UNIVERSITY OF HAWAII UBRAR,(

Isolation and characterization of l-deoxy-D-xylulose 5-phosphate reductoisomerase

(DXR) and putrescine N-methyl transferase (PMT) complementary deoxyribonucleic

acid (eDNA) in Nicotiana benthamiana using cytoplasmic inhibition of gene expression

(CIGE) technology

A THESIS SUBMIl lED TO THE GRADUATE DMSION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DEGREE OF

MASTER OF SCIENCE

IN

MOLECULAR BIOSCIENCES AND BIOENGINEERING

AUGUST 2006

By J Malkeet Singh

Thesis Committee:

Monto Hiroshi Kumagai, Chairperson Dulal Bortbakur

Winston Su

We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Molecular Biosciences and Bioengineering.

THESIS COMMlITEE

ii

ACKNOWLEDGEMENTS

This research was funded by U.S. Department of Agriculture (USDA; TSTAR Gram:

Patented research materials were kindly provided by Dr. Monto Kumagai and Dr. Guy

della-Cioppa and Large Scale Biology Corpolation.

Sincere thanks to all who have rendered encouragement and help:

Dr. Monto Kumagai, Alain Ogura, Aaron Lani, Jessica Proberts, Naomi, Leah Tedder.

Jennifer Busto, Rujunko Pugh, Manning Taite, Beth Irikura, Jason Dexter, Joanne

Kurosawa, Anti Vesnefski, Dr. Dulal Bortbakur and Dr. Winston Suo Also to all family

members and friends for their kindness and wisdom and especially to God who is

sovereign over all.

iii

TABLE OF CONTENTS

Aclmowledgements .................................................................................. iii

List of Table ......................................................................................... vii

. f· ... List 0 Figures ....................................................................................... V111

Chapter 1. Literature Review ......................................................................... 1

1.1 Vual Vectors ..................................................................................... 1

1.1.1 The Use of Plant Vual Vectors .............................................................. 1

1.1.2 Tobacco Mosaic VIrUS Vectors ............................................................. 3

1.1.3 Vual Vector Design Construct ............................................................ .4

1.2 Nicotiona benthamtana: A Model Plant for Functional Genomics ...................... 7

1.3 Safety and ContAinment Issues ................................................................ 8

1.4 Post Translational Gene Silencing (PTGS) .................................................. 9

1.5 Metabolic Engineering ....................................................................... 13

1.6 Isoprenoid Biosynthesis and DXR. ........................................................... 14

1.7 Alkaloid biosynthesis and PMT ............................................................. .16

1.8 Aim ............................................................................................. 16

Chapter 2. Introduction .............................................................................. 18

2.1 DXR. and PMT ................................................................................. 18

2.1.1 DXR. in the MEP Pathway ................................................................ 18

2.1.2 PMT in the Alkaloid Pathway ............................................................ 20

iv

Chapter 3. Materials and Methods ................................................................ 21

3.1 Bacterial Strains and Plants .................................................................. 21

3.2 Recombinant VIral vector Assembly ...................................................... 21

3.3 eDNA Expression and Characterization .................................................... 24

3.4 Plant RNA Isolation and Analysis ........................................................... 25

3.5 Microarray ....................................................................................... 26

3.5.1 Labeling and Hybridization for eDNA Microarrays ................................... 26

3.5.2 Microarray Scanning ....................................................................... 27

Chapter 4. Results ................................................................................... 28

4.1 DXR and PMT Cloning and Characterization ............................................. 28

4.1.1lsolation and Annotation of N. benthamiana DXR. ................................... 28

4.1.2 DXR Sequeru:e Analysis .................................................................. 30

4.2 Construction of Recombinant VIral Vectors ............................................... 33

4.3 RT PCR for the Verification ofInserts ..................................................... 35

4.4 Applications ofCIGE ......................................................................... 35

4.4.1 Mimicking Herbicide Effects inN. benthamiana ...................................... 35

4.4.2 Manipulation of Alkaloid Production in N. benthamiana ............................. 38

Chapter 5. Discussion ............................................................................... 39

5.1 Mimicking Herbicide Treatment ............................................................ 39

5.2 Manipulation of Biochemical Pathways ................................................... 40

5.3 Gene Silencing Systems ...................................................................... .43

v

5.4 DXRforDrugDesign ........................................................................ .45

5.4.1 Functioual Conservation across Plant, Protoman and Bacterial DXR ............ .45

5.4.2 Future Goals and Potential ofDXR Research ......................................... 47

5.5 References ...................................................................................... 50

vi

USTOFTABLE

1 Genes in Putrescine related pathway ............ .......................................... ... 38

vii

LIST OF FIGURES

I The PTGS system ................................................................................ 12

2 Screening of eDNA libraries fur gene functions ............................................ 14

3 DXR in tile IPP biosynthesis pathway ...................................................................... 19

4 PMT in the nicotine biosynthesis pathway ................................................. 20

5 General flow chart of tile cloning of genes ................................................. 23

6 N. benthamiana DXR open reading frame .................................................. 29

7 Multiple alignment of plant, E.colt and P .jalcipan.nn meR .............................. 31

8 Recombinant viral vectors ..................................................................... 34

9 RTPCR ........................................................................................... 35

10 Mimicking the effects of herbicides .......................................................... 37

11 OXR+. PMT- and GFP plants ............................................................... .42

12 Alignment between N. beothamiana OXR and E. coli DXR consensus

sequence ........................................................................................ 46

13 N. benthamina (NBOXR) and P. falciparum (PFDXR) double alignment ............. 46

14 Molecular model of the conserved domain ofOXR ...................................... .48

15 Flow chart of functional studies ............................................................... 49

viii

Chapter 1 Uterature Review

1.1 Viral Vectors

1.1.1 The Use of Plant Viral Vectors

Transgenic crops have played a role in the production of pharmaceuticals and

other valuable biological molecules. A more efficient strategy has involved by

inoculating non-transgenic plants with virus-based vectors that carry foreign genes. As

research progressed in the development of transgenic plants, it was discovered that plants

have a built-in post-transcriptional gene silencing mechanism [8, 29]. The introduction

of a gene homologous to an endogenous plant gene was found to cause a decrease in

expression of both genes sucb as sbown in the case of controlling gene expression in

transgenic petunia flowers [37]. This effect was also observed in response to

homologous genes from viruses, implicating the gene silencing mechanism as a possible

defense mechanism against viral genes [1].

With the development of infectious DNA clones, single-stranded RNA plant

viruses have become key players in gene function discovery, metabolic engineering, and

biomanufacturing. Viral expression vectors provide epigenetic expression of foreign

sequences throughout infected plants, leading to gain-or loss of function phenotypes due

to overexpression or cytoplasmic inhibition of gene expression.

1

Plant viruses are powerful transfection tools in molecular farming, producing

pure, properly folded and gIycosylated proteins in plants faster and more economically

than other expression systems [12, 13]. They are a highly desirable alternative to

transgenic systems that require protracted periods to transform and regenerate whole

plants, and that have variation in the expression levels of heterologous proteins. In

transgenic systems, once a particular construct is inserted into the plant genome, it may

take several crosses to establish a stable line in an elite cultivar. In contrast, plant viral

vectors employed in the large-scale production of therapeutic drugs in greenhouse and

field-grown crops directly yield high levels of foreign protein due to the rapid rate of

viral replication. In plants transfected with a recombinant tobamovirus, alpha­

trichosanthin, a potential anti-AIDS drug, accumulated to approximately 2% of total

soluble [23].

Therapeutic compounds stably produced in transfected plants are numerous, and

include anti-viral drugs such as human interferon-alpha 2 as well vaccines, proteins, and

secondary metabolites. Plant-derived anti-cancer vaccines have been produced for

treatment of human papillomavirus induced cancer by expressing recombinant E7

oncoproteins in N. benthamiana [15, 16]. The mv p24 nUcleocapsid protein, used as an

antigen in the development of mv vaccines, has been produced in plant protoplasts using

tomato bushy virus (TNSV) tombvirus vector [49].

For viruses that cannot be grown in tissue culture, such as hepatitis C (HCV),

tobamoviral vectors are under development to produce a plant-derived vaccine [33].

2

Recombinant proteins for use in diagnostics have also been expressed in plants. Full­

length recombinant monoclonal antibodies (rAbs) directed against a colon cancer antigen

and recombinant allergens have been expressed in N. benthamiana leaves using a TMV

vector [4]. The binding ofIgE from sera from birch pollen-and latex allergic patients

suggested that the plants produced allergens were properly folded.

1.1.2 Tobacco Mosaic Virus Vectors

Several viral groups have been under investigation for design as recombinant

plant viral vectors including geminiviruses; potyviruses; potexviruses; comoviruses;

tombusviruses; tobraviruses; alfaviruses; and hordeviruses. Members of the tobamovirus

group [14, 24, 33], are the most widely studied; the autonomously replicating RNA viral

vectors based on the tobacco mosaic vi1US (TMV) genome have been particularly

successful as research and commercial tools.

TMV possesses a positive sense, single stranded genome of 6396 nucleotides,

which encodes replicase enzymes, and movement and coat proteins. Viral genes were

expressed through the production of both genomic and subgenomic RNA. Essentially

designed as eDNA plasmids, TMV vectors were modified to contain a foreign gene

sequence. Viral vectors were originally constructed with the gene of interest replacing the

capsid protein [2]. It was later recognized that these viral vectors did not move

efficiently. TMV vectors have since been designed as hybrid versions of several different

strains of tobamoviruses [9, 28] that included all the essential viral genes and an

3

antibiotic resistance marker. Dual heterologous subgenomic promoters from related

tobamoviruses have enhanced stability, while an internal ribosome entry site sequence

(IRES) [45] has been incorporated into the design to enable expression of multiple

proteins.

The transfection process involves mechanically inoculating recombinant in vitro

RNA transcripts derived from viral cDNA clones onto plants. Recombinant virions that

are assembled in the plant move systemically by associating with the plasmodesmata and

intercellular cytoplasmic channels [22]. One to two weeks after inoculation, recombinant

proteins can be isolated from transfected plants. Interstitial fluid CODtaining the desired

product can be quickly separated from other cellular proteins by vacuum infiltration and

gentle centrifugation. For large agronomic applications, virions can be purified from

transfected plants and used for sUbsequent inoculations using high-pressure sprayers in

the field.

1.1.3 Viral Vector Design

For vaccine production, TMV vectors have been developed as coat protein fusions

[46], with the viral coat providing a flexible framework to attaching recombinant

proteins. TMV CP fusions include human immunodeficiency virus type I (IHV-I)

peptide, influenza virus hemagglutinin epitope, malaria parasite peptide, and hepatitis C

virus peptide [33]. A key technical advance in design has enabled TMV vectors to

produce ''free'' proteins that are not fused to the coat protein. Instead, genes encoding

4

bioactive compounds are fused to signal peptide sequences that cause the translated

protein to be processed via the endoplasmic reticulum and Golgi complex and to be

targeted for cellular secretion. It is recognized that the faithful and efficient expression of

such heterologous proteins is influenced by the choice of the signal peptide.

One of the most highly efficient TMV vector constructs fuses the gene of interest

to a sequence encoding a rice a,- amylase signal peptide adjacent to a 5' untranslated

leader [24). Viral vectors containing the rice a. - amylase signal peptide have been used

to express a wide variety of heterologous proteins including mammalian peptides, blood

products, glycoproteins and cytokines. TMV vectors that incorporate this signal peptide

have been used successfully to secrete single chain variable fragment (Fv) antibodies for

the treatment of Non-Hodgkin's Lymphoma [32). Phase I olinical trials have since been

completed [32).

The rice 0.- amylase 5' untranslated leader sequence may help to enhance

translation of the heterologous protein. The highly expressed viral coat sUbgenomic RNA

has a 5' cap (m7GpppN) and terminates with a tRNA-like structure instead of a poly (A)

tail. The 3' untranslated region (UTR) has two domains, which contain five RNA

pseudoknots. It is possible that interactions between the 34 base pair 5' leader of rice a,-

amylase and the 3' UTR may cause synergistic regulation of translation in transfected

plants. Significantly, the rice 0.- amylase signal peptide has been recognized and

processed in other transformed organisms, including Escherichia coli, Saccharomyces

cerevisiae, and Yarrowia lipolytica, as well as transgenic rice cell suspension [27, 34).

5

Subtle differences in the size, source, and sequence of the rice 0.- amylase signal peptide

can greatly affect the secretion process.

Striving for higher protein yields, researchers have collaborated to develop

improvements in vector design for increased production. It is recogniUld that inclusion of

foreign genes into TMV vectors reduces efficiencies of replication and movement

compared to the wild type virus [38]. Studies are aimed at improving the ability of

vectors to move and to replicate through "gene shuffling" of the 30K movement gene

[38]. Visible markers for heterologous gene expression in plants have been developed.

TMV vectors have been engineered to overexpress an enzyme involved in

carotenoid biosynthesis in N. benthomiana and other solaneaceous plants [25]. As the

viral vector replicates, the encoded enzyme, phytoene synthase (pay), is targeted to the

choloroplast causing an accumulation of carotenoids. Transfected plants develop an

orange phenotype in the leaves, as early as 4 days post inoculation (dpi) [25]. Whenpay

is inserted in a recombinant vector, pay may serve as a useful marker for gene expression,

being particularly useful for field applications.

Recent work has focused on improving the efficiency and expanding the

functionality of viral vectors. Toth et aL used a viral vector with an internal ribosome

entry site sequence to express a foreign gene while avoiding the problem of homologous

recombination that occurs when duplicate sUbgenomic promoters are present in the viral

vector [45]. Holtzberg et al. demonstrated that virus-induced gene silencing can be used

6

in monocot species as well as dicots [19]. The rate-limiting step in the discovery of the

function of genes is relating nucleic acid sequences to phenotypic changes. Since

systemic expression or cytoplasmic inhibition of genes in transfected plants can cause a

dramatic alteration in metabolism, plant color or morphology, a viral based Arabidopsis

thaliana eDNA was constructed and then screened transfected N. benthamiana for

changes in phenotype. Individual plants having altered characteristics have been

identified and their corresponding genes have been obtained by simply sequencing the

viral inserts.

1.2 Nicotitma benthamiana: A Model Plant for Functional Genomics

N. benthamiana is susceptible to numerous plant viruses especially TMV. It

showed systemic infection with viral vectors within a short period of time. The earliest

symptoms can be seen as early as 3 dpi. These plants are easily maintained at a low cost

due to quick growth and development Thus, using plant based viral vectors in N.

benthamiana can allow the foreign gene product to be harvested for maximal product

yield from infected plants within several weeks of inoculation. AIl these features render

this plant a good and efficient plant based system to study gene expression.

7

1.3 Safety and Containment Issues

Plant RNA viral vectors have become intensively utilized in several different

plant species for large scale production of high value therapeutic proteins and secondary

metabolites. The United States Food and Drug administration (FDA) has developed a

guidance document on ''plant-derived biologics" and thus has strengthened field-testing

controls for permits on those bioengineered traits that are not intended for commodity

uses, such as pharmaceuticals, veterinary biologics, or certain industrial products. The

humao safety of TMV based expression systems has been documented; plant viruses are

non-pathogenic to humans. TMV is only transmitted to other plants through mechanical

means; tools and machinery that might have been contaminated with viruses and TMV

based vectors can be sterilized through washing with bleach. In addition, the demands for

recombinant product purity by the FDA are rigorous.

Recombinant viruses are deemed uncompetitive against wild type viruses. As a

result, the recombinant viruses will eventually have to COlllpete for representation with

wild type viruses. A strategy employed by recombinant viruses is that all non-essential

coding domains are readily lost [36]. The recombinant viral vectors, therefore, delete

foreign genes and retain only those sequences required for optima\ replication and

movement, which therefore reduces the concern for virus-virus transfer of foreign gene

sequences in a competitive environment

8

A study done by Rabindran and Dawson [38] showed that the recombinants had

reduced vigor and were less competitive and pathogenic than wild type TMV. Thus,

initially the foreign product can be produced in large amounts but would not persist in the

environment. At 3 dpi, the recombinant TMV showed systemic infection of the leaves

with the Jellyfish green fluorescent (GFP) protein foreign gene. However, at 7 dpi, there

was a 'mottled' pattern on the leaves suggesting that the foreign gene was being deleted.

Furthermore, the recombinant TMV vector does not move efficiently into upper

leaves as compared to the wild type TMV in tobacco plants. Also, as the recombinant

viral vector moved to the upper leaves, it was in correlation with the loss of the GFP

gene. This suggests that the recombinant vector reverts to the wild type by losing the

foreign gene overtime and is also not as competitive. Additionally, work concerning

TMV in glasshouse conditions has been completed and field applications for TMV have

also been widely applied.

1.4 Post-Translational Gene Silencing (PTGS)

PTGS refers to a sequence-specific RNA degradation process (Figure 1). These

sequences can include viral RNA, transposon RNA and dsRNA [48]. PTGS-like

phenomena have been studied for many years in several diverse organisms but it was

only recently that these phenomena were found to be related to each other and to PTGS.

PTGS is now known to be significant in many other organisms. The PTGS-like systems

seen in other organisms have had different names such as quelling in the fungi

9

Neurospora crassa [35], also noted in the nematode Caenorhabditis elegans, RNAi, co­

suppression, and RNA-mediated resistance, but now all of them are generally referred to

as a common mechanism known as RNA silencing [43].

PTGS was found to be a mechanism which is believed to have been developed by

plants for protection from virus infection. Plants thus utilize this defense system to

degrade viral RNA [47]. Based on this mechanism, it can be suggested that the antisense

cDNA could work very well and had already been shown to be effective in the silencing

of genes as shown in the cytoplasmic inhibition of carotenoid biosynthesis [25] where the

system was shown to be successful in introducing antisense cDNA fragments that

targeted the specific gene transcripts. The antisense RNA transcripts produced from this

system could bind to the complementary mRNA transcripts and thus prevent these

transcripts from being translated into functional proteins. The formation of dsRNAs also

caused it to become a target for degradation by the defense mechanism of the plants.

Gene silencing was also observed in constructs that contained partial cDNA sequences in

the sense direction.

The PTGS system is dependent on homologous sequences, which will mean that

once viral RNA triggers PTGS, the PTGS system is directed only at that particular viral

RNA due to the formation of the dsRNA which is then degraded resulting in the

formation of small interfering RNAs. These degraded RNAs are also known as siRNAs

which are produced through a set of nucleases known as the Dicer enzymes [21]. Viral

10

RNA is thus degraded by the PTGS system. resulting in the loss of RNA to make protein.

This system will then prevent the virus from continuing to multiply and spread.

It is suggested that dsRNA of the virus is the strongest inducer of PTGS but the

double-stranded regions of both positive and antisense single stranded RNA (ssRNA)

also induce PTGS. As such, the formation of dsRNA can be exploited for silencing

purposes by introducing antisense RNA that are targeted to the specific gene transcripts

to be silenced in the cytoplasm of the cells.

It is interesting to note that the silencing signal has also been found to be mobile

whicb means that it can be transmitted to other parts of the planL This might seem similar

to the immune system in mammalian systems where antibodies are able to travel around

the circulatory system prevC1lting infection from proceeding. In plants, this system has

been extensively used against many kinds of viruses including TMV, Tobacco Rattle

Viruses (TRy) and Potex Viruses (PYX) [3].

11

dsRNA

siRNA (short inlerfering

RNAs, 21·24 nt long)

(note that both the + and - strands of siRNA can anneal to viral RNA . therefore both + and - strand viral RNAs can be targeled)

Viral ssRNA target

II ": I \I 1\\~l'IlT .ll J. T IIII! J.J. .1

,

siRNA/protein complex

Rise complex (RNA.lnduced

silencing complex)

(0)

Cleavage of dsRN A by Diccr·like enzyme

(Dicer contains dsRNA cleavage activity, helicase and dsRNA

binding activity)

siRNA is unwound. s ingie-stranded RNA formed

sequence-sped fie larget recognition

andlor (b)

(Specificity is due to the facllhal the siRNA can only bind \0 complementary RNA (whi ch can only be viral RNA)

Degraded viral RNA Newly synthesized viral RNA Host RNA dependent

polymerase single-stranded siRNA -

ewly synthesized viral dsRNA

p

The siRNAs feed back in to the cycle and further degradation of viral RNA occurs. New siRNAs

.. , . . , .etc

DICER

Figure 1 : The PTGS system. Adapted from Roth et aI., (2004) Virus Research 102: 97-108[41].

12

•

•

1.5 Metabolic Engineering

While plant viruses that are engineered to produce pharmaceutically relevant

proteins have proved to be powerful gene expression tools, they are also valuable tools

for use in gene discovery and in the metabolic engineering of existing pathways in plants.

The biosynthesis of leaf carotenoids in transfected N. benthamiana was altered by forced

"re-routing" of the pathway, resulting in the synthesis of capsanthin. a non-native

chromoplast-specific xanthophyll [25). The ectopic expression of the capsanthin­

capsorubin synthase (Ccs) eDNA caused the plant to develop an orange phenotype and

accumulate high levels of capsanthin, up to 36% total carotenoids. By redirecting the

existing pathways of plants that produce biologically active compounds, plant viral

expression systems can potentially be used to alter the production of secondary

metabolites, or cause the accumulation of non-native bioactive compounds [26).

The technology of virus-induced gene silencing has also been refmed and adapted

as a high throughput procedure for functional genomics in plants [3). This technology had

been particularly useful to identify and characterize uncharacterized cDNAs especially

since the construction of cDNA libraries generates a collection of uncharacterized

sequences, many of which represent genes that are highly conserved across the plant

kingdom. The antisense cDNA derived from the eDNA library has been applied for the

rapid identification of these genes by transfecting infectious transcripts into N.

bentluzmiana via a tobamoviral vector that contains a transcription site and subgenomic

promoters for the expression of transgenes. The phenotypic change observed in the plants

13

due to gene silencing was easily observed for the characterization of novel sequences.

This was aided through high-throughput robotic screening. This technology has also been

adapted for the purpose of overexpressing PSY that resulted in the production of an

orange phenotype. In the present project it is being used for the overexpression of DXR

to study developmental and growth changes in plants.

Viral Vectors

Known or unknown Genes

DNA

eDNA Libraries

Righ-Throughput Robotic Screening

Figure 2: Screening of cDNA libraries for gene functions.

1.6 Isoprenoid Biosynthesis and DXR

Biochemical Assays Phenotypic screens

Novel Gene Functions

Transgenic manipulations of the mevalonate-independent (DXP) pathway in

Escherichia coli have indicated that IPP and DMAPP likely arise independently by

branching of pathway [39] and that overexpression of the first pathway gene, for DXP

14

synthase (dxps), increases carotenoid and ubiquinone biosynthesis [31] [17]. The

manipulation of the mevalonate pathway that operates in yeast also resulted in increased

carotenoid production [18] . Studies conducted on the overexpression and

underexpression of dxps in Arabidopsis also indicated that this enzyme catalyzes a slow

step in the mevalonate-independent pathway to plastidial isoprenoids (chlorophylls and

carotenoids) [11].

Since DXP has been found to be an intermediate not only for IPP and DMAPP

biosynthesis but also for the biosynthesis of thiamine and pyridoxol [20] , it can be

suggested that the conversion of DXP to methylerythritol phosphate (MEP), catalyzed by

DXP reductoisomerase (OXR) [44], could represent the first committed step in the

production ofIPP.

A study has also shown that modifying the expression level ofDXR had an

influence on essential oil and mint production yield [30] . There have been several reports

on the use of dxps to increase the essential oils in plants; however using DXPS will also

cause an imbalance in the monoterpenes in essential oils [5]. As such, it has been

suggested that DXR which constitutes the first committed step in the DXPS pathway of

terpenoid synthesis can be used instead to increase essential oils without the side effect of

altering the balance of terpenoids in the production of higher yield essential oils. DXR is

an an important enzyme to study as it is involved in the production of secondary

metabolites and the overexpression of DXR can also be studied for its contribution as an

assay for herbicide compounds which may include compounds that are also suitable for

15

becoming drug candidates against the malaria causing pathogen Plasmodiumfalciparum

since DXR is also found in this pathogen.

1.7 Alkaloid biosynthesis and PMT

Plant alkaloids, being one of the largest groups of natural products, are of great

interest as they provide researchers with many pharmacologically active compounds to

explore and investigate for functional analysis. In one study [42], PMT was expressed in

transgenic plants of Atropa beilado1l1UJ and Nicotiana sylvestris. The overexpression of

pmt gene increased the nicotine content in N. sylvestris, whereas suppression of

endogenous PMT activity severely decreased the nicotine content and induced abnormal

morphologies. The phenotypic changes in transfected plants with the tobamovira1 vector

containing the antisense N. benthamiana pmt cDNA will be investigated to see whether

these results are consistent with previous conclusions.

1.8 Aim

The main goal of this thesis is to show that functional genomics studies can be

conducted through gene silencing and overexPlessing genes via vira1 vectors. This project

thus involves the cloning and subcloning of cDNA fragments in the sense and antisense

direction for the study of phenotypic changes due to the overexpression and silencing

effects of these inserts in N. benthamiana. The cDNAs that are used in this study are

DXR (sense), DXR- (antisense), EPSP+ (sense), EPSP- (antisense) and PMT-

(antisense). This project will thus seek to confirm the presence of the inserted cDNA

16

fragments in the viral vector at 15 dpi through reverse transcription polymerase chain

reaction (RT PCR). This will show the presence of intact recombinant viral vectors

containing the cDNA inserts at 15 dpi At some point the cDNA inserts will be deleted

causing the recombinant viral vectors to revert back to the wild type. RNA silencing

methods can also be used to mimic known herbicides such as norflurozon and a

glyphosate compound (RoundupTM) (Monsanto, Minnesota) that inhibits the enzyme

EPSP synthase in the shikimate pathway. N. benthamiana DXR was shown to be a new

herbicide target to a known anttbiotic fusmidomycin which is being represented as an

herbicide in this project.

Recombinant viral vector containing antisense pmt was made to decrease the

levels of nicotine in the N. bentlUlmiana plants. A recombinant viral vector containing the

sense dxr insert was also made to overexpress DXR in N. benthamiana to observe its

effects on the growth and development of the plant. In summary, the fullowing

objectives will help achieve the main goal of this thesis.

• Isolate and characterize plant DXR and N. bentlUlmiana PMT.

• Create recombinant viral vectors containing cDNA inserts.

• Conduct RT PCR of the recombinant viral vectors from infected plant tissues.

• Illustrate the use of antisense methods to mimic the effects of herbicides.

• Show the developmental and growth changes due to DXR overexpression.

17

2.1 DXR and PMT

Chapter 2 Introduction

Due to the considerable interest in carotenoids and alkaloids, it was decided that

this project will utilize the viral based gene silencing method to show its efficacy by

silencing two important enzymes in each of the pathway. The first is DXR in the MEP

pathway and the second is PMT in the alkaloid biosynthetic pathway, specifically

involved in the production of nicotine. N. benthamiana DXR and PMT have not been

isolated and characterized in N. benthamiana prior to this project.

2.1.1 DXR In the MEP Pathway

The enzyme DXR that is involved in the first committed step in the MEP pathway

was chosen based on the importance of producing carotenoids, as a possible drug

screening assay and fur its medicinal value in essential oils. In the present study, dxr was

modified by site-directed mutagenesis and placed under the transcriptional control of a

tobamovirus sUbgenomic promoter in a plant viral vector. Infected plants with partial

fragment of the antisense dxr and full length sense dxr inserts were observed for

phenotypic changes. The ptesence of the infectious transcripts containing·the dxr was

confmned using reverse transcription (RT) PCR. Transient viral based expression

systems had been used to silence endogenous genes in transfected plants to show that the

silencing ofphytoene desaturase (PDS) and S-enolpyruvylsbikimate-3-phosphate (EPSP)

18

synthase (U.s. Patent No. 5,312,910) that lead to phenotypes that mimic herbicide

treatment. The resuIts of the present study and these findings allude to the fact that DXR

can be used as a possible herbicide target and as an assay fur herbicide compounds

including drugs that inhIbit DXR that can be used against P. falciparum.

Pyruvate + G1yceraldehyde-3-Phosphate

!DXS _----. Thiamine, Pyridoxal

l-deoxy-D-xylulose-5-Phosphate ---(DXP) -r- ¢::::::J fosmidomycinlantisense dxr

DXR

2-C-methyl-D-erythritol-4-P (MEP)

ISOpentl diphosphate (IPP)

! _--. Chloroplast, Gibbere1Iins,Vitamin E GeranylGeranylPhosphate -----

(GGPP)

1 Phytoene synthase (PSy)

Phre

! Phytoene desaturase (PSD)

Q:!~~ Lycopene a-CYcI7" ~ycopene (kycIase (LCY)

a-carotene ~carotene

LJin :zelnthin

Figure 3: DXR in the IPP biosynthesis pathway. DXR specifically converts DXP to MEP thus resulting in as the frrst committed step in the production of IPP.

19

2.1.2 PMT in the Alkaloid Pathway

The second enzyme, PMT, was chosen to illustrate the ability of using this same

strategy to manipulate nicotine production. PMT being a branching point enzyme that is

involved in the committed step in producing nicotine became a primary target of interest.

Phenotypic observations fur evidence of any abnonnal growth of plants that were

inoculated with the antisense pmt constructs were not observed. The persistence of the

antisense pmt viral vector was confirmed using RT PCR.

Arginine and Proline Metabolism

1 ....... ,..... ..... .,..... Putrescine ____________________ • homospermidine

_I _ <:= antisense pmt r Puterscine N-Methyltransferase

N-Methyl Putrescine

1 Amine Oxidase

___ --. Cocaine I-Methyl Pyrrolinium ---

1 Nicotine

Figure 4: PMT in the nicotine biosynthesis pathway. PMT is involved in the conversion of Putrescine to N-Methyl Putrescine that leads to the production of nicotine.

20

Chapter 3 Materials and Methods

3.1 Bacterial Strains and Plants

Wild type N. benthamiana plants were germinated from seeds in soil (Premier,

ProMix BX General Purpose Growth Medium) in a growth chamber (Whatlow 1500,

dispatch Industries) WIder a 16 hour light and 8 hour dark photoperiod or in continuous

light in growth room E. coli C600 was generously provided by Large Scale Biology

Corporation (LSBC) and Dr. Monto Kumagai.

3.2 Recombinant Viral Vector Assembly

The first step in creating a recombinant viral vector was to amplifY the partial

clone with designed primers. As shown below, the primers were designed to incorporate

site directed mutagenesis so as to use restriction enzymes to "cut and paste" into the viral

vector.

Primers were designed based on sequence alignments between DXR homologous

sequences using the blast alignment program from the National Center fur Biotechnology

Infurmation (NCBI) website. The 5 prime end primer included the AvrIl restriction digest

site while the 3 prime end included the Xho 1 site.

21

The TIOSAl Ape pBAD viral vector was exeised with Xhol/Avrn and replaced

with the DXR eDNA. which was amplified from the eDNA N. benthamiana leaflibrary

by PCR using forward primer:

(5'-TACCTAGGACITGGGATGGTCCAAAGCCTATCTCA-3') and reverse primer:

(5'-GCACTCGAGGCGCTGAATGTICTGAATCTGCAGGAAGA -3') to introduce the

respective5'-Avrn site upstream and 3'-Xhol site downstream of the partial eDNA clone.

which was subsequently isolated by precipitation using chloroform/phenol extraction.

The resulting precipitated dxr eDNA was digested with Avrn and Xhol. and ligated into

similarly prepared and gel purified TIOSAl Ape pBAD viral vector to replace the

original GFP insert so as to generate antisense partial dxr mRNA. This enabled us to

create a viral vector containing the partial fragment of dxr clone in the antisense

orientation for the purpose ofperfonning a cytoplasmic inlnbition of the endogenous

DXR gene in N. benthamiana.

Figure 5 illustrates how antisense and sense cDNA fragments can be cloned to

produce recombinant viral vectors. Restriction enzymes that do not have internal sites

within the cDNA fragment are ehosen to digest and produce non-blunt ends to ligate to

the TIOSAl Ape pBAD viral vector that contains the GFP protein which itselfwill be

digested with the same restriction enzymes to produce a linearized non-blunt vector.

Infectious transcripts are made in order to infect the plants so they will express or silence

the specified endogenous genes. Phenotypic changes are then observed to detect whether

these genes are involved in the growth and development of the plant.

22

TIOSAI Ape pBAD#5

pmt-Idxr-Idxr+

Xho I Avr II

Recombinant plasmid

E.coli transformants

Amplified plasm ids

Inoculated plant

InfeClious RNA

Figure 5: General flow chart of the cloning of genes. The initial viral vector had a green fluorescent protein CGFP) cDNA inserted into a multiple cloning site region. The gfp insert was then removed through double restriction digests and subsequently ligated to the cDNA fragments of interest. The transfolll1ants were then selected through restriction digest maps and subsequent recombinant viral vectors were isolated and purified for inoculation purposes.

The second step in the project was to overexpress the full length sense DXR

cDNA in N. benthamiana. The TIOSAI Ape pBAD viral vector was excised with

Sph lIAvrIl and replaced with the DXR full length cDNA containing both the start and

stops codons, which was amplified from the N. benthamiana cDNA leaf library by PCR

using forward primer C5'-AGGCATGCCCCTCAA TITGCTITCTCCTGCTGAA-3') and

reverse primer (5'-TACCTAGGTCATACAAGAGCTGGACTCAAACCAG -3') to

introduce the respective5'-Sphl site upstream and 3'-AvrTI site downstream of the

sequence. The above-mentioned procedure was successfully used to generate the full

length cDNA sequence and cloned into the TIOSAI Ape pBAD viral vector creating a

recombinant viral vector expressing dxr.

23

The antisense PMT eDNA bad previously been eloned into a pbluescript TM vector

(Stratagene, Califurnia) by Manning Taite (unpublished) from the N. benthamiana eDNA

library. The antisense pmt was then subcloned into the viral vector fur expression studies.

The antisense epsp synthase eDNA was subcloned from the full length sense

eDNA present in previously prepared recombinant viral vector.

The forward primer (5'-TACCT AGGGGAGCGA TTTGGTGTCTCTGTGGA-3,) and

reverse primer (5'-CACTCGAGATGCTTGGAGTACTGCTGGAGAAC-3') was used to

introduce the respective5'-AvrII site upstream and 3'-Xhol site downstream of the partial

eDNA clone, which was subsequently isolated through precipitation using

cblorofurmlphenol extraction. The resulting precipitated epsp synthase eDNA was

digested with AvrIl andXhol, and ligated into similarly prepared and gel purified

TTOSAl Ape pBAD viral vector to replace the original GFP insert so as to generate anti

sense partial epsp synthase mRNA.

3.3 cDNA Expression and Characterization

In vitro transcription was performed using KpnI digested TTOSAIDXR

TTOSAlDXR-, TTOSAIPMT- and TTOSAIEPSPS- templates in an SP6 polymerase

driven reaction for the production of infectious RNA, and then used to infect N.

benthamiana by mechanical inoculation [25]. The open reading frame (ORF) of dxr was

placed under the control of a tobamoviral subgenomic promoter.

24

During in vitro transcription, a capped RNA is synthesized, mimicking most

eukaryotic mRNAs found in vivo with a 7-methyl guanosine cap structure at the 5' end.

The following protocol was used to successfully infect N. benthamiana plants with the

recombinant viral vector ITOSAlDXR. ITOSAlDXR-. TIOSAIPMT - and

ITOSAIEPSPS-

In vitro Transcription Ambion mMESSAGE mMACHINE™ for SP6 or T7 reactions (Ambion The RNA Company, Austin, Texas)

For viral vectors with SP6 promoter: (25 pI)

2XNTP/CAP lOX Reaction Buffer Linearized plasmid

N.B. Use 8 pI restriction digest Enzyme Mix (RNA Polymerase)

12.5 pI 2.5 pI

1!Jg

Incubate in hot water bath, 2 hrs @ 37°C

For T7 Promoter with ribozyme: Build a 20 pI reaction and incubate in water for I hr @

37°C

3.4 Plant RNA lsoJadon and Analysis

Harvested leaf material was pulverized using liquid nitrogen and a mortar and

pestle. Total RNA was extracted from the powdered leaf material using an RNeasyTM

Plant Mini kit (QIAGEN, Valencia, California) following the manufacturer's

specifications. cDNA was synthesized using a RETROScript™ kit (Ambion The RNA

Company, Austin, Texas). The reaction took place in a two tube system.

25

The primer sequences used in the RT PCR included the restriction enzyme sites

TOICP (5' TAATACGAATCAGAATCCGCG 3') and Clal (5' ATCGAT 3'). which

were present on the multiple cloning site of the vira1 vector and which flanked the

upstream and downstream regions of the inserted fragment of the cDNA.

The reaction was incubated at 42"C for 2 hours. In a 20 iii reaction, up to 2 ~ of

isolated RNA was used as the template material. 20% of the PCR reaction volume

contained the synthesized cDNA from the RT PCR reaction. The PCR reaction

parameters were as follows: 95"C. 5.0 min; 95"C. 1.0 min; 6O"C. 1.0 min; 72"C. 1.0 min

(30 cycles); 72°C. 7.0 min (1 cycle).

3.5 Mieroarray

3.5.1 LabeUng and Hybridization for eDNA Mieroarrays

Total RNA was extracted using TRIzoI (INvitrogen Cat. No. 15596-018) and

cleaned using Qiagen RNeasy Clean -Up protocol Invitrogen SuperScript Direct cDNA

Labelling System (Cat# L101IS-01) was used to differentially label with flurescent

cyanine-3(cy3) or cyanine-5 (cyS) dyes (Amersham, Cat Nos. PA53022). Quality and

quantity of RNA was checked on a 1 % agarose gel, and on a Sbirnadm

spectrophotometer. Cy-dye labeled eDNA was quantified using a Beckmann

spectIophotometer. As a control for both microarray studies, RNA from GFP- transfected

plants was isolated, quantified, reverse transcribed with cy5 using the same procedures.

26

•

Equal amounts of1abeled treated and control eDNA were mixed and hybridized to 10K

potato eDNA microarrays from TIGR (The Institute fur Genomic Research).

Hybridization and post-hybridization washes were perfurmed using recommended

hybridization conditions fuund in protocols developed by TIGR. A detailed description of

TIGR Potato Microarray optimized methods, the array design and the eDNA description

file used fur this study (GenePix Array List, GAL file version 1 and 2) can be fuund at

the fullowing URL:bttp:/Iwww.tigr.org/tdb/potato/microarray_comp.shtml

3.5.2 Mieroarray Seannlng

Scanning of the microarrays for the experiments was performed using the

BioRAd VersArray Chip reader. Raw signal data was combined into text meso A

Microsoft1M Excel me was created for studying the genes that were related to the

A1ka1oid pathway relating to putrescine.

27

Chapter 4 Results

4.1 DXR and PMT Cloning and Characterization

4.1.1 Isolation and Annotation of N. benth"",illna DXR

A partial fragment of N. benthamiana dxr was obtained through screening the N.

benthamiana cDNA leaf library. Primers were designed based on tomato dxr sequence to

perform PCR reactions on the library. A fragment of amplified DNA was subsequently

loaded for gel electrophoresis that approximated to 490 base pairs. The amplified PCR

fragment was then subsequently isolated through phenol-chloroform extraction and

digested with the restriction enzymes AvrIl andXhol. The digested product was then

ligated to a similarly digested viral vector. A PCR was also conducted on the N.

benthamiana cDNA library with primer sets as described in Chapter 3 and similarly a

phenol chloroform extraction was performed to isolate the fu\llength N. benthamiana dxr

and digested with the restriction enzymes Sphl and AvrIl. The digested product was then

ligated to a similarly prepared viral vector.

Sequence analysis was conducted through multiple sequence alignments that

produced a consensus sequence using the Sequencher™ software. Sequencing reactions

thus produced a consensus sequence of 1422 base pairs encoding a mature DXR protein

with a chloroplast transit signal peptide. Comparison of N. benthamiana amino acid

sequence to the Genbank database showed 90 to 98% similarity to other plant DXR

enzymes.

28

ATG GeG CTG AAT TTG CTG TCA CCT TCT GAA ATT AAG ACC ATC TCT TTC TTG GAC ACC TCC Met ala leu asn leu leu ser pro ser qlu ile lys thr ile ser phe leu asp thr ser

AAA TCC AGC TAC AAC CTT AAC CAC CTT AAG TTC CAA GGT GGA GTG GCT ATC AAA AGA AAG lys ser ser tyr asn leu asn his leu lys phe qln qly qly val ala ile lys arq lys

GAG TGG AGT GGG ATT TCT GGT AAG AGG GTT CAG TGT TCA GTT CAG GCA CCT CCT CCT GeC qlu trp ser qly ile ser qly lys arq val qln cys ser val qln ala pro pro pro ala

TGG CCT GGA AGA GCT GTT GCT GAA GCA CGG AAA ACT TGG GAT GGT CCA AAG CCT ATC TCA trp pro qly arq ala val ala qlu ala arq lys thr trp asp qly pro lys pro ile ser

ATT GTT GGG TCC ACT GGC TCT ATT GGA ACT CAG ACA TTG GAT ATA GTC GCT GAG AAT CCG ile val qly ser thr qly ser ile qly thr qln thr leu asp ile val ala qlu asn pro

GAT AAG TTT AGA GTT GTC GCA CTA GCT GeT GGT TCA AAT GTT ACT CTT CTC GCT GAT CAG asp lys phe arq val val ala leu ala ala qly ser asn val thr leu leu ala asp qln

GTC AAA ACA TTC AGA CCA AAA CTA GTT GCT GTT AGA AAT GAG TCA TTG GTT GAG GAA CTC val lys thr phe arq pro lys leu val ala val arq asn qlu ser leu val qlu qlu leu

AAA GAT GCT CTG GeC GAT ATG GAA GAC AAG CCT GAG ATT ATA CCT GGT GAG CAG GGT ATC lys asp ala leu ala asp met qlu asp lys pro qlu ile ile pro qly qlu qln qly ile

ATC GAG GTT GeC CGC CAT = GAT GCT GTC ACT GTA GTT ACA GGA ATA GTT GGT TGe GCA ile qlu val ala arq his pro asp ala val thr val val thr qly ile val qly cys ala

GGT TTA AAG CCT ACA GTG GCT GeC ATA GAA GCA GGA AAG GAC ATT GeC TTG GeC AAT AAA qly leu lys pro thr val ala ala ile qlu ala qly lys asp ile ala leu ala asn lys

GAG ACT TTA ATT GCT GGT GGT CCA TTT GTC CTT CCT CTT GeG CAC AAG CAT AAG GTG AAG qlu thr leu ile ala qly qly pro phe val leu pro leu ala his lys his lys val lys

ACT CTT = GCA GAT TCA GAA CAT TCA GCT ATA TTC CAG TGe ATA CAA GGC TTG CCA GAG thr leu pro ala asp ser qlu his ser ala ile phe qln cys ile qln qly leu pro qlu

GGT GeC CTT CGT CGC ATT ATA TTA ACT GCA TCT GGA GGG GCT TTC AGG GAC TTA CCA GTT qly ala leu arq arq ile ile leu thr ala ser qly qly ala phe arq asp leu pro val

GAG AAG TTG AAA GAA GTT AAA GTA GCT GAT GCT TTG AAG CAT CCC AAT TGG AAC ATG GGG qlu lys leu lys qlu val lys val ala asp ala leu lys his pro asn trp asn met qly

AAA AAG ATT ACT GTT GAT TCT GeC ACC TTA TTC AAT AAG GGT CTT GAA GTT ATT GAA GCT lys lys ile thr val asp ser ala thr leu phe asn lys qly leu qlu val ile qlu ala

CAC TAC CTT TTC GGA GeT GAG TAT GAT GAC ATT GAA ATT GTC ATC CAT CCC CAG TCC ATC his tyr leu phe qly ala qlu tyr asp asp ile qlu ile val ile his pro qln ser ile

ATA CAT TCA ATG GTG GAA ACA CAG GAT TCA TCA GTA TTG GCA CAG CTG GGG TGG CCT GAT ile his ser met val qlu thr qln asp ser ser val leu ala qln leu qly trp pro asp

ATG CGT TTG CCC ATC CTT TAT ACT TTA TCC TGG CCC GAC AGA ATT TAC TGT TCG GAG GTT met arq leu pro ile leu tyr thr leu ser trp pro asp arq ile tyr cys ser qlu val

29

ACT TGG CCG CGG CTT GAT CTT TGC AAG CTC GGA TCA TTG ACA TTT AAA GTC CCT GAT AAT thr trp pro arg leu asp leu cys lys leu gly ser leu thr phe lys val pro asp asn

GTA AAA TAC CCA TCC ATG GAT CTG GCT TAC GCT GCT GGG CGC GCT GGA GGG ACC ATG ACT val lys tyr pro ser met asp leu ala tyr ala ala gly arg ala gly gly thr met thr

GGA GTT CTA AGT GCA GCA AAC GAG ATG GCA GTG GAA TTG TTT ATT AGT GAG AGA ATT AGC gly val leu ser ala ala asn glu met ala val glu leu phe ile ser glu arg ile ser

TAC TTG GAC ATT TTC AAG ATT GTG GAA CTA ACA TGC GCG AAG CAT CGG GAA GAG TTG GTG tyr leu asp ile phe lys ile val glu leu thr cys ala lys his arg glu glu leu val

TCT TCT CCA TCG CTG GAG GAA ATC ATA CAT TAC GAT TTG TGG GCT CGG GAT TAT GCA GCC ser ser pro ser leu glu glu ile ile his tyr asp leu trp ala arg asp tyr ala ala

AGT TTG GAA ACC ACG GCT GGT TTG AGT CCA GCT CTT GTA TGA ser leu glu thr thr ala gly leu ser pro ala leu val OPA

Figure 6: N. benthamiana OXR open reading frame containing the chloroplast transit peptide. The chloroplast signal peptide spans 66 residues with a cleavage site between residues GInSI and CysS2.

4.1.l DXR Sequence Analysis

Multiple amino acid sequence alignments with other plant OXRs produced

several conserved amino acids as shown in Figure 7 suggesting a common enzymatic

function in the MEP pathway in all plants. The inclusion of the E. coli and P.falctparum

proteins in the multiple alignment also showed several conserved regions implicating

conserved function in distant species. Using the CbloroP program [10] together with the

multiple alignment showed the N. benthamiana OXR as having a chloroplast transit

peptide and a cleavage site between residues position GInS} (marked with an asterix *)

and CysS2. The processing site of the transit peptide was predicted at the N-terminus ofa

conserved Cys-Ser-X mo~ where X means any of the hydrophobic residues Ala, Val, or

Met. The regions at the N-terminal or C terminal side of the putative processing site have

30

different structural features. At the N-terminal side, the sequence is poorly conserved but

enriched in Ser residues features that are common in chloroplast transit peptides. In

contrast, the extended region at the C-terminal side (positions 50-80 of N. benthamiana

DXR) is highly conserved and particularly rich in Pro residues. The Pro residues csn

range between 6 residues to 8 residues in the conserved region. The consensus motif

P(P/Q) PA WPG(Rff or Q in the case of Pip erIKa va DXR) A csn be defined in the Pro

rich region of plant DXR (positions 56-65 of the Nicotiana sequence).

Analysis of all the plant DXR sequences suggested that all plant DXRs have a

transit peptide fur plastids, and are processed at a conserved cleavage site and also

contain an extended Pro-rich region at the N terminus of the mature protein that was not

fuund in both prokaryote and P.fa/ciparum DXRs.

Nicotiana Lycopersicon catharanthus Antirrhinum Picrorhiza Mentha PlectranthuB Linum Arablciopsis Zea Oryza piper Escherichia Plasmodium

Nicotiana Lycopersicon Catharanthus Antirrhinum Picrorhiza Mentha Plectranthus Linum Arabldopsis Zea Oryza piper Escherichia Plasmodium

• -MALNLL-SPSEIKTISFLDT-SKSSYNLNHLKFQGGVAIKRKEiiSGISGKRVQCSV())\.- 56 -MALNLL-SPAEIKSISFLDN-SKSSYNLSIILKF'l'GGLSIRRKECSGAFAKRVQCSAQL- 56 -MALNSL-SPPKIKTISFLDS-SKSNYNLNLLKLPGGFAFKKKDFGASGGKKIQCSVQP- 56 -MALNML-SPSEIKSLSFLDS-SKSNYNLNLFKLQG---LKRKENGCSAVKRVQCLAQT- 53 -MALNLL-SPSEIKSLSFLES-SKSKSNFNSFKLQGGFSLKRKENGRTAALRVQCSASA- 56 -MAP------TEIKTLSFLDS-SKSNYNLNPLKFQGGFnEKRKDSGCTAAKRVHCSAQSQ 52 -MALN-----LElKALSFLDS-SKSSYNLNPLKLHGGFAFKRKDSRCSAPNRVHCSAQ-- 51 -MSLNML-SPAEVKSISFLDT-SKSFHSHALPKLPGGFSVKKK-SSALSLRRIQCSVQQS 56 MMTLNSL-SPAESKAISFLDT-SRFN---PIPKLSGGFSLRRRNOGRGFGRGVRCSVKVQ 55 MAALKAS-FRGELSAASFLDS-SRG----PLVQHKVDFTFQRKGKRAISLRRTCCSMQQ- 53 MALKVVS-FPGDLAAVSFLDS-NRGG---AFNQLKVDLPFQTRDRRAVSLRRTCCSMQQ- 54 -MALRLLHFPSELRGASFAESHGLVNHPIIKIILSAGETIFllRRKGNGVTSVKSVRCCSAQR 59

--------MKKYlylyFFFITITINDLVINNTSKCVSIERRKNNAYINYGIGYNGPDNKI 52

---PPP-AWPGRAVAEA-RKTWDGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 108 ---PPPPAWPGRAVAEPGRQSWDGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 110 ---PPP-AWPGRAVAEPGYKTiIEGQKPISIVGSTGSVGTQTLDlVAEN---PDKFRVVAL 109 ----PPPAWAGRAVADPGHKRWEGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 106 ---QPPPAWPGRAVANSGHKSWEGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 110 --S-PPPAWPGRAFPEPGRMTWEGPKPISVIGSTGSIGTQTLDlVAEN---PDKFRIVAL 106 --P-PPPAWPGRAVYEPGRKTWEGPKPISVIGSTGSIGTQTLDlVAES---PDKFRVVAL 105 --QQPPSAWPGTAIPEPGRKIWDGPKPISIVGSTGSIGTQTLDIVSEN---PDKFKVVAL 111 QQQQPPPAWPGRAVPEAPRQSWDGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 112 ---APPPAWPGRAVAEPGRRSWDGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 107 ---APPPAWPGRAVVEPGRRSWDGPKPISIVGSTGSIGTQTLDlVAEN---PDKFRVVAL 108 --QAPPPAWPGQAVPEPDKMRWDGPKPISIVGSTGSIGTQTLDIVAEN---PDKFEVVAL 114 ------------------------MKQLTILGSTGSIGCSTLDVVRHN---PEHFRVVAL 33 TKSRRCKRIKLCKKDLIDlGAIKKPINVAIFGSTGSIGTNALNllRECNKIENVFNVKAL 112

:::.*****:* .:*::: . *.: .* 31

Nicotiana Lycopersicon Catha ran thus Antirrhinum. Picrorhiza Mentha Plectranthus Linum Arabidops1s Zea Oryza Piper Escherichia Plasmodium.

Nicotiana Lycopersicon catharanthus Antirrhinum Picrorhiza Mentha Plectranthus Linum Arabidopsis Zea Oryza Piper Escherichia Plasmodium

Nicotiana Lycopersicon Catharanthus Antirrhinum Picrorhiza Mentha Plectranthus Linum Arabidopsis Zea Oryza Piper Escherichia Plasmodium

Nicotiana Lycopers!con catharanthus Antirrhinum Picrorhiza Mentha Plectranthus Linum Arabidopsis Zea Oryza piper Escherichia Plasmodium

Nlcotlana Lycopers!con Catharanthus Antirrhinum

AAGSNVTLLADQVKTFRPKLVAVRNESLVEELKDALADM-EDKPEIIPGEQGIIEVARHP 167 AAGSNVTLLADQVKTFRPKLVAVRNESLVEELKDALADM-EDKPEIIPGEQGVIEVARHP 169 AAGSNVTLLADQVKTFKPQLVSVRNESLVNELKEALSDV-DDKPEIIPGEQGVVEVVRHS 168 AAGSNVTLLADQIRTFKPQLVSVRDESLINELKEALFDV-EDKPEIIPGEQGIIEVARHP 165 AAGSNITLLADQIKTFKPELVSVRDESLIDELKEALADL-EHKPEIIPGEQGIIEVARHP 169 AAGSNVTLLADQVKAFKPKLVSVKDESLISELKEALAGF-EDMPEIIPGEQGMIEVARHP 165 AAGSNVALLADQVKAFKPKLVSIKDESLVSELKEALADV-EDKPEIIPGEQGMIEVARHP 164 AAGSNIALLADQIRTFKPQLVSVKNESLAKELKEALAGL-EVMPEIIPGEEGlVEVARHP 170 AAGSNVTLLADQVRRFKPALVAVRNESLlNELKEALADL-DYKLEIIPGEQGVIEVARHP 171 AAGSNVTLLADQVKTFKPKLVAVRNESLVDELKEALADC-EEKPEIIPGEQGVIEVARHP 166 AAGSNVTLLADQVKTFKPKLVAVRNESLVDELKEALADC-DlIKPEIIPGEQGVIEVARHP 167 AAGSNV'l'LLADQIKTFKPRLVSIKNEALLNELKDAlADA-DYKPEIIPGEEGVIEVARHP 173 VAGKNV'l'RMVEQCLEFSPRYAVMDDEASAKLLKTMLQGQ-GSRTEVLSGQQAACDMAALE 92 YVNKSVNELYEQAREFLPEYLCIHDKSVYEELKELVKNIKDYKPIILCGDEGMKEICSSN 172

: :. • • : ::: .. :: .::.

DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPINLPLAHRHK-VKTLPADS 226 DAVTVVTGIVGCAGLKP'l'VAAIEAGKDIALANKETLIAGGPFVLPPAHKHK-VKILPADS 228 DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGXPFVLPLAHKHK-VKILPADS 227 DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLAHKHK-VKILPADS 224 DAVTVVTGIVGCAGLKPTVAAlEAGKDIALANKETLIAGGPINLPLAHKHN-VKILPADS 228 DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLAKKHN-VKILPADS 224 DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLAHKHN-AKILPADS 223 DAATVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLAHKHK-VKILPADS 229 EAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLANKHN-VKILPADS 230 DAVTVVTGIVGCAGLKPTVAAlEAGKDIALANKETLIAGGPFVLPLAHKHK-VKILPADS 225 DAVTVVTGIVGCAGLKPTVAAIEAGKDIALANKETLIAGGPFVLPLAQKHK-VKILPADS 226 DAVTVVTGIVGCAGLRPTVAAlKAGKDIALANKETLIAGGPFVLPLAHEHK-VRILPADS 232 DVDQVMAAIVGAAGLLPTLAAIRAGKTILLANKESLVTCGRLFMDAVKQSK-AQLLPVDS 151 SIDKIVIGIDSFQGLYSTMYAIMNNKIVALANKESIVSAGFFLKKLLNIHKNAKIIPVDS 232

.• • * .... : .. * : ••••• :::: : .: :*. **

EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDLPVEKLKEVRVADALK 273 EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDWPVEKLKEVRVADALK 275 EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDWPVEKLEEVKVADALK 274 EHSAIFQCIQG-------------LPEGALRRVILTASGGAFRDLPVEKLKEVKVADALK 271 EHSAIFQCIQG-------------LPEGALRRVILTASGGAFRDLPVEKLKEVRVADALK 275 EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDLPVEKLEEVKVADALK 271 EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDLPVEKLKDVKVADALK 270 EHSAIFQCIQG-------------LPEGALRKIILTASGGSFRDLPVEKLKDVKVADALK 276 EHSAIFQCIQG-------------LPEGALRKIILTASGGAFRDWPVEKLKEVKVADALK 277 EHSAIFQCIQG-------------LSEGALRRIILTASGGAFRDWPVDRLKDVKVADALK 272 EHSAIFQCIQG-------------LPEGALRRIILTASGGAFRDWPVDKLEEVKVADALK 273 EHSAIFQCIQG-------------LPEGALRRIILTASGGAFRDWPVEKLKDVKVADALK 279 EHNAIFQSLPQP---IQHNLGYADLEQNGVVSILLTGSGGPFRETPLRDLATMTPDQACR 208 EHSAIFQCLDNNKVLKTKCLQDNFSKINNINKIFLCSSGGPFQNLTMDELKNVTSENALK 292 ........ : .: :: ...... ::.: . :. :.:

HPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYDDIEIVIHPQSIIHSMVETQDSSVL 333 HPNWNMGKKITVDSATLFNKGLEVIEAHYLFGAEYDNIEIVIRPQSIIRSMVETQDSSVL 335 HPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYONIDIVIHPQSIIHSMVETQDSSVL 334 RPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYODIEIVIHPQSIIHSMIETQDSSIL 331 HPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYODIDIVIHPQSIIHSMIETQDSSIL 335 HPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYODIEIVIHPQSIIHSMVETQDSSVL 331 HPNWNMGKKITVDSATLFNKGLEVIEAHYLFGAEYDDIEIVIRPQSIIHSMVETQDSSVL 330 HPNWSMGKKITVDSATLFNKGLEVlEAHYLFGADYDNIDIVIHPQSIIHSHIETQDSSVL 336 BPNWNMGKKITVDSATLFNRGLEVlEAHYLFGAEYDDIEIVIHPQSIIHSMIETQDSSVL 337 HPNWNMGRKITVDSATLFNKGLEVIEAHYLFGAEYDDIEIVIHPQSIIHSMVETQDSSVL 332 HPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYODIEIVIHPQSIIHSMIETQDSSVL 333 RPNWNMGKKITVDSATLFNKGLEVlEAHYLFGAEYODIEIVIHPQSIIHSMIETQDSSVL 339 HPNWSMGRKISVDSATMMNKGLEYIEARWLFNASASQMEVLIRPQSVIRSMVRYQDGSVL 268 HPKWKMGKKITID5ATMMNKGLEVIETHFLFDVDYNDIEVIVHKECIIHSCVEFIDKSVI 352 .. : .... : .. :: .... :: ....... ::: ...... ::::::. :.: ... : ... ::

AQLGWPDMRLPILYTLSWPDRIYCSEVTWPRLDLCKLGSLTFKVPDNVKYPSMDLAYAAG 393 AQLGWPDMRLPILYTLSWPDRVYCSEITWPRLDLCKLGSLTFKAPDNVKYPSMDLAYSAG 395 AQLGWPDMRLPILYTLSWPDRISCSEITWPRLDLCKLGSLTFKTPDNVKYPSMDLAYAAG 394 AQLGWPDMRLPILYTLSWPDRVHCSEITWPRLDLCKLGSLTFKVPDNVKYPSMDLAYAAG 391

32

Picrorhiza Mentha Plectranthus Linum. Arabidopsis Zea Oryza Piper Escherichia Plasmodium

Nicotiana Lycopersicon catharanthus Antirrhinum. Picrorhiza Mentha Plectranthus Linum. Arabidopsia Zea Oryza Piper Escherichia Plasmodium.

Nicotiana Lycopers!con Catharanthua Ant!rrhinum. Picrorhiza Mentha Plectranthus Linum Arabidopsis Zea Oryza Piper Escherichia Plasmodium.

AQLGWPDMRLPILYTLSWPDRIYCSEITWPRLDLCKLESLTFKSPDNVKYPSMDLAYAAG 395 AQLGJPDMRLPILYTLSWPERIYCSEITWPRLDLCKVD-LTFKKPDNVKYPSMDLAYAAG 390 AQLGJPDMRLPILYTLSWPDRVYCSEVTWPRLDLCKVS-LTFKTPDHVKYPSMALAYAAG 389 AQLGJPDMRLPILYTMSWPDQVPCSEVTWPRLDLCKLGSLTFRAPDNVKYPSMNLAYAAG 396 AQLGJPDMRLPILYTMSWPDRVPCSEVTWPRLDLCKLGSLTFKKPDNVKYPSMDLAYAAG 397 AQLGJPDMRLPILYTLSWPDRIYCSEVTWPRLDLCKLGSLTFRAPDNVKYPSMDLAYAAG 392 AQLGJPDMRIPILYTMSWPDRIYCSEVTWPRLDLCKLGSLT~DNVKYPSMDLAYAAG 393 AQLGJPDMRLPILYTLSWPERIYCSEKTWPRLDLCKLGTLTFKTPDNVKYPSMNLAYSAG 399 AQLGEPDMRTPIAHTllAWPNRVNSGVRP---LDFCKLSALTFAAPDYDRYPCLKLAIIEAF 325 SQMYYPDMQIPILYSLTWPDRIKTNLKP---LDLAQVSTLTFRKPSLSHFPCIKLAYQAG 409 :*: *.*: ** ::::**::: *.:.:: *.* * ::*.: •••

RAGGTMTGVLSAANEMAVSLFISERISYLDIFKIVSLTCAKllREELVSSPS---LEEIIR 450 RAGGTMTGVLSAANEKAVSLFISERISYLDIFKIVELTCAKHREELVSSPS---LEEIIH 452 RAGGTMTGVLSAANEKAVSLFIDEKISYLDIFKVV&LTCAKllQAELVTSPS---LDEIIH 451 RAGGTMTGVLSAANEKAVEMFIDEKISYLDIFKVV&LTCDRHRAELVTAPS---LEEIVH 448 RAGGTMTGVLSAANEKAVEMFIAEKIGYLDIFKVAELTCTKRQAELVTTPS---LEEIVH 452 RAGGTMTGVLSAAHEKAVEMFIDEKIGYLDIFKVV&LTCDKRRSEMAVSPS---LEEIVH 447 RAGGTMTGVLSAANEKAVEMFlNE-IGYLDIFKVVELTCDKRRAELVASPS---LEEIVH 445 RAGGTMTGVLSAANEKAVELFIDEKIAYLDIFKIV&LTCAKllllEELVTSPS---LEEIIR 453 RAGGTMTGVLSAAHEKAVEMFIDEKISYLDIFKVVELTCDKRRNELVTSPS---LEEIVH 454 RAGGTMTGVLSAANEKAVSLFIDEKISYLDIFKVVELTCNAHRNELVTSPS---LEEIVH 449 RAGGTMTGVLSAANEKAVSLFIDEKIGYLDIFKVVELTCDAHRNELVTRPS---LEEIIR 450 RAGGTMTGVLSAANEKAVEMFIDERINYLDIFKVVELTCEQRMNDIVTSPS---LEEIIH 456 EQGQAATTALNAANEITVAAFLAQQIRFTDlAALNLSVLEK--MllMREPQC---VDDVLS 380 IKGNFYPTVLNASNEIANNLFLNNKIKYFDISSIISQVLESFNSQKVSENSEDLMKQILQ 469

• . .•.• :*. : .: : . : .. YDLlIARDYAASLETTAG-LSPALV 473 YDLWARDYAASLEPSSG-LSPALV 475 YDLGARDYAASFQNSLG-LSPALV 474 YDLllAREYAANVQPIWl-LSPALV 471 YDLWARDYASNLKLATG-LSPALV 475 YDQIIAlUlYAATVLKSAG-LSPALV 470 YDQIIAlUlYAAELRRSAAGLSPALV 469 YDLWAKIlYAASLQQAHG-LSPALV 476 YDLlIAREYAANVQLSSG-ARPVHA 477 YDLliARRYAASLQPSSG-LSPVPA 472 YDLllAREYAASLQPSTG-LSPVPV 473 YDLWARDYAANYKTLSSGLNPVPV 480 VDANAREVAR--KEVMR-LAS--- 398 IHSWAKDKATDIYNKHN--SS--- 488

*: *

: . : : :

FIgure 7: Multiple alignment ofP1ant, E. coli and P.falciparum DXR. ••• means that the residues or nucleotides in that column are identical in all sequences in the alignment. n:n

means that conserved substitutions have been observed. n. n means that semi-conserved substitutions are observed. Gaps in the sequence is represented with a dash.

4.2 Construction of Reeomblnant Viral Vectors

Recombinant viral vectors were successfully constructed that incorporated the

cDNAs fur the purpose of studying gene silencing and overexpression in N. benthamioruI.

33

Four different recombinant viral vectors were successfully made and named

TIOSAlDXR- for silencing DXR, TIOSAlDXR for the overexpression ofDXR,

TIOSAIEPSPS- for the silencing ofEPSPS and TIOSAI PMT - for the silencing of

PMT.

TIOSAIDXR-

TMV RNA (.1

(a)

""",'"

TIOSAIPMT-

TMVBNA(+I

(c)

-' ....

• ..u , "'"

..", .....

pBII3"

-..

...

Sph1(31

TIOSAlDXR+

ThN RNA (+1

(b)

... ,,,

TIOSAlEPSP-

ntVRNA(+1

(d)

. nG!)

".,.,.", _ ..

...,22

AvrD C f1U)

..,.. .....

Figure 8: Recombinant viral vectors. (a) Recombinant viral vector inserted with partial antisense DXR eDNA fragment from N. benthamiana of size 452 bp. (b) The viral vector inserted with ful11ength DXR eDNA fromN. benthamiana of size 1422 bp. (c) Recombinant viral vector inserted with partial antisense PMT eDNA fragment of size 722 bp. (d) Recombinant viral vector with partial EPSP- eDNA fragment of size 710 bp.

34

4.3 RT PCR for the Verification of Inserts

To confirm the persistence of the production of the dxr-, dxr+, epsp-, gfp and pmt-

inserts in the recombinant viral vectors, RT PCR was carried out. The RT PCR confirmed

that the tobamovira1 vector with the insert was present thus producing the transcripts at

least until 15 days post inoculation.

1.8kb _

750b _

1234567

.', .' -'1, _. L.I..I'.' I .... ,

II';"" - I . .. ' I ~

(a)

1234567 1 2 3 4

950bp

I.Okb 1.0kb (b) (c)

Figure 9: RT PCR (a) Lane I: Control from non-infected plant. Lane 2: TTOSAlDXR+. Lane 3: RTPCR product from dxr overexpressed plant. Lane 5: RT PCR product from dxr antisense infected plant. Lane 6: TTOSAlDXR-. Lane 4 contains I kb ladder. (b) Lane I: Control from non-infected plant. Lane 2: TTOSAIAPEpBAD. Lane 3: RTPCR product fromgfp overexpressing product. Lane 4: I kb ladder. Lane5: RTPCR frompmt antisense infected plant. Lane 6: TTOSAIPMT-. (c) Lane I: TTOSAIEPSP. Lane 2: RT PCR product from EPSP synthase silenced plant. Lane 3: I kb ladder. Lane 4: Control from non-infected plant.

4.4 AppHcations of CIGE

4.4.1 Mimicking Herbicide Effects in N. benthamiana

Cytoplasmic inhibition of EPSP synthase genes in plants has been used to blOCk

aromatic amino acid biosynthesis and caused a systemic bleaching phenotype similar to

35

the RoundupTM herbicide. A dual subgenomic promoter vector encoding 1097 base pairs

of an antisense EPSPS gene from Nicotiana tabacum (Class I EPSP synthsase) was

previously developed by Kumagai and della-Cioppa. Systemic expression of the

Nicotiana tabacum Class I EPSP synthase gene in the antisense orientation caused a

systemic bleaching phenotype similar to the herbicide RoundupTM. To confirm these

fmdings, partial epsp synthase cDNA was subcloned through site directed mutagenesis

and placed under the transcriptional control of a tobamovirus subgenomic promoter in a

plant recombinant viral vector in the antisense direction. Infectious transcripts were

produced and subsequently inoculated onto N. benthamiana plants. The resulting

phenotype confrrmed previous fmdings by Kumagai and della-Cioppa in showing its

effect in mimicking the herbicide RoundupTM after 21 dpi

The N. benthamiana dxr was also modified by site-directed mutagenesis and

placed under the transcriptional control of a tobamovirus subgenomic promoter in a plant

recombinant viral vector that expressed the anti-sense partial dxr RNA transcripts. A

white phenotype plant was observed after three weeks post inoculation with the

recombinant viral vector that mimicked the effects offusmidomycin, a commercial

antibiotic that inlu"bits P.falciparum DXR. Specifically, it caused photobleaching at

approximately 21 dpi in newly formed leaves and eventuaIly in stems and other leaves.

The recombinant tobamoviral vector containing the antisense pds construct was

supplied by Monto Kumagai, which was used as another example to show the effect of

using CIGE to mimic another herbicide, norflurozon. After 21 dpi, the plants showed

36

phenotypic changes which were typical of the white phenotype produced by treatment of

plants with the herbicide, norflurozon.

antisense pds antisense dxr antisense epsp

norflurozon fosmidomycin RoundupTM

Figure 10: Mimicking the effects of herbicides. The plants infected with the antisense constructs started to turn white as the infection progressed. The infected plants were also compared with plants that were applied with the herbicide, norflurozon and RoundupTM and also with plants treated with the antibiotic fosmidomycin.

Plants were conferred partial res istance to the treatment of fosmidomycin and

norflurozon as compared to control plants through the recombinant viral vector

express ing dxr and pds in the sense direction suggesting that functional DXR and PDS

can be produced using transient expression of full length cDNAs. The synth.etically

37

derived version of epsp synthase had also conferred resistance to RoundupTM when

expressed in the plants using the recombinant tobamoviral vectors.

4.4.2 Manipulation of Alkaloid Production In N. benthamilma

N. benthamiana plants that were infected with the antisense pmt tobamoviral

vector construct TIOSAIPMT - were harvested for a bioassay study to elucidate the gene

silencing effect on the production of nicotine, one of many alkaloids in plants.

Preliminary investigation of the microarray data also revealed two poss~ble genes of

interest that was affected in pmt silenced plant

Table 1: Genes in Putrescine related pathway

Log Ratio Fold Gene8ank Name (594/635) Change Number

Spermidine/putrescine ABC transporter permease protein putative 0.415 2X 8Q507797 Spermidine synthase 1 (Putrescine amioopropyltransferase 1) (SPDSY 1) 1.322 4X 8Q121566

The Log ratio corresponding to log cy3/cy5 correlates to the log PMT silenced

plantlGFP plant which shows that both genes were upregulated in the PMT silenced plant

showing that increasing the level of putrescine can possibly upregulate these two genes.

Spermidine ABC transporter was shown to be upregulated two fold and spermidine

synthase was shown to be upregulated four fold due to the silencing effect of PMT.

38

Chapter Five Discussion

5.1 Mlmieking Herbicide Treatment

Many herbicides act by iohibiting a key plant enzyme or protein necessary for

growth. For example, the herbicide RounduplM destroys plants by iohibiting the activity

of an enzyme necessary for synthesizing aromatic amino acids. Some bacteria also

contain enzymes that confer resistance to herbicides. A gene encoding an herbicide

resistant enzyme from bacteria may be cloned, modified for expression in plants, and

inserted into crop plant genomes. When sprayed with such an herbicide, plants containing

the bacterial gene grow as well as unsprayed control plants. This strategy was utilized by

Monsanto to produce the RounduplM tolerant plants by inserting modified EPSP synthase

genes into the plant genome (U.S. Patent No. 5,312,910).

The strategy that was employed in this study involved the targeting of a specific

enzyme in a different pathway (MEP) that similarly affects the growth of the plant that

mimics the effects of an herbicide. Besides showing DXR as a potential herbicide target

by gene silencing, two other know examples were also used to show the ability of

antisense constructs to produce plants that mimicked the effect of herbicide treatments.

The expression of antisense pds mimicked the effect of plants being treated with

the herbicide nuroflurozon, the antisense rlxr mimicked the effect of plants being treated

39

with the antibiotic fosmidomycin and the antisense epsp synthase mimicked the effect of

plants being treated with the herbicide RoundupTM. Just as the RoundupTM resistant plants

were created using EPSP synthase as an herbicide target, it can be assumed that other

potential herbicide targets such as DXR can be used as substitutes for producing other

herbicides and engineered plants.

Farmers in the United Kingdom had been using eight different types of herbicides

to control weeds in sugarbeet thus creating unfavorable conditions to the environment.

Making more efficient herbicides and herbicide resistant plants will allow a wide

spectrum of weeds to be controlled thus limiting the use of many herbicides. Farmers can

thus optimize the survivability and reproduction capability of their crops by eliminating

the weeds efficiently [7). Herbicides that will target DXR as shown in this example using

an antibiotic that targets DXR will be non-toxic to animals thus providing a useful

strategy in increasing farm produce and at the same time keeping animals safe.

S.2 Manipulation of Bioehemieal Pathways

The fIrSt step of the plastidial mevalonate-inciependent pathway for the production

of isoprenoid precursors is catalyzed by DXPS [40), which also supplies precursor DXP

for the synthesis of thiamine and pyridoxol. The second step of the pathway is catalyzed

by DXR (for the conversion of DXP to methylerythritol phosphate, which is considered

the committed step in the supply of terpenoid and carotenoid precursors [44) and thus a

potential target for control of flux through this branch of the pathway. Based on this

40

understanding it was decided that the N. benthamiana DXR will be manipulated via

overexpression and silencing mechanism using viral vectors in order to evaluate the

influence of this gene on the MEP pathway and subsequent effect on carotenoid

biosynthesis.

By using the expression system of tobamoviral vectors, it was possible to note

genes such as dxr that can have an effect on the growth and development of the plant. An

accumulation of DXR most likely can affect other pathways. The overexpression of DXR

showed a phenotype that is suggestive of DXR's role in contributing to the regulation of

differential pathways in the plant that may contribute to plant developmental regulation.

An overexpressing DXR plant showed a unique phenotype with extensive lateral

branching. It did not escape our attention that such a phenotype is atypical and that DXR

could very well be important in the growth and development of the plant.

Although tobamovirus causes phenotypic changes in plants with leaf curling and

stunted growth, it cannot explain the atypical phenotype that we observed. A comparison

of the DXR overexpressing plant to that of the tobamovirus infected plant with GFP

insert and without any insert pointed to the contribution of the overexpressed DXR to the

phenotype. Figure 11 shows the overexpressing DXR plant, PMT silenced plant and GFP

expressing plant.

41

The PMT silenced plant using our recombinant viral vector showed no

developmental phenotypic difference as compared to the GFP expressing plant. Both

plants appeared stunted which is attributable to the effect of infection by the TMV.

(a) (b)

•

(c)

Figure 11: DXR+, PMT- and GFP Plants. (a) The DXR overexpressing plant shows extensive lateral branching implicating DXR's role in plant growth and development. (b) The GPF producing plant does not show the phenotypic changes observed in the DXR overexpressed plant. (c) The PMT silenced plant shows no drastic morphological distinctions between the GFP producing plant and itself. The mottled appearance and slight di coloration regions of PMT silenced plant hows where infection has progressed.

The N. benthamiana plant infected with the recombinant viral vector with no

transgene insert exhibited severe symptoms and died off fairly early within 2 weeks.

However, the tobamovirus that carries a transgene insert had a slower infectivity, the

42

viral symptoms were less severe and the plant could survive well above a month. The size

of the transgene insert may thus playa role in the ability of the recombinant vital vectors

to replicate efficiently and perhaps slow down the infection time and also reduce the virus

induced symptoms.

The white phenotype observed in transfected plants with the recombinant viral

vector containing the antisense dxr was a characteristic of the effect of silencing of the

OXR gene. Thus, it can be concluded that the recombinant viral vector was successfully

made and that it was effective in silencing the DXR gene through cytoplasmic inhibition.

This also conimns the effect of OXR as an important committed step in the production of

plastid isoprenoids and therefore the formation of chlorophyll and carotenoids. It is

interesting that OXR has also been found to be present in both plants and human

pathogens but not in humans. OXR can thus be used as a target for the screening of novel

pharmaceutical drugs such as against the malarial parasite P.falciparum.

5.3 Gene SDenclng Systems

Reverse genetics, in which a gene is made non-functional by inserting fragments

of ON A within its translated region, may cause an observable loss on an organism,

thereby providing a simple way to investigate its effect on the organism. Many strategies

have been employed in relation to mutagenizing genes in this manner. Some methods,

such as introducing T -DNAs and other transposon tagging methods have been developed

in plants [6].

43

Although these methods have enabled researchers to develop an extensive

resource fur gene function studies, a reverse genetics approach does not address the issue

of duplicated genes. As such, there have been many recent developments of various

VIGS strategies. Using such gene silencing strategies can circumvent limitations by

reducing the cost of gene knock-out studies and in studying duplicated genes to I'liminate

the issue of gene redundancy. Investigators usually will select a particular method

depending on their research interest.

In light of the RT PCR results, we have fuund the tobamovirus to be a very robust

vira1 vector. We would also like to find out how much longer could tobamoviruses with

transgene inserts persist in the plants. The RT peR results showed that the use of a

tobamoviral vector was suitable fur this project as it allowed us to see the effects of gene

silencing and overexpression over a prolonged period oftime.

The findings in this project are indicative of the potential use of our strategy in

studying gene function in plants and also in providing a quick and easy way to open up

avenues in producing herbicide and disease resistant plants. It also becomes a candidate

in the manipulation of nicotine modification which we believe can in the future lead to

the production of significant amounts of a1ka1oids or the decrease of a1ka1oids that will be

useful in pharmaceutical and medical applications.

44

From preliminary microarray data, it was shown that by silencingpmt two other

genes were upreguIated which are involved in a competing pathway as putrescine is

utilized both in making nicotine as well as in polyamines. Gene silencing methods used

to inhtbit PMr might possibly increase other alkaloids through differential pathways. An

assay is being conducted to measure the levels of nicotine which we hope to show

reduced levels in the plants that were infected with the recombinant viral vector

containing the antisense pmt construct.

5.4 DXR for Drug Design

It is now known that the antibiotic fusmidomycin acts on the DXR in P.

falciparum, the ma1arial causing agent. In light of this, it will be useful to study and

characterize the function ofDXR, its catalytic activity and perform molecular modeling

fur newer and more effective drugs. As such, a finding of the conserved region ofDXR

was investigated through the NCBI website and the fullowing comparison through double

alignment between N. benthamiana DXR and P. falciparum DXR was also conducted.

5.4.1 Functional Conservation across Plant, Protozoan and Bacterial DXR

The investigation and finding of conserved domains brings out an interesting

principle that although distinct organisms might not be closely related at the nucleotide

level, they may still share sufficient amino acid homology to retain useful functions such

as in the case ofDXR. This led us to perfurm a comparison between the protozoan,

45

bacterial and the plant DXR, which highlighted the similarity between the DXR at the

protein level.

10 20 30 40 50 60 10 80 ... . ~ .... 1_ . ....... r.·· . ····· I·~ · • .... 1_ . • .... 1_ . · _.1 .... · _ .1 ... . ' _ · 1

NBDXR 79 IsrVGSTGSIGTQTLDIVAENPOKFRWALAAGSNVTL LACQVKTFRPKLVAVRNE SLVEELKDALAOHEd kpelIPGEQ 1 58 con sensu s 1 LWLGSTGS T Gf<QT LQWRR FPDRFE LVGLAAGRNVELLLEQ IREFKPKYVAVY DI::TAYKDLKELPPHTE----VLLGEE 76

90 100 110 120 130 140 150 160 _ . • .. ·. 1- ... · ... 1_ · · .·.· 1_ . ~ ... . 1_ . • .... 1_. ' _ ·1 .. , · ' _ .1 _. · ' _ .,

NBDXR 159 GIIEVARHPDAVTWTGIVGCAGLKPTVAAlEAGKDIALANKETLlAGGPfVLPLAHKHKVKTLPAOSEHSAlfQCrQGL 238 consensus 17 GLKELAEECEADVVVNAIVGAAGLLPTLAAlKAGKTLALANKESLVAAGELVLKAARESGVQILPVDSEHNALFOCLOGV 156

170 180 190 200 210 220 230 2 40 _ . ' ._ . 1_ . ' _ .. 1_ . ' _ . 1_ . ' _ . 1_ . ' _ . 1 _ _ · _ .1 ... . · _ . 1 ... . · _ . r

NBDXR 239 PEGALRR tIL TASGGAflI.DLPVEKLKEVKVAOALKH PNWNMGKKITVDSATLFNKGLEVIEAIIY LfGAEYOO I E IVIH PO 318 consensus 151 KALGVKKLILTASGGPFRDKSLEELPHVTVEDALKHPNWSMGSKITVDSATLHNKGLEV lEAHW LFGIPYEEIDVVIHPQ 236

250 260 210 280 290 300 310 320 _ . ' - . 1- . · _ . I_ .~ _. I_ . ' _ . 1_. ' _ · 1_· · _ . 1 .... · _ . 1 .. · · · _ · 1

NBDXR 319 S r r HSMVETQOSSVLAQLGWPDHRLP 1 LYTLSWPDRI YCSevtWPRLDLCKLGSLTn<VPDN'lKY PSMDLAY AAGRAGGT 398 consensus 231 SIIHSMVEFIDGSVIAQLGPPDMRLPIQYALTYPERSPAP---AKPLDFLKLSS LTfEPPDTDRFPCLRLAKDAGLAGGA 313

330 340 350 360 3"10 380 ... . · _ . 1 ... . ·_ . 1 ... . ' _ · I'H ' · _ . 1 ... . · _ . IH .. • .... I .

NBDXR 399 MTGVLSAANEHAVELFISERISYLDIFKIVELTCAKHReeLVSSPSLEEIIHYDLWARDYA 459 consensus 31 4 MG1VLNAANEEAVAAFLAGEIGFLDIVDLIEQALEE HW- PYKPQSLEEVLEADAEMERA 372

Figure 12: Alignment between N. benthamiana DXR and E. coli DXR consensus region

The function between prokaryotic DXR and plant DXR in the terpenoid pathway

for the enzyme DXR has been conserved. It can thus be postulated that the bacterial

DXR can function in a plant. Based on this understanding, a double alignment between

the P.falciparum and the N. benthamiana DXR was also conducted. The alignment

between the two DXR showed high homology between them exhibiting 40% identity in

amino acids with a p value of 2e·73. This thus shows that the dxr genes have high

homology between plants, bacteria and P.falciparum.

NBDXR 76 PKPIS I VGSTGSIGTQTLDI VAEN---PDKFRVVALAAGSNVTLLADQVKT FRPKLVAVR 132 P ++1 GSTGS1GT L+1+ E + F V AL +V L +Q + F P+ + +

PFDXR 77 P1NVA1FGSTGS1GTNALNI IRECNKIENVFNVKALYVNKSVNELYEQARE FL PEYLCIH 136

NBDXR 133 NESLVEELKDALADME D- KXXXXXXXXXXXXVARHPDAVTV VTGI VGCAGLKPTVAAIEA 191 ++ S+ EELK+ + +++ 0 KP 1+ G++G+ E+ +V GI GL T+ AI

PFDXR 137 DKSVY EELKELVKNIKDYKP1 1LCGDEGMKEI CSS NS1DK1 V1 G1 DSFQGL YSTMYAIMN 196

NBDXR 192 GKDI ALANKETLIAGGPFVLPLAHKH K-VKTLPAOSEHSA1FQC1QGLPE---------- 240 K +ALANKE++++ G F+ L + HK K +P OSEHSAIFQC+

PFOXR 197 NK1VALANKES1VSAGFFLKKLLNI HKNAKI1 PVOSEHSAIFQCLONNKVLKTKCLQDNF 256

46

NBDXR 241 ---GALRRI I LTAS GGAFRDLPVEKLKEVKVADALKHPNWNMGKKITVDSATLFNKGLEV 297 + +1 L +SGG F++ L +++ LK V +ALKHP W MGKKIT+DSAT+ NKGLEV

PFDXR 257 SKINNINKI FLCSSGGPFQNLTMDELKNVTSENALKHPKWKMGKKITI DSATMMNKGLEV 316

NBDXR 298 IEAHYLFGAEYDDIEIVI HPQS IIHSMVETQDSSVLAQLGWPDMRLPILYTLSWPDRIYC 357 IE H+LF +Y+DIE+++H + IIHS VE D SV++ Q+ +PDM++PILY+L+WPDRI

PFDXR 317 IETHFLFDVDYN DIEVIVHKEC IIHSCVEFI DKSVIS QM YY PDMQI PILYSLTWPDR1-- 374

NBDXR 358 SEVTWPRL DLCKLGSLTFKVPDNVKY PSMDLAYAAGRAGGTMTGVLSAANEMAVELFISE 41 7 + LDL ++ +LTF P +P + LAY AG G VL+A+NE+A LF++

PFDXR 375 - KTNLKPLDLAQVSTLTFHKPSLEHFPC IKLAYQAGIKGNFYPTVLNAS NE I ANNLFLNN 433

NBDXR 418 RI SYLDI FKI VELTCAKHREELVSSPS---LEEI I HY DLWARDYAASL 462 +1 Y D1 1+ + VS S +++ 1+ WA+D A +

PFDXR 434 KI KYFDISS I ISQVLESFNSQKVS ENSEDLMKQI LQIHSWAKDKATDI 481

Figure 13: N. benthamina (NBDXR) and P./alciparum (PFDXR) double alignment Score = 280 bits (716), Expect = 2e-73. Identities = 166/408 (40%), Positives = 247/408 (60%), Gaps = 24/408 (5%). Red colored residues show identical amino acids.

5.4.2 Future Goals and Potential of DXR Research

The goal in future is to study the expression using the P./alciparum dxr.

Furthermore, based on the amino acid alignment above, we noticed that there is a strong

conserved domain between the prokaryotic, protozoan and eukaryotic DXRs wlllch we

believe can complement mutant plants lacking DXR. We therefore deduce that the

expression of P.falciparum DXR in plants can facilitate the discovery of novel

compounds for antibiotic production. Conservation is only at the amino acid level as a

double aI ignment between the N. benthamiana dxr nucleotide sequence and the P.

/alciparum nucleotide sequence did not reveal any significant similarity thus preventing

us from proceeding to u e an antisense construct of P.falciparum dxr to silence

endogenous N. benthamiana dxr expression.

47

Figure 14: Molecular model of the conserved domain of DXR. The 3-dimensional conformation shows a groove pred iCling a catal ytic site that can be used for drug des ign.

A possible method for future research might be to find E. coli mutants thaI are

resistant against fosmidomcyin. These resistant DXRs can then be modified and cloned

into plants to create transgenic herbicide resistant plants. An alternative approach is also

to look at molecular modeling and to design oligonucleotides to make similar varying

protein structures based on the conserved regions that will be effective in its catalytic

activity as well as resistant to herbicides.

The main conclusion for this project is that we had modified our initial strategy in

using a high throughput system to characterize uncharacterized genes to selectively target

two genes to study its silencing effect as well as to study the overexpression of DXR in

N. benthamiana using viral vectors. This is to show that viral vectors can be used for

functional genomic studies. This led to the discovery of a possible herbicide target gene,

48

which is DXR in N. benthamiana. The overexpression ofDXR was also shown to have

produced a unique phenotype that showed it had affected the growth of N. benthamiana

with extensive lateral branching of the leaves. This result is thus suggestive of DXR's

role in the growth and development of plants.

Another important function of this system that we believe could be useful in the

future is in the targeting of genes that are involved in cell signaling pathways. This can

lead us to learn more about protein conservation across plant and animal systems and also

provide insights into how homologous proteins might have evolved differential functions

in organisms.

Gene silencingloverexpression

1 Herbicide target (DXR)

Growth and Identification of Genes --_~ Development

Cell Signaling

Human diseases Evolutionary studies Drug design

Figure 15: Flow chan of functional studies. The flow diagram shows how by using gene silencing and overexpression, we might learn about identification of gene functions and which may lead to several downstream functional studies.

49

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