initiated when rhizobia

secrete nodulation (Nod) factors upon sensing (iso)flavonoids produced by compatible

legumes. The Nod factors consist of N-acetyl-D-glucosamine oligomers linked by β-1, 4

bonds, where the N-acetyl group on the nonreducing end is replaced by a fatty acid (Lerouge

et al. 1990; Spaink et al. 1991). Genetic studies of loss-of-function and gain-of-function

mutants in Lotus japonicus (Lotus) and Medicago truncatula (Medicago) show that the Lotus

Nod factor receptors NFR1 and NFR5 and the corresponding proteins LYK3 and NFP in

Medicago (Limpens et al. 2003; Madsen et al. 2003; Radutoiu et al. 2003; Arrighi et al. 2006;

Mulder et al. 2006; Smit et al. 2007) are pivotal for perception of rhizobial Nod factors, and

in vitro NFR1 and NFR5 bind Nod factors from compatible bacteria with high affinity

(Broghammer et al. 2012). In the susceptible root zone, Nod factor perception then leads to

initiation of a nodulation signaling cascade, which bifurcates into branches promoting

epidermal root hair infection and cortical nodule organogenesis, respectively (Kouchi et al.

2010; Madsen et al. 2010). Promotion of epidermal infection relies on the activities of the

pectate lyase NPL, the ubiquitin ligase CERBERUS, the NAP1, PIR1 and ARPC1 proteins

required for actin rearrangement, and the transcription factors NIN, NSP1 and NSP2

(Schauser et al. 1999; Kalo et al. 2005; Smit et al. 2005; Heckmann et al. 2006; Murakami et

al. 2006; Marsh et al. 2007; Yano et al. 2008; Yano et al. 2009; Yokota et al. 2009; Hossain

et al. 2012; Xie et al. 2012).

An overlapping set of proteins act downstream of NFR1 and NFR5 to trigger the

organogenesis process in the root cortex. The LRR receptor kinase SymRK, three

nucleoporins, NUP133, NUP85 and NENA, together with CASTOR and POLLUX cation

channel proteins (Schauser et al. 1998; Szczyglowski et al. 1998; Endre et al. 2002;

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Kawaguchi et al. 2002; Stracke et al. 2002; Imaizumi-Anraku et al. 2005; Kanamori et al.

2006; Saito et al. 2007) are essential for the nuclear calcium spiking signal that is in turn

interpreted by CCaMK (Levy et al. 2004; Mitra et al. 2004; Tirichine et al. 2006a).

Subsequently, CCaMK activates organogenesis via cytokinin signaling through the LHK1

receptor (Tirichine et al. 2006b; Murray et al. 2007; Tirichine et al. 2007), but also cross-

signals to the infection pathway by phosphorylating and activating the transcription factor

CYCLOPS (Yano et al. 2008; Liao et al. 2012; Singh et al. 2014). Further downstream, the

transcription factors NIN, NSP1 and NSP2 (Schauser et al. 1999; Kalo et al. 2005; Smit et al.

2005; Heckmann et al. 2006; Murakami et al. 2006; Marsh et al. 2007) relay the

organogenesis signal (Madsen et al. 2010).

Several of the key nodulation genes mentioned above are associated with ubiquitination,

either because they are putative targets for ubiquitination, or because they share sequence

similarity with ubiquitin ligases. CERBERUS belongs to the latter category, whereas the

Medicago Nod factor receptor LYK3 phosphorylates the E3 ubiquitin ligase Plant U-box

Protein 1 (PUB1) (Mbengue et al. 2010), and SymRK was suggested to interact with the E3

ubiquitin ligases SymRK INTERACTING E3 UBIQUITIN LIGASE (SIE3) in Lotus, and

SEVEN IN ABSENTIA (SINA) in Medicago (Den Herder et al. 2008; Hervé et al. 2011; Den

Herder et al. 2012; Yuan et al. 2012).

Ubiquitination is a reversible process, which regulates protein degradation, trafficking, DNA

repair, apoptosis and signal transduction, making it an important post-translational regulatory

mechanism. It involves linking the carboxy-terminal glycine residue of ubiquitin to lysine

residues of specific protein targets, in many cases followed by ubiquitin polymerization

(Kimura and Tanaka 2010). The fate of target protein can be determined by the identity of the

lysine residue involved in polyubiquitin chain formation. If the poly-Ubiquitin chain is

generated by K48 linkage, the substrate is targeted to the proteasome for degradation. On the

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other hand, formation of K63-linked chains promotes DNA repair, endocytosis and vesicular

trafficking or ribosomal protein synthesis (Mukhopadhyay and Riezman 2007; Sato et al.

2008; Ye and Rape 2009).

Accurate control of protein ubiquitination status is crucial for the cell, which is why the

deubiquitinating enzymes (DUBs) play an important role in cellular homeostasis (Kimura and

Tanaka 2010). DUBs are isopeptidases, which detach ubiquitin from target proteins. They are

divided into five groups: ubiquitin-specific proteases (USP), ubiquitin carboxy-terminal

hydrolases (UCH), Otubain proteases (OTU), Machado–Joseph disease proteases and

JAB1/MPN/Mov34 metalloenzymes (JAMM) (Isono and Nagel 2014). All DUBs belong to

the cysteine protease group, except for the JAMMs, which are zinc metalloproteases (Amerik

and Hochstrasser 2004; Nijman et al. 2005; Sato et al. 2008; Isono and Nagel 2014). The

metalloproteases contain an MPN+ domain with a conserved JAMM motif (EXnHXH10D),

which is believed to be the active site involved in metal binding (Maytal-Kivity et al. 2002;

Tran et al. 2003). In Arabidopsis three ASSOCIATED MOLECULE WITH THE SH3

DOMAIN OF STAM (AMSH) homologs, which belong to the JAMM zinc metalloproteases

class were identified: AMSH1, AMSH2 and AMSH3. Their catalytic MPN+ domain is

homologous to MPN+ in human AMSH. AMSH1 and AMSH3 interact with Endosomal

Sorting Complex Required for Transport III (ESCRT III), which is involved in sorting of

ubiquitinated membrane proteins to multivesicular bodies (MVB) (Isono et al. 2010;

Katsiarimpa et al. 2011; Katsiarimpa et al. 2013). Recently it was shown that AMSH3 is

involved in the degradation of AvrPtoB-activated CHITIN ELICITOR RECEPTOR KINASE

1 (CERK1) (Katsiarimpa et al. 2014).

Despite the aforementioned associations between proteins from the nodulation pathways and

the ubiquitination machinery, the role of ubiquitination in nodulation signaling is not well

understood. Using an unbiased forward genetics approach, we screened the LORE1 collection

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(Fukai et al. 2012; Urbanski et al. 2012) for mutants with aberrant nodulation phenotypes,

and found that loss of a gene encoding the deubiquitinating enzyme AMSH1 prevented

nodulation. Our analysis showed that Lotus AMSH1 is an active DUB with ubiquitin K63-

link specificity and highlights the importance of post-translational protein modification and

ubiquitin signaling in the developmental events leading to root infection and nodule

organogenesis.

Results

Identification of two independent LORE1 mutants with similar aberrant

nodulation phenotypes

Following the successful establishment and annotation of a small-scale LORE1 insertion

mutant population (Fukai et al. 2012; Urbanski et al. 2012), we initiated a forward genetic

screen for nodulation deficient mutants. We identified a LORE1 family segregating plants

without mature nodules, where no insertions in known nodulation genes were registered in

the LORE1 database. In addition to displaying arrest of nodule development at the

primordium stage, the mutant plants showed severely stunted shoot growth, while root

growth was less affected (Figure 1a-e). Additionally, the mutants failed to survive till seed

set.

Initially, we were unable to identify the causative insertion based on our LORE1 sequencing

data, since none of the annotated insertions co-segregated with the phenotype. After

performing Sequence Specific Amplification Polymorphism (SSAP) analysis optimized for

LORE1 insertion detection (Urbanski et al. 2013) followed by Sanger sequencing of

candidate PCR products, we detected a co-segregating insertion in exon 8 (amsh1-2) of the

Amsh1 gene, which was not part of the Lotus v. 2.5 reference genome sequence. We then

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cloned and sequenced the full length Amsh1 cDNA sequence and identified the Amsh1

genomic DNA sequence by aligning the cDNA sequence to a set of Lotus contigs assembled

from short Illumina reads (not shown). After re-running the analysis of the LORE1

sequencing data including the Amsh1 genomic sequence, we discovered an additional

insertion in the Amsh1 gene in exon 1 in an independent LORE1 mutant line (amsh1-1)

(Figure 1f). In addition to Amsh1, the Lotus v. 2.5 genome comprised two additional Amsh

homologs, which appeared to be putative orthologs of the Arabidopsis AMSH2 and AMSH3

genes (Figure S1).

LORE1 insertions in the Amsh1 gene cause the aberrant nodulation

phenotype

The phenotypes of the two independent amsh1 alleles were compared. Since the homozygous

amsh1 mutants did not produce seeds, offspring from heterozygous plants were used in all

analyses. We found that all plants with stunted shoots and small nodule primordia carried

homozygous LORE1 insertions in the Amsh1 gene. To confirm that the two LORE1 insertions

in the Amsh1 gene were the causal mutations, heterozygous amsh1-1 and amsh1-2 plants

were crossed to test for non-complementation in the F1 generation. amsh1-1/amsh1-2

heteroallelic individuals displayed phenotypes indistinguishable from the homozygous

amsh1-1 and amsh1-2 mutants (Figure S2). amsh1-1 and amsh1-2 are therefore allelic and we

conclude that the LORE1 insertions in the Amsh1 gene are the causal mutations.

To further validate correct identification of the causal mutations, we identified six

independent LORE1 lines following the expansion of the LORE1 population. These

additional amsh1-3 to amsh1-8 alleles (Fig. 1f) all displayed phenotypes very similar to the

amsh1-1 and amsh1-2 alleles, re-confirming correct identification of the causal mutations

(Figure S3).

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amsh1 accumulates ubiquitin conjugates in vivo

Alignment of the conceptual Lotus AMSH1 amino acid sequence with AMSH1 protein

sequences from human and Arabidopsis AMSH metalloproteases showed that they share 42%

and 64% overall amino acid sequence identity, respectively (McCullough et al. 2004; Isono et

al. 2010), which suggested that the Lotus AMSH1 protein might be a metalloprotease enzyme

with DUB activity (Figure S4). In contrast to ubiquitin ligases, DUBs cleave ubiquitin chains

off target proteins, and loss of DUB function can lead to accumulation of ubiquitin

conjugates (Wilkinson et al. 1995; Amerik et al. 1997). To test if amsh1 mutants accumulated

ubiquitinated proteins, we used an anti-ubiquitin antibody to detect ubiquitin conjugates in

total extracts from wild type, amsh1-2, heterozygous Amsh1/amsh1, nfr1-1, and nfr5-2

seedlings. Cyclin-dependent kinase (CDC2 kinase) (Riabowol et al. 1989; Madrid et al. 2007)

was used as loading control. amsh1 clearly stood out as the genotype with the highest level of

ubiquitin conjugates (Figure 2a), while there were no apparent differences between the

protein samples from the remaining genotypes.

Lotus AMSH1 specifically cleaves K63-linked ubiquitin chains

Ubiquitin molecules can be linked through different lysine (K) residues to form poly-

ubiquitin chains, and signaling responses can be modulated by variations in the patterns of

lysine links (Komander and Rape 2012). DUBs act by cleaving the amide bond between the

lysine and carboxy-terminal glycine of a linked ubiquitin (Amerik and Hochstrasser 2004).

To examine if AMSH1 is an active DUB, the fusion protein GST-LjAMSH1 was expressed

in the E. coli Rosetta strain, purified and tested for DUB activity. K48-linked and linear

ubiquitin chains remained intact upon LjAMSH1 treatment, whereas K63-linked chains were

cleaved to ubiquitin monomers (Figure 2b). We conclude that Lotus AMSH1 is an active

DUB, which specifically cleaves K63-linked polyubiquitin chains in vitro. These results are

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consistent with the previously reported K63 chain specificities of human AMSH and

Arabidopsis AMSH1 (McCullough et al. 2004; Katsiarimpa et al. 2013).

AMSH1 acts in Lotus roots to promote nodulation

Since the amsh1 mutants displayed a severely stunted shoot phenotype, and since Amsh1 was

expressed strongly across all Lotus tissues (Figure S5), the amsh1 mutation could indirectly

affect root growth and progression of nodulation through its effect on shoot function and/or

delivery of photosynthates to the root. To determine whether this was the case, we first

examined root and shoot growth rates in the wild type and in the amsh1-1 and amsh1-2

mutants. The growth rate was measured at 1, 2 and 3 weeks for plate grown plants with or

without rhizobial inoculation (Figure 3, Figure S6). Although nitrogen starvation inhibited

shoot growth in uninoculated wild type plants, amsh1 shoot growth rate was significantly

lower than that of the wild type both with and without inoculation (Figure 3a, c). In contrast,

root growth rates were similar across all genotypes independent of inoculation, despite the

short initial lengths of the amsh1 roots (Figure 3b, d). With respect to lateral root formation,

there were no significant differences between amsh1 and the wild type in the number of

lateral roots produced per cm of main root, whereas the total number of lateral roots per plant

was reduced (Figure 3e-f). Despite the relatively severe amsh1 shoot phenotype, root growth

and development was thus not strongly affected, suggesting that the indirect shoot effect on

the root phenotype is minimal in plate-grown amsh1 mutants. As further testimony to the

relatively normal general functionality of amsh1 roots, they supported mycorrhizal

colonization (Figure S7), which distinguishes amsh1 from the Medicago vapyrin mutant

(Murray et al. 2011).

Although root growth was not severely affected, Nod factor signaling and/or nodule

development could potentially still be affected by limited shoot growth and functionality. To

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examine this possibility, we used Agrobacterium rhizogenes-mediated transformation to

generate transgenic roots expressing the wild type Amsh1 cDNA sequence. Using this

system, the transgene is exclusively expressed in the transformed roots, allowing us to

separate shoot and root effects. Expressing Amsh1 from the CaMV 35S promoter restored

development of large pink nodules in 17 out of 26 amsh1 mutants, while their shoots

remained stunted compared to the wild type controls (Figure 4, Table S1, Figure S8).

Vibratome sections (Figure 4) showed that these large pink nodules were fully infected and

appeared indistinguishable from wild type nodules at the same developmental stage.

However, closer inspection of the complemented nodules using electron microscopy showed

that, although symbiosomes were detected in the complemented amsh1 nodules, they were

less frequent than in wild type nodules and appeared to be partly degraded (Figure S8).

To determine if the degraded symbiosomes observed at 6 wpi in the complemented plants

had been functional at earlier stages, we quantified the shoot lengths of amsh1 plants in the

complementation experiment. Complemented amsh1 plants with pink nodules had

significantly longer shoots than the amsh1 mutants without pink nodules (p=0.0048,

Student’s t-test) (Figure 4e), indicating that the pink nodules on the complemented plants had

contributed fixed nitrogen to the amsh1 seedling shoots. Despite the premature symbiosome

senescence observed, transformation of roots with the 35S:Amsh1 construct thus restored

both infection and, at least partly, nitrogen fixation in the amsh1 mutants, and we conclude

that AMSH1 acts in the root to promote nodulation.

Infection thread progression is delayed in amsh1 mutants

To determine the exact nature of the amsh1 nodulation defect, we characterized the formation

of infection threads (ITs) in root hairs. These ITs represent the early stages of rhizobial

infection that allow the bacteria to traverse plant epidermal cells. The earliest morphological

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response to rhizobia is root hair curling, which we observed both in the wild type and amsh1

mutants (Figure S9a-c). We then used DsRed-labelled M. loti to track IT progression, and

categorized ITs into three groups: 1) Incipient (microcolonies/short ITs not progressing), 2)

Elongating (incomplete traversal of root hair cells) and 3) Long (completely traversing the

root hair cell) (Figure 5a-c).

At 8 dpi, the wild type had approximately 30 long ITs per cm (Figure 5d) and also showed a

number of incipient and elongating ITs. In contrast, the number of long ITs was significantly

reduced to ~one IT/cm in amsh1-1 and amsh1-2 (Figure 5d). Instead, we found mainly

incipient or elongating ITs in amsh1-1 and amsh1-2, some with aberrant morphology (Figure

S9d-g).

Subsequently, at 21 dpi, ~30 and ~10 long ITs per cm were observed in the wild type and in

amsh1-1 and amsh1-2, respectively. The number of incipient and elongating ITs was similar

in amsh1-1 and amsh1-2 and wild type plants, suggesting that the infection process was

progressing at a reduced speed in the amsh1 mutants (Figure 5d). It thus appears that amsh1-

1 and amsh1-2 are able to perceive rhizobial signals and initiate the infection process, but that

IT progression is aberrant and delayed, resulting in decreased infection.

amsh1 infection threads display aberrant morphology and bacterial release

The slower infection process in amsh1 mutants was also reflected at the level of nodule

organogenesis (Figure 5e). Wild type plants produced nodule primordia within days of

infection and functional pink nitrogen fixing nodules were present at 11 dpi. In contrast, none

of the amsh1 mutants had fully developed mature nodules at 6 wpi (Figure 5e and Figure 6).

To investigate if the infection status of the amsh1 primordia could explain the lack of mature

nodules, we visualized the infection of wild type nodules and amsh1 nodule primordia 6

weeks after inoculation with M. loti expressing a hemA::lacZ reporter gene. Dark blue

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staining was observed at the center of the wild type nodules, indicating full colonization. In

contrast, only weak staining near primordia surfaces was detected in amsh1-1 and amsh1-2

primordia, suggesting limited colonization (Figure 6a).

Next, we examined the amsh1 primordia and wild type nodules at 3 and 6 wpi using light and

transmission electron microscopy. At 3 wpi, wild type nodules were fully colonized with ITs

present within the nodules (Figure 6b, c). In some amsh1-1 and amsh1-2 primordia, ITs had

entered cortical cells and these ITs were irregular, bulbous, and enclosed in unusually thick

cell wall-like structures (Figure 6b-c). Release of bacteria into the plant cells was not

observed in the amsh1 mutants.

At 6 wpi, the infected cells of wild type nodules showed symbiosomes containing up to 2-3

bacteroids, while in the occasional larger amsh1-1 and amsh1-2 nodule primordia a higher

number of branching ITs with aberrant morphology were present (Figure 4c and Figure 6d-e).

For a few of the older amsh1 mutant primordia, release of bacteria could be observed (Figure

6d-e). These released bacteria did not differentiate and symbiosomes were not observed

(Figure 6d-e).

AMSH1 is required downstream of cytokinin signaling

The severe infection defects in the amsh1 mutants could explain the arrested progression of

organogenesis observed in response to rhizobia. However, amsh1 mutants could suffer

defects in nodule organogenesis independent of their infection deficiencies. To investigate

this possibility, we determined the extent of organogenesis induced by exogenous application

of cytokinin in the absence of rhizobia and infection (Heckmann et al. 2011). Following six

weeks of treatment with 10-8 M of the cytokinin analog 6-benzylaminopurine (BAP), no

apparent growth rate differences were observed between plants growing on medium with and

without BAP (Figure S10). BAP treatment induced formation of more than 5 white nodules

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per wild type plant, while only ~2 small primordia per plant were seen in amsh1-1 and very

few primordia were induced in amsh1-2 (Figure 7a). The amsh1 BAP-induced primordia

were much smaller than the white nodule-like structures observed on the wild type roots

(Figure 7b-c).

Additionally, we tested if application of BAP could rescue the amsh1-1 and amsh1-2

nodulation phenotype. We did not observe a difference in primordium number compared to

the plants grown on medium without BAP (Figure S10). The wild type plants formed

approximately three pink nodules while amsh1 mutants produced only two nodule primordia.

Likewise, we found no effect of BAP treatment on the amsh1 infection phenotype, and the

cytokinin-treated amsh1-1 and amsh1-2 primordia were not properly colonized, but had a

high abundance of branching and bulbous infection threads enclosed in thick cell wall-like

structures as previously observed without BAP treatment (Figure 7d-g). In conclusion, amsh1

mutants appear to have organogenesis defects that are independent of infection, suggesting a

requirement of AMSH1 downstream of cytokinin signaling.

Discussion

The LORE1 resource facilitated efficient identification and validation of the

amsh1 mutants

The LORE1 mutant collection represents a substantial reverse genetics resource, providing

access to loss-of-function alleles of more than 20,000 unique Lotus genes (Fukai et al. 2012;

Urbanski et al. 2012; Urbanski et al. 2013). Here, we exemplify the efficient use of the

LORE1 resource in a forward genetics approach aimed at identification of novel genetic

regulators of nodulation. We carried out a family-wise screen, where multiple mutant

individuals displaying the amsh1 phenotype were detected within distinct segregating

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families, facilitating reliable detection of the aberrant phenotype. In the case of amsh1, the

family-wise screen was also critical in allowing ready access to heterozygous amsh1

individuals, since the homozygous amsh1 mutants did not produce seeds. Following

identification of LORE1 insertions that co-segregated with the aberrant phenotype, the

LORE1 collection provided a large number of additional amsh1 alleles, which ensured rapid

validation of the causal mutation and the corresponding gene, allowing us to proceed quickly

with the biochemical and phenotypic characterization. In general, the two amsh1 alleles,

amsh1-1 and amsh1-2 characterized in detail here, showed very similar phenotypes, although

amsh1-2 had a tendency to show slightly more severe defects. The large number of amsh1

LORE1 lines with consistent phenotypes suggests that, at least in this case, background

mutations and/or variation in LORE1 insertion site position within the target gene did not

strongly influence the observed phenotypes.

AMSH1 acts as a K63-specific deubiquitinating enzyme

In Arabidopsis three AMSH1 homologues were identified, among which AtAMSH3 appears

to be the major DUB that cleaves both K48- and K63-linked ubiquitin chains (Isono et al.

2010). Whereas the homozygous Arabidopsis amsh3 mutant is seedling lethal (Isono et al.

2010), amsh1 and amsh3 mutants show synergistic interaction, suggesting partially redundant

functions (Katsiarimpa et al. 2013).

Our biochemical analysis revealed that Lotus AMSH1 is an active DUB that specifically

cleaves K63- but not K48-linked ubiquitin chains. A specificity it shares with human AMSH

and Arabidopsis AMSH1 (McCullough et al. 2004; Katsiarimpa et al. 2013). Previously, it

was suggested that K63 ubiquitin chains could act as a signals for endocytosis

(Mukhopadhyay and Riezman 2007; Woelk et al. 2007). Supporting this suggestion,

Arabidopsis AMSH1 and AMSH3 interact with ESCRT-III components involved in

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endocytosis (Katsiarimpa et al. 2013; Katsiarimpa et al. 2014). It is thus possible that Lotus

AMSH1 function could also be related to endocytosis.

amsh1 mutants display a pleiotropic phenotype including a severe

nodulation defect

At first glance, the amsh1 mutant phenotype seems pleiotropic because of the strong effects

on shoot growth, plant viability, and nodulation. Such global phenotypic deviations are

consistent with the ubiquitous expression of Amsh1 and with the biochemical function of

AMSH1 as a DUB, which presumably acts on a number of target proteins. AMSH1 therefore

clearly does not act exclusively in the nodulation pathways. Nevertheless, it still has a strong

nodulation phenotype, which can be decoupled from the shoot phenotype, as we have

demonstrated here using A. rhizogenes-mediated transgenic root complementation (Figure 4).

Within the root, the nodulation defect appears to be the most prominent phenotypic

aberration, since amsh1 roots are able to support mycorrhization (Figure S7), grow at rates

comparable to the wild type, and produce lateral roots (Figure 3). Despite this overall normal

appearance of amsh1 roots, they fail to produce functional nodules. Instead, amsh1 displays

the impaired IT progression as well as bacterial release from the ITs and amsh1 nodule

development is arrested at the primordium stage.

AMSH1 could target nodulation proteins

The most likely explanation for these observations is that one or more critical components of

the nodulation signaling machinery are highly sensitive to K63-linked ubiquitination, and

require AMSH1 DUB activity to retain functionality.

The simplest hypothesis is that AMSH1 promotes infection and organogenesis by acting on a

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single nodulation component, leaving the transcriptional regulators NIN, NSP1, and NSP2 as

candidates, because they are required for both processes (Madsen et al. 2010; Heckmann et

al. 2011). The remaining nodulation proteins are less likely candidates under this hypothesis,

because they either have more specific infection- or organogenesis-related functions, or

because their corresponding loss-of-function mutants, unlike amsh1, respond to cytokinin

treatment with formation of larger nodule-like structures. The infection mutants nap, pir,

arpC1, cyclops, and cerberus thus display abnormal IT development, but nodule

organogenesis that proceeds further than in amsh1. Conversely, loss of organogenesis-

specific proteins, such as the cytokinin receptor LHK1 (Tirichine et al. 2006b; Tirichine et al.

2007) and the DNA topoisomerase VI components VAG1 and SUNER1 (Suzaki et al. 2014;

Yoon et al. 2014), does not hinder epidermal infection, but result in absence of, or reduced,

organogenesis. Finally, the nfr1, nfr5, symrk, nup133, nup85, castor, pollux, ccamk, pir and

cyclops mutants all respond to cytokinin treatment with formation of nodule-like structures

(Heckmann et al. 2011), making it unlikely that AMSH1 targeting one of the corresponding

proteins could explain the requirement of AMSH1 downstream of cytokinin signaling.

The alternative hypothesis to AMSH1 targeting a single nodulation component required for

both infection and organogenesis is that AMSH1 targets multiple components distributed on

both signaling pathways, for instance LHK1 and one or more of the proteins required for

infection.

With the availability of multiple amsh1 mutant alleles, these hypotheses can now be tested

using biochemical approaches to compare the ubiquitination status and protein abundance of

known nodulation signaling components in amsh1 and wild type backgrounds. The AMSH1

characterization presented here thus opens new avenues of investigation with the potential for

greatly advancing the understanding of ubiquitin impact on nodulation signaling in legumes.

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Experimental procedures

Bacterial strains

Mesorhizobium loti strain NZP2235 was used for forward screening under greenhouse

conditions, while for most of the studies on plates and in Magenta containers, M. loti strain

MAFF303099 expressing DsRed was used (Maekawa et al. 2009). The M. loti strain

NZP2235 expressing hemA::lacZ reporter gene was used for lacZ staining (Schauser et al.

1998; Wopereis et al. 2000). An OD600 = 0.06 was used for all the studies involving rhizobia.

Agrobacterium rhizogenes strain AR1193 was used for hairy root transformation (Stougaard

et al. 1987).

Plant materials

amsh1 mutants were obtained from the LORE1 collection (Fukai et al. 2012; Urbanski et al.

2012). The isolation of nfr1-1 and nfr5-2 was described by Schauser et al. (Schauser et al.

1998). Lotus japonicus B-129 Gifu is the wild type for all these mutants (Handberg and

Stougaard 1992).

Seeds were scarified by treatment with 98% sulfuric acid for 15 min, or using sand paper, and

then sterilized with 0.5% sodium hypochlorite for 20 min. They were rinsed 5 times with

sterile water and incubated on a shaker for 3 h at room temperature. Seeds were germinated

on moist, sterile paper on vertical plates in growth chambers under the following conditions:

16/8h light/darkness and 21°C/18°C temperature for 3 days. Subsequently, for nodulation

tests they were moved to plates containing ¼-strength B&D medium (Broughton and

Dilworth 1971) and 1.4% agar noble slants without nitrate, or to Magenta containers (Sigma)

filled with Leca (Optiroc) and Vermiculite (3:1) and 80 ml of ¼-strength B&D medium

without nitrate. On plates, roots were shielded from light using a metal spacer. For cytokinin

experiments, ¼-strength B&D medium was supplemented with BAP to a final concentration

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of 10-8 M. For complementation, 3 days old seedlings were moved to ½-strength Gamborg’s

B5 basal salt mixture (Duchefa Biochemie) supplemented with Gamborg’s vitamin solution

(Sigma).

For each experiment, a segregating population of amsh1 plants was used. Preparation of 200

seeds usually yielded approximately 20 amsh1 plants. For root growth assays, 20 Gifu plants,

17 amsh1-1 and 15 amsh1-2 were counted. For nodule counts, 20 plants from each genotype

were used. For infection thread counts 10 plants from each genotype was used at 8 and 21

dpi. For western blot the protein extract from one seedling was used and it was repeated three

times. For hairy root complementation 1000 seeds from segregating population were used.

500 seeds were transformed with empty vector control and 500 with 35S::Amsh1 cDNA.

After 4 weeks the small plants were moved to Magenta containers and inoculated. 6 weeks

later plants were scored, pictures were taken and plants were genotyped.

Arbuscular Mycorrhiza

One week old plants from segregating population of amsh1 and wild type Gifu seedlings

were transferred to Rhizophagus irregularis-colonized chive nurse pots as previously

described (Chabot et al. 1992; Kosuta et al. 2005; Kruger et al. 2012). Plants were co-

cultivated for 5-6 weeks at 25°C. Each week they were fertilized with chive nutrient solution

supplemented with potassium nitrate to a final concentration of 5.5 mM nitrate. Plants roots

were scored for AM colonization by ink staining as previously described (Vierheilig et al.

1998).

Genotyping

DNA was extracted according to the standard CTAB method (Rogers and Bendich 1985).

The genotyping PCR reactions were performed using the primers designed by the FSTpoolit

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software as previously described (Urbanski et al. 2012), except that the touch-down step was

skipped and a three-step, 30 cycle reaction was performed with a 62°C annealing

temperature.

Cloning procedure

The Lotus Amsh1 coding sequence was cloned from a Lotus cDNA library obtained from

roots using the F1 and R1 primers (Table S2). The promoter region was amplified together

with the 5’UTR from genomic DNA of Gifu using the F2 and R2 primers. The promoter

region and cDNA were combined by using the F3 and R3 primers. The Phusion polymerase

(Thermo Fisher Scientific) was used to amplify all of the fragments. They were cloned into

pENTR/d-TOPO vector between attL1 and attL2 sites (Invitrogen). An LR reaction was

performed according to the manufacturer’s instructions (Invitrogen) in order to transfer the

constructs from pENTR/d-TOPO to a pIV10 expression vector containing a Gateway cassette

(Stougaard et al. 1987).

Ligation Independent Cloning (LIC cloning) was performed according to the Novagen

protocol to clone the Amsh1 cDNA into pET41 for recombinant expression in E. coli. The F4

and R4 primers were used to amplify the Amsh1 cDNA (Table S2).

Quantitiave RT-PCR analysis

Total RNA was isolated from different tissues at 3 wpi inoculated with M. loti MAFF303099

or mock using modified Lithium Chloride - TRIzol LS method as described by Holt and

associates (Holt et al. 2015). cDNA was synthesized using oligodT primer and MuLV RT

from Thermo Fisher Scientific as described in Holt et al. (Holt et al. 2015). The F5 and R5

primers were used to amplify Amsh1 transcripts. Ubiquitin-conjugating enzyme (LjUBC) was

used as a reference gene. Primers for LjUBC were F6 and R6 (Table S2). Real-Time PCR

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was performed using the LightCycler 480 II (Roche Molecular Biochemicals) using

LightCycler 480 SYBR Green I Master (Roche Molecular Biochemicals).

Protein extraction, DUB assays, and Western blots

The GST-AMSH1 fusion protein was expressed from a pET41 vector in the E. coli Rosetta

strains (DE3) (Merck Chemicals) at 18°C and purified using Glutathione Sepharose 4B (GE

Healthcare) beads. Protein was eluted from the beads by incubation for 30 min at 4°C with

100 mM Glutathione. DUB assays were performed according to Isono (2010). 8 pmol of

LjAMSH1 enzyme in DUB buffer were incubated with 125 ng µl-1 of K48-, K63-linked or

linear ubiquitin chains (2-7 ubiquitins, Enzo Life Sciences) for 120 min at 30°C. The reaction

was then stopped by adding 2.5 µl SDS of sample buffer. The samples were run on the

NuPAGE Novex Bis-Tris 4-12% gel (Invitrogen) in MES buffer according to the instructions

provided by Invitrogen. The antibodies used were: anti-GST (1:2000, raised in goat, GE

Healthcare), anti-CDC2 kinase (1:5000, raised in rabbit, Santa Cruz Technology), anti-Ub

P4D1 (1:2500, raised in mouse, Cell Signaling Technology), anti-mouse HRP conjugated

antibody (1:2000, Pierce), anti-goat AP conjugated antibody (1:1000, Sigma), anti-rabbit AP

(1:4000, Sigma). SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher

Scientific) was used for HPR reaction. The BCIP and NBT for Alkaline Phosphatase (AP)

detection were purchased from AppliChem.

Microscopic observation, staining and image processing

Characterization of nodulation phenotypes was carried out using M. loti MAFF303099

expressing DsRed. The confocal microscope Zeiss 780LSM was used for counting of ITs. ITs

were counted at 8 and 21 dpi. To visualize nodule colonization, plants were inoculated with

M. loti NZP2235 expressing hemA::lacZ, and lacZ staining was performed as previously

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described (Wopereis et al. 2000). The root lengths were measured using ImageJ. 6 wpi

nodules/primordia were sectioned using a Leica vibratome. Transmission electron

microscopy was performed on ultrathin sections (80 nm thick) of fixed and resin-embedded

nodules and nodule primordia according to Madsen et al. (2010), and were viewed and

photographed using a JEOL JEM1400 TEM.

Acknowledgements

This work was funded by DFG grant no. IS 221/2-2 [SPP1365] (EI), Danish National

Research Foundation grant no. DNRF79 (JS), the ERC Advanced Grant 268523 (JS), and

EMBO Short-Term Fellowship no. ASTF-408-2012 (AM). The authors would like to thank

Dennis Berg Holt and Katharina Markmann for supplying the cDNA used for Amsh1

transcription analysis.

Accession numbers The Amsh1 sequences have Genbank accession numbers KR270441.

Author contributions:

A.M., D.F.U., N.S. performed the forward screening. D.F.U. and A.M. carried out the

cloning and the SSAP analysis. A.M. characterized the mutant. E.K.J. carried out the light

and transmission electron microscopy. E.I. supervised the biochemical assays. S.U.A and J.S.

supervised the work. A.M. and S.U.A wrote the manuscript with input from JS.

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Supplemental Tables and Figures

Supplemental Figure 1. Alignment of Lotus and Arabidopsis AMSH homologs

Supplemental Figure 2. amsh1-1 and amsh1-2 are allelic

Supplemental Figure 3. Phenotypes of the amsh1-3 to amsh1-8 alleles

Supplemental Figure 4. Alignment of human AMSH (HsAMSH), Arabidopsis AMSH1

(AtAMSH1) and Lotus AMSH1 (LjAMSH) protein sequences

Supplemental Figure 5. Relative expression of Amsh1 in roots, nodules and leaves of

inoculated and uninoculated wild type plants

Supplemental Figure 6. Comparison of shoot and root growth in the wild type and amsh1

Supplemental Figure 7. amsh1 mutants are able to establish arbuscular mycorrhiza with

Rhizophagus irregularis

Supplemental Figure 8. Root specific complementation restores normal infection in amsh1-

1. 1

Supplemental Figure 9. amsh1-1 and amsh1-2 mutants respond to bacteria but fail to form

long infection threads

Supplemental Figure 10. The application of exogenous cytokinin does not revert the amsh1

shoot and nodulation phenotype

Supplemental Table 1. Complementation of the amsh1-1 mutant

Supplemental Table 2. Primers used in this study

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Figure legends

Figure 1. amsh1 nodulation phenotype. (a)-(b) Plants grown on ¼-strength B&D plates, 6

wpi with M. loti rhizobia. (c)-(e) Plant root systems at 6 wpi. Fully developed nodules are

indicated by arrows and nodule primordia by arrowheads. Scale bar indicates 0.5 mm. Plant

genotypes are indicated at the bottom of the panels. (f) Structure of the L. japonicus Amsh1

gene (GenBank accession KR270441). The exon-intron structure was obtained by alignment

of the sequenced full length Amsh1 cDNA to Lotus contigs assembled from short Illumina

reads (not shown). The Amsh1 gene has 14 exons, covers ~6,2 Kb of genomic sequence and

encodes a protein of 58 kD. The LORE1 insertion sites are shown for the amsh1 mutant

alleles.

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Figure 2. AMSH1 deubiquitination activity. (a) Western blot using an anti-ubiquitin

antibody (P4D1) to detect ubiquitin conjugates in protein extracts from plant seedlings. An

anti-CDC2 kinase antibody was used to detect the CDC2 kinase loading control. Wt – Gifu,

het L1 – heterozygous Amsh1/amsh1 plant derived from the amsh1-2 line, wt L1 – wild type

plant derived from the amsh1-2 line. (b) Deubiquitination assay using linear, K48- and K63-

linked ubiquitin chains as substrates. The ubiquitin chains were incubated with or without

Lotus AMSH1 for 0 or 120 min. Numbers from 1 to 5 indicate the number of ubiquitin units

in the chains. An anti-ubiquitin antibody (P4D1) was used to detect ubiquitin chains and an

anti-GST antibody was used to detect the presence of the GST-AMSH1 protein.

Figure 3. amsh1 shoot and root growth and growth rates. (a) Shoot lengths of plate grown

seedlings. (b) Root lengths of plate grown seedlings. (c) Average shoot growth rates from 1

to 3 weeks. (d) Average root growth rates from 1 to 3 weeks. (e) Average number of lateral

roots per plant at 3 weeks. (f) Average number of lateral roots per cm of root at 3 weeks. The

asterisks indicate statistically significant differences compared to the wild type (*:P < 0.05,

**: P< 0.01, ***: P<0.001, Student’s t-test). Error bars indicate SEM. 20 Gifu, 17 amsh1-1

and 15 amsh1-2 were assayed.

Figure 4. 35S::Amsh1 complementation of amsh1-1 in transgenic roots. (a) Plants with

transgenic roots at 6 wpi. Arrowheads indicate nodule primordia, while arrows indicate pink

well-developed nodules. Scale bars indicate 1 cm. (b) Close up views of root and

nodules/nodule primordia at 6 wpi. The top panels are light micrographs. The red signal in

the middle panels indicates DsRed-labeled M. loti rhizobia, and the lower panels are overlays

of the two panels above. Scale bars indicate 0.5 mm. (c) Vibratome sections of wild type

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nodules and amsh1 nodule primordia at 6 wpi. The purple signal indicates DsRed-labeled

rhizobia. The blue signal represents cell wall autofluorescence. A dashed white line indicates

a region with branched infection threads typical of amsh1, while dashed yellow lines indicate

groups of normally infected cells. Infected cells were rarely seen in amsh1-1. Scale bars

indicate 50 µm. (d) Close up views of the regions marked by white boxes in (c). Scale bars

indicate 100 µm. The construct used for transformation and the plant genotype are indicated

at the top and bottom of the panels, respectively. (e) Shoot lengths of amsh1 plants. The mean

shoot length of 35S::Amsh1 complemented amsh1 plants with pink nodules is significantly

larger than the mean shoot length of 35S::Amsh1 transformed amsh1 plants without pink

nodules and amsh1 plants transformed with an empty vector control ( p=0.0048, Student’s t-

test).

Figure 5. Root hair infection thread and nodule frequencies in wild type and amsh1 mutants.

(a)-(c) Representative examples of IT categories, i.e. incipient, elongating and long ITs. Scale

bars indicate 50 µm. (d) Number of infection threads of different categories at 8 and 21 days

post infection (dpi). Error bars indicate SEM. 10 plants of each genotype were assayed. (e)

Number of nodules and nodule primordia on wild type and amsh1-1 and amsh1-2 at 3 and 6

wpi. Error bars show SEM. 20 plants of each genotype were assayed.

Figure 6. Nodule infection thread phenotypes of amsh1 mutants. (a) Nodule colonization

visualized by lacZ staining (blue) of M. loti expressing a hemA::lacZ reporter gene. Scale

bars indicate 0.5 mm. (b) Micrographs of wild type nodules and amsh1-1 and amsh1-2

nodule primordia at 3 wpi. Scale bars indicate 100 μm. Red arrowheads indicate ITs. (c)

Transmission electron micrographs of wild type nodules and amsh1-1 and amsh1-2 nodule

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primordia at 3 wpi. Scale bars indicate 2 μm. The green arrow indicates symbiosomes.

Infection thread structures are indicated by dashed red lines and thickened cell wall structures

by dashed yellow lines. (d) Micrographs of wild type nodules and amsh1-1 and amsh1-2

nodule primordia at 6 wpi. Scale bars indicate 100 μm. Red arrowheads indicate ITs. (e)

Transmission electron micrographs of wild type nodules and amsh1-1 and amsh1-2 nodule

primordia at 6 wpi. Scale bars indicate 2 μm. Infection thread structures are indicated by

dashed red lines. Green arrows indicate symbiosomes, while blue arrowheads indicate free

bacteria. The genotypes are indicated at the bottom of the panels.

Figure 7. Cytokinin response in amsh1 mutants. (a) Number of BAP-induced nodules/nodule

primordia in wt and amsh1-1 and amsh1-2. Error bars indicate SEM. (b)-(g) Plant tissue six

weeks post BAP treatment. (b) Uninoculated wild type root with nodule-like structures

(arrows) induced by BAP treatment. (c) Uninoculated amsh1 roots with a primordium

(arrow) induced by BAP treatment. (d)-(g) Plant tissue six weeks post inoculation with

rhizobia. (d)-(e) Sections of amsh1 nodule primordia. The red signal indicates DsRed-labeled

rhizobia. The blue signal represents cell wall autofluorescence. Excessive IT branching

(dashed white lines) was seen in amsh1. Scale bars indicate 50 µM. (f)-(g) Transmission

electron micrographs of amsh1 primordia. Blue arrowheads indicate free bacteria. Infection

threads are marked by dashed red lines. Scale bars indicate 2 µm. The genotypes are

indicated at the bottom of the panels.

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20-mar-2015 accepted date : 22-jun-2015 article...

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