POKEWEED ANTIVIRAL PROTEIN-MEDIATED RESISTANCE TO CITRUS
PATHOGENS
By
YOLANDA PETERSEN
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2003
Copyright 2003
by
Yolanda Petersen
To my parents
ACKNOWLEDGMENTS
The completion of this dissertation would not have been possible without the help
and support of many people. First, I would like to express my sincere gratitude and
appreciation to my advisor Dr. Richard F. Lee for his support and encouragement, and for
having me in his lab. I would like to thank Dr. G. Moore, Dr. D. Pring and Dr. G. Wisler
for serving on my committee and reviewing this manuscript.
I would like to thank Dr. G. Moore and the members of her lab for always
answering my questions; and for their help in facilitating my research. Some people who
deserve special mention are Dr. K.L. Manjunath, Dr. V.J. Febres, A. Guerra, Dr. K.
Biswas. Their help and understanding during two lab relocations is much appreciated. I
want to thank Professor W.O. Dawson and the people in his lab for providing me with the
CTV infectious clone and for assisting me with my CTV replication experiment, which I
conducted in their laboratory. I thank E. Crawford and E. Philmon for taking care of my
plants in the greenhouse and growth rooms, in Gainesville. Without them, a lot more
plants would have died. I also want to thank Neil for helping me graft and inoculate my
transgenic citrus plants as well as for taking care of these plants in Lake Alfred. I would
like to thank the ladies in the main office for their patience and taking care of the
numerous problems that arose.
I want to thank all the wonderful friends I made in Gainesville as well as friends in
Germany, Colorado, New Zealand, and South Africa who were always ready to lend an
ear and give me their unconditional support.
iv
My most sincere thanks and appreciation go to my parents and family, for their
understanding and support, and for always believing in me.
v
TABLE OF CONTENTS Page ACKNOWLEDGMENTS ................................................................................................. iv
LIST OF TABLES............................................................................................................. ix
LIST OF FIGURES .............................................................................................................x
ABSTRACT...................................................................................................................... xii
CHAPTER 1 LITERATURE REVIEW .............................................................................................1
Introduction...................................................................................................................1 Citrus exocortis viroid (CEVd).....................................................................................1
Movement of Viroids ............................................................................................2 Replication of Viroids ...........................................................................................3
Citrus tristeza virus (CTV)...........................................................................................4 Classification and Genome Organisation of CTV.................................................5 Replication and Expression Strategy of CTV .......................................................7 Control of CTV......................................................................................................8 Genetic Engineering for Virus/Viroid Resistance...............................................10 Ribosome-Inactivating Proteins (RIPs) ...............................................................11 Enzymatic Activities of RIPs ..............................................................................12 Antiviral Activity of RIPs ...................................................................................13 RIPs in Transgenic Plants....................................................................................14
2 In vitro TRANSLATION OF A Citrus tristeza virus REPLICON AND THE
EFFECT OF POKEWEED ANTIVIRAL PROTEIN ON ITS TRANSLATION .....16
Introduction.................................................................................................................16 Materials and Methods ...............................................................................................19
Source of Viral Nucleic Acid ..............................................................................19 In vitro Transcription and Translation of CTV-∆Cla..........................................19 In vitro Antiviral Assay .......................................................................................20
Results.........................................................................................................................21 In vitro Translation of CTV-∆Cla .......................................................................21 PAP Inhibits Translation of CTV-∆Cla Transcripts............................................23
Discussion...................................................................................................................24
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3 TRANSFORMATION OF Citrus paradisi cv Duncan GRAPEFRUIT WITH POKEWEED ANTIVIRAL PROTEIN......................................................................27
Introduction.................................................................................................................27 Materials and Methods ...............................................................................................32
PAP Gene Constructs and Plasmid Expression Vectors .....................................32 Citrus Transformation and Regeneration ............................................................33
Seed germination..........................................................................................35 Transformation of epicotyl segments ...........................................................35 Selection and regeneration of plantlets ........................................................35
Analysis of Transgenic Plants .............................................................................37 Fluorescent microscopy................................................................................37 Polymerase chain reaction (PCR) analysis...................................................37
Southern hybridization ........................................................................................38 Reverse-transcription (RT) PCR .........................................................................39 Western blot analysis...........................................................................................39 Grafting and Challenge of Transgenic Plants with CTV.....................................40 Challenge of Transgenic Plants with Citrus exocortis viroid (CEVd) ................40
Results.........................................................................................................................42 Transformation and Regeneration .......................................................................42 Southern Blot Analysis and RT-PCR of Transgenic Plants ................................46 Protein Expression Analysis................................................................................48 Virus Challenge of Transgenic Plants .................................................................49 Viroid Challenge of Transgenic Plants................................................................51
Discussion...................................................................................................................51 4 THE EFFECT OF POKEWEED ANTIVIRAL PROTEIN ON THE
REPLICATION OF Citrus tristeza virus IN Nicotiana benthamiana PROTOPLASTS.........................................................................................................57
Introduction.................................................................................................................57 Materials and Methods ...............................................................................................59
Constructs ............................................................................................................59 Tobacco Transformation .....................................................................................59
Explant preparation ......................................................................................59 Transformation and regeneration of tobacco................................................60
Analysis of Transgenic Plants by PCR................................................................60 Southern Hybridization .......................................................................................61 Reverse-Transcription (RT) PCR ........................................................................61 Analysis of Protein Expression ...........................................................................62 Determination of Antiviral Properties of Transgenic Plants ...............................63 Viral Replication in Transgenic Protoplasts........................................................63
Results.........................................................................................................................64 Transformation and Regeneration .......................................................................64 Molecular Analysis of Transgenic Plants............................................................65 CTV Replication in PAPn-Transgenic N. benthamiana Protoplasts...................69
Discussion...................................................................................................................70
vii
5 SUMMARY AND CONCLUSIONS.........................................................................73
LIST OF REFERENCES...................................................................................................78
BIOGRAPHICAL SKETCH .............................................................................................90
viii
LIST OF TABLES
Table page 3-1. Expected properties of PAP and PAP mutants. ..........................................................31
3-2. Sequences of oligonucleotide primers used for amplification and mutagenesis of the PAP gene ............................................................................................................33
3-3. Summary of results for the transformation of Citrus paradisi cv. Duncan grapefruit with pokeweed protein (PAP) antiviral constructs. .................................45
3-4. CTV antigen levels four months post-inoculation.in transgenic Citrus paradisi plants expressing PAPn............................................................................................51
4-1. Summary of N. benthamiana transformation experiments with the PAP constructs..................................................................................................................65
4-2. TMV antigen levels in transgenic N. benthamiana expressing PAPn following infection with the virus.............................................................................................68
ix
LIST OF FIGURES
Figure page 1-1. Symptoms caused by Citrus tristeza virus ...................................................................6
2-1. Schematic representation of the Citrus tristeza virus (CTV) genome........................17
2-2. Optimization of CTV-∆Cla replicon translation in wheat germ extract.....................22
2-3. Optimization of CTV-∆Cla translation in rabbit reticulocyte lysate..........................23
2-4. Effect of wild type PAP on translation.......................................................................24
3-1. Cloning strategy of the PAP constructs for plant transformation...............................34
3-2. Agrobacterium-mediated transformation of etiolated Citrus paradisi cv. Duncan grapefruit and regeneration of plants .......................................................................36
3-3. Analysis of GFP expression in citrus shoots regenerated from epicotyl segments transformed with pCambia2202-PAPc and -PAPn ..................................................43
3-4. PCR analysis of potentially transgenic shoots containing the PAPn transgene ......44
3-5. Phenotypes of plants transformed with the PAPc and PAPv constructs ....................47
3-6. Southern analysis to determine transgene copy number in transgenic grapefruit plants transformed with PAPn..................................................................................48
3-7. RT-PCR analysis of transgenic citrus transformed with PAPn to determine gene expression at the level of transcription.....................................................................49
3-8. Western blot analysis of total soluble proteins from transgenic citrus to determine the expression the PAPn transgene ..........................................................................49
3-9. RT-PCR analysis of representative viroid-infected PAPn-transgenic plants. ............52
4-1. PCR analysis of selected potential transgenic N. benthamiana plants using primers CN444 and CN445 ...................................................................................................65
4-2. Southern analysis of R0 tobacco plants transformed with PAPn. ..............................66
x
4-3. RT-PCR analysis of RNA isolated from selected transgenic plants to determine PAPn expression at the transcriptional level............................................................67
4-4. Northern blot analysis of CTV RNAs in N. benthamiana protoplasts derived from PAPn transgenic R1 plants .......................................................................................69
xi
Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
POKEWEED ANTIVIRAL PROTEIN-MEDIATED RESISTANCE TO CITRUS PATHOGENS
By
Yolanda Petersen
December 2003
Chair: Richard F. Lee Major Department: Plant Pathology
Citrus production is adversely affected by a number of diseases caused by viruses
and viroids. This project has employed a ribosome-inactivating protein, pokeweed
antiviral protein (PAP) isolated from Phytolacca americana in an effort to control Citrus
tristeza virus (CTV) and citrus exocortis viroid (CEVd). PAP has antiviral activity and
can act directly on RNA. The applicability of PAP to inhibit CTV translation was
evaluated in vitro. In vitro transcription and translation assays using the CTV replicon,
CTV-∆Cla, and wild type PAP showed that viral translation decreased in the presence of
increasing concentrations of PAP. In vivo experiments required the production of non-
toxic N-terminal (Gly75-Ala), active site (Glu176-Ala) and C-terminal deletion mutants
(Trp237-Stop), which were then used to transform Citrus paradisi cv. Duncan by
Agrobacterium-mediated transformation. The overall transformation efficiency was low,
at 8.6% as determined by the percentage of regenerated plantlets that were PCR-positive
for the PAP transgene. Six surviving transgenic plants containing the N-terminal mutant
xii
were obtained and evaluated by virus/viroid challenge. Molecular characterization of
these plants showed that the plants contain one to four copies of the mutated PAP gene
and that the gene was being expressed at the transcriptional and translational levels. The
transgenic citrus plants were challenged with CTV and CEVd by graft-challenge
inoculation. Four months post-inoculation the transgenic plants had significantly lower
levels of CTV, in comparison to the non-transgenic control plants. All plants supported
viroid replication at levels equivalent to that of the non-transgenic controls. To obtain a
more accurate view of the effect of the N-terminal mutant on CTV replication, Nicotiana
benthamiana was transformed with the mutated PAP gene used for citrus transformation.
Five lines of transformed N. benthamiana were selected for protoplast experiments in
which CTV virions were used as inoculum for transfection. Results showed that the lines
TN5, TN11 and TN23a supported lower levels of CTV replication relative to the non-
transgenic control. Thus, the PAPn mutant does not inhibit CEVd replication; and this
mutant reduces, but does not completely inhibit the replication of CTV in citrus plants
and tobacco protoplasts.
xiii
CHAPTER 1 LITERATURE REVIEW
Introduction
Diseases caused by viruses and viroids have had major impacts on citrus
production. Citrus tristeza virus (CTV) alone has caused the loss of millions of trees
worldwide. The propagation of viroid-infected trees has caused economic losses in the
Florida citrus industry and is a major threat in regions where susceptible rootstocks are
used. Once trees are infected, the disease agents cannot be eliminated. This is of
particular economic importance, since citrus trees should have a long productive life.
Thus the best control measures would be to prevent the introduction of the disease or
propagate plants that are disease resistant.
Citrus exocortis viroid (CEVd)
Viroids are small unencapsidated circular, highly base-paired RNAs which adopt
rod-like structures in their native state (reviewed by Hull, 2001). Viroid RNAs do not
encode any proteins. Citrus viroids are grouped into six classes: Citrus exocortis viroid
(CEVd, 370-375 nucleotides (nt)), Citrus bent leaf viroid (CBLVd, 315-329 nt; formerly
called CVd-I), Hop stunt viroid (HSVd, 295-303 nt; formerly called CVd-II), Citrus
viroid III (CVd-III, 291-297 nt), Citrus viroid IV (CVd-IV, 284-286 nt) and Citrus viroid
OS (CVd-OS, 329-331 nt) (Duran-Vila et al., 2000; Ito et al., 2000, 2002). CEVd is
usually found in combination with other viroids; and in the field causes mild to severe
stunting and bark scaling on susceptible rootstocks. The disease is exacerbated on citrus
trees propagated on rootstock derived from Poncirus trifoliata or hybrids that have P.
1
2
trifoliata as one of their parents. These rootstocks are used in many citrus-growing
regions. The severe stunting results in a reduced canopy with an adverse effect on fruit
yield. Less severe symptoms occur on Citrus sinensis X P. trifoliata (citrange) and Citrus
limonia (Rangpur lime) and only mild symptoms are usually seen on Swingle citrumelo
(Lee et al., 2003). CEVd and viroids in general are spread primarily via infected
budwood and through the use of contaminated cutting tools and equipment. Thus these
agents are relatively easily controlled through budwood certification programs and good
hygienic practices. CEVd can be detected by biological indexing on the indicator host
Citrus medica cv. etrog (Etrog citron), a process that takes 3 to 6 months or via more
rapid molecular methods such as analysis by sequential polyacrylamide gel
electrophoresis and the reverse transcriptase-polymerase chain reaction.
Movement of Viroids
From experiments involving fractionating components of viroid-infected cells,
confocal laser scanning microscopy, transmission electron microscopy, and in situ
hybridisation, it became evident that most viroids are located in the cell nucleus; with the
exception of Avocado sunblotch viroid (ASBVd), which is found in the chloroplasts
(reviewed by Hull, 2001). CEVd accumulates in the nucleoplasm (Bonfiglioli et al.
1996). Systemic movement of viroids that replicate in the nucleus follows four distinct
steps: (1) import into the nucleus via the nuclear pores (2) export out of the nucleus (3)
cell-to-cell movement and (4) long-distance movement.
Experiments with potato spindle tuber viroid (PSTVd) led to the conclusion that
viroids move rapidly from cell to cell via the plasmodesmata; and that the movement is
mediated by a specific sequence or structural motif (Ding et al., 1997). The spread of
PSTVd in tomato and Nicotiana benthamiana followed the pattern of photoassimilate
3
transport, suggesting that PSTVd moves long distance through the phloem. (Palukaitis et
al., 1987; Zhu et al., 2001). Maniataki et al. (2003) and Owens et al. (2001) showed that
PSTVd interacts with host cell proteins and suggested that these proteins might facilitate
systemic movement of the viroid. Maniataki et al. (2003) also showed that the region
interacting with the host cell protein consisted of eight nucleotides in the TR (terminal
right) region of the viroid RNA. The TR region has a bulged hairpin secondary structure
reminiscent of micro-RNA precursors, and it is thought that the micro-RNA precursors
are capable of systemic movement.
Replication of Viroids
Despite their small size, the elucidation of viroid replication is remarkably
complex. Viroid-infected tissue contains a variety of multimeric RNAs, and replication is
thought to proceed via a ‘‘rolling circle’’ mechanism. There are two models for rolling
circle replication: (1) the symmetric pathway in which both circular forms of the (+) and
(-) strands serve as templates for the synthesis of complementary multimers that are
cleaved into monomers and ligated to form circular molecules, and (2) the asymmetric
pathway in which the circular (+) strand serves as a template for synthesis of multimeric
linear (-) strands, which in turn serve as templates for the synthesis of multimeric (+)
strands that are cleaved and ligated to produce the final circular forms. CEVd and other
viroids in the family Pospiviroidae replicate via the asymmetric pathway (reviewed by
Hull, 2001).
Replication does not require a helper virus; and the viroid RNA does not encode
any proteins necessary for replication. This suggests that the information necessary for
replication resides in the unusual secondary structure of the viroid genome. Localization
of CEVd in the nucleus suggests that a host nuclear RNA polymerase may be involved
4
(Bonfiglioli et al., 1996). The fact that viroid RNA synthesis is inhibited by α-amanitin
indicates that a host-encoded RNA polymerase II may be required for replication
(reviewed by Hull, 2001). Warrilow and Symons (1999) obtained direct evidence for the
association of CEVd with a host-encoded RNA polymerase II.
Citrus tristeza virus (CTV)
Citrus tristeza virus is one of the most important pathogens of citrus. CTV is
endemic to most citrus-growing regions and in the past has resulted in devastating
economic losses due to decline of sweet orange scions on sour orange rootstock (Bar-
Joseph et al., 1989). Hence, the name “tristeza” which in Spanish means “sadness”.
CTV causes a number of different symptoms depending on the virus strain and the
host/rootstock combination. Decline strains result in decline and death of citrus species
on sour orange rootstock as a result of phloem necrosis at the graft union which prevents
the movement of nutrients from the canopy to the root system (Figure 1-1 A; Garnsey et
al., 1987). Other strains express stem pitting symptoms on either sweet orange and/or
grapefruit scions irrespective of the rootstock. Stem pitting reduces tree vigor, fruit yield
and quality (Figure 1-1 B and C). Vein corking (Figure 1-1 D) is induced by some strains,
and a syndrome called seedling yellows, which is expressed as stunting and chlorosis of
grapefruit, lemon and other susceptible varieties.
The virus is spread by aphid vectors including Toxoptera citricida (brown citrus
aphid), T. aurantii, Aphis gossypii, and A. spiraecola, and by propagation of infected
tissue (Bar-Joseph et al., 1989; Roistacher and Bar-Joseph, 1987). T. citricida is the most
efficient vector of CTV; and its introduction into Brazil and Argentina in the 1930s
caused the death of 16 million trees on sour orange rootstock as a result of the efficient
5
transmission of decline strains of CTV (Carver, 1978). The brown citrus aphid was first
reported in Florida in 1995 (Halbert and Evans, 1996). This vector has been shown to
transmit CTV strains that cannot be transmitted by other vectors (Roberts et al., 2001).
Thus the presence of the brown citrus aphid may cause a shift in the major viral
populations in the state and result in the appearance of CTV strains causing more severe
symptoms than in the past.
Classification and Genome Organisation of CTV
Citrus tristeza virus belongs to the family Closteroviridae within the order
Nidovirales. The virus structure is a long flexuous rod 2000 x 11 nm in size. The viral
genome consists of a single-stranded, positive-sense RNA and at approximately 19 kb,
this genome is one of the largest known for plant viruses (Karasev et al., 1995). The RNA
is encapsidated by two coat proteins. Ninety-five percent of the genome is encapsidated
by the major coat protein (p25); the remaining 5% is protected by the divergent minor
coat protein (p27), which gives the virus a characteristic “rattlesnake” appearance (Febres
et al., 1996).
The CTV genome has 12 open reading frames with the potential to code for 17
proteins (Karasev et al., 1995; Pappu et al., 1994). Some of the genes have been assigned
functions based on sequence homology to other viral genomes. The arrangement and
expression strategy of the replicase region of CTV (Karasev et al., 1995) resembles that
of the animal Coronaviruses (Lai et al., 1990). ORF1a is expressed as a 349 kDa
polyprotein and encodes two papain-like proteases, a methyltransferase-like and a
helicase-like domain. ORF1b encodes a RNA-dependent RNA polymerase (RdRp). The
protein encoded by the p20 gene is involved in the accumulation of amorphous inclusion
bodies in the phloem (Gowda et al., 2000).
6
The p65 and p61 proteins share homology with the cellular heat shock protein
HSP70 and the cellular chaperone HSP90, respectively (Pappu et al., 1994). There is
evidence that these proteins are required for efficient virus assembly (Satyanarayana et
al., 2000). The gene product of p23 was shown to be an RNA-binding protein involved in
the asymmetric accumulation of RNA (Lopez et al., 2000; Satyanarayana et al., 2002b).
The functions of other genes remain to be elucidated. The ten 3' genes are expressed via
3' co-terminal subgenomic RNAs (Karasev et al., 1997).
Figure 1-1. Symptoms caused by Citrus tristeza virus. A) Decline of sweet orange on
sour orange rootstock (R. F Lee). B) Stem pitting on Pera sweet orange (R. F. Lee). C) Reduced fruit quality of a Marsh grapefruit tree infected by severe stempitting isolates, grafted onto rough lemon rootstock (R. F. Lee). D) Vein corking on leaves of a Mexican lime seedling inoculated with a severe seedling yellows isolate (C. N. Roistacher). Photographs were downloaded from http://www.ecoport.org. The names of the authors are given in parentheses.
7
Replication and Expression Strategy of CTV
The small genome size of viruses has led to the evolution of a number of different
expression strategies that maximize the number of products encoded by the genome. The
genome organization of CTV has similarities with both the animal Coronaviruses and the
alpha-supergroup of viruses. The amino acid sequence of the replicase-associated
domains and the lack of a common 5' leader sequence in the subgenomic RNAs are
similar to the alphavirus supergroup (Dolja et al., 1994). Like the Coronaviruses, the
replicase-associated proteins of CTV are expressed as a polyprotein, which is then
proteolytically processed into equimolar amounts of nonstructural proteins. The
proteolytic activity of the two proteases has been demonstrated in vitro (Vazquez-Ortiz,
2001). The RdRp is expressed via a +1 frameshift that occurs at a frequency of 1-5%
(Cevik, 2001). The 3' ORFs are expressed via positive and negative sense 3' co-terminal
subgenomic RNAs (Karasev et al., 1995), which are temporally and quantitatively
regulated (Navas-Castillo et al., 1997).
The phloem-limited nature of the virus, low concentrations in trees, and large
complex genome has hindered progress towards understanding the replication strategy of
CTV. The development of a full-length cDNA clone and a smaller replicon, designated
CTV-∆Cla (Satyanarayana et al., 1999), has clarified the function of some of the
replication-associated genes such as p23, which is involved in the asymmetrical
accumulation of RNA (Satyanarayana et al., 2002b); and elucidated replication signals
present in the 3' UTR (Satyanarayana et al., 2002a). Also, Satyanarayana et al. (1999)
showed that p23, ORFs 1a and 1b and the 5' and 3' UTRs are sufficient to support viral
replication. Additionally, the infectious cDNA clones have proved useful in determining
8
the gene products required for virion assembly (Satyanarayana et al., 2000). CTV
produces a complex array of RNAs, including a genome-length negative-sense RNA
which acts as a template for further transcription; subgenomic RNAs, both single and
double-stranded (Hilf et al., 1995; Mawassi et al., 1995a); positive-sense low molecular
weight transcripts (LMTs) and large molecular weight transcripts (LaMTs) (Che et al.,
2001; Mawassi et al., 1995b). In total, more than 35 RNA species have been shown to be
produced during the replication of CTV.
Control of CTV
CTV has a complex population structure in its perennial hosts. In nature any one
tree is infected by a CTV population that is made up of a number of genetic variants,
some of which may exhibit great variation in sequence similarity (Ayllόn et al., 1999;
Brlansky et al., 2003; Hilf et al., 1999). One of these variants might predominate, but it is
not known whether the symptoms exhibited by the infected host are due to the
predominant strain or a result of an interaction among the different strains. The structure
of these populations is subject to change with time, since some CTV strains are more
efficiently transmitted by certain aphid species.
A number of strategies have been developed to control CTV. The strategies include
eradication programs to prevent the spread of the virus, quarantine and budwood
certification to prevent the introduction of CTV, the use of CTV-tolerant rootstocks, mild
strain cross protection, breeding for resistance, and genetic engineering (reviewed by
Ferguson and Garnsey, 1993). Mild strain cross protection is the phenomenon in which a
plant is deliberately infected with a mild strain of a virus which then serves to prevent or
delay subsequent infection or expression of more severe strains of that virus (Fulton,
1986). This strategy has been used in a number of countries including Australia, Brazil,
9
South Africa and Florida (Barkley et al., 1990; Costa and Muller, 1980; Lee and Rocha-
Peňa, 1987). However, this type of protection is not the same as resistance and will
eventually break down, resulting in the loss of groves due to the expression of severe
strains that may or may not have been present in the original isolate used to protect the
trees.
A few citrus relatives have been found to be immune to CTV; that is, they do not
support replication of the virus (Garnsey et al., 1987). However, only one species
(Poncirus trifoliata) is sexually compatible with commercial citrus species. Introgression
of the resistance found in P. trifoliata into commercial cultivars has not produced viable
scion varieties since genetic breeding introduced undesirable fruit characteristics along
with CTV-resistance (Ferguson and Garnsey, 1993). Resistance to CTV has been mapped
and shown to be controlled by a single dominant gene, Ctv (Gmitter et al., 1996; Yoshida
et al., 1996). The mechanism of action of this gene is currently unknown, since
protoplasts of resistant plants support viral replication (Albiach-Marti et al., 1999). The
progress that has been made toward mapping the location of Ctv (Deng et al., 1996, 2001;
Fang et al., 1998) makes cloning of the gene and using it to transform commercially
important citrus cultivars a reality. With the development of an efficient Agrobacterium-
mediated citrus transformation protocol (Luth and Moore, 1999), it is now possible to use
molecular methods to engineer resistance to CTV. Thus far an attempt at engineering
pathogen-derived resistance, using the coat protein gene of various CTV isolates as well
as other CTV genomic sequences, has been made (Dominguez et al., 2002; Febres et al.,
2003; Gutierrez, 1997).
10
Genetic Engineering for Virus/Viroid Resistance
Recent advances in plant transformation, the study of the molecular genetics of
both plants and their pathogens, and the elucidation of the interacting components of the
plants’ natural defense mechanisms have given rise to a number of different ways in
which to create virus- or viroid-resistant plants. Pathogen-derived resistance (PDR), a
concept pioneered by Sanford and Johnston (1985), makes use of the information derived
from viral genomes to generate specific host resistance. Since many virus genes involved
in the important functions of replication and encapsidation have been elucidated, PDR
can be achieved by interfering with these processes within host plants. One of the first
successful instances of PDR was achieved through the transformation of tobacco with the
coat protein (CP) gene of Tobacco mosaic virus (TMV) (Powell-Abel et al., 1986).
Successful CP-mediated resistance has since been achieved for a number of RNA viruses
including Potato virus X (PVX), Alfalfa mosaic virus, Cucumber mosaic virus (CMV),
Tobacco rattle virus, with Papaya ringspot virus being one of the most recent
commercial successes (Baulcombe 1996; reviewed in Wilson 1993; Ferreira et al., 2002).
Next to CP-mediated resistance, replicase-mediated resistance technology is the most
widely applied. Different forms (sense/antisense, translatable/nontranslatable) of the
replicase genes from diverse viruses have been used to transform plants and have
exhibited a wide range of host responses to viral infection. In certain virus-host systems,
the full-length sense construct of the replicase gene provides an adequate level of
resistance; while in others it is the antisense, mutated or truncated versions of the
replicase that provide a higher level of resistance (Brederode et al., 1995; Chen et al.,
2001; Huet et al., 1999; Vazquez Rovere et al., 2000; Wintermantel et al., 1997, 2000).
The antisense approach has also been tried for CEVd (Atkins et al., 1995). Instead of
11
complete resistance, the antisense construct, targeting the negative strand of CEVd,
resulted in only a moderate decrease in viroid RNA accumulation, while the construct
targeting the positive strand RNA had no effect.
Ribozymes, which are RNA molecules that have enzymatic activity and can cleave
RNA, have also been used to transform plants in an attempt to produce virus- or viroid-
resistant plants. Resistance to CEVd and PSTVd has previously been achieved (Atkins et
al., 1995; Yang et al., 1997). Varying degrees of resistance were seen in
ribozyme-expressing plants infected with TMV (de Feyter et al., 1996), CMV (Kwon et
al., 1997) and Rice dwarf virus (Han et al., 2001). Sano et al. (1997) and Ishida et al.
(2002) produced plants resistant to PSTVd and chrysanthemum stunt viroid by
expressing a double-stranded RNA-specific ribonuclease (pacI) from
Schizosaccharomyces pombe in transformed potato and chrysanthemums. Thus
transformation of plants with proteins or enzymes that act directly on RNA would
provide protection against virus or viroid infection.
Another approach to engineer resistance is to modify the expression or introduce
genes that activate, regulate or directly contribute to defense responses in plants. These
genes could be resistance (R) genes, general regulators of signal-transduction pathways
or genes that are activated further downstream and confer the actual resistance (for
example, PR genes) (reviewed by Campbell et al., 2002). Ribosome-inactivating proteins
are another group of plant defense-related genes that have been used to transform
heterologous species and shown to confer resistance to viruses.
Ribosome-Inactivating Proteins (RIPs)
Ribosome-inactivating proteins have been reported from over 50 plant species and
include both monocotyledonous and dicotyledonous plants (reviewed by Nielsen and
12
Boston, 2001). The only plant known to definitely not have RIP activity is Arabidopsis
thaliana. RIPs are a group of plant proteins that are toxic to ribosomes as a result of their
specific N-glycosidase depurination of the conserved α-sarcin loop of large rRNAs (Endo
et al., 1987). Depurination of the α-sarcin loop interferes with both the elongation factor
EF-Tu/EF-1-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of
EF-G/EF-2 to ribosomes, arresting protein synthesis at the translocation step and
ultimately resulting in cell death (Montanaro et al., 1975; Osborne and Hartley, 1990).
This translational inhibitory effect was seen as having potential in the medical field, for
use as selective cell-killing agents in anti-tumor therapy (Pastan and Fitzgerald, 1991;
Spooner and Lord, 1990).
RIPs can be divided into three classes based on their physical properties. Type 1
RIPs, such as pokeweed antiviral protein (PAP) from Phytolacca americana and
trichosanthin from Trichosanthes kirilowii, consist of a single polypeptide chain. Type 2
RIPs, such as ricin (from Ricinus communis) have a type 1-like catalytic subunit (A
chain) linked to a galactose-binding lectin (B chain) that facilitates adhesion to and
penetration of cell membranes (Batelli and Stirpe, 1995). As a result, type 2 RIPs are
much more toxic to living cells. Type 3 RIPs, found in maize and barley, are synthesized
as inactive precursors that require proteolytic cleavage for activation (Chaudhry et al.,
1994; Walsh et al., 1991).
Enzymatic Activities of RIPs
Extensive studies of the enzymatic activities of RIPs revealed that RNA N-
glycosidase activity on the α-sarcin loop of rRNA is by no means the only property of
these proteins. All RIPs have the ability to act as polynucleotide:adenosine glycosidases.
That is, they can remove multiple adenine residues from RNA or DNA (Barbieri et al.,
13
1997). In addition to removing adenine residues from nucleic acids, PAP is capable of
deguanylating ribosomal and viral RNA (Rajamohan et al., 1999a, b). The type 1 RIPs
gelonin (from Gelonium multiflorum) and PAP, and the type 2 RIP, ricin, possess DNA
glycosylase/AP lyase activity. These proteins, however, do not act in the traditional
protective manner of DNA glycosylase/AP lyases. Instead of removing potentially
cytotoxic or mutagenic bases, these proteins remove undamaged, non-mispaired bases
(Nicolas et al., 1998). Two novel properties have been demonstrated for ricin and
trichosanthin (from Trichosanthes kirilowii). Ricin has been shown to act as a
phosphatase on lipids and nucleotides (Helmy et al., 1999; reviewed in Peumans et al.,
2001), and trichosanthin possesses chitinase activity (Shih et al., 1997).
Antiviral Activity of RIPs
Perhaps as a consequence of some of the known enzymatic activities, a number of
RIPs have also been shown to possess antiviral and/or antifungal properties. The ability
of RIPs to inhibit viral infection in plants has been known since 1925, when extracts from
Phytolacca americana (pokeweed) were found to inhibit transmission of TMV (reviewed
by Nielsen and Boston, 2001; Peumans et al., 2001). The purified forms of many RIPs
have proven to be effective against both plant and animal viruses (Barbieri et al., 1993).
The antiviral property of the RIPs from pokeweed is being exploited in the medical field
as anti-human immunodeficiency virus (HIV) agents, especially since it was found that
PAP can inhibit viral replication at concentrations which do not inhibit the translation
machinery of the host cells (Rajamohan et al., 1999b; Ucken et al., 1998).
Several explanations have been put forward to explain the antiviral activities of
RIPs. The first is that RIPs act directly on the viral nucleic acid via their
polynucleotide:adenosine glycosidase activity. Second, RIPs could gain selective entry
14
into virus-infected cells when the cell is damaged upon vector feeding and thus inhibit the
protein synthesis machinery of only the infected cell. Third, RIPs may act indirectly
through the activation of signal-transduction pathways involved in plant defense.
Although there is in vitro evidence for the direct action of RIPs on viral nucleic acid
(Rajamohan et al., 1999b), it has not been shown in vivo. Many RIPs are sequestered in
the cell wall or cellular storage compartments from host ribosomes. It is not known how
the RIPs move from these compartments into the cytosol where they can act on either the
viral nucleic acid or the host-cell translational machinery. The most probable theory is
that RIPs are defense-related proteins regulated by signalling pathways in plants. It has
been demonstrated that RIPs from Phytolacca insularis and barley are induced in vivo by
jasmonic acid, a key signalling molecule in the expression of a number of plant defense-
related proteins (Chaudhry et al., 1994; Song et al., 2000). Two type 1 RIPs present in
Beta vulgaris accumulate in response to treatment of plants with salicylic acid and
hydrogen peroxide (two mediators of induced acquired resistance) (Girbés et al., 1996).
RIPs in Transgenic Plants
The role of RIPs in plant defense and promising in vitro results of RIPs as antiviral
proteins prompted exploration into their use as a source of microbial resistance in
heterologous plant hosts. Transgenic plants expressing different isoforms (different gene
products with the same function and similar or identical sequence) of PAP, and other
RIPs including trichosanthin, dianthin, IRIP (Iris-RIP), MOD1 (modified b-32 RIP) and
SNA-I' (Sambucus nigra agglutinin I') have been produced and tested for resistance to
viruses and fungi (Chen et al., 2002; Desmyter et al., 2003; Hong et al., 1996; Kim et al.,
2003; Krishnan et al., 2002; Lodge et al., 1993; Wang et al., 1998). Most researchers
chose to transform the model plants N. benthamiana or N. tabacum and study the
15
antiviral properties of the RIPs by inoculating the plants with TMV. PAP, MOD1 and
trichosanthin have been expressed in economically important crops (such as potato and
rice) to which they conferred resistance to Potato virus Y (PVY) (Lodge et al., 1993),
Rhizoctonia solani (Kim et al., 2003), and Pyricularia oryzae (Yuan et al., 2002),
respectively. PAP has been shown to confer resistance to a number of plant viruses
including TMV, PVY, PVX, and CMV (Lodge et al., 1993); while the expression of
trichosanthin resulted in complete resistance to infection by Turnip mosaic virus (Lam et
al., 1996). Dianthin has proven very effective against the geminivirus, African cassava
mosaic virus (Hong et al., 1996). Transgenic plants expressing PAP-II and MOD1 are
resistant to the fungus, R. solani, with PAP-II also conferring resistance to Sclerotinia
homoeocarpa in a turfgrass species (Wang et al., 1998; Kim et al., 2003; Dai et al.,
2003).
The main objective of this research was to determine the efficacy of PAP as an
antiviral agent against CTV and CEVd. More specifically, the efficacy would be
evaluated using an in vitro system prior to using PAP mutants to transform Citrus
paradisi cv. Duncan grapefruit, which would then be challenged with CTV and CEVd by
graft inoculation. The same PAP mutants would be used to transform Nicotiana
benthamiana for the purpose of studying the effect of PAP in protoplasts on the
replication of CTV.
CHAPTER 2 In vitro TRANSLATION OF A Citrus tristeza virus REPLICON AND THE EFFECT
OF POKEWEED ANTIVIRAL PROTEIN ON ITS TRANSLATION
Introduction
Citrus tristeza virus (CTV), the largest positive stranded RNA virus of plants, has
properties that are intermediate between the animal Nidovirales group, which contains the
family Coronaviridae and the alphavirus supergroup, which contains the Tobamoviruses.
CTV resembles members of the Coronaviridae because of the similarity of the genes,
gene arrangement of ORF1a and 1b, the expression strategy of the RNA-dependent RNA
polymerase (RdRp), and the accumulation of double-stranded (ds) RNAs corresponding
to subgenomic (sg) RNA. The relationship with the alphaviruses is based on amino acid
similarity in the replicase-associated domains and on the lack of a common 5' leader on
the sgRNAs (Karasev et al., 1997; Snijder and Horzinek, 1993). The CTV genome is
organized into 12 open reading frames (Pappu et al., 1994), with ORF1a being expressed
as a 349 kDa polyprotein and ORF1b expressed via a +1 frameshift (Cevik, 2001). The
ten 3' genes are expressed via 3' co-terminal sgRNAs (Hilf et al., 1995; Karasev et al.,
1997).
The study of the biological properties and detailed characterization of CTV at the
molecular level have been hampered by the limited host range, the lack of local lesion
hosts, the fact that CTV is phloem-limited and not easily mechanically transmissible, and
by the large, complex genome of this virus. A full-length infectious cDNA clone was
constructed and amplified in E. coli (Satyanarayana et al., 1999). However, the large size
16
17
of the clone impeded the ready manipulation of the construct and the study of mutants for
elucidation of gene functions. As a result, a smaller, more manageable replicon
designated CTV-∆Cla, was generated (Satyanarayana et al., 1999). This clone, retaining
only the 5' and 3' UTRs, ORFs 1a and 1b, and a gene fusion between ORF11 and part of
ORF2 (Figure 2-1), was able to replicate efficiently in Nicotiana benthamiana mesophyll
protoplasts. The use of the infectious CTV clones has, thus far, been used to study
replication and gene expression at the transcriptional level in a protoplast system.
Figure 2-1. Schematic representation of the Citrus tristeza virus (CTV) genome. A) Full
length CTV genome. B) CTV-∆Cla replicon. Shown are the two papain-like proteases (Pro), the methyltransferase (Met), Helicase (Hel), RNA dependent RNA polymerase (RdRp) and ORFs 2-11. Diagrams were adapted from Satyanarayana et al., 1999.
Although the cDNA clone has been used to examine the effect of the product of the
ORF11 (p23) on viral replication (Satyanarayana et al., 2002), examination of the events
occurring at the translational level in protoplasts is more difficult since visualization of
the viral proteins requires the use of antibodies to distinguish viral proteins from other
plant proteins. Additionally, only one protein can be studied per western blot.
18
Development of an efficient in vitro model system would allow the examination of the
expression levels of viral proteins and also the effect of exogenous agents, such as other
proteins, on the level of translation without the use of antibody-detection methods.
Studies at the translation level have been conducted in an in vitro transcription and
translation system (Cevik, 2001; Vazquez-Ortiz, 2001). However these experiments used
CTV sequences (the two papain-like protease domains and the RdRp, respectively) out of
the viral context. There is a possibility that additional viral sequences further upstream or
downstream of the gene of interest may have an effect on expression. Thus transcription
and translation of the CTV-∆Cla replicon (which allows for the insertion of other genes)
in an in vitro translation system would facilitate the analysis of multiple viral proteins.
This would circumvent the need to dually transfect protoplasts with both CTV-∆Cla and
another transcript to examine the effect of the product of the latter transcript on replicon
replication. When transfecting protoplasts with two different transcripts, it is unlikely that
both transcripts will enter all the cells. This might result in the erroneous interpretation of
data. The in vitro approach should facilitate the study of the effect of potential
resistance/antiviral gene products on viral translation, and give an initial indication of the
efficacy of these proteins without the need for plant transformation and screening of the
resultant plants for resistance to CTV (a lengthy and time-consuming process for citrus).
Pokeweed antiviral protein (PAP) isolated from the leaves of Phytolacca
americana (pokeweed) is a 29 kDa ribosome-inactivating protein that has antiviral
properties (reviewed in Nielsen et al., 2001; Tumer et al., 1999). This antiviral effect is
not limited to a particular virus or virus group, and is effective against both plant and
animal viruses (Chen et al., 1991; Irvin and Uckun, 1992). There is evidence that the
19
antiviral activity is not solely dependent upon the inactivation of ribosomes (Tumer et al.,
1997). In vitro experiments showed that PAP can inhibit translation by directly
depurinating the RNA (Rajamohan et al., 1999a); and that although PAP preferentially
depurinates capped RNA, it is still capable of inhibiting translation of uncapped viral
RNAs by an, as yet, unknown mechanism (Vivanco and Tumer, 2003).
The objective of this study was to express the CTV replicon, CTV-∆Cla, in an in
vitro translation system and to test the efficacy of PAP as a potential inhibitor of the
translation of this replicon.
Materials and Methods
Source of Viral Nucleic Acid
The CTV-∆Cla replicon (kindly supplied by Dr. W.O. Dawson) was transformed
into E. coli JM109 cells. Large-scale DNA isolation was performed using the Qiagen
Midiprep kit according to the manufacturer’s instructions.
In vitro Transcription and Translation of CTV-∆Cla
The first attempt to translate the CTV replicon was done in a wheat germ extract
TNT® coupled in vitro transcription and translation system (Promega, Madison, WI).
Different concentrations of DNA were tested in order to optimize the system. A 20 µl
reaction containing 10 µl of wheat germ extract, 0.75 µl reaction buffer that contained all
the amino acids except leucine, 0.75 µl SP6 RNA polymerase, 0.75 µl RNAsin
ribonuclease inhibitor, and 0.5 µCi/ml 3H-leucine was incubated at 30°C for 1 h. The
reactions were then subjected to SDS-PAGE in 12% Tris Ready gels (Bio-Rad) at 150 V
for 1.5 h. The radio-labeled proteins were detected using the method described by Bonner
and Laskey (1974). Briefly, the gels were fixed in dimethyl sulfoxide (DMSO) and
DMSO-2, 5-diphenyloxazole (PPO), dried under vacuum at 70°C for 40 min, exposed to
20
X-ray film overnight at -80°C, and developed using a Kodak X-Omat Clinic 1 film
processor.
Alternatively, the transcription and translation reactions were conducted separately.
The plasmid DNA was linearized with the restriction enzyme, NotI (New England
Biolabs, Beverly, MA) and transcribed using the RibomaxTM large scale in vitro RNA
production system (Promega, Madison, WI) according to the manufacturer’s instructions.
Briefly, the transcription reaction was done in a final volume of 25 µl, containing 5 µl
SP6 transcription buffer, 5 µl of 25 mM rNTPs, 5 µg DNA template, 2 µl SP6 enzyme
mix and nuclease-free water. The reaction was incubated at 37°C for 3 h, stopped by the
addition of RNAse-free DNAseI and further incubation at 37°C for 15 min. Further
purification of the RNA transcripts was done according to the manufacturer’s
instructions.
Different concentrations of the transcripts were then translated in either wheat germ
extract (Promega, Madison, WI) at 25°C for 2 h or the Flexi® Rabbit reticulocyte lysate
system (Promega, Madison, WI) at 37°C for 1 h. The transcripts were either capped using
the m7GpppG cap analogue (Promega, Madison, WI) or uncapped. For the wheat germ
system, different concentrations of potassium acetate were tested; while for the rabbit
reticulocyte system, potassium chloride and magnesium acetate concentrations were
optimized. The final concentrations of potassium and magnesium used in the translation
reactions were established based on the guidelines stipulated by the manufacturer.
In vitro Antiviral Assay
To determine the direct effect of PAP on CTV-∆Cla RNA, the transcripts were
treated with 0 ng, 20 ng, and 100 ng of wild type (wt) PAP (Worthington, Lakewood, NJ)
21
per 500 ng of RNA in RIP buffer (50 mM Tris, 50 mM KCl, 10 mM MgCl2 pH 7.5) and
incubated at 30°C for 30 min. PAP was removed by phenol-chloroform extraction, and
the translation reaction performed in the rabbit reticulocyte lysate system. RNA from
Brome mosaic virus (BMV) supplied with the translation kits was used as a control.
Results
In vitro Translation of CTV-∆Cla
The first attempt at translating CTV-∆Cla using a wheat germ-based TNT®
coupled transcription and translation system was unsuccessful. This could be due to the
absence of specific co-factors or conditions required by the replicon for translation.
Factors that might have a positive effect on translation include varying salt and
magnesium concentrations as well as the presence of a capped transcript. For this reason,
the transcription and translation reactions were performed separately in two different
systems (plant and animal) to optimize the translation reaction.
The wheat germ system allowed for optimization of the concentration of the salt,
potassium acetate. The combination of 2 µg of the DNA template and 53 mM potassium
acetate yielded translation products (Figure 2-2). The band of approximately 28 kDa in
size might be the product of the fusion of ORF11 with part of ORF2, while the faint high
molecular weight band might be the polyprotein or part of the polyprotein since the
translation system has an upper limit of approximately 200 kDa. The band at 48 kDa does
not correspond to any expected size of products translated from ORF 1a and 1b.
Due to the unexpected sizes of the protein bands in the wheat germ system,
optimization in a rabbit reticulocyte system was undertaken. The rabbit reticulocyte
system allowed for optimization of salt and magnesium concentrations in addition to
supporting the translation of larger RNA templates. Two different RNA concentrations
22
were translated in the absence or the presence of either 70 mM potassium chloride (KCl)
or 0.25 mM magnesium acetate (Figure 2-3).
Figure 2-2. Optimization of CTV-∆Cla replicon translation in wheat germ extract. Lane:
1, 1 µg RNA with 53 mM potassium acetate; 2, 1 µg RNA with 133 mM potassium acetate; 3, 2 µg RNA with 53 mM with potassium acetate; 4, 2 µg RNA with 133 mM potassium acetate; 5, 4 µg RNA with 53 mM potassium acetate; 6, 4 µg RNA with 133 mM potassium acetate.
Although the standard translation reaction (Figure 2-3, lanes 1 and 4) supported
translation of the CTV-∆Cla transcript, with the higher RNA concentration of 2 µg
producing more protein, the addition of KCl to 1 µg RNA increased translation to the
same level produced by 2 µg of RNA without any additives. A strong band of
approximately 56 kDa was observed in all translation reactions. This band fit the
expected size of the proteases and the RdRp expressed by ORF 1a and 1b. The high
molecular weight bands seen on the gel were most likely artifacts since they were not
consistently observed in subsequent experiments. Due to the higher level of translation,
23
the appearance of bands closer to the sizes of expected proteins and the reproducibility of
the results, it was decided to continue using the rabbit reticulocyte system with the
addition of KCl to all further translation reactions.
Figure 2-3. Optimization of CTV-∆Cla translation in rabbit reticulocyte lysate. Lane 1, 1
µg RNA; 2, 1 µg RNA + 70 mM potassium chloride; 3, 1 µg RNA + 0.25 mM magnesium acetate; 4, 2 µg RNA; 5, 2 µg RNA + 70 mM potassium chloride. RNA was transcribed from the NotI-linearized plasmid containing the CTV-∆Cla replicon.
PAP Inhibits Translation of CTV-∆Cla Transcripts
To determine whether PAP would act as an antiviral agent against CTV translation
in vitro, capped CTV-∆Cla transcripts were treated with commercial wtPAP prior to
translation. After incubation with CTV-∆Cla transcripts, PAP was removed by phenol-
chloroform extraction to eliminate the possibility that any observed decrease in
translation might be due to the ribosome-inactivating properties of wtPAP. BMV RNA
was used as a positive control to confirm the inhibitory effect of the wtPAP preparation.
SDS-PAGE analysis of the translation products of wtPAP-treated BMV RNA showed
24
that 20 ng of wtPAP caused a visible decrease in translation (Figure 2-4 A), indicating
that wtPAP had enzymatic activity. The CTV-∆Cla transcripts treated with 20 ng wtPAP
exhibited decreased levels of translation as indicated by the decreased intensity of the
major product of the approximately 56 kDa expressed from the CTV-∆Cla transcript
(Figure 2-4). The translation of both BMV and CTV-∆Cla was completely inhibited by
100 ng of wt PAP (data not shown).
Figure 2-4. Effect of wild type PAP on translation. A) BMV translation products B)
CTV-∆Cla translation products. Lane 1, untreated RNA; 2, RNA treated with 20 ng PAP/500 ng RNA. All RNA samples were incubated at 30°C for 30 min in RIP buffer followed by a phenol-chloroform extraction.
Discussion
In vitro translation has played an important role in the elucidation of requirements
for viral gene expression. For example, in vitro systems have been used to investigate
translational enhancement in Hibiscus chlorotic ringspot virus (Koh et al., 2002),
translational frameshifting in Barley yellow dwarf virus (Di et al., 1993; Paul et al., 2001)
and the mechanism of cap-independent translation in Tomato bushy stunt virus (Wu and
25
White, 1999). In addition, in vitro systems are useful when the translation products of
viral genomic or subgenomic RNAs are not known (Dinesh-Kumar et al., 1992). Schulte
and Kearney (2000) used an in vitro translation system to determine the antiviral
potential of a variety of antisense oligonucleotides (ASO). They were able to identify the
most effective ASOs before doing more time consuming plant transformation
experiments.
The objective of this study was to use CTV-∆Cla transcribed RNA as a reliable
template for in vitro translation followed by rapidly testing the efficacy of PAP as an
inhibitory agent of CTV translation before proceeding with plant transformation
experiments. The results showed that CTV-∆Cla transcripts can be translated in an in
vitro rabbit reticulocyte lysate system. However, the inherent limitations of the system
may have prevented the expression of the large polyprotein. Alternatively the two
proteases located at the N-terminal end of the polyprotein could have processed the 349
kDa polyprotein into functional proteins of a smaller size (Vazquez-Ortiz, 2001).
Using an in vitro translation assay, Hudak et al. (2000) showed that one of the
mechanisms by which wtPAP was able to inhibit viral translation was by acting directly
on the RNA of BMV and Potato virus X (PVX), removing adenine residues at multiple
sites. Therefore, to determine whether PAP could inhibit translation of CTV-∆Cla RNA
by acting directly on the RNA, capped CTV-∆Cla transcripts were treated with
commercial wtPAP prior to translation. A reproducible decrease in the translation of
PAP-treated RNA was observed at a concentration of 20 ng of the antiviral protein
(Figure 2-4 B). At a concentration of 100 ng, wtPAP completely inhibited translation of
BMV and the CTV-∆Cla replicon (data not shown). However, previous research has
26
shown that wtPAP is able to almost completely inhibit the translation of 250 ng BMV
RNA at a concentration of 5 ng (Hudak et al., 2000). The major difference between that
result and the one from this experiment might be explained by differences in the
enzymatic activity of the PAP preparation used. Based on the results obtained from the
translation of BMV RNA and the possibility that the PAP preparation did not have
optimal enzymatic activity, one could extrapolate that a lower concentration of PAP
might also have an inhibitory effect on translation. Since the viral RNA will be exposed
to the intracellular environment in plants, the in vitro translation result demonstrating the
direct effect of PAP on the RNA suggests that transgenic citrus correctly processing and
expressing PAP may exhibit reduced levels of CTV.
CHAPTER 3 TRANSFORMATION OF Citrus paradisi cv Duncan GRAPEFRUIT WITH
POKEWEED ANTIVIRAL PROTEIN
Introduction
Diseases caused by viroids and viruses have had a major impact on citrus
production world-wide. Viroids are the smallest known plant pathogens. They consist of
unencapsidated, circular, single-stranded RNA molecules that exist as highly base paired
rod-like structures. Viroids found in citrus plants can be classified into the following
classes: Citrus exocortis viroid (CEVd, 370-375 nucleotides (nt)), Citrus bent leaf viroid
(CBLVd, 315-329 nt; formerly called CVd-I), Hop stunt viroid (HSVd, 295-303 nt;
formerly called CVd-II), Citrus viroid III (CVd-III, 291-297 nt), Citrus viroid IV (CVd-
IV, 284-286 nt) and Citrus viroid OS (CVd-OS, 329-/331 nt) (Duran-Vila et al., 2000; Ito
et al., 2000, 2002).
In the field, CEVd causes severe stunting and bark scaling of citrus trees on
Poncirus trifoliata rootstock. Less severe symptoms occur on Citrus sinensis x P.
trifoliata (citrange) and Citrus limonia (Rangpur lime) and only mild symptoms are seen
on Swingle citrumelo (Lee et al., 2003). The severe stunting on trifoliate rootstock leads
to reduced canopy size and ultimately a reduction in yield. CEVd is spread primarily via
infected budwood and contaminated cutting tools and equipment.
Resistance to CEVd and potato spindle tuber viroid (PSTVd) has previously been
achieved by transforming plants with ribozymes, which are RNA molecules that have
enzymatic activity (Atkins et al., 1995; Yang et al., 1997). Sano et al. (1997) and Ishida et
27
28
al. (2002) produced potato and chrysanthemum plants resistant to PSTVd and
chrysanthemum stunt viroid, respectively, by expressing a double-stranded RNA–specific
ribonuclease, pacI, from Schizosaccharomyces pombe. Thus transformation of plants with
proteins or enzymes that act directly on RNA may provide protection against viroid
infection.
Citrus tristeza virus (CTV), a member of the genus Closterovirus, is one of the
most economically important virus diseases of citrus and has resulted in widespread
losses in many citrus-growing regions. The virus is transmitted by six aphid vectors, with
Toxoptera citricida being the most efficient vector, as well as by propagation of infected
buds (Bar-Joseph et al., 1989). CTV isolates differ in the symptoms they cause depending
on the scion/rootstock combination. Certain isolates result in decline and death of citrus
species on sour orange rootstock; stem pitting of scions which reduces tree vigour, fruit
yield and quality; and a syndrome called seedling yellows which is expressed as stunting
and chlorosis of susceptible varieties such as lemon and grapefruit.
CTV isolates exist as complex populations consisting of a number of different CTV
genotypes, with large sequence variation among the genotypes. Thus CTV isolates are
populations of CTV genotypes, in which one genotype may predominate (Ayllόn et al.,
1999; Hilf et al., 1999). A number of strategies have been developed to manage the
disease. These strategies include eradication programs, quarantine and budwood
certification to prevent the introduction of CTV into a country, the use of CTV tolerant
rootstocks, and mild strain cross protection (reviewed by Ferguson and Garnsey, 1993).
A source of genetic resistance to CTV has been found in Poncirus trifoliata. This
resistance is known to be controlled by a single dominant gene, Ctv (Yoshida et al.,
29
1996). Although the introgression of the Ctv gene into rootstock species is possible via
sexual hybridization, commercially acceptable scions have not yet been produced as a
result of the concomitant introgression of undesirable fruit characteristics (Ferguson and
Garnsey, 1993). Currently the mechanism of action for Ctv is not known, since
protoplasts of virus resistant plants are still able to maintain viral replication (Albiach-
Marti et al., 1999). However, cloning the Ctv gene and using it to transform commercially
important citrus species should be a successful strategy to develop genetic resistance to
CTV.
Genetic transformation of woody fruit plants is a promising alternative to
conventional breeding practices for the introduction of new traits. This technology
circumvents the limitations imposed on breeding by the heterozygosity, long juvenile
periods and auto-incompatibility of the plants. With the development of an efficient
Agrobacterium-mediated citrus transformation protocol (Luth and Moore, 1999), it is
now possible to use molecular methods not only to engineer resistance to CTV, but also
to introduce other commercially favorable traits. For example, citrus was successfully
transformed with the Arabidopsis thaliana genes LEAFY and APETALA1, both of
which reduced the juvenile period of the resultant plants (Peňa et al., 2001). Thus far
attempts to engineer resistance to CTV has centered on the pathogen-derived resistance
approach (PDR); transforming plants with the coat protein gene of various CTV isolates
as well as other CTV genomic sequences, (Dominguez et al., 2000, 2002; Febres et al.,
2003; Ghorbel et al., 2001; Gutierrez, 1997). Given the complexity of CTV populations,
it is possible that a PDR approach may not produce the expected “CTV resistance”.
30
Ribosome-inactivating proteins (RIPs) with additional antiviral or antifungal
properties have been isolated from many different plant species and are enzymes that
inhibit protein synthesis in many prokaryotic and eukaryotic cells. This inhibition is due
to N-glycosidation activity, which causes the removal of a specific adenine residue from
the α-sarcin loop, a region conserved in the large rRNAs of both prokaryotes and
eukaryotes (Endo et al., 1987; Endo and Tsurugi, 1987). Depurination of the α-sarcin
loop interferes with both the elongation factor EF-Tu/EF-1-dependent binding of
aminoacyl-tRNA and the GTP-dependent binding of EF-G/EF-2 to ribosomes, arresting
protein synthesis at the translocation step (Montanaro et al., 1975; Osborne and Hartley,
1990). RIPs can be divided into three classes based on their physical properties. Type 1
RIPs, such as pokeweed antiviral protein (PAP; from Phytolacca americana) and
trichosanthin (from Trichosanthes kirilowii) consist of a single polypeptide chain. Type 2
RIPs, such as ricin (from Ricinus communis), have a type 1-like catalytic subunit (A
chain) linked to a galactose binding lectin (B chain) that facilitates adhesion to and
penetration of cell membranes (Batelli and Stirpe, 1995). As a result, type 2 RIPs are
much more toxic to living cells. Type 3 RIPs, found in maize and barley, are synthesized
as inactive precursors which require proteolytic cleavage for activation (Chaudhry et al.,
1994; Walsh et al., 1991).
PAP is a 29-kDa protein produced by Phytolacca americana (pokeweed) and has
been shown to exhibit strong antiviral activity against a broad spectrum of plant and
animal viruses, including Southern bean mosaic virus, Potato virus X, Cucumber mosaic
virus, Tobacco mosaic virus, influenza virus (Tomlinson et al., 1974), poliovirus (Ussery
and Hardesty, 1977), herpes simplex virus (Aron and Irvin, 1980) and HIV (Zarling et al.,
31
1990). A number of PAP mutants have been isolated and tested for toxicity to ribosomes
and retention of antiviral properties (Table 3-1; Hur et al. 1995). The most effective
mutants with respect to reduced toxicity and inhibition of viral infection are a C-terminal
deletion mutant, due to a nonsense mutation (Trp237stop), and an N-terminal mutant
(Gly75-Asp). The reason for their efficacy is thought to be due to the fact that the C-
terminal deletion removes a region responsible for PAP toxicity to ribosomes, while the
N-terminal mutation has been shown to adversely affect the binding of PAP to ribosomes
(Zoubenko et al. 2000). Other mutants which have been used include a PAP variant
(PAPv) in which a two amino acid change, Lys20-Arg and Trp49-His, causes a slight
reduction in toxicity to ribosomes while maintaining the wild type ability to inhibit viral
infection, and an active site mutant (PAPx) in which the mutation, Gly176-Val abolished
enzymatic activity.
Table 3-1. Expected properties of PAP and PAP mutants.
Information presented in this table was obtained from Lodge et al. (1993), Tumer et al., (1993) and Zoubenko et al., (2000).
Construct Toxicity to ribosomes Translation inhibition Pathogen resistance Wild type PAP Yes Yes Yes PAPv Yes Yes Yes PAPn No Yes Yes PAPc No Yes Yes PAPx No No No
The broad-spectrum virus resistance displayed by wild type PAP and its mutants
makes it an ideal candidate for the development of virus-resistant plants. To date, PAP
has been used to transform Nicotiana benthamiana, N. tabacum cv. Samsun, Solanum
tuberosum cv. Russet Burbank and Agrostis stolonifera L. (creeping bentgrass), and the
plants have been evaluated for resistance to viruses as well as fungi (Dai et al., 2003;
Lodge et al., 1993; Tumer et al., 1997; Zoubenko et al., 2000). Although the mechanism
32
mediating the antiviral activity of PAP is still under investigation, a number of insights
have been gained. PAP has been shown to inhibit translation by directly depurinating
viral RNA at multiple sites (Rajamohan et al., 1999a), preferentially depurinating capped
RNAs (Hudak et al., 2000; Vivanco and Tumer, 2003), and inhibiting +1 frameshifting in
the yeast retrotransposon, TY1 (Tumer et al., 1998). Since CTV has an RNA genome that
most probably has a 5' guanosine cap and a RdRp that is expressed via a +1 frameshift, it
presents an ideal subject for translational inhibition by PAP in a non-strain specific
manner.
In this study, four PAP mutants (PAPv, PAPc, PAPn and PAPx), the properties of
which are summarized in Table 3-1, were used to transform Citrus paradisi cv. Duncan
grapefruit by Agrobacterium tumefaciens-mediated transfer. The resultant transgenic
plants were characterized by molecular methods and evaluated for resistance to CTV and
CEVd by graft challenge inoculation.
Materials and Methods
PAP Gene Constructs and Plasmid Expression Vectors
All PAP constructs were derived from the PAPv sequence which contains two
mutations, Lys20-Arg and Trp49-His. These mutations make the protein slightly less
toxic to ribosomes than the wild type (wt) PAP (Tumer et al., 1997). Using a series of
oligonucleotide primers (Table 3-2), two mutants, PAPn containing a point mutation that
changed residue Gly75 to Ala, and PAPx with Glu176 and Arg170 changed to Ala, were
constructed by overlap PCR, while the C-terminal deletion mutant was constructed by
changing Trp237 to a translation termination codon, thereby truncating the PAP coding
region. The PCR products were cloned into pGEM-T (Promega, Madison, WI) and
sequenced to verify the presence of the introduced mutations. The PAP insert was
33
removed from pGEM-T by digestion with restriction enzymes ApaI and NotI, flanking
the PAP coding sequence, and subcloned between the Figwort mosaic virus (FMV)
promoter and the Cauliflower mosaic virus (CaMV) 35S termination sequence contained
in the pUC118 vector provided by Dr V. J. Febres (Figure 3-1). The PAP expression
cassettes containing the PAPv, PAPn, PAPx and PAPc genes were released from pUC118
by digestion with PstI and ligated into the PstI-digested binary expression vector,
pCambia2202 (provided by Dr V. J. Febres) which has the kanamycin resistance gene,
nptII, as the selection marker and the green fluorescent protein (GFP) as the reporter gene
(Figure 3-1). The binary vector containing the PAP constructs were introduced into
Agrobacterium tumefaciens strain AGL1 by the cold shock transformation method
(Birnboim and Doly, 1979). The bacteria were incubated on LB-agar plates supplemented
with 50 µg/ml carbenicillin and 100 µg/ml chloramphenicol, at 28°C for two days to
screen for transformants.
Table 3-2. Sequences of oligonucleotide primers used for amplification and mutagenesis of the PAP gene
Primer Sequence Orientation CN442 GGTATCAGcGGCAGCtgcATTCAAGTAC Sense CN443 GTACTTGAATgcaGCTGCCgCTGATACC Antisense CN444 ATTAGGGCCCGGGAAGATGAAGTCAATGCTTGTG Sense CN445 AATTGCGGCCGCTCAGAATCCTTCAAATAGATC Antisense CN446 AATTGCGGCCGCTCACTTGGCACC Antisense CN447 GTATGTGATGGATTATTCTG Sense CN448 CAGAATAATCCATCACATAC Antisense Lower case letters indicate the mutated nucleotides Citrus Transformation and Regeneration
An Agrobacterium-mediated transformation protocol previously developed for
Carrizo citrange and Swingle citrumelo (Moore et al. 1992) and subsequently modified
for Citrus paradisi cv. Duncan (Luth and Moore, 1999) was followed.
34
Figure 3-1. Cloning strategy of the PAP constructs for plant transformation. The
ApaI/NotI-digested gene fragments were ligated into ApaI/NotI-digested pUC118 between the FMV promoter and CaMV 35 terminator. The resulting expression cassettes were cloned into the PstI restriction site of the binary vector pCambia2202. The black lines on the PAP constructs indicate positions of mutations. All constructs had the mutations Lys20-Arg and Trp49-His. Additional mutations were Gly75-Ala for PAPn, Glu176-Ala and Arg170-Ala for PAPx , and Trp237-Stop for PAPc.
35
Seed germination
Seeds were extracted from mature fruit of Citrus paradisi cv. Duncan grapefruit.
The seedcoats were removed from seeds, and the seeds were then sterilized first in 70%
ethanol for 2 min, then in a 0.525% sodium hypochlorite solution with 0.05% Tween 20,
followed by 3 rinses of 2 min each in sterile water. One or two seeds were placed into test
tubes containing half strength MS medium (2.15 g/L MS salts, 50 mg/L myo-inositol, FM
stock, 15 g/L sucrose) and 7 g/L agar. The seeds were germinated in the dark for
approximately six weeks.
Transformation of epicotyl segments
Agrobacterium tumefaciens strain AGL1 containing pCambia2202 with the PAPv,
PAPn, PAPx or PAPc construct was grown overnight at 28°C, in YEP medium (10 g/L
bactopeptone, 10 g/L yeast extract, 5 g/L sodium chloride) supplemented with the
appropriate antibiotics, to an OD620nm between 0.4 and 1.0. The cultures were centrifuged
at 5000 rpm in a Beckman GSA rotor for 5 min and resuspended to a final concentration
of 5x108 cfu in MS medium supplemented with 100 µM acetosyringone.
The epicotyl portions of the etiolated seedlings were cut into 1 cm2 segments and
incubated in the Agrobacterium solution for 1-2 min. The segments were then transferred
to co-cultivation medium (MS medium, 7 g/L agar and 100 µM acetosyringone) and kept
in the dark at room temperature for 2-3 days (Figure 3-2 A and B).
Selection and regeneration of plantlets
Following co-cultivation, the epicotyl segments were placed onto shoot-inducing
medium (MS medium, 7 g/L agar and 0.5 mg/L benzylaminopurine) supplemented with
75 mg/L kanamycin to select for transformed shoots and 500 mg/L claforan to prevent
further growth of Agrobacterium (Figure 3-2 C and D).
36
Figure 3-2. Agrobacterium-mediated transformation of etiolated Citrus paradisi cv.
Duncan grapefruit and regeneration of plants. A) Inoculation of stem segments with Agrobacterium strain AGL1 containing pCambia2202 with the PAP construct. B) Co-cultivation. C-D) Regeneration of shoots on selective medium. E) Shoots on root-inducing medium. F) Transgenic plant transferred to soil in the greenhouse.
37
The plates were incubated at 28°C with a 16 h photoperiod provided by cool-white
fluorescent lights. The segments were transferred to fresh medium every 4 weeks. Shoots
were transferred directly to root-inducing medium (MS medium, 8 g/L agar, 100 mg/L
myo-inositol, 0.5 mg/L naphthalene acetic acid), where they remained until they had
produced roots (Figure 3-2 E). Upon root production, the plantlets were transferred to
covered containers with sterile soil and half strength MS medium and kept at 28°C with a
16 h photoperiod until they outgrew the containers. The plants were acclimated by
placing them in pots covered by clear plastic bags in a humid growth room with a 16 h
photoperiod and a temperature of 28°C. After 1-2 weeks, the plastic bags were slashed
and after another 1-2 weeks, the bags were completely removed, whereupon the surviving
plants were transferred to the greenhouse (Figure 3-2 F).
Analysis of Transgenic Plants
Fluorescent microscopy
The regenerated shoots were examined for GFP expression using a dissecting
microscope (Zeiss) equipped with a fluorescent light source with a 515 nm long pass
emission filter transmitting red and green light and a 450-490 nm excitation filter. The
fluorescent images were captured with a 35 mm camera attached to the microscope.
Polymerase chain reaction (PCR) analysis
All shoots were tested for the presence of the PAP gene by PCR using gene-
specific primers. Some of the shoots were also analysed for the presence of the GFP gene
using the gene-specific primers CN462 5'-ATGGTGAGCAAGGGCGAGGAG-3' and
CN463 5'-CTTGTACAGCTCGTCCATGCC-3'. DNA was extracted from the plants
using a modification of the protocol by Oliveira et al. (2000). Briefly, 0.1 g of tissue was
ground in liquid nitrogen. The tissue was resuspended in 300 µl of extraction buffer
38
(100 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 500 mM NaCl, 140 mM
ß mercaptoethanol, 80 µl of 10% SDS), incubated at 65°C for 10 min, after which, 100 µl
of potassium acetate was added and the samples further incubated on ice for 20 min. The
samples were centrifuged at 12000 x g for 30 min at 4°C. The supernatant was then
transferred to a new tube and an equal volume of isopropanol added. The DNA was
precipitated by centrifugation at 12000 x g for 20 min at 4°C. The DNA pellet was
washed with 80% ethanol, dried and resuspended in sterile water containing 2 µg/µl
RNAse. PCR was carried out using primers CN444 and CN446 or CN444 and CN445
(Table 3-1) in a Biometra thermocycler using a profile of 94°C for 2 min initial
denaturation, 30 cycles of denaturation at 94°C for 30 sec, primer annealing at 55°C for
45 sec, primer extension at 72°C for 45 sec and a final primer extension step of 72°C for
5 min. The PCR products were analysed by electrophoresis in 1% agarose gels in TAE
buffer.
Southern hybridization
For Southern hybridization 10 µg of total genomic DNA was isolated from
transgenic plants and non-transformed control plants using a modification of the protocol
by Oliveira et al. (2000). The DNA was digested with BglII (New England Biolabs,
Beverly, MA). After agarose gel electrophoresis, the DNA was transferred to nylon
membrane (Hybond N+, Fisher Scientific) by capillary action overnight (Sambrook et al.
1989). Using the gene specific primers CN444 and CN446, a digoxigenin (DIG) -labeled
probe was produced by PCR amplification according to the manufacturer’s instructions
(Roche, Indianapolis, IN). The DIG-labelled probe was denatured and used for a 16 hour
hybridisation after 2-4 hours of prehybridisation in DIG Easy Hyb Buffer (Roche).
Following hybridization, the membrane was washed twice for 5 min in 2x SSC + 0.1%
39
SDS at room temperature, and twice for 15 min in 0.5x SSC + 0.1% SDS at 65°C. The
blot was exposed to X-Ray film and developed using a Kodak X-Omat Clinic 1 film
processor.
Reverse-transcription (RT) PCR
PAP gene expression at the transcriptional level was analysed in the transgenic
plants by RT-PCR. Total RNA was isolated from the plants with TRIzol reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Prior to RT-
PCR, the RNA was treated with DNAse I (Promega, Madison, WI) to eliminate any DNA
contaminants, and then phenol-chloroform cleaned to remove the DNAse I. cDNA was
prepared using 5 µg of RNA, the reverse primer CN446, and MMLV reverse
transcriptase (Promega). RNA isolated from a non-transgenic plant was used as a
negative control in the RT-PCR reaction. The parameters for the RT reaction were: 1 h at
42°C and 15 min at 70°C. One tenth of the volume of the RT reaction was used for PCR
and the products were analysed on a 1% agarose gel in TAE buffer.
Western blot analysis
To determine the expression levels of PAP in the transgenic plants, 200 mg of
tissue was homogenized in an equal volume of phosphate buffered saline (PBS, 137 mM
NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4 pH 7.4). Total protein
concentration was determined by the Bradford Assay (BioRad, Hercules, CA). Aliquots
of the soluble plant extracts containing 100 µg total protein were mixed with equal
volumes of dissociation buffer (140 mM SDS, 160 mM Tris-HCl pH 7.8, 1% v/v
glycerol, 142 mM β-mercaptoethanol), boiled for 2 min and separated on a precast 12%
polyacrylamide Tris-HCl gel (BioRad). Proteins were transferred to nitrocellulose
membrane using a BioRad semi-dry horizontal electrotransfer apparatus according to the
40
manufacturer’s instructions.Colorimetric immunodetection of the protein was carried out
using a 1:500 dilution of 0.5 mg/mL anti-PAP-IgG kindly supplied by Dr Maria
Kanieskowski (Monsanto) and the chromogenic substrates ρ-nitro blue tetrazolium
(NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine (BCIP).
Grafting and Challenge of Transgenic Plants with CTV
Individual transgenic lines were grafted onto Swingle citrumelo rootstock. For
challenge with CTV, eight to twenty-eight plants from each replicated line were graft
inoculated with the severe strain T66 sub-isolate E (Tsai et al., 2000). Two and four
months after inoculation, CTV-inoculated plants on which the grafts had survived were
analyzed for infection by direct antibody sandwich indirect-ELISA (DASI-ELISA) (Tsai
et al., 2000) using the antibodies, CREC1052 and G604 (provided by R. F. Lee). Plates
were coated with a 1:5000 dilution of CREC1052, followed by the addition of 10 mg/100
µl of virus-infected tissue, and then a 1:20,000 dilution of G604. Each antibody
incubation step was carried out overnight at 4°C and was separated from the next step by
vigorous washing with water and phosphate-buffered saline supplemented with 0.05%
Tween 20. The results were analysed using a BioRad Model 680 ELISA plate reader. The
mean absorbance values of the different transgenic citrus lines were compared using the
1-way analysis of variance with Fisher’s Protected Least Significant Difference test.
Statistical analysis was done using the SPSS version 8.0 software (Chicago, Illinois).
Challenge of Transgenic Plants with Citrus exocortis viroid (CEVd)
Ten plants from the transgenic lines, N3, N11, N25, N63 and N71, were challenged
with CEVd by graft inoculation. N9 was omitted from the experiment due to a shortage
of replicated plants. After one month, RNA was extracted from the viroid-inoculated
plants. Briefly, 0.2 g of tissue was ground in liquid nitrogen. Four hundred microliters of
41
STE (0.01 M Tris-HCl pH8.0, 0.1 M NaCl, 0.01 M EDTA) and phenol, chloroform-
isoamyl alcohol (24:1), and 91.4 µl of 10% SDS was added to each sample which was
briefly vortexed, shaken at room temperature for 10 min and centrifuged at maximum
speed at 4°C. The supernatant was transferred to a new tube to which 0.02 g of CF-11
powder and ethanol (final concentration of 35%) were added. The samples were shaken
at 4°C for 30 min, centrifuged and the supernatant discarded. The resultant pellet was
washed four times with 1x STE + 35% ethanol. The RNA was precipitated at -20°C with
0.1 volume of 3 M sodium acetate pH 5.2 and 2.5 volumes of 95% ethanol. The RNA
pellets were resuspended in 20 µl of RNAse-free water. Five microlitres of RNA was
used in RT reactions with primers CEV D-5 5'-ACGAGCTCCTGTTTCTCCGCTG-3'
and CEV D-7 5'CCGGGCGAGGGTGAAAGCCC-3' (Sieburth et al. 2002), and MMLV
reverse transcriptase (Invitrogen, Carlsbad, CA). The following parameters were used for
the RT reaction: 42°C for 15 min, 99°C for 5 min and 5°C for 5 min. One tenth of the
volume of the RT reaction was used for PCR with primers CEV D-5 and CEV D-7. The
PCR parameters were as follows: 92°C for 5 min, and 30 cycles of 92°C for 30 sec, 55°C
for 45 sec and 72°C for 45 sec, with a final extension step at 72°C for 3 min. The
products were analyzed on a 2% agarose gel.
To determine whether there was any difference in the level of viroid in the plants,
1 µl of a 1:10, 1:100 and 1:1000 dilution of each RNA sample was spotted onto a
Hybond+ membrane, and the procedure for Northern blotting as described in the Roche
hybridization manual was followed. The DIG-labeled probe used for hybridization was
synthesized by PCR according to the manufacturer’s instructions (Roche) using the
primers CEV D-5 and CEV D-7.
42
Results
Transformation and Regeneration
For the transformation experiment a total of 10,850 epicotyl segments were used,
of which only 3% produced shoots (Table 3-3). Once the regenerated shoots had been
transferred to root-inducing medium, they were examined for GFP expression. Only
1.44% of the shoots from the PAPc transformation experiment expressed GFP (Figure
3-3), and this expression was not uniform throughout the shoots. The percentage of PAPn
and PAPx-transformed shoots expressing GFP was slightly higher, with two PAPn and
one PAPx seedling expressing GPF in all tissues (Table 3-3).
Since the process for visualization of GFP expression in citrus tissue was not yet
optimized, all shoots were also tested for the presence of the PAP and GFP genes by PCR
(Figure 3-4). It was found that there was a comparatively good association between GFP
expression and the presence of the GFP transgene. PCR analysis for the presence of the
PAP gene in PAPc transformants showed that 9.4% of the regenerated shoots carried the
transgene as opposed to 1.44%, which had both PAP and GFP genes (Table 3-3).
Shoots that produced roots were transferred from the growth chamber to a growth room
with a temperature of 28ºC and light cycle of 16 h light followed by 8 h of darkness.
Once the plants had been acclimatized and transferred to the greenhouse, they were
periodically screened for the presence of the PAP transgene by PCR. Results showed that
all of the PAPc plants that had initially tested positive for the PAP gene were negative.
None of the PAPx and PAPv plants survived after being transferred to soil.
Phenotypically, the PAPn plants were indistinguishable from the
non-transformants. The PAPc-transformed plants did not exhibit any abnormality except
that they grew more slowly than the non-transformed plants (Figure 3-5 A). The PAPv
43
PCR-positive plants that had been transferred to soil exhibited a severely stunted
phenotype (Figure 3-5 B) and did not survive.
Figure 3-3. Analysis of GFP expression in citrus shoots regenerated from epicotyl
segments transformed with pCambia2202-PAPc and -PAPn. A-C) Transgenic shoots expressing GFP. D) Non-transgenic plant exhibiting auto-fluorescence.
44
Figure 3-4. PCR analysis of potentially transgenic shoots containing the PAPn transgene.
The names of the plants are indicated above the lanes. A) PCR amplification of the PAPn gene using the primers CN444 and CN445. B) PCR amplification of the GFP gene using the primers CN462 and CN463. N14, N15, N17, N18, N19, N20 and N21 were negative for GFP. The plasmid used as a positive control was pCambia2202 containing the PAPn gene.
Table 3-3. Summary of results for the transformation of Citrus paradisi cv. Duncan grapefruit with pokeweed protein (PAP) antiviral constructs.
Construct No. Segments used in transformation experiment
No. Segments producing shoots
Total no. shoots regenerated
No. shoots expressing GFP
No. PCR Positive shoots PAP GFP
% shoots PAP and GFP positive
No. Transgenic survivors in soil
PAPc 5000 120 (2.40%) 138 2 13 2 1.44 0 PAPn 4400 196 (4.45%) 215 17 17 22 7.90 6 PAPx 850 11 (1.29%) 14 1 2 1 7.14 0 PAP 600 6 (1.00%) 6 0 2 0 0.00 0 Total 10,850 333 373 20 32 25 6
45
46
Southern Blot Analysis and RT-PCR of Transgenic Plants
Initial information on transgene integration was provided by the PCR results (Table
3-3 and Figure 3-4). However, information on the number of copies of the transgene can
only be provided by Southern blot analysis. Nine surviving plants that were PCR-positive
for the PAPn gene were analyzed for stable integration of the transgene and its copy
number by Southern blot analysis. The plant genomic DNA was digested with BglII,
which does not have a cleavage site within the transgene sequence. There was no
hybridization of the PAP-derived probe to the non-transformed control (Figure 3-6, lane
1). All the plants were found to be stable transformants resulting from independent
integration events and having one to four copies of the PAP sequence present, with the
totally GFP-expressing plant, N101, having just one copy of the gene.
The four plants, N8, N10, N16 and N101 died. The remaining plants were analyzed for
transcript production by RT-PCR. Traditionally, transgene transcripts are analyzed by
Northern blotting. However, RT-PCR is increasingly being used as an alternative method,
especially when only a qualitative result is sought. To eliminate the possibility that PCR
positives might be due to the amplification of contaminating genomic DNA, the RNA
was DNAse-treated prior to the RT reaction, and this RNA was then also used as a
negative control (No-RT reaction) in the PCR amplification of the PAP gene. As seen in
Figure 3-7 no amplification was obtained for the non-transformed control and the No-RT
controls. This confirmed that the RT-PCR amplified products observed for N3, N9, N11,
N63 and N71 were the result of amplification of DNA complementary to PAP mRNA
that had been transcribed in these plants. Plant N25, which has the PAP gene integrated
into its genome, did not yield an RT-PCR product, suggesting that the gene had been
silenced.
47
A
B
Figure 3-5. Phenotypes of plants transformed with the PAPc and PAPv constructs. A)
PAPc transformants are on the right and non-transformed plants are on the left. B) A non transformed plant is on the left and a plant transformed with PAPv is on the right.
48
Figure 3-6. Southern analysis to determine transgene copy number in transgenic
grapefruit plants transformed with PAPn. A) Lane 1, non-transformed negative control; 2, N3; 3, N63; 4, N101; 5, plasmid positive control; 6, N10; 7, N16; 8, N8; 9, N11; 10, N9. B) Lane 1, N25; 2, N71; 3, plasmid positive control.
Protein Expression Analysis
The five plants for which PAP expression had been demonstrated at the level of
transcription were further analysed by western blotting for expression of the recombinant
protein. Faint bands corresponding to the 29 kDa protein obtained for the wild type
positive control were detected for the plants N3, N9, N11, N63, and an extremely faint
band was observed for N71, indicating that the protein was being expressed in the
transgenic plants, albeit at a very low level (Figure 3-8).
49
Figure 3-7. RT-PCR analysis of transgenic citrus transformed with PAPn to determine
gene expression at the level of transcription. Lane 1, lambda-HindIII; 2, plasmid control; 3, no nucleic acid control; 4, non-transgenic-RT; 5, non-transgenic no-RT; 6, N3-RT; 7, N3 no-RT; 8, N9-RT; 9, N9 no-RT; 10, N11-RT; 11, N11 no-RT; 12, N25-RT; 13, N25 no-RT; 14, N63-RT; 15, N63 no-RT; 16, N71-RT; 17, N71 no-RT. Total RNA was isolated from the plants and cDNA was prepared using 5 µg of RNA.
Figure 3-8. Western blot analysis of total soluble proteins from transgenic citrus to
determine the expression the PAPn transgene. Lane 1, molecular weight marker; 2, 250 ng purified commercial PAP; 3, non-transgenic control; 4, N3; 5, N9; 6, N11; 7, N63; 8, N71.
Virus Challenge of Transgenic Plants
In vivo antiviral activity of the five transgenic lines expressing PAP, as well as line
N25 which contained the PAP transgene but did not express it at detectable levels, was
50
tested by graft inoculating the multiplied lines with the severe CTV strain, T66
sub-isolate E. Plant samples were tested for viral antigen levels by ELISA two and four
months after inoculation. Absorbance values twice that of the healthy control were scored
as positive reactions for the presence of CTV.
ELISA results after two months showed that almost all of the multiplied plants
within each of the five PAP-expressing lines as well as line N25 were positive for the
virus as determined by comparison of antigen levels in these lines with that of the
non-infected control (mean = 0.03125). Absorbance values (A405 nm) ranged from
0.0335-1.3235 for N3, 0.0445-1.3915 for N9, 0.0165-0.585 for N25, 0-1.541 for N63 and
0.002-2.165 for N71. Despite scoring an overall positive ELISA reaction for the presence
of CTV, approximately 50% of the replicates of the lines N11, N25, and N71 and 25% of
those in lines N3, N9, and N63 tested negative for CTV. However, statistical analysis of
the absorbance data showed that the viral antigen levels of the transgenic plants were not
significantly different from that of the non-transgenic control.lines.
After four months, CTV antigen levels decreased in comparison to the antigen
levels after two months in lines N3 (87.4%), N9 (105.1%), N11 (115.4%) and N63
(22.3%). An increase in antigen levels was observed for lines N71 (2.4%) and N25
(54.8%) in comparison to the level of CTV antigen present in the plants after two months.
Despite the increased antigen level, line N25 maintained one of the lowest levels of CTV
after four months, when compared to the non-transgenic control. The mean absorbance
values for the transgenic plants were lower than that of the non-transgenic control. The
line N9 showed the lowest CTV antigen level as compared to the control (344.1%),
followed by N11 (270.9%), N25 (251.6%), N63 (142.8%), N3 (90.0%) and N71 (63.0%).
51
Statistical analysis showed that the mean absorbance values obtained after four months
were significantly lower than that of the non-transgenic control (Table 3-4).
Table 3-4. CTV antigen levels four months post-inoculation.in transgenic Citrus paradisi plants expressing PAPn
Plant Line¶ Mean Absorbance at 405 nm§ Non-transgenic control 0.675 a N71 0.414 b N3 0.355 b c N63 0.278 b c d N25 0.192 c d N11 0.182 d N9 0.152 d Mean absorbance values of the different transgenic citrus lines were compared using Fisher’s Protected Least Significant Difference test. Means with the same letter are not significantly different at P=0.05. ¶ Eight to twenty-eight plants per transgenic citrus line were graft-inoculated with CTV strain T66 sub-isolate E § CTV antigen levels were determined by DASI-ELISA four months post-inoculation. Viroid Challenge of Transgenic Plants
Plants were assayed for the presence of viroid RNA by RT-PCR one month after
inoculation. The plants from transgenic lines N3, N11, N24, N63 and N71 tested positive
for the presence of CEVd (Figure 3-9). To determine whether there were differences in
the levels of viroid present in the transgenic plants versus the controls, a Northern dot
blot was done using three different 10-fold dilutions of each RNA sample. The transgenic
and control plants appeared to have similar levels of viroid RNA, indicating that PAPn
had no effect on viroid replication (data not shown).
Discussion
The broad-spectrum virus resistance displayed by wild type PAP and its mutants
makes it an ideal candidate for the development of virus-resistant plants. PAP-expressing
transgenic plants have been obtained for N. benthamiana, N. tabacum cv. Samsun,
Solanum tuberosum cv. Russet Burbank and Agrostis stolonifera L. (creeping bentgrass).
52
Figure 3-9. RT-PCR analysis of representative viroid-infected PAPn-transgenic plants.
A) Lane 1, 100 bp marker; 2, no DNA control; 3, healthy control; 4, positive control; Lanes 6, 9, 11, line N25; 7, 15, line N3; 8, 13, 16, line N71; 10, 14. line N63. B) Lane 1, 100 bp marker; 2, line N63; 3, 4, line N11; 5-11, infected non-transgenic controls. The plants were graft-inoculated with CEVd and assayed for the presence of the viroid RNA one month later.
These plants have been evaluated for resistance to RNA viruses such as TMV,
CMV, PVX and PVY as well as the fungi Rhizoctonia solani and Sclerotinia homeocarpa
(Dai et al., 2003; Lodge et al., 1993; Tumer et al., 1997; Zoubenko et al., 2000). The
objective of this research was to transform Duncan grapefruit with PAP mutants and
53
determine the efficacy of these mutants as antiviral agents against CTV and CEVd. Both
pathogens have RNA-based genomes, albeit with different structures.
The percentage of shoots produced in the transformation experiment as a whole
was very low at 3%. This value is considerably lower than the 23-29% obtained in C.
paradisi transformation experiments by Rosales (2001), Cevik (2001), and Luth and
Moore (1999) using the same protocol. Lower percentages of regeneration, ranging
between 4% and 13%, were obtained by Febres et al. (2003). Collectively very few
transformed shoots were obtained. The only surviving transformants contained the PAPn
construct. PAP mutants, PAPn and PAPc, reportedly retain their antiviral activity without
the toxic rRNA depurination activity, while PAPv retains both properties and PAPx is an
inactive form of the protein (Tumer et al., 1997; Zoubenko et al., 2000). Therefore, one
would expect that only the PAPv construct would produce a low number of
transformants. Tumer et al. (1997) and Zoubenko et al. (2000) were able to obtain
transformation frequencies of approximately 13% in N. tabacum transformed with the
non-toxic forms of PAP. Very few transformed tobacco plants and only one transformed
potato plant containing the PAPv gene were obtained, and only at low expression levels
of PAPv did the plants resemble non-transformed plants (Lodge et al., 1993).
Transformation of creeping bentgrass with PAPc produced very few transgenic plants,
which ultimately did not exhibit the same level of pathogen resistance as their N.
tabacum counterparts (Dai et al., 2003). Therefore, it is possible that the nature of the
gene, combined with the inherent problems of transforming a heterologous host such as
citrus, may have had an impact on the number of transgenic plants produced.
54
PCR analysis of the transgenic plants revealed that not all plants had both PAP and
GFP genes (Table 3-3). Most of the plants contained only the PAP gene. This
discrepancy may be due to the partial integration of T-DNA. It is known that transfer
proceeds from the right border of the T-DNA to the left border (reviewed by Gelvin,
2003). Thus one would expect a higher proportion of plants to be positive for the reporter
gene, which is closest to the right border. However, transfer can occasionally be initiated
from the left border, and the efficiency of transfer depends upon the binary vector and
Agrobacterium strain used. All of the PAPc shoots that had been PCR positive for the
transgene appeared to eventually have lost the gene. This may be due to the fact that
these plants had been chimeras, and new tissue growth did not contain the gene of
interest. Alternatively, the gene may have been deleterious to the plants, and
chromosomal rearrangements may have led to the loss of the PAP gene.
Before evaluating transgenic plants for their performance in field trials, it is
important to first characterize the plants with respect to transgene integration and
transcript production. A number of stably transformed grapefruit containing 1-4 copies of
the transgene per genome were obtained (Figure 3-6) and of the six surviving plants, five
had detectable levels of PAPn transcript and protein. Except for N9, all these plants as
well as the plant, N25, which retained the transgene in its genome but did not express it at
a detectable level, were replicated and challenged with both CEVd and CTV strain T66
sub-isolate E by graft inoculation. The replication of CEVd was not inhibited in the
transgenic plants. A possible reason for this result is that the site of replication for viroids
in the Pospiviroidae group is the cell nucleus. Thus the replicating viroid RNA may have
been protected from PAPn. Vivanco (1999) showed that spraying plants with a RIP from
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Mirabilis jalapa prior to inoculation with PSTVd, prevented infection of the plants by the
viroid. Thus in this case the RIP had access to the viroid RNA and could have inactivated
it via its N-glycosidase activity.
Using a rule that an A405 nm two times higher than the non-inoculated control
constituted a positive result for the presence of CTV, 25-50% of the transgenic plants
tested negative for the presence of the virus after two months. This result was not
completely unexpected, since in many cases PAP-expressing transgenic plants have been
shown to accumulate lower levels of virus rather than showing total resistance to
infection (Lodge et al., 1993; Tumer et al., 1997). In antifungal tests PAP-expressing
plants also showed a lower percentage of disease incidence (Dai et al., 2003; Zoubenko et
al., 2000) and in virus and fungal-infected plants, the onset of disease was delayed.
Statistical analysis of the ELISA data showed that there was no significant difference in
CTV antigen levels in comparison to the non-transgenic controls after two months.
Surprisingly, line N25, for which no PAPn transcript was detected, had the lowest level
of CTV. After four months, viral antigen levels decreased in lines N3, N9, N11 and N63
and overall, the transgenic plants had significantly lower levels of CTV than the non-
transgenic control. After four months, line N25 still had one of the lowest levels of CTV,
despite a 54.8% increase in antigen levels. Dai et al. (2003) found that two creeping
bentgrass transformants expressing PAPII, an isoform of PAP, showed no detectable
protein expression and only a very low level of PAPII mRNA, and yet these plants
exhibited restance to Sclerotinia homeocarpa. Thus, the levels of PAPn mRNA and
protein in line N25 may have been below the limits of detection by the methods used,
since a low level of CTV antigen was detected, indicating aresistance phenotype. The
56
results obtained suggest that the expression of the PAPn gene in Citrus paradisi causes an
antiviral response. From these results, it can be concluded that the PAPn construct does
not provide complete resistance to CTV. However, the decrease in viral antigen with time
would need to be monitored for a longer period to ascertain the full and accurate effect of
PAPn on CTV infection.
CHAPTER 4 THE EFFECT OF POKEWEED ANTIVIRAL PROTEIN ON THE REPLICATION OF
Citrus tristeza virus IN Nicotiana benthamiana PROTOPLASTS
Introduction
Early attempts to produce virus-resistant plants were based on the pathogen-derived
resistance (PDR) approach, which utilizes genes and defective interfering RNAs or
DNAs of viral origin (reviewed by Baulcombe, 1996). Since then, researchers have tried
to exploit plant genes conferring natural resistance to particular pathogens by
transforming these genes into heterologous plant hosts, and also altering the expression of
genes involved in plant defense-related signal transduction pathways. A group of proteins
that have shown promise as naturally occurring antiviral agents are ribosome-inactivating
proteins (RIPs). However, as a result of their N-glycosidase activity, which results in the
inactivation of ribosomes and ultimately the cessation of translation, many wild type
RIPs cause major phenotypic aberrations when expressed in heterologous hosts.
Pokeweed antiviral protein (PAP) is a RIP that adversely affects plant development
when expressed in tobacco at concentrations higher than 5 ng/mg of total protein (Lodge
et al. 1993). PAP was shown to inhibit infection of plants by seven different viruses, each
representing a different virus group (Chen et al., 1991); thus PAP might confer a broad-
spectrum of resistance to plants. The potential agronomic value of PAP as a
broad-spectrum resistance agent prompted the search for non-toxic mutants that retained
their antiviral activity. Several such mutants were identified by random mutagenesis and
selection in Saccharomyces cerevisiae (Hur et al., 1995). Two of these mutants, viz.
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58
PAPc (Trp237-stop) and PAPn (Gly75-Val), have been used successfully to transform
tobacco and produce phenotypically normal plants that exhibited resistance to certain
viruses (Tumer et al., 1997; Zoubenko et al., 2000). Two other mutants that were
produced, viz. PAPv and PAPx showed reduced toxicity and no enzymatic activity,
respectively.
Citrus tristeza virus (CTV) is one of the more complex plant viruses. For many
years the fragile virions, the limited host range, lack of local lesion hosts,
phloem-limitation of virions and a single-stranded RNA genome of approximately 20 kb
in size hampered the in depth study of its biology and molecular characteristics. To
facilitate the study of CTV replication, two protoplast systems, one from a CTV host,
Citrus sinensis cv. Hamlin and the other from a non-host, Nicotiana benthamiana, were
developed (Price et al., 1996; Navas-Castillo et al., 1997). Although the general pattern of
CTV replication, with respect to the time of accumulation of the viral products was the
same in both protoplast systems, the final yields of viral RNAs, proteins and virions were
approximately 40 times higher in N. benthamiana (Navas-Castillo et al., 1997). Virions
were found to be a better source of inoculum than purified genomic RNA. The lower
level of replication observed with RNA as the inoculum is most probably due to the
reduced efficiency of protoplast inoculation as a result of the large genome size. The
development of a full-length infectious cDNA clone of CTV and smaller CTV replicons
has further increased the progress towards understanding the gene functions and
replication strategy (Satyanarayana et al., 1999). The smaller replicons are much more
efficient at transfecting N. benthamiana protoplasts, indicating that the large genome size
may have been a limiting factor in previous work.
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The study of the effect of different genes and genetic mutations on replication of
CTV can be done in either citrus or N. benthamiana protoplasts. However, if the
objective is to study the effect of a stably integrated heterologous gene on CTV
replication, it would be more efficient to use the N. benthamiana protoplast system. The
relatively short generation time and an established transformation protocol make N.
benthamiana an ideal model system in which to study the effect of potential resistance
agents on viral replication.
The objectives of this study were to transform N. benthamiana with PAP mutants,
characterize the resultant transgenic plants by molecular methods and to examine the
effect of the expressed recombinant proteins on CTV replication in a protoplast system.
Materials and Methods
Constructs
The four constructs used for transformation, viz. PAPn, PAPc, PAPv and PAPx
were described in Chapter 3. The plasmids were transformed into Agrobacterium
tumefaciens strain AGL1 by the cold-shock transformation method (Birnboim and Doly,
1979).
Tobacco Transformation
Explant preparation
Nicotiana benthamiana seeds were sterilized by shaking them in 70% ethanol for
10 min, 0.042% sodium hypochlorite solution plus Tween 20 for 20 min and 3 x 2 min in
sterile deionized water. The seeds were germinated on half strength MS medium
(2.15 g/L MS salts, 50 mg/L myo-inositol, 10 g/L sucrose pH 5.8) supplemented with
6 g/L of agar. The plates were kept in a growth chamber that had a constant temperature
of 28°C and white fluorescent lighting with a 16 h day and 8 h night cycle. Once the
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seedlings had a well developed root, they were transferred to sterile soil in jars and
magenta boxes, and grown until the upper leaves reached a size of 2-3 cm across.
Transformation and regeneration of tobacco
The transformed Agrobacterium cultures were grown overnight at 28°C in YEP
medium (10 g/L yeast extract, 10 g/L Bacto peptone, 5 g/L sodium chloride)
supplemented with 50 µg/ml carbenicillin and 100 µg/ml chloramphenicol. The cultures
were centrifuged at 5000 rpm in a Beckman GSA rotor for 5 min and the cell pellet
resuspended in MS medium containing 100 µM acetosyringone to give a final
concentration of 5x108 cfu/ml. The leaves from sterile explants were cut into 1 cm2,
transferred to the Agrobacterium solution for 1 min, and the excess liquid removed prior
to being transferred to regeneration medium (4.3 g/L MS salts, 100 mg/L myo-inositol,
4 mg/L thiamine, 30 g/L sucrose, 1 mg/L IAA, 2 mg/L kinetin). The plates were
incubated in the growth chamber for 48 h and then transferred to regeneration medium
supplemented with 200 mg/L mefoxin and 50 mg/L kanamycin. The leaf pieces were
transferred to fresh medium every two weeks. Shoots were transferred to rooting medium
(R1/2N) supplemented with 200 mg/L mefoxin and 50 mg/L kanamycin. After the
appearance of the first root, shoots were transferred to soil in pots and covered with a
plastic bag which was slashed after a few days to allow the plants to become acclimated
to the conditions in the growth room (28°C, 16 h photoperiod).
Analysis of Transgenic Plants by PCR
All the regenerated plants were tested for the presence of the transgene by PCR
using either of two gene-specific primer pairs, CN444, 5'
ATTAGGGCCCGGGAAGATGAAGTCAATGCTTGTG 3' and CN445 5'
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AATTGCGGCCGCTCAGAATCCTTCAAATAGATC 3', or CN444 with CN273 5'-
TTATCTGGGAACTACTCACAC 3’, which is specific for the CaMV 35S termination
sequence. DNA was extracted from the plants using a modification of the protocol by
Oliveira et al. (2000), previously described in chapter 2. PCR was carried out in a
Biometra thermocycler using a profile of 94°C for 2 min initial denaturation, 29 cycles of
denaturation at 94°C for 30 sec, primer annealing at 55°C for 45 sec, primer extension at
72°C for 45 sec and a final primer extension step of 72°C for 5 min. The PCR products
were analysed by electrophoresis in 1% agarose gels in TAE buffer.
Southern Hybridization
For Southern hybridization 10 µg of total genomic DNA from transgenic plants and
non-transformed control plants was digested with BglII (Invitrogen, Carlsbad, CA). After
agarose gel electrophoresis the DNA was transferred to nylon membrane (Hybond N+,
Fisher Scientific) by capillary action overnight (Sambrook et al., 1989). Using the
primers CN444 and CN273, a DIG-labeled probe was produced by PCR amplification
according to the manufacturer’s instructions (Roche, Indianapolis, IN). The DIG-labeled
probe was denatured and used for a 16 h hybridization following 2-4 hours of
prehybridization in DIG Easy Hyb Buffer (Roche). The blot was prepared for
visualization on X-ray film according to the protocol provided by Roche.
Reverse-Transcription (RT) PCR
PAP gene expression at the transcriptional level was analysed in the transgenic
plants by RT-PCR. Total RNA was isolated from the plants with TRIzol reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Prior to RT-
PCR, the RNA was treated with DNAse (Promega, Madison, WI) to eliminate any DNA
contaminants, and then phenol-chloroform cleaned to remove the DNAseI. cDNA was
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prepared using 5 µg of RNA, the reverse primer CN273 and MMLV reverse transcriptase
(Promega). The parameters for the RT reaction were: 1 hour at 42°C and 15 min at 70°C.
One tenth of the volume of the RT reaction was used for PCR with the primers CN273
and CN444 and the products were analysed on a 1% agarose gel run in TAE buffer.
Analysis of Protein Expression
PAP expression was analyzed by both Western blotting and ELISA. Protein
concentration was determined by the Bradford Assay (BioRad, Hercules, CA). For
Western blotting, aliquots of the soluble plant proteins in phosphate buffered saline (PBS,
137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), were mixed
with equal volumes of dissociation buffer (140 mM SDS, 160 mM Tris-HCl pH 7.8,
1% v/v glycerol, 142 mM β-mercaptoethanol), to give a final concentration of 100 µg
total protein, which was then boiled for 2 min and separated on a precast 12%
polyacrylamide Tris-HCl gel (BioRad). Proteins were transferred to nitrocellulose
membrane using a BioRad semi-dry horizontal electrotransfer apparatus according to the
manufacturer’s instructions. Colorimetric detection of the protein was carried out using a
1:500 dilution of 0.5 mg/ml anti-PAP-IgG (provided by Dr. Maria Kaniewski) and the
substrates NBT and BCIP (Sigma).
For ELISA analysis the 96 well plates were coated with either a 1:500 or 1:1000
dilution of anti-PAP-IgG. The plant tissue was extracted in an equal volume of PBS-
Tween 20 containing polyvinylpyrrolidone (PVP-40). The PAP-alkaline phosphatase
conjugate (provided by Dr Maria Kaniewski) was used at a 1:5000 dilution. All
incubations were carried out overnight at 4°C. The substrate, ρ-nitrophenyl-o-phosphate
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was used at a concentration of 1 mg/ml. The plates were analyzed using a BioRad Model
680 plate reader and associated software.
Determination of Antiviral Properties of Transgenic Plants
To determine whether the transformed plants exhibited antiviral activity, five plants
from the R0 and one from the R1 generation along with a non-transgenic control plant
were challenged with Tobacco mosaic virus (TMV), a virus known to be inhibited by the
antiviral activity of PAP (Lodge et al., 1993). Infected plant material (0.5 g) was ground
in liquid nitrogen, resuspended in 5 ml of PBS containing carborundum, and applied to
the leaves. Leaf samples were collected and analysed by ELISA at 7 and 14 days post-
inoculation. The 96 well plates were coated with the antigen for 2 h at room temperature,
followed by incubations with the TMV-U strain antibody (provided by Dr. Ernest
Hiebert) and goat-anti-rabbit-alkaline phospatase conjugate (Sigma) used at a 1:5000 and
1:30 000 dilution respectively. Results were analyzed using a Bio-Rad Model 680
microplate reader. The mean absorbance values obtained from the different tobacco lines
were compared using the 1-way analysis of variance with Fisher’s Protected Least
Significant Difference test. Statistical analysis was done using the SPSS version 8.0
software (Chicago, Illinois).
Viral Replication in Transgenic Protoplasts
R1 plants from lines TN5, TN11, TN18, TN23a and TN27a were grown for six
weeks under controlled environmental conditions. Fully expanded leaves were sterilized
in 70% ethanol and 10% sodium hyperchlorite and washed 3 times in sterile water. The
lower surface of the leaves were slashed at approximately 1 mm intervals and incubated
overnight in the dark at 25°C, in an enzyme solution containing 0.5% Onozuka cellulase
RS (Yakult Honsha, Japan) and 0.25% macerase pectinase (Calbiochem, USA) dissolved
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in 13% MMC buffer (13% D-mannitol, 5 mM MES, 10 mM CaCl2, pH 5.8). The
mesophyll protoplasts were isolated and transfected with CTV virions using polyethylene
glycol (PEG) mediated transfection as described by Navas-Castillo et al. (1997) and
Satyanarayana et al. (1999). The macerated leaves were centrifuged at 100 x g for 3 min
and the supernatant was discarded. The pellet was resuspended in 15 ml of 0.6 MM
buffer (0.6M D-mannitol, 5 mM MES, pH 5.8) and centrifuged for 3 min at 100 x g. The
pellet was resuspended in 0.6 MM buffer, layered over 20.5% sucrose, centrifuged for 7
min at 100 x g. The protoplasts were collected from the top layer and washed twice with
0.6 MM. Ten microliters of purified CTV virions were added to 200 µl of protoplasts.
Five hundred microliters of PEG 1540 was then added, and the solution was gently mixed
for 20 sec, after which 0.6 MM bufferwas added. The protoplasts were allowed to recover
for 10 min and centrifuged at 100 x g for 3 min. The pellet was resuspended in 0.6 MM
AOKI (0.6 M D-mannitol, 5 mM MES, 100 ml of 10x AOKI salts to make 1 L of
solution, pH 5.8) containing a 1:200 dilution of antibiotic/antimycotic solution (Sigma,
St. Louis, MO), centrifuged at 100 x g for 3 min and resuspended in a final volume of 3
ml of 0.6 MM AOKI with antibiotic/antimycotic. The protoplasts were incubated under
light and harvested three days post-inoculation (dpi). Total RNA was isolated and
Northern hybridization was performed as described by Satyanarayana et al. (1999), using
positive and negative sense 3' terminal probes.
Results
Transformation and Regeneration
A total of seventy-four shoots were obtained from all the N. benthamiana
transformation experiments. The majority of the shoots produced had been transformed
with the PAPn construct and no shoots were obtained from leaf disks transformed with
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PAPv (Table 4-2). The potentially transgenic plants were screened only by PCR for the
presence of the transgene (Figure 4-1). Although 55 plants initially tested positive for the
presence of the transgene, subsequent PCR screens as the plants matured revealed that
only 27 continued to test positive. The plants that maintained the transgene after three
months (as determined by PCR) had all been transformed with the PAPn mutant. No
phenotypic abnormalities were observed for the surviving plants.
Figure 4-1. PCR analysis of selected potential transgenic N. benthamiana plants using
primers CN444 and CN445. Lane 1, 1 kb marker; 2, no DNA control; 3, plasmid control; 4, non-transgenic control; 5, N5; 6, N6; 7, N7; 8, N8; 9, N9; 10, N10; 11, N11; 12, N12; 13, N13.
Table 4-1. Summary of N. benthamiana transformation experiments with the PAP
constructs. Construct Total number of
shoots Initial PCR Positive
PCR positive after 3 months
PAPv 0 0 0 PAPc 24 12 (50.0%) 0 PAPx 9 4 (44.4%) 0 PAPn 55 39 (70.9%) 27 (49.1%) Molecular Analysis of Transgenic Plants
All the transgenic plants that survived and had been transferred to soil were
analyzed by Southern blotting to determine the transgene copy number. No hybridization
of the PAP-specific probe was detected in the genomic DNA sample from the
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non-transformed control (Figure 4-2). The transgene was stably integrated with the copy
number of the PAPn transgene varying from 1-4 copies per genome (Figure 4-2). Having
established the genomic integration of the transgene, the plants were analyzed for PAPn
transcript production by RT-PCR (Figure 4-3). Total RNA isolated from the transgenic
plants was first treated with DNAseI to remove any contaminating DNA that might result
in a false positive result. The results showed that the transgene was transcribed in 48.1%
of the surviving plants.
Figure 4-2. Southern analysis of R0 tobacco plants transformed with PAPn. The names of
the plants are indicated above the lanes. TUT is a non-transformed control plant. The positive control is a plasmid containing PAPn, digested with PstI.
Since the protein product of the PAP gene is required for antiviral activity, the
plants were further analysed by western blotting and ELISA in order to determine
whether the protein is being expressed. No protein was detected when the plants were
analyzed by western blotting (data not shown). Since ELISA is more sensitive than
67
western blotting, the plants were screened for protein expression using this technique. In
comparison to the colorimetric reaction of purified wild type PAP (Worthington) at
concentrations as low as 1 ng, the level of PAPn in the plants tested was not visually
detectable. Including purified PAP at different concentrations, and using the ELISA
analysis software to construct a standard curve, PAPn levels of 0-25 pg/mg were
determined (Table 4-2).
Figure 4-3. RT-PCR analysis of RNA isolated from selected transgenic plants to
determine PAPn expression at the transcriptional level. Lane 1 is the λ HindIII molecular weight marker. Lanes 2, 4, 6, 8, 10, 12 and 14 are the no-RT controls for each sample during the PCR reaction. Lane 3, non-transformed control; 5, TN5; 7, TN7; 9, TN11; 11, TN18; 13, TN23a; 15, TC27.
The low or undetectable levels of PAPn protein in the transgenic plants raised
questions as to whether it would be viable to continue with the CTV replication studies in
protoplasts derived from the progeny of these plants. Therefore, it was decided to
challenge five R0 plants and one R1 plant with TMV, a virus known to be inhibited by
PAP (Lodge et al., 1993). The second and fourth systemically infected leaves were
evaluated for virus antigen levels by ELISA at 7 dpi and 14 dpi respectively (Table 4-2).
The absorbance values obtained showed that there was no detectable level of TMV in all
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the test plants after one week, as compared to the non-transgenic control. Statistical
analysis of the mean absorbance values obtained for each plant 14 dpi, using Fisher’s
Protected Least Significant Difference test showed that all five transgenic plants had
significantly lower levels of virus antigen compared to the control and that the level of
virus in TN5, TN11 and TN18 was significantly different from TC27, TN7 and TN23a
(Table 4-2). Comparison of the ELISA data obtained 7 and 14 dpi using a one tailed t-test
assuming equal variance showed that there was no significant difference in virus antigen
levels after the two week period for TN5, TN11, TN18 (Table 4-2). Thus these plants
containing the PAPn transgene showed resistance towards TMV, although this resistance
was not complete since a low level of viral antigen could be detected by ELISA. These
results were the basis for proceeding to study the effect of PAPn on CTV replication.
Table 4-2. TMV antigen levels in transgenic N. benthamiana expressing PAPn following infection with the virus.
Plant Line PAP (pg/ml)§ Mean Absorbance¶ 7 dpi 14 dpi
Infected non-transgenic control 0 0.0705 0.230a TC27* nd 0.008 0.154b TN23a nd 0.005 0.121b TN7 nd 0.0055 0.117b TN11 25.1 0.006f 1.067x10-2cf TN18 5.05 0.0055e 9.33x10-3ce TN5 nd 0.006d 8.667x10-3cd Mean absorbance values of the different plants 14 dpi were compared using the 1-way analysis of variance with Fisher’s Protected Least Significant Difference test. Means with the same letter are not significantly different at P=0.05. dpi: Days post-inoculation. § PAPn levels were quantitated by ELISA. ¶ The second and fourth systemically infected leaves were assayed for virus antigen levels by ELISA 7 and 14 dpi, respectively. *This plant was from the R1 generation. All other plants were from the R0 generation. nd=not detectable
69
CTV Replication in PAPn-Transgenic N. benthamiana Protoplasts
The replication of CTV in the protoplasts was assessed by Northern blot analysis.
Analysis of RNA extracted from the CTV virion-transfected protoplasts, using a 3' minus
sense probe showed no difference between the levels of positive sense RNA in the
transgenic plants when compared to the control (Figure 4-4 A). This result may have
been influenced by the presence of the positive- sense input RNA, which masked the true
level of replication.
Figure 4-4. Northern blot analysis of CTV RNAs in N. benthamiana protoplasts derived
from PAPn transgenic R1 plants. A) Positive sense 3' probe. B) Negative sense 3' probe. Lane 1, inoculated non-transgenic control; 2, TN5; 3, TN7; 4, TN11; 5, TN18; 6, TN23a. Protoplasts were transfected with CTV virions and RNA isolated 3 dpi. The numbers to the left of the figures indicate the subgenomic mRNAs corresponding to ORFs 4-10. The arrow indicates the genomic RNA.
Thus, to obtain a more accurate view of replication, a 3' positive-sense probe was
used to measure the accumulation of minus strand RNA (Figure 4-4 B). The Northern
hybridization results showed that the production of negative-sense subgenomic RNAs
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and thus replication was considerably reduced in protoplasts derived from TN11 and
TN23a, while replication in TN5 was comparable to that of the control and TN7 and
TN18 had higher levels of replication. These results indicate that two PAPn transgenic N.
benthamiana lines are able to suppress CTV replication.
Discussion
N. benthamiana was transformed with four mutants of PAP, a ribosome-
inactivating protein reported to confer broad-spectrum resistance to viruses (Lodge et al.
1993). In two of the mutants (PAPc and PAPn) the antiviral activity of PAP has been
separated from the toxic ribosome-inactivating activity. The greater proportion of
transgenic shoots obtained were from transformation experiments with these two
constructs. However, repeated PCR analysis showed that the PAPc plants did not retain
the transgene as the plants matured. A similar result was obtained when Citrus paradisi
cv. Duncan grapefruit was transformed with the same construct (Chapter 3). There are
two possible reasons for this result. First, it is possible that this gene construct is
deleterious to the plants. The second reason could be that the plants were chimeras that
did not express the transgene in every cell. Lodge et al. (1993) showed that PAPc
expressed in transgenic N. tabacum did not have a toxic effect on ribosomes nor did it
cause phenotypic abnormalities. Therefore, the most likely reason for the loss of the
transgene appears to be that the PAPc plants had been chimeras. Not obtaining any
transformants for the PAPv construct was not surprising since PAPv, which has antiviral
activity, is only slightly less toxic to ribosomes than wild type PAP.
Approximately 48% of the plants produced the PAPn transcript. However, PAPn
expression at the protein level was almost undetectable for most of the plants. In other
studies where PAP or PAP mutants were expressed in N. tabacum, N. benthamiana or
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Solanum tuberosum, PAP levels were usually found to be above 0.6 ng/mg (Lodge et al.,
1993; Tumer et al., 1997). However, expression of PAP mutants in creeping bentgrass
was either non-existent or undetectable, yet field trials with those plants resulted in the
observation of different levels of resistance against the test-pathogen (Dai et al. 2003). To
determine whether the transgenic N. benthamiana plants might have any antiviral
activity, the plants were inoculated with TMV. These plants behaved similarly to the
creeping bentgrass, showing very low levels of accumulation of TMV antigen with time
despite having undetectable levels of PAPn.
Protoplast systems have played a pivotal role in elucidating viral infection events
occurring at the single cell level. Using protoplasts, the effects of salicylic acid on
different cell types during the infection process of TMV and Cucumber mosaic virus
(CMV) were demonstrated (Murphy and Carr, 2002). Events involved in cell-to-cell
movement of CMV and the replication of Cowpea mosaic virus (CoMV) have also been
studied in protoplast systems (Carette et al., 2002; Mise et al., 2001). An N. benthamiana
protoplast system was developed to facilitate the study of CTV replication (Navas-
Castillo et al., 1997). The R1 generation of transgenic plants which had shown inhibition
of TMV accumulation was used to produce protoplasts for the study of the effect of
PAPn on CTV replication. This approach was undertaken to quickly determine whether
PAPn affects CTV replication, as well as to decrease the inoculum pressure as compared
to graft inoculation of a plant. Northern blot analysis showed that three lines, TN5, TN11
and TN23a, were able to inhibit replication of CTV. This suggests that PAPn could be an
effective antiviral agent against CTV and provides further evidence that the PAPn
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construct expressed at very low to undetectable levels does not completely inhibit CTV
replication. A higher level of PAPn expression might completely inhibit CTV replication.
CHAPTER 5 SUMMARY AND CONCLUSIONS
Genetic engineering for resistance is often specific for a particular pathogen, e.g.
pathogen-derived resistance. Natural selection and the fact that the genomes of RNA
viruses are subject to changes as a result of errors introduced by the RNA polymerase
suggest that PDR may not be effective as a durable form of resistance. Targeting signal
transduction pathways that play a role in the plants’ general defense response, expressing
more than one gene in transformed plants or expressing proteins that can function as
antiviral agents by more than one mechanism, may prove effective in the longterm.
The efficacy of a ribosome-inactivating protein (RIP) from Phytolacca americana,
as an antiviral/antiviroid agent against two citrus pathogens was investigated. The RIP,
pokeweed antiviral protein (PAP), was reported to have broad-spectrum antiviral activity
against plant and animal viruses. The protein was reported to inhibit viral accumulation in
a number of ways. In vitro it inhibited viral translation by removing adenine and guanine
residues directly from the viral RNA; it recognised and preferentially depurinated RNA
that had a 5' methyl-guanosine cap structure; and it inhibited +1 frameshifting in the yeast
retrotransposon, TY1. All three of these mechanisms would be applicable to Citrus
tristeza virus (CTV), while only the first could be predicted to inhibit replication of a
viroid, viz. Citrus exocortis viroid (CEVd). CTV alone has caused the loss of millions of
trees worldwide and the propagation of viroid-infected trees has caused economic losses
in the Florida citrus industry, and is a major threat in regions where susceptible
rootstocks are used.
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To determine whether it would be viable to transform citrus with PAP, a
preliminary experiment to observe the effect of PAP on the translation of CTV was
conducted in vitro. The translation of a CTV replicon, CTV-∆Cla, which retains only the
5' and 3' UTRs, ORFs 1a and 1b and a gene fusion between ORF11 and part of ORF2,
was first optimized. CTV-∆Cla RNA was then incubated in the presence of wild type
PAP prior to translation in a rabbit reticulocyte lysate translation system. A decrease in
translation was observed at 20 ng PAP per 500 ng of RNA. Compared to previous
research, the concentration of PAP required to inhibit translation is very high. However,
since a similar result was obtained with the Brome mosaic virus RNA (a virus whose
translation in known to be inhibited by PAP) used as a control in this experiment, it was
concluded that the preparation of wild type PAP that was used may not have had optimal
enzymatic activity or that optimal activity may have been affected by some component of
the antiviral assay. Since the viral RNA will be exposed to the intracellular environment
in plants, the in vitro translation result demonstrating the direct effect of PAP on the
RNA, suggested that transgenic citrus correctly processing and expressing PAP might
exhibit reduced levels of CTV.
Epicotyl segments of etiolated Duncan grapefruit seedlings were transformed with
four PAP mutant constructs by Agrobacterium-mediated transfer. Two of the mutants,
PAPn and PAPc reportedly retained their antiviral activity without the deleterious
ribosome-inactivating property, while PAPx was an inactive form of the protein, and
PAPv which had antiviral activity was only slightly less toxic to ribosomes than wild type
PAP. Very few transformed plants were obtained, and those that survived to the
pathogen-challenge stage contained only the PAPn transgene. It is possible that the nature
75
of the gene, combined with the inherent problems of transforming a heterologous host
such as citrus, may have had an impact on the number of transgenic plants produced.
Southern analysis of the transgenic plants showed them to be stably transformed with 1-4
copies of the PAPn gene per genome. Of the six plants that survived, only five (N3, N9,
N11, N63 and N71) expressed PAPn at the protein level. However, all six transgenic
plants were replicated in preparation for the graft-challenge experiment. Due to a
shortage of plants, all lines except N9 were challenge-inoculated with CEVd. Plants
assayed for the presence of the viroid by RT-PCR one month post-inoculation were all
positive, indicating that PAPn did not inhibit viroid replication. There has been one other
published report of a RIP being tested for antiviroid activity (Vivanco, 1999). However,
the research did not involve the in vivo expression of the RIP in transgenic plants.
Instead, it was shown that an exogenous application of the RIP prevented viroid
infection. Therefore, a possible reason for the failure of PAPn to inhibit CEVd replication
may be due to the fact that the site of replication for CEVd is the cell nucleus. Thus the
viroid RNA may have been inaccessible to PAPn.
All six transgenic plant lines were graft challenged with CTV and viral antigen
levels were determined by DASI-ELISA two and four months post inoculation. Using a
rule that an A405 two times higher than the non-inoculated control constituted a positive
result for the presence of CTV, 25-50% of the transgenic plants tested negative for the
presence of the virus after two months. This result was not completely unexpected, since
in many cases PAP-expressing transgenic plants have been shown to accumulate lower
levels of virus rather than showing total resistance to infection. Statistical analysis of the
ELISA data showed that virus antigen levels were significantly lower than the non-
76
transgenic control plants for the lines N9, N11, N25 and N63. Surprisingly, the plants of
line N25, in which the PAPn transcript was not detected, accumulated the lowest level of
virus. The reason for this may be the result of the location of the transgene in the plant
genome or the RNA transcript levels may have been below the level of detection.
However, after four months CTV antigen levels increased in all the lines, indicating a
delay in virus accumulation in the plants that had tested negative after two months.
Although all the transgenic plants tested positive for the presence of CTV after four
months, the levels of CTV present in the transgenic lines were significantly lower than
that of the control line. In addition, the mean level of CTV antigen present in each
transgenic line was lower than level present after two months. Thus these results indicate
that the PAPn construct does not provide complete resistance to CTV. However,
continued analysis of the transgenic plants for the presence of CTV is required to
determine whether the CTV levels in these plants will continue to decrease or whether
they will maintain a consistently lower level of CTV in comparison to the non-transgenic
control plants.
Replication of CTV in PAPn-transgenic Nicotiana benthamiana protoplasts
indicated that three lines, TN5, TN11 and TN23a supported a lower level of replication
than the non-transgenic control. This was further evidence that the PAPn construct
expressed at very low to undetectable levels did not completely inhibit CTV replication.
Thus, a larger pool of transgenic grapefruit plants expressing higher levels of PAPn may
have produced plants that did not support CTV replication. Alternatively, expression of
PAPn from a phloem-specific promoter or the expression of other RIP candidates that
77
have antiviral activity, in citrus, may provide resistance to CTV and other pathogens of
citrus.
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BIOGRAPHICAL SKETCH
Yolanda Petersen was born in 1973 in Cape Town, South Africa. She earned a
Bachelor of Science degree in Microbiology and Botany at the University of Cape Town,
in December 1994. Her Bachelor of Science (Honors) and Master of Science theses were
entitled: “The Characterisation of a novel bacteriophage from the the causal agent of
chocolate spot disease of cabbage” and “The Characterisation of a novel Xanthomonas
bacteriophage”. The degrees were awarded at the University of Cape Town in 1995 and
1999, respectively. In 1999 she was awarded a scholarship by the USDA-ARS
International Programs to pursue the Doctor of Philosophy degree in the field of plant
pathology at the University of Florida.
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