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This is to certify that the

dissertation entitled

RNA Recombination between Viral Transgenes
and Infecting Viruses

presented by

Min Deng

has been accepted towards fulfillment
of the requirements for

Ph. D. , Plant Pathology
degree in

 

 

W¥m

Major professor

Date 9/21/99

 

MSU is an Affirmative Action/Equal Opportunity Institution 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
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RNA RECOMBINATION BETWEEN VIRAL TRANSGENES AND INFECTING
VIRUSES

By

Min Deng

A DISSERTATION

Submitted to
Michigan State University
in partial fiilfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1999

ABSTRACT

RNA RECOMBINATION BETWEEN VIRAL TRANSGENES AND INFECTING
VIRUSES

Min Deng

RNA recombination is a natural process that occurs during viral RNA replication,
and it contributes to RNA virus evolution. In transgenic plants expressing viral genes,
RNA recombination between viral transgenes and infecting viruses could generate
chimeric viruses with distinct properties. Using the bromovirus model system, Greene
and Allison (1994) demonstrated that a bromovirus capsid transgene with a complete 3’
untranslated region (UTR) was involved in RNA recombination with an infecting virus
and the recombinant recovery rate was 3%. A decrease in recombinant recovery rate was
found when the 3’ UTR was deleted (Greene and Allison, 1996). Experiments carried out
in this study were built upon these two studies and attempted to expand our
understanding of RNA recombination in transgenic plants expressing viral genes. These
experiments have demonstrated that 1) a complete 3’ UTR in the viral transgene allowed
the synthesis of minus strand copies of the viral transgene, and thus permitted RNA
recombination to happen more frequently, presumably during both plus and minus strand
synthesis; 2) a polymerase transgene could also be involved in RNA recombination with
an infecting virus, and the recombinant recovery rate (4%) of the polymerase transgene is
comparable with that of the capsid transgene; 3) a translatable transgene is less likely to
be involved in RNA recombination, especially if the transgene provides resistance to the

infecting virus; 4) truncated bromovirus capsid and polymerase transgenes provided virus

resistance to transgenic plants. Such smaller viral transgenes may be more desirable than
complete genes as they should less likely contribute to viral evolution through RNA

recombination.

Copyright by
MIN DENG
1 999

ACKNOWLEDGMENTS

I would like to thank my major professor Dr. Richard Allison for his support and
guidance during the last four years. I would like to acknowledge my guidance committee
members Dr. Gustaaf de Zoeten, Dr. Rebecca Grumet, and Dr. Suzanne Thiem for
challenge me to become a better scientist. I would like to say thanks to Dr. Barbara Sears
for helping me to change my career to molecular sciences. I would like to thank Dr. Jihad
Skaf, Dr. Heng Zhong, and Dr. Sergei Mekhedov for valuable discussions and help on lab
techniques. I would like to say thanks to Jeanne, Al and Bill for their help. Thanks to
current lab members Adam, David and Cheryl for their suggestions and corrections on
my thesis. Thanks to Jan Szyren for her help on clonally propagating transgenic plants. I
would like to thank Shirley Owens and Nancy Veenstra, who always care about me. I
would like to thank MSU writing center, especially Cathy Lydia Fleck, for helping me
with my thesis writing. Thank you, Cathy, for your encouragement and help. I would like
to thank Dr. Aureal Cross and Dr. Ralph Taggart for their kindness to me. I would like to
thank Haibo for his support and encouragement. Thanks to Joshua, who understand that
Mommy is busy and has to work at such an early age. I am so proud of him. I would like

to thank my parents, who always wanted me to reach high and do the best I could.

TABLE OF CONTENTS

List of Tables ........................................................................................ vii
List of Figures ..................................................................................... viii
Introduction ........................................................................................... 1

Chapter 1. Synthesis of minus strand copies of the viral transgene during viral infections
of transgenic plants

Introduction ........................................................................ 16
Materials and Methods ............................................................ 18
Results .............................................................................. 23
Discussion .......................................................................... 3 1

Chapter 2. RNA recombination between a brome mosaic virus deletion mutant and a
polymerase transgene

Introduction ..... ' ................................................................... 36
Materials and Methods ............................................................ 38
Results .............................................................................. 46
Discussion .......................................................................... 57

Chapter 3. RNA recombination in transgenic plants expressing translatable or
untranslatable viral transgenes

Introduction ........................................................................ 66
Materials and Methods ............................................................ 67
Results .............................................................................. 74
Discussion .......................................................................... 80

Chapter 4. Test of virus resistance in transgenic plants expressing bromovirus transgenes

Introduction ........................................................................ 85
Materials and Methods ............................................................ 86
Results .............................................................................. 92
Discussion .......................................................................... 98
Summary and Conclusions ....................................................................... 102
Appendix ............................................................................................ 107
List of References ................................................................................. 1 18

vi

LIST OF TABLES

Chapter 3
Table 1. Test of virus resistance in transgenic plants .......................................... 78
Chapter 4
Table]. Test of virus resistance ................................................................... 97

vii

LIST OF FIGURES

Chapter 1
Figure 1. Plasmid construction .................................................................... 22
Figure 2. Dot blot assay quantifying the sensitivity of radiolabeled RA518(-) probe. . . ...25
Figure 3. Northern blot analysis of dsRNA ...................................................... 27
Figure 4. Agarose gel electrophoresis showing RT-PCR amplified

negative-sense CCMV RN A3 .......................................................... 29
Figure 5. Agarose gel showing Noll treated RT-PCR products ............................... 30
Chapter 2
Figure 1. Plasmid construction .................................................................... 39
Figure 2. Detection of viral transgenic RNA transcripts in transgenic plants ............... 48
Figure 3. Test of BMV resistance in transgenic plants ......................................... 50
Figure 4. Northern blots showing the detection of recombinants ............................. 53
Figure 5. RT-PCR results of recombinants established in

nontransgenic N. benthamiana ........................................................ 55
Figure 6. Sequence analysis of the recombinants ............................................... 58
Figure 7. Representative mutations recovered in the polymerase gene

of recombinant viruses ................................................................ 59

Figure 8. Polymerase amino acid sequence of recovered recombinants ..................... 60
Chapter 3
Figure 1. Plasmid construction .................................................................... 69
Figure 2. Northern blot analysis ................................................................... 75
Figure 3. Schematic diagram of CCMV RNA3 and the position of the two primers ...... 79
Figure 4. Diagram showing mutations recovered in the RN A3 of recombinants ........... 82
Figure 5. Capsid protein amino acid sequence of recovered recombinants .................. 83
Chapter 4
Figure 1. Plasmid construction for plant transformation ....................................... 88
Figure 2. Northern blot analysis reflecting viral transgene transcription in

transgenic plants ......................................................................... 93
Figure 3. Dot blot analysis of virus resistance in transgenic plants ........................... 96
Appendix
Figure 1. Infection of cowpea with virus A69-1 ............................................... 1 11
Figure 2. Model for RNA recombination to generate recombinant A69-1 ................. 112
Figure 3. Nucleotide and amino acid changes in virus A69-1 ............................... 114
Figure 4. Plasmid construction for making infectious transcripts .......................... l 15

viii

INTRODUCTION

Since the advent of Agrobacterium-mediated transformation techniques to
genetically modify tobacco plants (Matzke and Chilton, 1981), a variety of genetic
modification technologies have been developed. This has enabled plant breeders to
develop new lines of crops at a faster pace than was possible with traditional methods. A
large range of plants, including cereals, vegetables, trees, and ornamental crops, was
genetically modified for the purpose of introducing novel and economically important
traits. For example, the Flavr SavrTM tomato contains an antisense copy of the
polygalacturonase gene, which delays the ripening of the tomatoes (Kramer and
Redenbaugh, 1994), and ZW-20 squash contains the coat protein genes from watermelon
mosaic virus 2 (WMV2) and zucchini yellow mosaic virus (ZYMV), which provide
resistance to these two viruses (Fuchs and Gonsalves, 1995; Tricoli et al., 1995). These
two varieties, plus dozens of other genetically modified (GM) major crop species, have
reached or are in the process of reaching the market. The appearance of GM food in the
market gives rise to concerns and controversy. One major concern in the plant virology
community is the risk associated with the viral transgenes, which includes
transcapsidation and RNA recombination (de Zoeten, 1991; 1995). The following study
assesses the possibility that all or part of a viral transgene may be usefully incorporated
into a challenging virus through RNA recombination. Additionally, this study begins to
elucidate the mechanism by which the transgenes become involved in viral RNA

recombination events.

Dynamic features of RNA virus population contributes to RNA virus evolution

The genetic information of most plant viruses is stored in the form of single-
stranded positive sense RNA. These plant RNA viruses encode their own replicative
proteins which, through association with cellular proteins and membranes, form
replication complexes which carry out the replication of the viral RNA genome (Kao et
al., 1992; Restrepo-Hartwig and Ahlquist, 1996). The central part of this replication
complex is the viral encoded RNA-dependent RNA polymerase (RdRp). RdRp has two
unique characteristics which differentiate it from its counterpart in DNA-based
organisms, the DNA-dependent DNA polymerase (Dde); these characteristics also
contribute to the dynamic features of RNA virus population and the rapid evolutionary

drifi of RNA viruses.

One unique characteristic of RdRp is its high error rate. Error rates of RdRps have
been estimated to be in the range of 10“ to 10 ‘5, whereas those of Ddes are between 10'8
to 10'9 (Drake, 1993). This is due to the lack of 3’—->5’ exonucleolytic proofreading
activity in RdRp. Theoretically, for an RNA virus with a genome of 10 Kb, one error
could be introduced during each replication cycle. Unlike the mutations in DNA-based
organisms which may be repaired, these RNA mutations are incorporated into the
genome of the progeny viruses during RNA replication. Thus, the population of an RNA
virus is not genetically homogeneous, but represents a dynamic distribution which has

been described as mutant swarms (Domingo et al., 1995).

Another characteristic of RdRp is its ability to switch templates during the process
of RNA replication, which is referred to as RNA recombination (for review, see Lai,
1992; 1995). RNA recombination has been observed frequently in animal RNA viruses,
such as poliovirus and coronaviruses (Lai, 1992). The recombination fi'equency was 2%
for a particular segment of the poliovirus genome (Cooper, 1968). For plant RNA viruses,
RNA recombination was observed in members of several different virus groups,
including bromoviruses (Bujarski and Kaesberg, 1986; Bujarski and Dzianott, 1991;
Allison et al., 1990), turnip crinkle virus (Cascone et al., 1990), and alfalfa mosaic virus

(Huisman et al., 1989; Van der Kuyl et al., 1991).

RNA recombination is thought to be a three-step process, during which the
replication complex first pauses at a strong secondary structure in the template RNA.
Secondly, the nascent RNA-replication complex dissociates from the original template,
and reassociates with a different RNA template. Following the polymerization, a
chimeric progeny RNA, which results from copying two distinct templates, is created.
This chimeric RNA is referred to as a recombinant (Lai, 1992; 1995). The generally
favored model for RNA recombination is the copy-choice model, which proposes that the
viral RNA polymerase switches templates during negative strand synthesis (Kirkegaard

and Baltimore, 1986).

RNA recombination is believed to play a major role in RNA virus evolution.
Sequence similarities among RNA viruses and arrangements of viral genetic segments

indicate that RNA viruses evolved through the exchange of functional modules between

related and possibly unrelated viral RNAs. This evolutionary process, which is referred to
as modular evolution of viruses (Koonin and Dolja, 1993; de Zoeten, 1995), is obviously

dependent on RNA recombination.

In addition to high polymerase error rates and RNA recombination, other
mechanisms also reshape the RNA virus population. Gene segment reassortment and
transcapsidation perturb the populations of multipartite RNA viruses (Domingo and
Holland, 1994; de Zoeten, 1991). During host-to-host transmission, bottleneck events
happen frequently in nature due to random sampling of viral genomes (Domingo et al.,
1996). For a bottlenecked RNA virus p0pulation which has high mutation rate,
deleterious mutations will accumulate in an irreversible manner. Repeated bottlenecked
transmission of RNA viruses will fix sub0ptimal classes of genomes and decrease the
viral fitness. Only compensatory mechanisms such as RNA recombination can restore the

initial, mutation-free class of a genome (Domingo et al., 1996).

Natural selection plays a major role in reshaping the perturbed RNA virus
populations, since most of the mutations generated during the process of RNA replication
and recombination result in such dramatic fitness loss that the variants fail to compete
well with their wild type progenitors, and eventually disappear (Domingo et al., 1978;
Duarte et al., 1994). This explains why, despite high mutation rates, RNA viruses
maintain major consensus sequences that typify each virus (Domingo et al., 1985).
Current studies on viral population genetics show that an RNA virus population is not a

genetically defined population, but rather a dynamic genetic organization in which

individual genomes differ in one or more positions from the consensus or average
sequence of the population (Domingo and Holland, 1994). Thus, an RNA virus species is
considered a quasispecies, rather than a true species consisting of a more homogeneous

population (Duarte et a1, 1994).

A striking feature of the RNA virus quasispecies is its ability to not only create
variations, but also to tolerate them. The genetic flexibility of quasispecies provides a
selective advantage, and allows RNA viruses to penetrate various types of host tissue
under normal conditions (Morimoto et al., 1998), and upon fast environmental changes,
to adapt to new ecological niches (Domingo and Holland, 1994). Thus, RNA viruses,

including bromoviruses, are different from all other organisms.

Bromoviruses

Bromoviruses are among the most extensively studied RNA viruses, and they
have been used as a model system to study viral RNA replication, encapsidation,
movement, and RNA recombination. Bromoviruses include brome mosaic virus (BMV)
and cowpea chlorotic mottle virus (CCMV), which are closely related and share
extensive sequence similarities (Allison et al., 1989). The natural hosts of BMV are
monocots such as barley (Hordeum vulgare L.), whereas those of CCMV are dicots,
especially legumes. A common host for both viruses is Nicotiana benthamiana Domin

(Lane, 1981).

Both BMV and CCMV are single-stranded positive sense RNA viruses, which
belong to the plant RNA virus family Bromoviridae. Bromoviruses have three genomic
RNA3: RNA 1 (3.2 Kb), RNA 2 (2.9 Kb) and RNA 3 (2.1 Kb), and a subgenomic RNA 4
(0.8 Kb). Genomic RNA] and 2 are monocistronic and in BMV they encode for the 1a
(109 KDa) and 2a (94 KDa) proteins, respectively, which together are sufficient to direct
viral RNA replication in barley protoplasts (French et al., 1986; Kiberstis et al., 1981;
Allison et al., 1988). The 1a protein contains two conserved domains within the N-
terrninal and C-terminal regions. The N-terminal domain has sequence homology to the
Sindbis virus nsPl methyltransferase protein, which is believed to be involved in capping
the viral RNAs (Ahlquist et al., 1985; Mi and Stollar, 1991). The C-terminal portion of
the la protein has homology to viral and cellular helicases, which typically unwind
double-stranded nucleic acid molecules (Evans et al., 1985). The 2a protein, a putative
RNA-dependent RNA polymerase (RdRp), is characterized by the GDD (Gly-Asp-Asp)
motif which is present in all known RdRps. The RdRp of BMV has a central polymerase
core region flanked by 5’ and 3’ regions. The central polymerase core region is highly
conserved and is indispensable for RNA replication, whereas the 5’ and 3’ flanking
regions are not required for replication (Traynor et al., 1991).; The la and 2a proteins,
through association with cellular proteins and membranes, form a replication complex
which is responsible for viral RNA replication (Kao et al., 1992; Restrepo-Hartwig and

Ahlquist, 1996).

.Genomic RNA 3 of bromoviruses is dicistronic and encodes for a putative

movement protein 3a (32 KDa) and the coat protein (CP) (20 KDa). The 3a protein is

translated directly from genomic RNA 3, whereas the coat protein is translated from the
subgenomic RNA 4, which is derived from the minus-strand RNA 3 during replication
(Allison et al., 1988, Ahlquist, 1994). Molecular and genetic studies revealed that, for
both BMV and CCMV, the movement protein is critical for cell-to-cell movement (Mise
et al., 1993; Misc and Ahlquist, 1995). However, despite the sequence similarities
between these two viruses, BMV and CCMV appear to utilize different mechanisms to
move from cell to cell. BMV requires a fimctional coat protein for cell to cell movement
in addition to the movement protein (Schmitz and Rao, 1996), whereas CCMV moves

between epidermal cells without a coat protein (Rao, 1997).

The replication of bromoviruses, like most other positive sense RNA viruses, can
be divided into three distinct steps: minus-sense RNA synthesis, positive-sense genomic
RNA synthesis, and subgenomic RNA synthesis (Dinant et al., 1993). Each step is
differentially regulated at various stages of viral infection. Evidence suggests that the 3’
untranslated region (UTR), which is conserved for all three genomic BMV RNAs,
contains the replication initiation site for minus-sense RNA synthesis (Ahlquist et al.,
1981a; Dreher and Hall, 1988). The viral replication complex first utilizes the positive-
sense viral RNA as a template to make a small amount of minus-sense RNA, which
serves as a template for synthesis of large quantities of positive-sense genomic RNA
(Marsh et al., 1991b). The minus-sense copy of the genomic RNA 3 has an intercistronic
region, which bears the promoter for synthesis of subgenomic RNA 4 (French and

Ahlquist, 1987; Pogue et al., 1992).

Minus-sense RNA is associated with viral positive-sense RNA in the cytoplasm.
Upon extraction, they form a double-stranded viral RNA called replicative form, which is
resistant to mild RNase digestion at high salt concentrations, but susceptible at low salt
concentrations (Hardy et al., 1979). In BMV infected barley protoplasts, negative-strand
RNA accumulation plateaus at 8 hours post inoculation (hpi), while positive-strand
genomic RNA and subgenomic mRN A continue to accumulate until 20 hpi (Kroner et al.,
1989). In the presence of all three genomic BMV RNAs, positive and negative strand
RNA synthesis is asymmetric. The negative-sense RNA accumulates 100-fold lower than
the positive sense RNA in BMV infected barley protoplasts at 24 hpi (Rao et al., 1990b;
Marsh et al., 1991a). The quantity of negative-strand RNA in wt CCMV infections is

even lower (Pacha and Ahlquist, 1991).

The 3’ terminal sequences of BMV and CCMV are distinct from each other and
are highly conserved within each virus. However, the predicted secondary structures of
the 3’ terminal 200 bases of BMV and CCMV are strikingly similar (Ahlquist et al.,
1981a). A genomic RNA reassortment experiment in N. benthamiana protoplasts
indicated that the replication complex of BMV could replicate CCMV RNAs, and vice
versa (Allison et al., 1988). The genomic RNA reassortment experiment also indicated
that, using either BMV or CCMV replicative proteins, BMV genomic RNAs are
preferentially replicated and the accumulation level of BMV RN As was higher than that

of CCMV RNAs (Allison et al., 1988).

Using an in vitro replication system of BMV, Miller et a1. (1986) found that the 3’
terminal 134-nt fragment, which folds into a tRNA-like structure, contains all of the
signals required for initiation of minus-strand replication. Minus-strand initiation occurs
opposite the penultimate 3'-nucleotide (the 3’ C of CCAOH), and the terminal adenosine is
added to the plus-strand RNA afier replication through interaction of viral RNA and the

host tRNA nucleotidyl transferase (Miller et al., 1986).

Virus resistant transgenic plants

Following the theoretic prediction of pathogen-derived resistance in 1985
(Sanford and Johnston), virus resistant transgenic plants were first generated in Roger
Beachy’s laboratory in 1986 (Powell-Abel et al., 1986). In this experiment, tobacco
plants were transformed with a cDNA copy of the coat protein (CP) gene of tobacco
mosaic virus (TMV). These transgenic plants had an increased level of resistance to TMV
virion inoculation. This phenomenon was named coat protein-mediated resistance
(CPMR). CPMR has since been demonstrated for many other plant RNA viruses,
including alfalfa mosaic virus (Loesch-Fries et al., 1987), cucumoviruses (Cuozzo et al.,
1988), potyviruses (Lawson et al., 1990; Stark and Beachy, 1989), potexviruses
(Hemenway et al., 1988), tobravirus (van Dun and B01, 1988), tospoviruses(Gie1en et al.,
1991), and luteoviruses (Kawchuk et al., 1990; for review on CPMR, see Grumet, 1995).
In a majority of the cases, virus accumulation is reduced or absent, in some cases
however, the level of protection is related to the level of CP expression. The transgenic
plants were protected against only the virus from which the CP gene was derived, with

some exceptions where the transgenic plants were also protected against closely related

viruses (Nejidat and Beachy, 1990). Currently, several coat protein-mediated virus
resistant crops are on the market, and numerous others are being field tested prior to

commercial release (Fuchs et al., 1997; Yeh et al., 1998). The ZW-20 squash mentioned

above was the first commercialized CPMR crop.

The theory of pathogen-derived resistance was also applied to several other non-
structural viral protein genes, including the replicase gene and the movement protein
gene. Replicase-mediated resistance was first demonstrated with TMV (Golemboski et
al., 1990) and later in several other virus groups, including potexviruses (Braun and
Hemenway, 1992; Longstaff et al., 1993), tobraviruses (MacFarlane and Davies, 1992),
cucumoviruses (Carr et al., 1994; Zaitlin et a1, 1994), tombusviruses (Rubino and Russo,
1995), and potyviruses (Audy et al., 1994; Guo and Garcia, 1997). In many cases, the
level of resistance provided by the replicase transgene is greater than that provided by the
coat protein gene (Braun and Hemenway, 1992). Compared to CPMR the spectrum of
replicase-mediated resistance was sometimes broader (Donson et al., 1993), with some
exceptions where it was restricted (Rubino and Russo, 1995). In some cases, a
translatable replicase transgene is required before virus resistance is observed
(MacFarlane and Davies, 1992), while in other cases, the resistance is believed to be

mediated by the transgenic mRN A only (Guo and Garcia, 1997).

An interesting phenomenon associated with replicase-mediated resistance is that a

higher level and/or a broader spectrum of resistance was observed when the replicase

gene used for generating transgenic plants was modified or truncated. For example, a

10

truncated cucumber mosaic virus (CMV) replicase gene provided absolute resistance to
CMV virion and RNA inoculum at concentrations up to 500 ug/ml or 50 ug/ml,
respectively (Anderson et a1, 1992). In another study, tobacco plants transformed with
the replicase gene of potato virus X (PVX) containing a modified GDD motif were
extremely resistant to PVX in that no virus systemic movement was observed at

inoculum concentrations as high as 250 ug/ml PVX RNA (Longstaff et al., 1993).

Although the mechanism of pathogen-derived resistance remains unknown, many
studies indicate several distinct mechanisms. Based on the observation that transgenic
tobacco plants expressing TMV CP were more resistant to the TMV virion than to the
TMV RNA inoculum, the inhibition of virion disassembly in the initially infected cells
was thought to be responsible for CPMR (Register and Beachy, 1988). Specific
interactions between the transgenic CP and the CP of the challenging virus as well as an
interference with virus movement could also be necessary components of CPMR against

TMV (Bendahmane et al., 1997)

Replicase mediated resistance is likely RNA-based (Mueller et al., 1995), and
post-transcriptional gene silencing may provide the mechanism (Baulcombe, 1996).
Plants naturally use homologous or complementary nucleic acid sequences to control
excess production of host mRNA or the replication of RNA pathogens (Matzke and
Matzke, 1995). Overproduction of a transgene can trigger post-transcriptional gene
silencing of both the transgene as well as the homologous host gene (Wassenegger and

Pelissier, 1998). If the transgene is derived from part of the genome of an RNA virus,

11

plants show post-transcriptional silencing of the viral transgene as well as the RNA of
infecting viruses (Lindbo et al., 1993). The consequence is that the accumulation of the
virus in the cytoplasm is strongly reduced and plants display resistance against the

corresponding virus.

Risk assessment and the possibility of RNA recombination between viral transgene
and infecting viruses

Although pathogen-derived resistance appears to provide a promising way for
combating virus infection in transgenic crops, the dynamic features of RNA viruses
suggest that potential risks may be associated with the release of virus resistant transgenic
plants in the environment. In 1991, de Zoeten proposed two possible risks of the release
of transgenic plants expressing the viral CP gene, namely transcapsidation and RNA
recombination. Transcapsidation occurs in mixed infections when the genomic RNA of
one virus is encapsidated into the capsid of another virus, or a phenotypically mixed
capsid with protein subunits from both viruses (de Zoeten, 1991). For the viruses that
require aphids for their transmission, transcapsidation may lead to an altered virus-vector
specificity because the viral RNA of an aphid-nontransmissible virus could be
encapsidated into the capsid of an aphid-transmissible virus, and subsequently, be

transmitted to a new host (Creamer and Falk, 1990).

RNA recombination, as discussed previously, occurs when the viral replication

complex changes templates during the natural process of RNA replication (Kirkegaard

and Baltimore, 1986). If CP-transgenic plants become infected with viruses related to the

12

one which provides the CP transgene, or unrelated viruses, template switching might
occur between the transgenic mRNA and the replicating viral RNA (de Zoeten, 1991).
The progeny virus would have segments of genes from both the viral transgene and the
infecting virus and thus have properties different fiom the parental virus. Template
switching and transcapsidation, coupled with the other factors which perturb the RNA
virus population, could generate “new” viruses with altered vectors, host ranges, and new

combinations of genes (de Zoeten, 1991).

RNA recombination in transgenic plants expressing viral genes was first discovered on
red clover necrotic mosaic dianthovirus (RCNMV) and was reported during a conference
in 1991 (Lommel and Xiong, 1991). In this study, the RCNMV movement protein
transgene lacking the 5’ untranslated region (UTR) acquired a fimctional 5’ UTR through
RNA recombination with the inoculated RNA 1, which has a complete 5’ UTR. The
second demonstration of transgene recombination was in a plant DNA pararetrovirus
cauliflower mosaic virus (CaMV), where template switching occurred between the gene
VI transgene and a movement defective CaMV during reverse transcription of the CaMV

RNA to DNA (Gal et al., 1992; Schoelz and Wintermantel, 1993).

In 1994, Greene and Allison conducted a sensitive bioassay experiment using the
bromovirus model system and further demonstrated that RNA recombination involving
viral transgenes is a frequent phenomenon. In their experiment, three single nucleotide
substitutions, which also generate a unique N011 restriction site, were introduced near the

3’ end of the CCMV coat protein ORF. The 3’ 2/3 of the CCMV CP ORF with the NotI

l3

restriction site, along with the complete 3’ UTR was introduced into the N. benthamiana
plants using Agrobacterium-mediated transformation. Transgenic plants with a high
accumulation of transgene transcripts were selected for the recombination experiments
and were clonally propagated to ensure genetic homogeneity of the plant materials. These
transgenic plants were inoculated with a movement defective mutant, AG3, which lacked
the 3’ 1/3 of the CCMV CP ORF. The design of this experiment allowed Greene and
Allison to screen for recombinants by looking for the systemic movement of the virus in
transgenic plant cuttings, since the systemic movement of the virus indicated the recovery
of the deletion in the CP ORF through RNA recombination between the CP transgene and
the deletion mutant AG3. Out of the 235 transgenic plant cuttings inoculated, seven
recombinants were recovered, which gave a 3% rate of recombinant recovery. Sequence
analysis indicated that all recombinants had restored the deletion at the 3’ end of the CP
ORF, and the molecular marker N011 site was also present. These recombinants also bore
nucleotide and amino acid substitutions, deletions, and insertions, and several

recombinants produced atypical symptoms in the cowpea host.

In a similar study, Greene and Allison (1996) inoculated this same movement
defective mutant AG3 on a set of transgenic plants transformed with the same 3’ 2/3 of
the CP ORF but an incomplete 3’ UTR. No recombinant was recovered from any of the
479 transgenic plant cuttings. The lack of recombinant recovery in this experiment leads
to the conclusion that the transgene 3’ UTR played a significant role in RNA

recombination in transgenic plants (Greene and Allison, 1996).

14

My research is built upon the results of Greene and Allison (1994; 1996) and attempts

to answer four general questions:

1) Why is there a decrease in the RNA recombination rate when the 3’ UTR is
incomplete? What is the role of 3’ UTR in RNA recombination?

2) Can RNA recombination occur between a polymerase transgene and an infecting
virus? Is the RNA recombination rate in the polymerase transgene comparable to
that in the CP transgene?

3) Is a translatable transgenic mRNA more or less likely to be involved in RNA
recombination?

4) Does a full-length CCMV CP transgene confer virus resistance to transgenic

plants?

We hope that answers to these questions will lead to a better understanding of RNA
recombination in transgenic plants expressing viral genes. The ultimate goal of this
research is to provide information to genetically modified (GM) food regulators and
developers for the purpose of developing safer GM products, especially virus resistant
transgenic crops in which the transgenes are less likely to be involved in RNA

recombination events.

15

CHAPTER 1

Synthesis of Minus Strand Copies of the Viral Transgene during Viral Infections of

Transgenic Plants

INTRODUCTION

Coat protein and replicase mediated resistance are effective in limiting viral
infections in a variety of plant species (F itchen and Beachy, 1993; Grumet, 1995). While
the mechanisms of pathogen-derived resistance remain unsolved, many studies indicate
that transcription of the viral transgene and in some cases expression of the viral protein
is required for effective resistance (Fitchen and Beachy, 1993). While several studies
have shown that a full-length coat protein gene is sufficient to provide virus resistance
(Cuozzo et al., 1988; Smith et al., 1995; Tricoli et al., 1995; Hefi‘eron et al., 1997),
numerous constructs have included the 3’ UTR along with the full-length coat protein
gene (Powell-Abel et al., 1986; Stark and Beachy, 1989; Zaccomer et al., 1993).
Presumably inclusion of the 3’ UTR was intended to stabilize the transgene transcript in

the cytoplasm.

The 3’ UTR of most RNA viruses bears the replicase recognition site and also
serves as an initiation site for negative-sense RNA synthesis. This has been demonstrated
in bromoviruses (Ahlquist et al., 1981a; Bujarski et al., 1986), potexviruses (Sriskanda et
al., 1996; White et al., 1992), and ilarviruses (Bachman et al., 1994). Our previous data

indicated that exclusion of the 3’ UTR in the viral transgene discouraged RNA

16

recombination between viral transgenic transcripts and infecting viruses (Greene and
Allison, 1996). When transgenic N. benthamiana plants transcribing the 3’ two-thirds of
the capsid gene and the complete 3’ UTR of CCMV were inoculated with a movement
defective CP deletion mutant, recombinant virus was recovered from 3% of the
inoculated plants (Greene and Allison, 1994). However, when transgenic N. benthamiana
plants, transcribing the same viral gene fiagment but lacking all or part of the 3’ UTR,
were inoculated with the same CP deletion mutant, no recombinant was recovered from
479 transgenic plants (Greene and Allison, 1996). One hypothesis to explain these results
is that inclusion of replication recognition sequences in the transgene leads to partial
amplification of the viral transgene by the viral replicase. This amplification produces a
minus-strand copy of the transgene which is also available for recombination within the

transgenic plants.

In this chapter, we report the use of Northern blot analysis and RT-PCR
techniques to detect the complementary copy of a viral transgene in transgenic plants
infected with either BMV or CCMV. Two transgenic plant lines, one with an intact 3’
UTR, and one with a deleted 3’ UTR, were inoculated with either wild type BMV or
CCMV. In the PCR experiment, we took advantage of the uniqueness of the 3’ UTRs of
BMV and CCMV genomic RNA and also the ability of BMV to recognize and replicate
CCMV RNAs (Allison et al., 1988). Our data indicated that a fiall-length complementary
cOpy of the viral transgene was synthesized only when the 3’ UTR of the viral transgene

was intact.

17

MATERIALS AND METHODS
Plasmid construction

Full-length cDNA clones of wild type CCMV RNAI, RNA2 and RNA3 are
maintained in plasmid pCClTPl, pCC2TP2, and pCC3TP4, respectively (Allison et al.,
1988). Infectious transcripts, namely C1, C2, and C3, were synthesized from the Xbal
digested DNA templates using T7 RNA polymerase (Epicenter). Plasmid pCC3AG1 was
generated fi'om plasmid pCC3TP4 by introducing three single nucleotide substitutions
including a Natl restriction site near the 3’ end of the CP ORF (Greene and Allison,
1994; Fig. 1). Full-length cDNA clones of wild type BMV RNAl, RNA2 and RNA3
were maintained in plasmids pBlTP3, pB2TP5, and pB3TP8, respectively (Janda et al.,
1987). Infectious transcripts, namely B1, B2, and B3, were synthesized from EcoRI

digested DNA templates using T7 RNA polymerase (Epicenter).

Plant materials

The transgenes in transgenic plants 3-57 and A69 were derived from plasmid
pCC3AG1 which bears three marker mutations and the unique Natl restriction site near
the 3’ end of the CCMV CP gene (Fig. 1). Transgenic N. benthamiana plant 3-57 was
transformed with the 3’ 2/3 of the modified CCMV CP gene and the complete 3’ UTR.
Transgenic N. benthamiana plant A69 was transformed with the same 3’ 2/3 of the
CCMV CP gene, but the 3’ terminal 69-nucleotide of the 3’ UTR were deleted (Greene
and Allison, 1996; Fig. 1). Transgenic 3-57 and A69 plants used in this study were either

clonally propagated F0 plants, or their F1 seedlings. To ensure the transgenic status of the

18

seedlings, they were maintained in MS media containing 100 mg/L kanamycin for one
month before being transferred to soil. Nontransgenic N. benthamiana plants were grown

in soil.

Plant inoculation

Wild type BMV or CCMV infection was established using the full-length
infectious transcripts. Infected N. benthamiana leaf tissue was ground in a 10 mM
sodium phosphate buffer (pH 7.0) with a mortar and pestle. This viral inoculum was
applied to two fully expandedleaves dusted with carborundum. All of the plants were
inoculated at the 5-leaf stage. Newly inoculated plants were left in the dark for one day

before transferring to the grth chamber maintained at 26°C under florescent light with

a 16-hour day period.

Detection of viral infection

To determine the most appropriate time to sample viral RNA, dot blot analysis
was used to determine the progression of the infection and the viral RNA concentrations
in BMV and CCMV inoculated N. benthamiana. Leaves above the inoculated leaves
were numbered and two leaf discs 8 mm in diameter were collected from each leaf at 10,
12, 14, and 16 days post inoculation (dpi). The leaf samples were ground in 40 [.11 of
deionized distilled water in a microcentrifiige tube. After centrifugation at 13,000 rpm for
two minutes, the supernatant was collected and treated in 1% SDS at 65°C for five
minutes. The tubes were again centrifuged at 13,000 rpm for two minutes to remove all

the plant debris. Five microliters of the supernatant was dotted directly onto Hybond N

19

nylon membrane (Amersham). . Afier baking the membrane at 80°C for two hours under
vacuum, the blot was hybridized with dig-labeled RA518(+) (Allison et al., 1990) or HEl
RNA probe (Ahlquist et al., 1981). These probes are specific for the 3’ UTR of CCMV or
BMV RN As, respectively. The hybridization, washing and immunodetection were as

recommended by the manufacturer (Boehn'nger-Mannheim).

Total RNA extraction and treatment

Total RNA was extracted from BMV or CCMV inoculated leaf tissue at 14 days
post inoculation (dpi) (Puissant and Houdebine, 1990). To recover the double-stranded
RNA (dsRNA), total RNA fiom one gram of plant tissue was treated in a 100 ul solution
containing 10 mM Tris-HCl (pH 7.2), 10 mM MgCl;, 1 mM EDTA, 300 mM NaCl,
10ug/ml RNase A, 30 units/ml RNase T1 and 500 units/ml RNase fiee DNase I at 30°C
for 2 hours (Hardy et al., 1979; Sethna et al., 1989). The added nucleases were destroyed
by adding 2.5 pl 20% SDS and 2.5 pl 20 mg/ml proteinase K to each tube and incubating
them at 37°C for one hour. After seven extractions with phenol-chloroform-
isoamylalcohol, and twice with chloroform-isoamylalcohol, the double-stranded RNA

was precipitated with 2.5 volumes of ethanol.

Gel electrophoresis and Northern blot analysis

The double-stranded RNA pellets were resuspended in 11 ul of RNase-free water
and denatured at 55°C for 15 minutes in a 50 ul solution containing 1X MOPs buffer (pH
7.0), 2.2M formaldehyde, 50% forrnamide. Gel preparation, electrophoresis, and

Northern transfer were as described by Brown and Mackey (1997). Digoxigenin- or 32P-

20

labeled RA518(-) probes were synthesized using Pvul linearized pCC3RA518 (Allison et
al., 1990) plasmid DNA in the presence of T7 RNA polymerase (Epicenter). RA518(-)
anneals to a 179-nucleotide region in the 3’ UTR of all four negative-sense CCMV RNAs
(Allison et al., 1990). The procedure of hybridization and washing was as described by
Allison et a1. (1990). Plasmid DNA pCC3TP4 (Allison et al., 1989) was denatured by the
same method as double-stranded RNA and dotted directly on nylon membrane. The
position of negative sense RNA 4 and the viral transgene were calculated according the

migration pattern of negative sense RN As 1, 2, and 3 (Maniatis et al., 1982).

RT-PCR analysis and restriction digestion

The primer used for first strand cDNA synthesis was a negative sense CCMV
RNA3 specific primer RA83 (5’ AAGTGGATCCCCTCTTGTGCGGCTGC 3’), which
anneals at nucleotides 1519-1544. An additional primer used for PCR analysis was RA84
(5’ ACTCCAAAGAGTTCTTCCG 3’), which anneals at nucleotide 2072-2090 of
positive sense CCMV RN A3. Total RNA extracted from the plant tissue was first treated
with DNase I in a buffer containing 50 mM Tris-Cl, pH 7.5, 10 mM MgC12, 50 1.1ng
BSA, and 0.05 U/ul RNase fiee DNase I (Boehringer—Mannheim) at 37°C for an hour
prior to first strand cDNA synthesis. Reverse transcription was carried out as described
by the manufacturer (Boehringer-Mannheim). Complementary DNA synthesized from

lug of total RNA was used for each PCR reaction as described by Greene and Allison
(1994). For the control reaction, 0.1 ug pCC3AG1 (Fig. 1) plasmid DNA was used in

each reaction. RT- PCR products were purified by extraction twice in phenol-chloroform-

isoamylalcohol followed by ethanol precipitation. NotI restriction enzyme digestion was

21

pCC3TP4 1476

 

 

 

 

 

 

 

 

 

 

StyI
, l
I 3a | L | CF I
SM :33;
, I I
| 3a | I | or |
Natl
I
Transgenic plant 3-57 [ I
+ ’4-
RA83 RA84
Not]
I
Transgenic plant A69
-> <-
RA83 RA84

Figure 1. Plasmid construction. Wild type CCMV RNA3 has a 3a gene ORF and a
coat protein (CP) ORF, and it is maintained in plasmid pCC3TP4. A Not I restriction
site was introduced near the 3’ end of the CP gene ORF in plasmid pCC3AG1 as well
as the viral transgenes in transgenic plants 3-57 and A69 (Greene and Allison,
1994;1996). Transgenic plant 3-57 was transformed with the 3’ 2/3 of the CP ORF and
the full-length 3’ UTR. Transgenic plant A69 was transformed with the same viral
gene, but the 3’ UTR bears a 69-nucleotide deletion at the 3’ end. The negative sense
RNA specific primer RA83 (5’ AAGTGGATCCCCTCTTGTGCGGCTGC 3’)
anneals at nucleotides 1519-1544, and was used for first strand cDNA synthesis. An
additional primer RA84 (5’ ACTCCAAAGAGTTCTTCCG 3’) anneals at nucleotides
2072-2090, and was used for PCR.

22

as described by the manufacturer (Boehringer-Mannheim).

RESULTS

Three sets of plant materials, namely nontransgenic N. benthamiana and
transgenic 3-57 and A69 N. benthamiana, were used in this study. As indicated in Fig. 1,
a Natl restriction site was engineered into mutant AG] and was therefore included in the
viral transgenes of transgenic plants 3-57 and A69 (Greene and Allison, 1994; 1996).
Both 3-57 and A69 transgenic plants cany the 3’ two thirds of the CP ORF with the
molecular marker Natl site. The only difference between these plants is that 3-57 plants
have an intact 3’ UTR, whereas A69 plants have a 69 nucleotide deletion at the 3’ end of

the 3’ UTR (Greene and Allison, 1994; 1996).

Detection of minus-sense viral RNA in planta

As single-stranded positive sense RNA viruses, bromoviruses replicate in the
cytoplasm, and a small amount of minus-sense RNA is synthesized prior to the synthesis
of positive-sense RNA (Marsh et al., 1991a). The minus-sense RNA is constantly
associated with a plus-sense RNA, and upon extraction, becomes a double stranded form,
which is resistant to mild ribonuclease treatment under high salt conditions (Hardy et al.,
1979). The minus-sense RNA accumulates to a level 100-fold lower than the positive-
sense RNA in the total RNA extracted from protoplasts at 24 hour post inoculation (Rao

et al., 1990a; Marsh et al., 1991b).

23

For in planta detection of minus strand genomic RNAs, it was important to
determine the time when most plant cells were at an early stage of infection. To track the
movement of BMV and CCMV in N. benthamiana, leaf tissue was collected from each
leaf above the inoculated leaves at 10, 12, and 14 days post inoculation (dpi) and viral
RNA accumulation was analyzed in dot blots. Our data indicated that at 10 dpi, viral
RNA was only present in the youngest visible leaves, but absent in the other fully
expanded leaves. Viruses moved to the first fully expanded leaf at 12 dpi. By 14 dpi, viral
RNA was present in all leaves tested (data not shown). Thus, in this study, total RNA was

extracted at 14 dpi.

At the initial stage of this study, attempts were made to detect minus-sense viral
RNA directly from total RNA extracted from leaf tissue. However, none of them was
successfiJl. This is probably because minus-sense RNA represents such a small fraction
of the total RNA that the majority single-stranded RNA prevented it from hybridization
with the RNA probe. RNase treatment was used later to remove the single-stranded
cellular and viral RNAs, which make up the majority of the total RNA. The double-
stranded RNA which was resistant to RNase-treatment was denatured and analyzed in the
Northern blots using 32P-labeled RNA probe RA518(-), which recognizes the 3’ UTR of
CCMV RN As (Allison et al., 1990). The sensitivity of this probe was optimized to detect
10 pg of denatured plasmid DNA pCC3TP4 (Fig. 2). Using this RNA-RNA hybridization
system, we were able to routinely detect the negative-sense genomic RNAs of CCMV

from 0.5 to 1.0 gram of the infected nontransgenic N. benthamiana plant tissue at 14 dpi.

24

100 ng 10 ng 1 ng 100 pg 10 pg 1 pg

    

Figure 2. Dot blot assay quantifying the sensitivity of radiolabeled RA518(-)
probe. Serial dilutions of denatured plasmid pCC3TP4 were spotted mechanically
onto Hybond N nylon membrane. Each dot received 100 ng, 10 ng, 1 ng, 100 pg,
10 pg or 1 pg denatured plasmid DNA, respectively.

25

The complementary copy of the viral transgene accumulated at a much lower level
than that of the genomic RNAs

A total of four experiments were carried out to detect the complementary copy of
the viral transgene by Northern blot analysis. Fig. 3 shows the representative results.
Double-stranded RNA fi'om two grams of plant tissue was loaded in each lane; lanes 1-6
and lanes 7-10 contain double-stranded RNA extracted from CCMV and BMV infected
3-57 plant tissue, respectively. Negative-sense RNAs l, 2, and 3 of CCMV were detected
by the negative-sense CCMV RNA specific probe in CCMV infected plants, but not in
BMV infected plants. The amount of negative-strand RNA migrating at the position of
subgenomic RNA4 is very small compared with the negative-strand genomic RNA3 of
CCMV. This data agrees with the negative-sense RNA accumulation patterns in CCMV

infected barley protoplasts (Pacha and Ahlquist, 1991).

The expected site of the complementary strand of the viral transgene was
calculated according to the migration patterns of the negative sense RN As 1, 2, and 3 of
CCMV. This fi'agment was not observed in either BMV or CCMV infected plants (Fig.
3). Although F1 seedlings of 3-57 plants were used in this experiment, similar results
were obtained when F0 plant samples were used in repeated experiments. Despite the fact
that in this Northern blot, the probe was optimized to detect 10 pg of denatured plasmid
DNA, and dsRNA from two grams of the plant tissue was loaded in each lane, the
complementary copy of the viral transgene was not observed. This indicates that if the

complementary copies of the viral transgene were present in the double-stranded form,

26

RNA3 (-)

RNA4 (-)

 

Figure 3. Northern blot analysis of dsRNA. Double-stranded RNA extracted from
either CCMV or BMV infected transgenic plant 3-57 was probed with the
negative-sense CCMV RNA specific probe RA518(-). Lanes 1-6 contain samples
from CCMV infected plant tissue, lanes 7-10 contain samples from BMV infected
plant tissue. Positions of negative-sense CCMV RNAs l, 2, 3, and 4 were labeled
on the left of the picture. The arrow, —>, indicates the calculated position of

negative-sense viral transgene which was not detected.

27

they were beyond the detection limit of our conventional RNA-RNA hybridization

technique and below the concentration of ten picograms per two grams of leaf tissue.

Complementary copies of the viral transgene were detected in the RT-PCR analysis
To better search for the complementary copies of the viral transgene, a more
sensitive technique, RT-PCR, was used. In this procedure, total RNA from the above
mentioned plant tissue was first treated with RNase free DNase I to remove the plant
genomic DNA including the chromosomal copy of the viral gene. A minus-strand CCMV
RNA3 specific primer RA83 (Fig. l) was used for first-strand cDNA synthesis. PCR was
completed using an additional primer RA84, which anneals near the 5’ terminus of the
69-nucleotide deletion (Fig. 1). The size of the expected RT-PCR fi'agment was 572 bp.
As shown in Fig. 4, lanes 7, 10 and 13, a minus-strand RNA was amplified in all of the
CCMV infected 3-57, A69, and nontransgenic plants. This minus-sense RNA was not
observed in any mock inoculated transgenic plants or in BMV infected A69 or non-
transgenic plants (Fig. 4, lanes 5, 6, 8, 9 and 11). A band of the correct size was also

present in the BMV infected 3-57 transgenic plants (Fig. 4, lane 12).

A Natl restriction digestion was used to determine the origin of the amplified
minus-strand CCMV RNAs. As mentioned before, the Natl site was present only in the
viral transgene and not the wild-type CCMV inoculum (derived from pCC3TP4). As
shown in Fig. 5, lane 3, RT-PCR product of AGI mutant containing the Natl site was
completely cleaved by the Natl restriction enzyme, but that of wild type CCMV was not

cleaved (Fig. 5, lane 5). The RT-PCR product of CCMV infected A69 plants was not

28

Nontransgenic A69 3—57
AGl neg E le M B C
l 2 3 4 5 6 7

“E
cu:
m
3
w
n

 

Figure 4. Agarose gel electrophoresis showing RT-PCR amplified negative-sense
CCMV RNA3. Total RNA from virus infected or mock-inoculated transgenic and
nontransgenic N. benthamiana plant tissue was treated with RNase free DNase I
to remove the genomic DNA. A negative-sense CCMV RNA3 specific primer
RA83 (5’ AAGTGATCCCCTCTTGTGCGGCTGC 3’), which anneals at
nucleotide 1519-1544, was used for first-strand cDNA synthesis. An additional
primer M84 (5’ ACTCCAAAGAGTTCTTCCG 3’), which anneals at nucleotide
2072-2090, was used in PCR. Lanes 5-7, 8-10, and 11-13 were from
nontransgenic, transgenic A69, and 3-57 plants, respectively. Samples in lanes 5,
8, and 11 were mock-inoculated (M); lanes 6, 9, and 12 were inoculated with
BMV (B); lanes 7, 10, and 13 were inoculated with CCMV (C). Lane 1 contains
PCR product using 0.1 pg pCC3AG1 plasmid DNA. Lane 2 is the negative
control of PCR where water was added to the PCR mixture. Lane 3 is empty (E).
Lane 4 contains 1 Kb size marker (GibcoBRL).

29

nontransgenic A69 3-57 3-57
M pCC3AG1 CCMV CCMV BMV CCMV
1 2 3 4 5 6 7 8 9 10 ll

 

Figure 5. Agarose gel showing Natl treated RT-PCR products amplified from
total RNA extracted from virus infected plant tissue. Lane 1 contains the 1 Kb
size marker (GibcoBRL). Lanes 2-3 contain PCR products amplified from
pCC3AG1. RT-PCR amplified products were from CCMV infected nontransgenic
N. benthamiana plants (lanes 4-5); CCMV infected A69 plants (lanes 6-7); BMV
infected 3-57 plants (lanes 8-9); CCMV infected 3-57 plants (lanes 10-11). RT-
PCR products in lanes 2, 4, 6, 8, and 10 were not treated with Natl restriction
enzyme, whereas those in lanes 3, 5, 7, 9, and 11 were treated with Natl. Arrow
“(—” indicates undigested fragments. Arrow “4 ” indicates digested fragments.

Note the small digested bands in lane 11.

3O

cleaved by Natl (Fig. 5, lane 7), which indicates that the negative strand CCMV RNA
amplified in CCMV infected A69 transgenic plants was from the wild type CCMV. The
RT-PCR product of CCMV infected 3-57 plants was partially cleaved by Natl (Fig. 5,
lane 11), indicating that for CCMV infected 3-57 transgenic plants, the negative CCMV
RNA3 which was PCR amplified was of two origins: most of the RNA was from wild
type CCMV inoculum, while some was from the transgene mRNA. For BMV infected 3-
57 transgenic plants, the RT-PCR product was completely cleaved by Natl, which
indicates that this RT-PCR amplified fragment was derived from the viral transgene, the
only CCMV RNA fi'agment with the marker mutation Natl site. Note that no PCR
fragment is present in the BMV inoculated A69 plants (Fig. 4). The only explanation for
this data is that the replication complex of BMV recognized the CCMV viral transgene

and synthesized a complementary copy.

DISCUSSION

Previous studies revealed that negative-sense RNA of BMV and CCMV
accumulates to levels 100-fold' lower than positive-sense RNA in protoplasts at 24 hour
post inoculation (Marsh et al., 1991a; Pacha and Ahlquist, 1991). Considering that
synchronized infection of viruses is impossible in a plant, and the negative-strand
synthesis happens at the very initial stage of viral infection in a single cell, the negative-
sense RNA could accumulate at a much lower level in planta than in protoplasts. Thus in
planta detection of negative-sense viral RNA of bromoviruses usually requires total RNA
extracted from several grams of the plant tissue rather than a small amount of protoplast,

and a series of treatments to remove the single-stranded cellular and viral RNA from the

31

double-stranded replicative forms (Bastin and Kaesberg, 1976). More recent studies on
RNA replication of bromoviruses were exclusively conducted in protoplasts rather than
in planta (Loesch-Fries and Hall, 1980; Marsh et al., 1991b). However, since our
previous RNA recombination experiments implied that initiation of negative RNA
synthesis on the viral transgene in planta could increase the incidence of RNA
recombination in transgenic plants (Greene and Allison, 1994; 1996), we chose to
demonstrate in planta synthesis of the complementary copies of the viral transgene

during viral infections of transgenic plants.

Viral transgene transcripts were recognized by viral replication complex, and a
complementary copy of the viral transgene was synthesized

The RT-PCR experiment provided convincing data that the viral transgene can be
recognized by the viral replication complex when the viral transgene contains an intact 3’
UTR. The minus-strand promoter on the CCMV viral transgene is not only recognized by
the viral replication complex of wild type CCMV, but also by that of another closely
related virus, BMV. Thus in both cases, a full-length complementary copy of the viral

transgene was obtained.

Effects of deletion on the recognition of viral transgene

Minus-strand promoter was mapped to within the 134 3’ terminal bases of
genomic BMV RNAs (Miller et al., 1986), and the minus-strand promoter of CCMV
RNA is expected to reside in a similar location (Ahlquist et al., 1981a; Bujarski et al.,

1986). Deletions within the 3’ terminal sixty bases can cause dramatic loss of the minus-

32

strand promoter activity in BMV (Marsh et al., 1991b). In this study, the complementary
copy of the viral transgene with 69 nucleotides deleted 3’ UTR was not detected in either
BMV or CCMV infected plants. This indicated that the minus-strand promoter at the 3’
UTR of the viral transgene is essential for viral transgenic mRN A recognition and

synthesis of minus-sense RNA by the viral replication complex.

The viral transgene as well as the subgenomic RNA4 were not the preferred
templates for negative-sense RNA synthesis in planta

Despite the high accumulation level of the viral transgene in 3—57 transgenic
plants (Greene and Allison, 1994; 1996), the nearly 700 bp viral transgene is not
proportionately represented in the pool of negative-sense RNA. Even though the
detection limit of the RNA-RNA hybridization system was optimized to detect 10 pg of
denatured DNA and the negative-sense copies of the genomic RNAs 1, 2, and 3 showed
strong signals, the complementary copy of the viral transgene was still not observed in
the Northern blot (Fig. 3). The negative-sense copy of the subgenomic RNA 4 provided a
weak band (Fig. 3). This agrees with the observation of Pacha and Ahlquist (1991), that
the amount of negative-sense subgenomic RNA 4 is small for both BMV and CCMV.
The negative-sense RNA 4 of CCMV was barely visible in the Northern blot when either
BMV or CCMV replicative proteins in combination with CCMV RNA3 were used to
infect barley protoplasts (Pacha and Ahlquist, 1991). An explanation for these
observations is that, negative RNA synthesis in planta is a regulated process. Although
bearing the same minus strand promoter, subgenomic RNA 4, as well as the CCMV

transgenes, were not recognized as readily as the genomic RNA 3.

33

Miller et al. (1986) reported that polyadenylation of either the 3’—terminal 161-
nucleotide fragment of BMV RNA or the full-length RNA4 had little effect on minus
strand promoter activity in vitra when the poly(A) tail was between 15-30 nucleotides
long. Since the size of the replication product was the same as that obtained from
unmodified templates, Miller et a1. (1986) concluded that initiation occurred at the same
site in both cases. The poly(A) tail added to the viral transgenic mRNA in the 3-57 plants
may be up to several hundred nucleotides long. Whether the length of the poly(A) tail had
any effect on the viral RNA recognition and initiation during minus strand synthesis

remains unknown.

This study provides an explanation on the difference in recovery of recombinant
virus in transgenic plants with a complete or incomplete 3’ UTR. Several studies on
different viruses indicated that RNA recombination can happen during either plus
(Carpenter et al., 1995) or minus (Kirkegaard and Baltimore, 1986; Mueller and
Wimmer, 1998) strand synthesis. Detection of the complementary copy of the viral
transgene in transgenic plants with full-length 3’ UTR offers an explanation for the
reason why the recombination rate is higher when the 3’ UTR of the viral transgene is
intact. The presence of both plus and minus strand copies of the viral transgene in a
single infected cell increases the incidence of the interaction between the viral transgene
and the infecting viruses, and also allows the recombination event to happen during both
plus and minus strand RNA synthesis. This study also offers fiirther support to the

conclusion of Greene and Allison (1996), that excluding the viral replication initiation

34

site from the viral transgene is an effective way of reducing, although not abolishing,

RNA recombination in transgenic plants.

35

CHAPTER 2

RNA Recombination betWeen a Brome Mosaic Virus Deletion Mutant and a

Polymerase Transgene

INTRODUCTION

Transgenic plants that express a viral capsid or a polymerase gene are, in many
cases, resistant to infection by the virus from which the genes were derived, and closely
related viruses (Grumet, 1995). Following the discovery of coat protein-mediated
resistance (CPMR) (Powell-Abel et al., 1986), replicase-mediated resistance was
demonstrated in tobacco plants transformed with the TMV replicase gene (Golemboski et
al., 1990). In many cases, it appears that replicase mediated resistance is stronger than CP

mediated resistance (Braun and Hemenway, 1992; Donson et al., 1993).

While virus resistant transgenic plants (VRTPs) appear to be an effective means
of controlling viral diseases, several studies have indicated that the viral transgene
transcripts provide a unique opportunity for RNA recombination with challenging viruses
(reviewed in Allison et al., 1997). The case study by Greene and Allison (1994) indicated
that a capsid transgene of CCMV was involved in RNA recombination with a movement
defective CCMV CP deletion mutant used to infect transgenic plants, and 3% of the
plants yielded recombinants in which the deletion was repaired through recombination

with the transgene. Examination of the recovered recombinants revealed numerous

36

nucleotide and amino acid substitutions, deletions, and additions. Several recombinant

viruses caused symptoms that were distinctly different from wild type CCMV.

In this study, we sought to determine if RNA recombination could also involve
polymerase transgenes, and if so, to compare the recovery rate of polymerase
recombinants with that of the capsid recombinants obtained by Greene and Allison
(1994). The design of this experiment was similar to that of Greene and Allison (1994).
Nicatiana benthamiana Domin were transformed with a segment of the BMV polymerase
gene which bore a silent marker mutation which provided an Eca47III restriction site.
The challenging BMV included a 307-nt deletion near the 3’ end of the polymerase gene
open reading fi'ame (ORF) in BMV RNA 2. This deletion mutant, PTSO, remained
capable of replication in both barley (Hordeum vulgare L.) and N. benthamiana (Traynor
et al., 1991; Smirnyagina et al., 1996), but systemic movement in N. benthamiana was
inhibited. Transgenic lines contained the segment of the BMV polymerase gene that was
absent in the BMV deletion mutant. Thus, systemic movement of the virus in PTSO
infected transgenic plants signaled the restoration of the polymerase gene through RNA
recombination. Our data indicates that the viral polymerase transgene is actively involved
in RNA recombination events and the recovery of polymerase recombinants is

comparable with that of capsid recombinants.

37

MATERIALS AND METHODS
Plasmids for the synthesis of infectious transcripts

Full-length cDNA clones of wild type BMV RNAl, RNA2 and RNA3 were
maintained in plasmids pBlTP3, pB2TP5, and pB3TP8, respectively (Janda et al., 1987).
Transcripts from these clones are referred to as B], B2, and B3 hereafier. The BMV
RNA2 deletion mutant clone pBZPTSO was a generous gift from Dr. Patricia Traynor
(Virginia PolyTech, Blacksburg, Va.). This clone lacks 307 nucleotides at the 3’ end of
the 2a gene ORF which results in a 125 amino acid deletion at the C-terminal of the 2a
protein (Traynor et al., 1991; Figure 1). Transcripts from pB2PT50 are referred to as
PT50 hereafter. Plasmid pB2MD4 is a full-length BMV RNA2 clone containing an A—9G
silent mutation at position 2458. This mutation was generated through site-directed
mutagenesis of pB2TP5 using primers RA76 and B2. Mutagenesis primer RA76 (5’
ATCGTCGACCTTCTTTTTCWGTGTGGTC 3’) anneals at nucleotides 2449-
2482 and it introduces the A——>G silent mutation (bold and italicized) which also creates a
unique Eco47HI restriction site (double underlined). The existing restriction site Sail
(single underlined, Fig. 1) is also included in RA76 for the convenience of cloning.
Primer B2 (5’ GTAAACCACGGAACG 3’) anneals at nucleotides 1-15. Plasmid
pB2MD4 was constructed by replacing the 800 bp MIul-SalI fragment of pBZTPS with
the same restriction fragment containing the PCR generated silent mutation (Fig. 1).
Transcripts from pB2MD4 are referred to as MD4 hereafter. The mutation in plasmid
pB2MD4 and the 307-nucleotide deletion in plasmid pB2PT50 were confirmed by

dideoxy-sequencing (Amersham).

38

1252 1681
104 Kpnl MM 2195 San 2502 2569 2865

PBZTP5 I 1 1 3 ' /
I fir-if l /
I
/
/
I
I

 

 

 

pB2PT50 1 1 ’ UAA

 

pB2MD4

 

 

Transgene

 

Figure 1. Plasmid construction. Plasmid pB2TP5 is the full-length wild type BMV
RNA2 clone. Plasmid pB2PT50 is a deletion mutant of BMV RNA2, which lacks 307-
nt at the 3’ end of the polymerase gene (deleted nucleotides 2195-2501) (Traynor et al.,
1991). Plasmid pB2MD4 is the full-length BMV RNA2 clone with an A-—>G marker
mutation at position 2458. The primer used for site-directed mutagenesis was RA76 (5’
ATCGTCGACCTTCTTTTTCWGTGTGGTC 3’), which adds a unique
Eca47III restriction site (double underlined) through this single A->G substitution at
position 2458 (bold and italicized). The 3’ 1.6 Kb KpnI-BamH I fragment was excised
from pB2MD4 and inserted into binary vector pTETl downstream of the CaMV 358
promoter to make pTETMD3. This 1.6 Kb transgenic mRNA was detected in several
transgenic plants selected for the recombination experiments (Fig. 2). The boxes
indicate the polymerase open reading frame (ORF). The shaded area indicates the

central polymerase core region.

39

Infectious transcripts, namely B1, B2, B3, PT50, and MD4, were synthesized
using T7 RNA polymerase (Epicenter) and EcoRI digested DNA plasmid templates

(Janda et al., 1987).

Plasmid for the Agrabacterium—mediated transformation

To construct an Agrabacterium-mediated transformation vector, the 3’ 1.6 Kb
KpnI-BamHI fragment of pB2MD4 was excised and inserted into Kpnl-BamHI digested
binary vector pTETl, which is a derivative of pBin19 (Frisch et al., 1995), at a position
downstream of the CaMV 358 promoter to make pTETMD3 (Fig. 1). The NPT II gene is
also located within the left (LB) and right (RB) border sequences of the binary vector and
it provides kanamycin resistance to regenerated shoots. The correct orientation and
joining of the KpnI-BamI-ll fragment in pTETMD3 was confirmed by dideoxy-

sequencing (Amersham).

Bacterial strains

Escherichia cali strain JM101 cells were grown at 37°C for 16 hours prior to
plasmid DNA extraction. LB medium containing 100 mg/L kanamycin was used to select
JMlOl cells containing pTETl or pTETMD3. For all other plasmids in this study, 50

mg/L ampicillin was included in the media.

Agrabacterium tumefaciens strain LBA4404, which contains the helper Ti

plasmid pRAL4404 (Hoekema et al., 1983), was maintained on solid YEP medium (10

mg/ml peptone, 10 mg/ml yeast extract, 5 mg/ml NaCl, 1.5% Bacto-Agar) containing 50

4O

myL rifampicin (Tavazza et al., 1988). The transformation vector pTETMD3 was
introduced into LBA4404 cells using the freeze-thaw method (Holsters et al., 1978).
Transformed cells were selected and maintained on solid YEP medium containing 50

mg/L rifampicin and 100 mg/L kanamycin. LBA4404 cell lines were grown at 28-30°C.

Agrabacten‘um—mediated transformation

The Agrabacterium-mediated transformation was adopted from Benvenuto et a1.
(1991), Tavazza et a1. (1988) and Horsch et al. (1985), with minor modifications. MS
basal salt mix (GibcoBRL, Cat. No. 11117-066) was used to make both MS liquid and
solid media, which were adjusted to pH 5.0 and pH 5.7, respectively, using KOH or HCl.
Three types of MS solid media were used in this study, and they all contained Gamborg’s
B-S vitamins (100 mg/L myo-Inositol, 1 mg/L nicotinic acid, 1 mg/L pyridoxine—HCI,
and 10 mg/L thiamine-HCI), 3% sucrose, and 0.8% agar. The cocultivation medium
contained 1 mg/L IBA (3-indolebutyric acid) and 1 mg/L BAP (6-benzylaminopurine),
but lacked the antibiotics. The shoot induction medium contained the same
phytohormones as the cocultivation medium, plus 100 mg/L kanamycin for the selection
of transgenic shoots and 200 mg/L cefotaxime for the elimination of bacterial
contamination. The root induction medium lacked the phytohormones, but contained both

kanamycin and cefotaxime at the same concentration as shoot-induction medium.
For plant transformation, leaves from four-week old N. benthamiana were first

sterilized in 10% Chlorox containing 0.1% Tween 20 for ten minutes. The leaves were

rinsed in sterilized water three times, and blotted dry between two sheets of sterilized

41

Whatman 3MM paper. Steriliz‘ed leaf tissue was cut into one centimeter leaf discs and
inoculated by floating them for 5 min in diluted LBA4404 cells containing pTETMD3
plasmids (one milliliter overnight cell culture diluted in 20 ml MS liquid medium, pH
5.0). Inoculated leaf discs were blotted dry on sterilized Whatman 3MM paper and
incubated upside down (abaxial surface up) on cocultivation medium in the dark for two
days. After incubation, the discs were inverted and transferred to shoot-induction
medium. Afler one to two months, the regenerated shoots were excised and transferred to
root induction medium. Transformed shoots would usually regenerate roots within twenty
days and could be transferred to soil. These plant cell cultures were maintained in petri

dishes or magenta boxes and were housed in a grth chamber which was maintained at

26°C with a 16-hour day period.

NPT II ELISA testing for transformants

Transformants were initially identified by ELISA screening for the NPT II marker
gene which provides kanamycin resistance. A leaf disc 8 mm in diameter was collected
fi'om each plant and ground in a microcentrifuge tube in 100111 of 0.25 M Tris-Cl, pH 7.8
containing 1.0 mM phenylmethylsulfonylfluoride (PMSF). After centrifirgation at 7500 g
for 30 minutes, eight microliters of supernatant were used for the ELISA test (NPT ll
ELISA Kit, 5 prime —> 3 prime, Inc). ELISA readings were collected using Minireader II

from Dynatech.

42

Plant maintenance and inoculation

Transgenic plants (F0) were maintained in soil and clonally propagated by
removing auxiliary shoots, dipping them into root induction mixture (200 mg IBA in 100
m1 Talc), and rooting them in medium-sized vermiculite. Transgenic plant cuttings were
incubated at 26°C under strong fluorescent light 24 hours each day for two weeks or until
the roots grew two centimeters or longer. Rooted cuttings were transferred to soil and

maintained in a 26°C growth room with a 16-hour day period.

For the recombination experiment, these clonally propagated F0 transgenic plants
and non-transgenic N. benthamiana seedlings (used as controls) were inoculated at the
five-leaf stage with 0.5 ug each of B1 and B3 transcripts plus 1.0 11g of either B2 or PT50
transcripts. These transcripts were first mixed in a buffer which contained 5 mg/L
bentonite, 0.25 M NaCl, 25 mM Tris-Cl, 25 mM NaHZPO4 and SmM EDTA (pH 8.0)
and rub-inoculated on two leaves dusted with carborundum (Traynor et al., 1991).
Inoculated plants were lefi in the dark for one day before being transferred to the growth

room.

To test the symptom development of the MD4 mutant, barley (Hordeum vulgare
L.) plants were inoculated with 0.2 11g of each B1 and B3 transcripts plus 0.5 ug of either
B2 or MD4 transcripts. The procedure was similar as for inoculating N. benthamiana

plants.

43

To transfer the potential recombinant viruses to nontransgenic N. benthamiana
and barley plants, 0.2 g of leaf tissue was ground in 1 ml of 10 mM sodium phosphate
buffer (pH 7 .0) with a mortar and pestle. This inoculum was rub-inoculated to two fiJlly
expanded leaves of nontransgenic N. benthamiana at the S-leaf stage, or the first leaf of

7-day-old barley plants.

RNA extraction and Northern blot analysis

To analyze for the transgene transcripts in regenerated shoots, total RNA was
extracted from plant leaf tissue using the technique of Puissant and Houdebine (1990).
Total RNA from approximately 0.2 g of leaf tissue was separated in a 1.0% agrose gel in
TBE buffer and transferred to a Hybond N Nylon membrane (Amersham). A
digoxigenin-labeled HEI probe (Ahlquist et al., 1981b) which anneals to the 3’ 200
nucleotides of BMV positive strand RN As was used for hybridization as described by the

manufacturer (Boehringer-Mannheim).

For dot blot analysis, one square inch of leaf tissue was ground in a
microcentrifiJge tube in 40111 of distilled water. After centrifugation at 13,000 rpm for two
minutes, the supernatant was transferred to a fresh microcentrifuge tube and treated in
1.0% SDS at 65°C for five minutes. Following centrifugation at 13,000 rpm for two
minutes, 5111 of the supernatant was dotted directly onto Hybond N nylon membrane. The

probe and the procedure of hybridization were the same as for the Northern blot analysis.

44

Screening and analysis of recombinant viruses

To screen for all potential recombinant viruses, viral RNA was extracted from
PT50 inoculated transgenic N. benthamiana showing a positive signal in the dot blot
analysis using the same procedure as Traynor et al. (1991). Plants with even the faintest
signals were included in the analysis. Viral RNA fi'om 0.2 g of plant tissue was loaded in
1.0% TBE gel and the procedure of RNA-RNA hybridization was similar as for

analyzing the total RNA.

RT-PCR was also used to screen for potential recombinant viruses. Viral RNA
from 0.2 g of leaf tissue was used in the first strand cDNA synthesis using primer QE (5’
CGGAATTCTGGTCTCTTTTAAGAGAT 3’) as described by the manufacturer
(Boehringer-Mannheim). Primer OE anneals at nucleotides 2849-2865, and it introduces
an EcoRI restriction site (underlined). Primer RA96 (5’
AGTTTCTAGACTCGTCGAAACTGAAATGGG 3’) was added for PCR amplification
as described by Greene and Allison (1994). Primer RA96 anneals at nucleotides 1895-
1914, and it adds an XbaI site (underlined). RT-PCR products were purified twice
through phenol-chloroform-isoamylalcohol extraction followed by ethanol precipitation,
and the products resulting from recombination with the viral transgene were identified by
Eca47III digestion (MBI Fermentas). ' These potential recombinant viruses were
transferred to nontransgenic N. benthamiana and viral RNA was extracted at 30 dpi. RT-
PCR amplified cDNA of viral RNA of potential recombinants was used for cloning.
Purified RT—PCR products were first digested with EcaRl and Xbal, and cloned into

pBS/KS+ using T4 DNA ligase (GibcoBRL). Clones were sequenced manually by

45

dideoxy sequencing (Amersham). Student’s t-test was used for comparison of the

recombinant recovery rate in the study with that in Greene and Allison (1994).

RESULTS

Several preliminary experiments were required to ensure the logistic design of the
recombination experiment. First, we needed to know the movement capability of the
PT50 mutant in N. benthamiana plants. Secondly, we wanted to ensure that the single
nucleotide mutation at position 2458, which generates the unique Eca47IH restriction
site, did not affect the BMV infectivity or symptom development. Thirdly, we wanted to
confirm the transgenic nature of the plant materials by detecting the polymerase
transgene transcripts within the total RNA. And lastly, we wanted to ensure that the
transgenic plants chosen for RNA recombination experiment were not resistant to BMV

RNA transcript inoculation.

The movement of PT50 mutant is inhibited in N. benthamiana plants

Traynor et al. (1991) demonstrated that, in spite of the 307 nucleotides deletion,
PT50 directed viral RNA replication in barley protoplasts at nearly wild type level.
However, the deletion affected the movement capability of PTSO and caused 20 to 30-
fold decrease in viral RNA accumulation in barley plants. In this experiment, we assessed
the systemic movement capability of PT50 mutant in non-transgenic N. benthamiana
plants. A total of ten non-transgenic N. benthamiana plants and ten barley plants were
inoculated with Bl, BB, and PT50 transcripts and the viral RNA was extracted from the

non-inoculated leaves at 30 dpi. Despite two tries, viral RNA accumulation was not

46

detected in any N. benthamiana plants, while it was detected in barley using RT-PCR

(data not shown).

Mutation introduced in the RNA2 gene did not affect the infectivity and symptom
development of BMV

Plasmid pBZTPS was successfully modified to contain an A—->G silent mutation at
position 2458, thus creating a unique Eca47III restriction site in the new plasmid
pB2MD4. To ensure that the introduced mutation in the ORF did not affect the infectivity
and symptom development of BMV, infectious transcript MD4, together with B1 and B3,
were rub-inoculated onto both N. benthamiana and barley. Control plants were
simultaneously inoculated with wild type transcripts B1, B2, and B3. At 14 dpi, MD4
inoculated barley plants showed the same symptoms as B2 inoculated barley plants.
Electrophoresis and Northern blot analysis of viral RNA indicated that mutant MD4
accumulated at the same level as wt BMV in both N. benthamiana and barley (data not
shown). The Eca47lII restriction site in the mutant RNA was confirmed by restriction
digestion of RT-PCR amplified products from MD4 inoculated plants only (data not

shown).

Transgene transcripts were detected from transgenic N. benthamiana plants.
One hundred shoots were regenerated from the tissue culture of Agrabacterium
inoculated leaf discs. ELISA tests using anti-NPT 11 antibody indicated that half of the

shoots were transformed. To compare these transgenic plants on the basis of viral

47

Transgenic lines 3-5 4-1 6-9 7-1 7-2 7-3 9-1
Lane 1 .2“ .. _ 3 4 5 6 7

 

Figure 2. Detection of viral transgenic RNA transcripts in transgenic N.
benthamiana plants. N. benthamiana plants were transformed with the 3’ 1.6
Kb fragment of BMV RNA2 using Agrabacterium-mediated transformation
techniques. This 1.6 Kb fragment was first modified using site-directed
mutagenesis technique to introduce a unique molecular marker Eca47IH
restriction site, and then cloned into a binary vector to make pTETMD3.
Total RNA was extracted from transformants 3-5, 4-1, 6-9, 7-1, 7-2, 7-3, and
9-1. Dig-labeled riboprobe HEl, which is specific for a 200-nt fragment at
the 3’ end of all four BMV RNAs, was used in RNA-RNA hybridization. The
arrow “ 5 ” indicates the site of the transgenic mRNA of 1.6 Kb in size. The

“*9,

asterisk marks the plant that was resistant to BMV.

4s

transgene transcript accumulation, total RNA was extracted from ten transgenic plants
with the highest ELISA readings, and analyzed by hybridization in a Northern blot. The
probe HEl (Ahlquist et al., 1981b), which is specific for the 3’ terminal 200 nucleotides
of BMV RNAs, detected a single RNA band of predicted size (~1.6 Kb). Seven
transgenic plant lines, 3-5, 4-1, 6-9, 7-1, 7-2, 7-3, and 9-1, had viral transgene

accumulation (Fig. 2) and were chosen for the resistance test described below.

Transgenic plants susceptible to BMV infections were selected for the
recombination experiments

To select transgenic plants susceptible to BMV viral RNA inoculation, clonally
propagated cuttings of these seven transgenic plants were challenged with wt BMV RNA
transcripts (B1, B2, and B3). Gel electrophoresis and Northern blot analysis of the viral
RNA extracted at 14 dpi showed that six of the seven transgenic plants accumulated
BMV RNAs in non-inoculated leaves similar to non-transgenic N. benthamiana plants
(Fig. 3, lanes 1-2, 4-8). Short exposure of the Northern blot indicated no sign of
resistance (data not shown). One transgenic plant, namely 4-1, did not show any viral
RNA accumulation in the Northern blot (Figure 3, lane 3). This experiment was repeated
twice using either viral RNA transcripts or virion as inoculum, and dot blot analysis was
used to detect the systemic movement of the virus at 30 and 60 dpi. Viral RNA
accumulation was not detected in any of the 4-1 transgenic plants at 60 dpi. Based on
these above observations, transgenic plant 4-1, which was highly resistant to BMV virion
and viral RNA inoculation, was excluded from the recombination experiments. Six

transgenic plants, namely 3-5, 6-9, 7-1, 7-2, 7-3, and 9-1, were chosen for the

49

.— Control

m—rasuNM—r
Plants «lessees.
2345678

 

Figure 3. Test of BMV resistance in transgenic plants. Transgenic plants with
the highest accumulation level of transgenic mRNA were inoculated with
infectious RNA transcripts of BMV. Viral RNA was extracted at 14 dpi and was
examined in Northern blot analysis using dig-labeled riboprobe HEl (Ahlquist
et al., 1981b). The gel was overloaded with viral RNA in order to detect virus
accumulation in the resistant plant 4-1, and each lane contained viral RNA from
0.2 gram of the plant tissue. Plant samples used were: nontransgenic control
(lane 1), transgenic 3-5 (lane 2), 4-1 (lane 3), 6-9 (lane 4), 7-1 (lane 5), 7-2 (lane
6), 7-3 (lane 7), and 9-1 (lane 8).

50

recombination experiments. This resistance data is also pertinent to Chapter 4 of this

thesis.

Recombinants detected in PT50 inoculated transgenic plants

The above mentioned transgenic plants were clonally propagated through
cuttings. At the 5-leaf stage, established cuttings were inoculated with wild type B1, B3,
and mutant PTSO transcripts. Dot blot analysis was used for the initial screening of
recombinants at 30, 45 and 60 dpi. Of the 202 transgenic plants inoculated, 75 provided
dot blot hybridization signals. Viral RNA from plants providing even the faintest
hybridization signals was further examined by Northern blot analysis, RT-PCR, or both.
Three potential recombinants, namely 711 (Fig. 4A, lane 5), 911 (Fig. 4A, lane 11), and
7239 (Fig. 4B, lane 1), were detected in the Northern blot analysis (Figure 4). Another
five potential recombinants, namely 7210, 7246, 733, 919, and 9110, were detected using
RT-PCR techniques. All eight recombinants were detected by 30 dpi. Viral RNA
extracted at a later time did not give rise to additional recombinants. For the convenience
of nomenclature, these potential recombinants were named after the transgenic plant lines
in which they developed, followed by the cutting number. For example, recombinant 911

was detected from the transgenic plant 9-1 cutting number one.

These eight potential recombinants were transferred to non-transgenic N.
benthamiana plants, and the viral RNA was extracted at 30 dpi. The BMV 3’ terminal
primer QE was used for first-strand cDNA synthesis. Primer RA96 was added for PCR

amplification (see Fig. SA). All eight potential recombinants provided 989 bp RT-PCR

51

fiagments (Fig. 5B, lanes 1-8), which are equal in size to the wild type BMV generated

fragment (Fig. 5B, lane 9).

Sequence analysis of the cDNA clones indicated that the 307-nucleotide deletion
at the 3’ end of the polymerase gene was restored in all eight recombinants, and seven of
the recombinants contained the single marker mutation at position 2458 (Fig. 6). In
addition to the marker mutation, several nucleotide substitutions were observed in the 2a
gene ORF (Fig. 7), which resulted in amino acid changes in all but one recombinant virus
(recombinant 9110) (Fig. 8). No deletion or insertion was present in the sequenced area
of any recombinant. For virus 711, the marker mutation was not present, but one
nucleotide substitution was found in the sequenced area of 2a gene ORF. This virus also
accumulated at a much lower level than wild type BMV (Fig. 4A, lane 5). Thus, virus
711 could either be a recombinant with the marker mutation reverted to wild type, or a
variant of wt BMV. Counting that eight recombinant viruses were generated from a total

of 202 challenged plants, 4% of the transgenic plants yielded recombinant virus.

RT-PCR from recombinants 711, 7246, and 911 resulted in an additional product
of around 700 bp (Fig. 5B, lanes 1, 4, and 6), the same size as the RT-PCR fragment of
the inoculated PT50 mutant (Fig. 5B, lane 10). This indicates that PT50, although
incapable of systemic movement by itself in N. benthamiana, moved systemically in N.
benthamiana in the presence of recombinant viruses, and was transmitted along with the

recombinant viruses to new hosts.

52

E:
a man

a“ :b

1 2345678910111213

 
 

2
m
a

4—3

 

 

4A

a % eVNb

oan

2
w
e

(-——3

(—4

 

4B

53

Figure 4. (A) & (B) Northern blots showing the detection of recombinant
viruses. Transgenic plants were inoculated with B1, PTSO, and B3 transcripts.
At 30 dpi, viral RNA was extracted from each plant and analyzed in Northern
blot using dig-labeled riboprobe HE] (Ahlquist et al., 1981b). Three
recombinants were detected in the Northern blot, and were designated
recombinant 711 (Fig. 4A, lane 5), 911 (Fig. 4A, lane 11), 7239 (Fig. 4B, lane
1). Recombinants 733 (4A, lane 10) and 7246 (4B, lane 2) did not show up in
the Northern blot, but were amplified from systemically infected leaves using
RT-PCR techniques (see Fig. 5). Positive control lanes, Fig. 4A, lane 13, and
Fig. 4B, lanes 4 and 5, contained virion RNA from wt BMV inoculated
nontransgenic N. benthamiana plants. Remaining lanes repreSent attempts to
recover recombinant viral RNA from PT50 inoculated transgenic plants. Arrows
“9” mark the position of the corresponding genomic and subgenomic BMV
RNAs. Asterisks “*” mark the recombinant viruses that carried PT50 mutant for
systemic movement. The sign “#” marks the recombinant viruses that did not

show up in the Northern blot.

54

 

 

(.-
2865
(2E

_)
1895
IUA96

5A

Lotta—
cmhh
arezm_rs
c——a
a—a
fi—a
nab
eVNb
«nah
c—Nb
-b

6 7 8 9 10 11

5

4

   

5B

55

Figure 5. RT-PCR results of recombinants established in nontransgenic N.
benthamiana plants. (A) Schematic drawing of the BMV RN A2 and the site of
two primers used for RT-PCR. Primer QE (5’
CGGAATTCTGGTCTCT’ITTAAGAT 3’) was used for first-strand cDNA
synthesis. For the PCR reaction, an additional primer RA96 (5’
AGTTTCTAGACTCGTCGAAACTGAAATGGG 3’) was used. (B) Agarose
gel electrophoresis of RT-PCR products amplified from viral RNA extracted
from recombinants. Order of loading: recombinant 711 (lane 1), 7210 (lane 2),
7239 (lane 3), 7246 (lane 4), 733 (lane 5), 911 (lane 6), 919 (lane 7), 9110

(lane 8), wt BMV (lane 9), and PTSO mutant (lane 10). Arrow f indicates

RT-PCR product from full-length BMV RNA2. Arrow >> indicates RT-PCR
product from BMV RNA2 deletion mutant PT50. Lane 11 contains 1 Kb size
marker (GibcoBRL).

56

Symptom development and virus accumulation of the recombinants

Systemic infection of all eight recombinants in barley was confirmed using RT-
PCR techniques (data not shown). All eight recombinants accumulated. at very low levels
in both barley and N. benthamiana as compared to wt BMV infections. Mild chlorotic
local lesions were present on the inoculated barley leaves, and sporadic chlorotic areas
were found in uninoculated leaves. No stunting of barley was observed in any of the
recombinant viruses. Thus, these recombinant viruses cause symptoms which are distinct
from the PT50 deletion mutant, the MD4 mutant carrying the Eca47III mutation, and the

wild type BMV in barley.

DISCUSSION
Involvement of polymerase transgene in RNA recombination

Previous studies indicated that viral coat protein transgene could be involved in
RNA recombination with an infecting virus (Greene and Allison, 1994). In this study, our
data indicated that the polymerase transgene could also be involved in RNA
recombination events. Since the PTSO mutant is defective in systemic movement in N.
benthamiana, systemic movement of the virus in transgenic plants indicates the recovery
of the 307-nucleotide deletion at the 3’ end of the polymerase gene through RNA
recombination. Sequence analysis of cDNA clones of the recombinant viruses clearly
indicated that the 307-nucleotide deletion was recovered through RNA recombination
between the polymerase transgene of BMV RNA2 and PT50 mutant, because the marker
mutation Eca47III site, which is only present in the transgene, was present in seven of the

eight recombinant viruses.

57

Recombinants 91 l 733 7239

GAUC GAUC GAUC

2458 +

 

Figure 6. Sequence analysis of the recombinants showing that the marker mutation
Eco47III is present. RT-PCR fragments amplified from viral RNA extracted fi'om
recombinant virus infected plant tissue were cloned into pBS/KS+, and sequenced
using the dideoxy sequencing technique. Sequence analysis indicated that seven out
of the eight recombinants have the marker mutation at position 2458. Recombinant
virus 711 is the only one without this silent mutation. Arrows indicate the single A

to G marker mutation found in the recombinant viruses.

58

BMV RNA2 1900 2865

 

—
—---J- - - -

 

 

 

 

 

 

 

 

711 l
2438
our: .
7210 l
2255
INK; -
7239 :
2318
WC U/A .
7246 I n
I I
1950 1958
NC AC/CA A/G U/C .
733 l l » l l
1991 2002 2104 2215
10m; . (3AA
911 g :
2331 2695
IVCI .
919 l
2082
lUflS .
9110 .

 

2215

Figure 7. Representative mutations recovered in the polymerase gene of
recombinant viruses. The open box represents the polymerase gene open reading
frame. The gray shaded box represents the polymerase core region. The nature and
extent of each nucleotide substitution is denoted above the horizontal line, and the
position is marked below the horizontal line. The asterisk, * , indicates the

Eca47III marker mutation present in the recombinant viruses.

59

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6O

In this study, eight recombinants were recovered from 202 transgenic plants
inoculated with the PTSO mutant. Sequence analysis of the 989 bp cDNA clones
indicated that the marker mutation Eca47III was present in seven of the recombinants.
Although virus 711 does not have this single nucleotide mutation, it accumulates at a
very low level compared to the wild type, and contains a point mutation typical of other
recombinant molecules. Thus, we are considering 711 among the recombinants and
assume that the marker mutation reverted to wild type. If 711 were a point mutant that
had generated in an accidental wild type inoculation, we would have expected the wild
type infection to have out competed 711 which accumulates at a lower titer than wild
type. Nucleotide substitutions were present in the sequenced area of all of the eight
recombinant viruses. Although the single U to C change did not result in any amino acid
change in virus 9110, the low accumulation level and the mild symptom of the virus in
barley suggested that other mutations could exist in the 5’ region of the RNA 2 ORF, or

other BMV RN As.

Our study gave a 4% recombinant recovery rate in the polymerase transgene.
Based on student’s t-test at the 95% confidence level, this is not significantly different
from the 3% recombinant recovery rate in the coat protein transgene (Greene and Allison,
1994). Previous studies indicated that the concentration of viral RNA and the size of
homologous fi'agment positively affect the rate of RNA recombination (Allison et al.,
1997; Lai, 1992). Since in a bromovirus infected plant, RNA3 accumulates several folds
higher than RNA2, it appears that RNA3 should be more frequently involved in RNA

recombination events. However, the polymerase transgene used in this study was 1.6 Kb,

61

900 bases longer than the coat protein transgene used in Greene and Allison’s study

(1994)

Many studies have indicated that a full-length polymerase gene could provide
high resistance to virus infection (Audy et al., 1994; Braun and Hemenway, 1992). In this
study, transgenic plants expressing a truncated BMV polymerase gene displayed two
distinct phenotypes. Most of the transgenic plants were completely susceptible to wt
BMV transcript inoculation, and one plant (4-1) was highly resistant to either BMV
virion or RNA inoculation (Fig. 3). This is similar to the result of Tenllado et al. (1996),
where a truncated 54 kDa gene of pepper mild mottle tobamovirus (PMMoV) provided

high resistance to PMMoV.

A recent study demonstrated RNA recombination between the replicase genes of
two related tripartite RNA viruses, cucumber mosaic virus (CMV) and tomato aspermy
virus (TAV) upon mixed infection (Masuta et al., 1998). The involvement of the viral
polymerase transgene in RNA recombination with infecting viruses indicates that further
cautions should be taken when designing virus resistant transgenic plants expressing
polymerase transgenes. Since truncated polymerase transgenes were found to provide
high resistance to viral infection (Tenllado et al., 1996 and this study), and the length of
the viral transgene could affect the involvement of the transgene in RNA recombination
(Lai, 1992), a truncated polymerase gene is a better choice for generating VRTPs. Thus

the results of this study firrther support the suggestion of Allison et al. (1997), which

62

recommends that the transgene should be the smallest viral fragment that provides useful

resistance.

For the purpose of comparing RNA recombination rates in coat protein and
polymerase genes, the complete 3’ UTR was included in the transgene in this study. The
3’ UTR of many plant RNA viruses, including bromoviruses, bears the replication
initiation site (Alquist et al., 1981a; Sriskanda et al., 1996; Bachman et al., 1994). Greene
and Allison (1996) demonstrated that a truncated 3’ UTR discouraged the involvement of
transgene in RNA recombination. The recovery of recombinant viruses in this study
further implied the importance of the 3’ UTR in RNA recombination in transgenic plants.

Exclusion of the regions for replication initiation is a must for generating VRTPs.

RNA 2 plays a role in the systemic spread of the virus

PTSO mutant was found to direct RNA replication at nearly wild type levels in
barley protoplasts (Traynor et al., 1991). Regarding the replication capability of PTSO
mutant in N. benthamiana protoplasts, Smirnyagina et al. (1996) showed that BMV 1a-2a
fusion protein, whose 2a protein had the same 125 a C-terminal truncation as PTSO
mutant, could support negative- and positive-strand RNA synthesis to approximately
30% of wt level in the presence of the la protein. This implied that, in the presence of
wild type 1a protein, PTSO mutant could support viral RNA synthesis in N. benthamiana.
The detection of several recombinant viruses in this study fithher demonstrates that PT50
could indeed replicate in N. benthamiana, because RNA recombination requires the

involvement of viral RNA polymerase (Kirkegaard and Baltimore, 1986).

63

The 307-nt deletion affected the movement capability of PT50 and caused a 20 to
30-fold decrease in viral RNA accumulation in barley (Traynor et al., 1991). In this
study, PTSO mutant was found to be defective in movement in N. benthamiana plants.
However, with the recovery of the 3’ deletion, recombinant viruses regained the
capability to move in N. benthamiana. This agrees with the results of previous studies,
which indicate that replicative proteins play roles in systemic spread and/or host
specificity of bromoviruses (Allison et al., 1988; Traynor et al., 1991). Thus, BMV
RNA2 is multifirnctional. The 3’ terminal 307-nucleotide RNA encodes firnctions

supporting virus movement.

A previous study indicated that, in most of the recombinant viruses generated
with the coat protein transgene, nucleotide deletions and insertions were fiequently
observed in the recovered region (Greene and Allison, 1994). However, in this study,
only nucleotide substitutions were present in the cDNA clones of the recombinants. This
agrees with the observation that BMV polymerase gene is very conservative. Deletions in
the central polymerase core region abolish RNA replication in the barley protoplasts, and
deletions in the 5’ and 3’ regions cause decrease in RNA replication, virus accumulation,

and symptom development in barley plants (Traynor et al., 1991).

Favorable interaction between PT50 mutant and recombinant viruses

A discovery of this study is the recovery of the deletion mutant PTSO along with

the recombinant viruses in the upper uninoculated leaves. PTSO was shown in this study

64

to be defective in systemic movement in N. benthamiana. However, in the presence of
the recombinant viruses, PT50 moved systemically in N. benthamiana, and it was also
transmitted along with the recombinant viruses to new hosts (both barley and N.
benthamiana). This indicates a favorable interaction between these two viruses. Whether
the PTSO mutant played a role in the replication of these recombinant viruses remains

unknown.

Thus, from a population genetics point of view, RNA recombination between the
polymerase transgene and PT50 mutant is a favored event because the generation of
viable recombinants which can support the movement of mutant PTSO could lead to a

dramatic fitness gain for the mutant virus PTSO.

65

CHAPTER 3

RNA Recombination in Transgenic Plants Expressing Translatable or

Untranslatable Viral Transgenes

INTRODUCTION

Transgenic expression of viral genes was proven to be an effective way of
combating viral infections (Grumet, 1995). However, the presence of viral transgenes
provides a unique opportunity for RNA recombination with infecting viruses (de Zoeten,
1991). Following the demonstration of RNA recombination between a CCMV coat
protein transgene and an infecting virus (Greene and Allison, 1994), studies were carried
out to look for ways of generating virus resistant transgenic crops (VRTPs) whose viral
transgene would be less likely to be involved in RNA recombination. The study of
Greene and Allison (1996) demonstrated that excluding the 3’ UTR in the viral transgene
is one effective way of discouraging the involvement of viral transgene in RNA
recombination events. Experiments carried out in this study were to determine if a
translatable transgene was more or less likely to be involved in RNA recombination than

a non-translatable transgene.

In the study of Greene and Allison (1994), the transgene transcripts were derived
from the 3’ 2/3 of the CCMV CP ORF with a complete 3’ UTR. This fiagment of viral
gene was directly excised from the cDNA clone of CCMV RNA3, and no AUG start

codon was added at the 5’ end of the viral gene. Thus, translation of the viral gene was

66

unlikely. In this study, N. benthamiana plants were transformed with two modified
constructs of the CCMV CP gene used in the RNA recombination experiments of Greene
and Allison (1994). One construct had an inframe start codon added at the 5’ end of the
truncated CP gene. Theoretically, this viral transgene was translatable. The other
construct had an out-of-frame start codon followed by a stop codon, and this viral
transgene was untranslatable. For both constructs, three single nucleotide substitutions
near the 3’ end of the CCMV CP gene characterized the transgene, and the 144-
nucleotide translational enhancer TEV leader sequence (Gallic et al., 1995) was added at
the 5’ end of the transgene. Our data indicated that transgenes with an initiation codon
were less likely to be involved in recombination events than the transgene containing no
initiation codon. Additionally, the translatable transgene was less likely to be involved in

a recombination event than a nontranslatable transgene.

MATERIALS AND METHODS
Plasmid construction

Full-length cDNA clones of wild type CCMV RNA], RNA2 and RNA3 are
maintained in plasmid pCClTPl, pCC2TP2, and pCC3TP4, respectively (Allison et al.,
1988). Infectious transcripts, namely C1, C2, and C3, were synthesized using T7 RNA
polymerase (Epicenter) fi'om the Xbal digested DNA plasmids. Plasmid pCC3AG1 was
generated through site-directed mutagenesis which introduced three single nucleotide
substitutions including a Natl restriction site near the 3’ end of the CP open reading
frame (ORF) (Greene and Allison, 1994). Plasmid pCC3 AG3 was generated by removing

the SacII-Natl restriction fragment from pCC3AG1 and ligating the Klenow filled ends

67

(Greene and Allison, 1994). The transcripts synthesized from Xbal linearized pCC3 AG3,

AG3, were used to inoculate the transgenic plants together with transcripts C1 and C2.

Plasmid construction for plant transformation

Plasmid pGAMD201(+) is the transformation vector containing the untranslatable
viral gene which bears a stop codon seven codons down stream of the start codon (Fig.
1). Primer RA38 (5’ CCATCGCTCCATGGCCAATGGGCAA 3’) was designed to
introduce a start codon (bold) and a NcaI restriction site (underlined) at position 1473 of
CCMV RNA3. Addition of two nucleotides (bold and italicized) provided a frameshift
and introduced a UAA stop codon seven codons down stream of the start codon. An
additional primer CCMV 3’ (5’ CAGTCTAGATGGTCTCCTTAGAGAT 3’), which
introduces an Xbal site at the 3’ end (underlined), was used in conjunction with RA38 to
amplify a 700-bp fragment from pCC3AG1. After Neal-Xbal treatment, the PCR
fragment was inserted into a modified plasmid pTL37 (Carrington and Dougherty, 1987),
which has an added Ncal site downstream of the tobacco etch virus (TEV) 5’ leader
sequence. The resulting plasmid has a 144-bp TEV leader sequence upstream of the 700-
nucleotide CCMV CP fragment (from nucleotide 1473-2173). Primer RG26 (5’
AAGTCTAGAAAATAACAAATCTCAACA 3’) anneals to the 5’ end of the TEV
leader sequence and it also adds an Xbal site (underlined). Using an additional primer
CCMV 3’, the 850-nucleotide PCR fi'agment was amplified and cloned into Xbal

digested binary vector pGA643 (An et al., 1988).

68

pCC3TP4 StyI SacH Xbal

 

 

pCC3AG1

I 3a I

 

 

pCC3AG3

l 3a I

 

 

 

 

 

SaeII vNatI
pGACCMV I r
Transgenic line :.-;.I.€ as:
3-57
pGAMD201(+) ’ * SacII vNatI

 

Transgenic A and B
lines, untranslatable

 

 

GATAGCC ATG GCC AAT GGG CAA GGC TAT IAA
NcaI STOP

’ SacH vNatI
pGAMD202(+)
Transgenic G and H

lines, traiTW/ZYK

ACCCATCGCTCC ATG GGC CAA GGC AAG GCT
NcaI

 

69

Figure 1. Plasmid construction. Plasmid pCC3TP4 is the full-length clone of CCMV
RNA3 (Allison et al., 1989). Plasmid pCC3AG1 was generated through site-directed
mutagenesis which introduced three single nucleotide substitutions introducing a Natl
restriction site near the 3’ end of the CP ORF (Greene and Allison, 1994). Plasmid
pCC3AG3 was generated by removing the SacH-Natl restriction fragment from
pCC3AG1 and ligating the Klenow filled ends (Greene and Allison, 1994). The
transcripts synthesized from Xbal linearized pCC3AG3, AG3, were used to inoculate the
transgenic plants together with wild type transcripts C1 and C2. Plasmid pGACCMV was
generated by inserting the StyI-Xbal fiagment of pCC3AG1 into pGA643 and it was used
generate transgenic line 3-57 (Greene and Allison, 1994). Plasmid pGAMD201(+) is the
transformation vector containing the untranslatable viral gene which bears a stop codon
seven codons down stream of the start codon. Primer RA38 (5’
CCATCGCTCCATfiCCAATGGGCAA 3’) was designed to introduce the start codon
(bolded) and a Ncal restriction site (underlined) at position 1473. Addition of two
nucleotides (bolded and italicized) provided a frameshift and introduced a UAA stop
codon. Plasmid pGAMD202(+) is the transformation vector containing the translatable
viral gene which bears an inframe start codon. It was constructed using a similar strategy,
and differed from pGAMD201(+) in that the introduced start codon was inframe, and no
stop codon was introduced. The primer used for mutagenesis was RA45 (5’
ACCCATCGCTCCATGGGCCAAGGCAA 3’) instead of RA38. Transgenic A and B
lines were generated using pGAMD201(+), and G and H lines were generated using
pGAMD202(+). Open boxes indicate CCMV 3a or CP ORF. Shaded area indicates the
central part of the CP ORF. Black boxes indicate the 5’ TEV leader sequence. “ I ”
indicates the introduced start codon. “v” indicates the introduced marker mutation in the
CCMV CP ORF. An asterisk, *, indicates the introduced stop codon. The nucleotide
sequences of the 5’ end of the tmncated viral gene are below the diagram of
pGAMDZO 1 (+) and pGAMD202(+).

7O

Plasmid pGAMD202(+) is the transformation vector containing the translatable
viral gene which bears an infi'ame start codon (Fig. 1). It was constructed using a similar
strategy, and is different from pGAMD201(+) in that the introduced start codon was
inframe with the remainder of the coat protein gene, and no stop codon was introduced.
The primer used for this mutagenesis was RA45 (5’
ACCCATCGCTCCATGGGCCAAGGCAA 3’) instead of RA38. Primer RA45

introduces a Ncal site (underlined) and a start codon (bold).

The final plasmids pGAMD201(+) and pGAMD202(+) were sequenced to
confirm the correct orientation and joining of the fragments and the correct start and stop
codons. The NPT II gene which is located within the left (LB) and right (RB) border

sequences of both binary vectors, provides kanamycin resistance to transformed shoots.

Bacterial strains

The bacterial cell lines used in this experiment were the same as those used in
Chapter 2. Since vector pGA643 was used in this study, tetracycline was added to LB and
YEP medium to select JM101 and LBA4404 cells containing pGA643 and its derivatives.
The tetracycline concentrations used were 10 mg/L for selecting JM101 cells, and 3 mg/L

for selecting LBA4404 cells.

Agrabacterium-mediated transformation and ELISA tests of regenerated plants

The procedures for Agrabacterium-mediated transformation and anti-NPT II

ELISA tests of the regenerated plants were the same as in Chapter 2. Four-week-old

71

Nicatiana benthamiana Domin was used for leaf disc transformation. For each
transformation construct, two LBA4404 colonies were used for plant transformation.
Colonies were designated A and B (untranslatable construct) or G and H (translatable
construct). Independent transformants fi'om each colony were numbered, eg. A7 (Fig. 2).
Transgenic A and B lines were generated from pGAMD201(+) and were referred to as
non-translatable lines. Transgenic G and H lines were generated using pGAMD202(+)

and were referred to as translatable lines.

Transgenic plant maintenance and inoculation

Transgenic plants were maintained and clonally propagated using the same
techniques as described in Chapter 2. To test the virus resistance of each transgenic line,
0.5ug (20ug/ml) or 311g (120ug/ml) of each C1, C2 and C3 transcripts was applied to
each plant in an inoculation mix of 25111 (see Chapter 2 for procedure). For the
recombination experiments, 311g of each C1, C2 and AG3 transcripts was applied to each
plant in a mix of 25111 (120ug/ml). The procedure of sap inoculation was the same as for

Chapter 1.

Viral RNA extraction, Northern and Western blot analysis

Procedures for total RNA extraction, viral RNA extraction, Northern blot and dot
blot analyses were as described in Chapter 2. To test the CCMV resistance in transgenic
plants, dot blot analysis was performed at 14 and 30 dpi. For screening for recombinants,

dot blot analysis was performed at 30 and 45 dpi. Dig-labeled probe RA518(+), which is

72

specific for the 3’ UTR of CCMV RNAs, was used to detect viral transgenes in the total
RNA, and the CCMV viral RNA accumulation in the dot blot analysis.

Procedures for total protein extraction, quantification, and Western blot analysis
were as described (Ausubel et al., 1987). Approximately 100 11g of total protein was
loaded in each lane. Crude anti-CCMV capsid protein polyclonal antibody was diluted

1:10,000 in the blotting reagent.

RT-PCR, cloning and sequence analysis

Potential recombinants detected by dot blot analysis were transferred to both
nontransgenic N. benthamiana and cowpea (Vigna sinensis (Tamer) Savi), and the viral
RNA was extracted at 14 dpi. Viral RNA from 0.2 g of plant tissue was used in the first
strand cDNA synthesis using primer CCMV 3’ and the procedure was as described by the
manufacture (Boehringer-Mannheim). Primer CCMV 3’ also adds a 3’ terminal Xbal site.
An additional primer CCMV 3L (5’ TAAATCGCCGTAACCGC 3’) which anneals at
nucleotides 1078-1095 was used for PCR amplification and the procedure was as
described (Greene and Allison, 1994). The RT-PCR fragment was first digested with
BglII and Xbal, and cloned into the BamI-H and Xbal digested pBS/KS+ using T4 DNA
ligase (GibcoBRL). Clones were sequenced manually by dideoxy-sequencing

(Amersham).

73

RESULTS
Viral transgenic mRNA detection

For each plasmid construct, fifty transgenic plants were regenerated from callus
and transferred to soil for further examination. Anti-NPT II ELISA tests were used to
screen for the expression of the NPT II marker gene which was located adjacent to the
viral gene in the transformation vector. Twenty nine transgenic plants with the highest
ELISA readings were selected to examine the viral transgene transcription in the total
RNA using dig-labeled RA518(+) probe, which is specific for the 3’ UTR of the CCMV
RNAs. As shown in Fig. 2, most of the plants gave a single band the size of the transgene
(~850 bp). For the untranslatable construct, plant lines A7, B2, B6, B8, and B9 were
selected for the recombination experiments. For the translatable construct, plant lines G1,

G18, H1, H3 and H4 were selected (Fig. 2).

Viral protein detection

Total protein was extracted from the ten transgenic plants mentioned above and
analyzed by Western blot analysis to evaluate the translation of the viral protein using
CCMV polyclonal antibody. The detection limit of the Western blot was quantified as 1
ng of CCMV virion (data not shown). However, when 100 11g of total protein was loaded
in each lane, no viral protein was detected from either translatable or untranslatable lines

(data not shown).

74

 

Figure 2. Northern blot analysis. Total RNA was extracted from N. benthamiana
transformed with the 3’ 700-nucleotide fragment of CCMV RNA3. Transgenic A and B
lines were generated using binary vector pGAMD201(+), whose 700-nucleotide viral
gene was modified to have a stop codon seven amino acids down stream of the start
codon. Thus transgenic A and B lines are referred to as non-translatable lines. Transgenic
G and H lines were generated using binary vector pGAMD202(+), whose 700-nucleotide
viral gene was modified to have an inframe start codon. Thus, transgenic G and H lines
are referred to as translatable lines. An equal amount of total RNA was loaded in each
lane. Dig-labeled RA518(+) probe which is specific for the 3’ UTR of CCMV RNAs was
used in the RNA-RNA hybridization. An asterisk, *, indicates the transgenic plants

selected for the RNA recombination experiments.

75

Test of virus resistance in transgenic plants

Selected transgenic lines with high levels of viral transgene expression were
tested for virus resistance prior to the RNA recombination experiments. Clonally
propagated cuttings of translatable lines A7, 82, B6, B8, and B9, and untranslatable lines
G1, G18, H1, H3, and H4 (indicated with “*” in Fig. 2), were inoculated with CCMV
RNA transcripts synthesized in separate reactions. Dot blot analysis was used to detect
viral RNA accumulation in the inoculated plants at 14 and 30 dpi. When 0.5ug of each
viral RNA was used to inoculate each plant and the concentration of the inoculum was 20
ug/ml of each viral RNA, 100% infection rate was achieved for the nontransgenic control
plants. However, for both translatable and untranslatable lines, the infection rate was 20%
(Table 1). When 3ug of each CCMV viral RNA was used to inoculate each plant and the
concentration of the inoculum was 120 11ng of each viral RNA, the infection rate was
50%, and in one of the three experiments, it reached 80% (Table 1). The CCMV viral
RNA in both translatable and untranslatable lines accumulated at a lower level than in the
nontransgenic control plants, with a 50% reduction in viral RNA accumulation in
untranslatable lines, and an 80-90% reduction in the translatable lines (Table 1). The
reduced level of virus accumulation was not observed in the transgenic plants generated
by Greene and Allison (1994; 1996), which was transcribing the same 3’ 2/3 of the
CCMV CP gene but without the addition of the AUG start codon (observed in Chapter 1

and data not shown).

76

Table 1: Test of virus resistance in transgenic plants. N. benthamiana plants were
transformed with the two modified CCMV viral gene which contained the 3’ 2/3 of the
CCMV CP gene with a complete 3’ UTR. The translatable construct has an inframe start
codon added at the 5’ end of the truncated CP gene. The untranslatable construct has a
stop codon following the added start codon. Five transgenic lines were chosen from each
construct and tested for CCMV viral RNA resistance. Both dot blot and Northern blot

analysis was used to assess the viral RNA accumulation at 14 and 30 dpi.

 

 

 

 

 

Inoculum CCMV vRNA CCMV vRNA
0.5 ug/plant at 3 ug/plant at concentration
concentration 20 ug/ml 120 ug/ml
Resistance tests Infection rate Infection rate Viral RNA
accumulation
level
Nontransgenic 100% 100% 100%
control
Translatable lines G1, 20% 50% 10-20%
G18, H1, H3, and H4
Untranslatable lines 20% 50% 50%
A7, B2, B6, B8, and B9

 

 

 

 

 

 

77

RNA recombination in transgenic plants

Based on the data obtained from the resistant test, 311g of each viral RNA
transcripts C1, C2, and AG3 was used at the concentration of 120 ug/ml to inoculate each
transgenic plant at the 5-leaf stage. A total of 232 cuttings from the untranslatable lines
and 221 cuttings from the translatable lines were inoculated with the AG3 mutant
together with wild type C1 and C2. Dot blot analysis at 30 and 45 dpi detected the
systemic movement of virus, which indicated potential recombinant viruses. Viral RNA
was extracted from plants showing hybridization signals in the dot blot analysis and was
examined using RT-PCR techniques. Out of the 453 transgenic plant cuttings inoculated
with AG3, around 150 were examined using RT-PCR techniques. Systemic movement of
the virus was detected in onlytwo cuttings from the untranslatable lines. No virus was

recovered for the translatable lines. Both viruses were detected at 30 dpi.

After passing and establishing the potential recombinant virus infections in the
nontransgenic N. benthamiana plants, viral RNA was extracted, and cDNA was
synthesized, amplified, and cloned. Sequence identification of the transgene marker
mutation in the resulting clones indicated that both viruses had recovered the deleted
region of the AG3 mutant fiom the transgene. Counting the number of plant cuttings
inoculated, the rate of recovery of recombinant virus was 0.85% for the untranslatable
lines, and 0% for the translatable lines. These two recombinant viruses were named after
the transgenic lines, followed by the number of the cutting fiom which they were derived,

B2-13 and B9-5.

78

BgIII Xbal
, l
l 3a 1

CCMV 3L CCMV 3’

 

 

 

Figure 3. Schematic diagram of CCMV RN A3 and the positions of the two primers used
for RT-PCR analysis. Boxes represent wild type CCMV movement protein gene (3a) and
capsid gene (CP). Single linesrepresent non-coding sequences. Oligonucleotide CCMV
3’ (5’ CAGTCTAGATGGTCTCCTTAGAGAT 3’) anneals at nucleotides 2158-2173
and was used for first strand cDNA synthesis, and it also introduces an Xbal site
(underlined). An additional primer CCMV 3L (5’ TAAAATCGCCGTAACCGC 3’)
which anneals at nucleotides 1078-1095, was used for the PCR reaction. Restriction site
BglII is at the 3’ end of the 3a gene ORF. RT-PCR fragment was first digested with BglII
and Xbal before being cloned into BamHI and Xbal digested pBS/KS+.

79

The complete CP genes and the intercistronic regions of the two recombinants
were sequenced. Four nucleotide substitutions and three nucleotide deletions were found
in recombinant B9-5, and two nucleotide substitutions were found in recombinant B2-13
(Fig. 4). The nucleotide changes also resulted in several deduced amino acid substitutions
(Fig. 5). Dot blot analysis indicated that both recombinant viruses accumulated to levels
several fold lower as compared to wild type CCMV in cowpea and N. benthamiana
plants (data not shown). In contrast to wt CCMV which provides symptoms in cowpea at
10 dpi, recombinant B2-13 caused mild chlorotic symptoms at around 20 dpi, and

recombinant B9-5 remained symptomless till senecence.

DISCUSSION

The 3’ 2/3 of the CCMV CP gene used by Greene and Allison (1994; 1996) did
not provide any virus resistance to transgenic plants. However, the reduced accumulation
of viral RNA in the translatable and nontranslatable lines of this study indicated that the
addition of an AUG start codon and the TEV translational enhancer sequence at the 5’ of

the truncated CP gene conferred moderate virus resistance to the transgenic plants.

The translatable lines appeared to be more resistant because the viral RNA
accumulated at a much lower level than in the untranslatable lines. This implied that
translation of the coat protein might play some role in providing virus resistance to the
transgenic plants. However, the truncated viral coat protein was not detected in any
transgenic plant in the Western blot when the detection limit was 1 ng CCMV virion.
This is similar to the observations of Huntley and Hall (1996), where no BMV coat

protein was detected in transgenic rice protoplasts expressing the full-length BMV capsid

80

gene. Possible explanations could be that the coat proteins were made at such low levels
that they were below the limit of detection, or the coat protein was very labile and rapidly
turned over in the cell, or the antigenic epitopes of the virions were not present on the C-

terminal portion of the protein. .

Despite considerable efforts to detect recombinant virus, only two recombinants
were recovered from the 232 cuttings from the untranslatable lines, and none was
recovered from the 221 cuttings from the translatable lines. This gives a 0.85%
recombinant recovery rate for the untranslatable lines, and 0% for the translatable lines.
Thus, there is a decrease in the recombinant recovery rate for both constructs compared
to the 3% recombinant recovery rate of Greene and Allison (1994), with a lower rate for

translatable lines.

One reason for the overall decrease in the recombinant recovery rate could be the
lower infection rate, which was around 50% for both constructs. Another reason could be
the strong interference of the viral transgenes on viral RNA accumulation. Huntley and
Hall (1996) observed an 80-90% reduction in viral RNA replication in transgenic rice
protoplasts expressing the full-length BMV CP gene. This is similar to our results, where
viral RNA accumulation was reduced to 50% in the untranslatable lines, and 80-90% in

the translatable lines.

The purpose of this study was to see if the association of the transgenic RNA with

ribosomes during translation would reduce the involvement of the viral transgene in RNA

81

1150 1360 1473 1812 1932 Xbal

 

 

 

 

\

 

U/C A/G
- l l
B2 13 I l
1649 1862

A3 A/U A/C U/A C/G

- I I ll l
B95 I I II l

1290 1315 1552 1587 1766

Figure 4. Diagram showing mutations recovered in the RNA3 of
recombinant viruses B2-13 and B9-5. The open boxes represent the 3a
gene and capsid gene open reading frames. Shaded box represent the
homologous regions between the transgene and inoculum AG3. The
nature and extent of each nucleotide mutation is denoted above the
horizontal line. “A” indicates deleted nucleotides. Substitution
mutations are separated by a slanted line with the wild type to the left

and substitution to the right

82

WT
89-5
82-13

WT
89-5
B2-13

60 ' 100
AAAEAKVTSAITISLPNELSSERNKQLKVGRVLLWLGLLPS

133 . 173
VVAAMYPEAFKGITLEQLTADLTIYLYSSAALTEGDVIVHL

Figure 5. Capsid protein amino acid sequence of recovered CCMV recombinants B2-13

and B9-5. The amino acid sequences of wild type CCMV (WT) are shown at the top. For

each recombinant, single letter amino acid codes are used to denote substitutions, and

dots represent unchanged amino acids.

83

recombination. Ribosomes are generally observed to protect mRNA from degradation,
and binding of ribosomes to mRNA could exclude the entry of endonucleases (Petersen,
1993). The association of the viral transgene with ribosomes may exclude the binding of
the replication complexes. Subsequently, the translatable viral transgene could be less
likely to be involved in RNA recombination. Data provided in this experiment did show a
decrease in the recombinant recovery rate in the translatable lines comparing to the
untranslatable lines. However, since the translatable lines are more resistant than
untranslatable lines to CCMV inoculation, it is unclear whether the lower recombinant
recovery rate in translatable lines was due to the association of ribosomes during the

process of translation, or due to. reduced viral RNA replication.

A second hypothesis is that RNA recombination is less likely when the transgene
provides resistance to the infecting virus, especially if this viral transgene interferes with
the replication of that virus. Transgenes that show no interference with virus replication
would be more likely to be involved in RNA recombination, such as the 3% recovery rate
of recombinants by Greene and Allison (1994). Transgenes that interfere with the virus
infection would be less likely to be involved in RNA recombination, which is shown in

the translatable lines in this study.

84

Chapter 4
Test of Virus Resistance in Transgenic Plants Expressing Bromovirus Transgenes

INTRODUCTION

Coat protein mediated resistance (CPMR) was first demonstrated in tobacco
plants transformed with the coat protein gene of TMV (Powell-Abel et al., 1986). Since
then, CPMR has been applied to a long list of viruses in a variety of difi‘erent plant
species. In the majority of cases, translation of the CP gene leads to high virus resistance,
with some exceptions where the virus resistance was mediated by the viral transgene

alone.

Generally, transcription of the viral transgene is driven by the constitutive 35S
promoter of cauliflower mosaic virus (CaMV), which leads to high accumulation of the
viral transgene transcripts. In 1991, de Zoeten suggested that, RNA recombination
between the constitutively transcribed viral transgene and an infecting virus could create
viruses with unique properties. Using bromoviruses as a model system, Greene and
Allison (1994) demonstrated that viral transgenes could be involved in RNA

recombination with challenging viruses.
The transgenic plants used in the Greene and Allison (1994) study were

transformed with the 3’ 2/3 of the CCMV CP open reading frame (ORF), and they were

not resistant to CCMV infection. However, the addition of an AUG start codon at the 5’

85

end of this same viral gene provided some CCMV resistance to the transgenic plants
(Chapter 3). In Chapter 2, a transgenic line (4-1) which was transformed with a truncated
BMV replicase gene was highly resistant to BMV viral RNA and virion inoculation. A
remaining question is, can the lull-length CP gene of CCMV provide virus resistance to
transgenic plants? This experiment was designed to answer this question. N. benthamiana
were transformed with either a translatable full-length CCMV CP gene, an antisense full-
length CCMV CP gene, or an antisense truncated CCMV CP gene. Twenty-three
transgenic lines were tested for CCMV resistance and the data indicated that CCMV CP
gene provided CCMV resistance to transgenic plants at various levels and in different
constructs. The results of this resistance test are compared with the results of previous

studies (Chapters 2 and 3; Greene and Allison, 1994).

MATERIALS AND METHODS
Plasmids for the synthesis of infectious transcripts

Full-length cDNA clones of wild type CCMV RNA], RNA2 and RNA3 are
maintained in plasmid pCClTPl, pCC2TP2, and pCC3TP4, respectively (Allison et al.,
1988). Infectious transcripts, namely C1, C2, and C3, were synthesized using T7 RNA
polymerase (Epicenter) and the Xbal digested plasmid DNA (Allison et al., 1988).
Plasmid pCC3 AGl was generated through site-directed mutagenesis of pCC3TP4 by
introducing three nucleotide substitutions which also create a unique Noll site near the 3’

end of the CCMV CP gene (Greene and Allison, 1994).

86

Plasmid construction for plant transformation

Plasmid pGAWS4 is the transformation vector containing the firll-length CCMV
CP gene. To generate plasmid pGAWS4 (Fig. 1), pCC3TP10 (Pacha and Ahlquist, 1992),
a full-length CCMV RNA3 cDNA clone which contains SaII site at the 3’ end of the CP
gene, was digested with SalI and the sticky ends were filled using Klenow DNA
polymerase. This plasmid was treated with Xbal to excise an 800-nucleotide fragment
containing the full-length CCMV CP and the complete 3’ UTR. This 800-nucleotide
fragment was cloned into pTL37 (Carrington and Dougherty, 1988), which was first
digested with NcoI, blunt-ended with Klenow, and further digested with Xbal
(underlined). Plasmid pTL37 contains the TEV leader sequence for translational
enhancement upstream of the polylinker. Molecular markers of pCC3AGl were
introduced into the resulting plasmid by replacing the SacH-Xbal fragment with that of
pCC3AG1. The resulting plasmid had the l44-nucleotide TEV leader sequence joined
with the full-length CCMV CP gene followed by the fiJll-length 3’ UTR. Primer RG26
(5’ AAGTCTAGAAAATAACAAATCTCAACA 3’) anneals to the 5’ end of the TEV
leader sequence and it also adds an Xbal site (underlined). Using an additional primer
CCMV 3’ (5’ CAGTCTAGATGGTCTCCTTAGAGAT 3’) which anneals to the 3’ end
of CCMV RNA3 and also adds an Xbal site, the 950-nucleotide PCR fi'agment was

amplified and cloned into Xbal digested binary vector pGA643 (An et al., 1988).

87

pCC3TP4

 

 

 

3a I

 

pCC3AGl Styl SacII Natl

r 3a 1

 

 

 

pGACCMV
Plant 3-57

 

pGAWS4
Transgenic M and N
lines, messenger-sense
full-length CP gene

 

 

 

 

pGAMD101(-) . I
Transgenic S lines,
antisense full-length
CP gene

 

 

 

 

pGAMD202(—)
Transgenic X and Y
lines, antisense truncated
CP gene

 

l [3:233:12
NotI Sad]

88

Figure l. Plasmids construction for plant transformation. Three nucleotide substitutions,
which also create a unique N011 site, were engineered into plasmid pCC3TP4 to create
pCC3AGl (Greene and Allison, 1994). Transformation vector pGACCMV was used in
Greene and Allison (1994) to generate transgenic plant 3-57. Plasmid pGAWS4, the
transformation vector containing the full-length CCMV CP gene, contains the 144-
nucleotide TEV leader sequence (black box) placed 5’ of the full-length CCMV CP gene.
The 3’ UTR of CCMV RNA3 was retained in pGAWS4, as were the marker mutations
which generate the unique NotI site. Plasmid pGAMDlOl(-) is an antisense construct of
pGAWS4 without the TEV leader sequence. Plasmid pGAMD202(-) is the

transformation vector containing the antisense 3’ 2/3 of the CCMV CP gene.

89

Plasmid pGAMD101(-) is an antisense construct of pGAWS4 without the
translational enhancing TEV leader sequence (Fig. 1). This plasmid was constructed by
inserting the Sail-Xbal fragment of pCC3TPlO (Pacha and Ahlquist, 1992) into HpaI and
Xbal treated pGA643, with the sticky SalI end blunted with Klenow. Plasmid

pGAMDlOl(-) contains no distinguishing marker mutations within the transgene.

Plasmid pGAMD202(-), the transformation vector containing the antisense
truncated CCMV CP gene, was generated by digesting pCC3AG1 with SIyI at nucleotide
1477 and filling the sticky ends using Klenow DNA polymerase (GibcoBRL). Following
phenol-chloroform extraction and ethanol precipitation, the DNA was treated with Xbal
to excise the 700-nucleotide fragment containing the 3’ two thirds of the CP gene plus the
3’ UTR. This 700-nucleotide fragment was purified fi'om an agarose gel using GeneClean

Kit (BiolOl), and ligated into HpaI-Xbal digested pGA643 (An et al., 1988).

These three transformation vectors have the NPT II marker gene adjacent to the
viral gene and within the lefi (LB) and right (RB) Agrobacterium border sequences. This
NPT II gene provides kanamycin resistance to the transformants. The correct nucleotide
sequence and orientation of the inserts of all three binary vector plasmids was confirmed

by dideoxy-sequencing (Amersham).
Bacterial strains

The bacterial cell lines used in this experiment were the same as those used. in

Chapter 2. Since vector pGA643 was used in this study, tetracycline was used to make

90

LB and YEP medium to select JM101 and LBA4404 cells containing pGA643 or its
derivatives. The concentrations used were 10 mg/L for selecting JM101 cells, and 3 mg/L

for selecting LBA4404 cells.

Agrobacten'um-mediated transformation and ELISA tests of regenerated plants

The Agrobacterium-mediated transformation procedure and the anti-NPT II
ELISA tests of regenerated plants were as described in Chapter 2. F our-week-old
Nicotiana benthamiana Domin plants were used for leaf disc transformation. For each
transformation construct, several LBA4404 colonies were selected for plant
transformation. Each colony was given a letter name, such as M. Independent
transformants generated using this colony were designated M followed by a transformant
number, such as transgenic line M2 (Fig. 2A and 3A). Transgenic M and N lines were
transformed with the full-length translatable CP gene using the transformation vector
pGAWS4. Transgenic S line was transformed with the antisense lull-length CCMV CP
gene using transformation vector pGAMDlOl(-). Transgenic X and Y lines were
transformed with the antisense 3’ 2/3 of the CCMV CP gene using transformation vector

pGAMD202(-).

Plant maintenance and inoculation for CCMV resistance test
Transgenic plants were maintained and clonally propagated as in Chapter 2. To
inoculate transgenic plants with infectious CCMV RNA transcripts, approximately three

micrograms of each viral RNA was applied to each plant at the concentration of

91

lZOug/ml (see Chapter 2 for procedure). The procedure of sap inoculation was the same

as for Chapter 1.

Total RNA extraction, Northern and Western blot analysis

Procedures for total RNA extraction, Northern and dot blot analysis were as
described in Chapter 2. Total RNA was extracted from transgenic plants and analyzed for
viral transgene transcripts by Northern blots. To test the CCMV resistance in transgenic
plants, dot blot analysis was performed at 14 and 30 dpi. Dig-labeled probe RA518(+),
which is specific for the 3’ UTR of CCMV RN As, was used in the Northern blot analysis
to detect the viral transgene in M and N lines, and in the dot blot analysis to detect the
viral RNA accumulation. Dig-labeled probe RA518(-), which is specific for the 3’ UTR
of negative sense CCMV RNAs, was used to detect the antisense viral transgene in X, Y,
and S lines. Total protein was extracted from the transgenic X and Y lines expressing the
full-length CCMV CP gene and analyzed by Western blot. The procedures of protein

extraction and Western blot analysis were as described in Chapter 3.

RESULTS
Viral transgenic mRNA detection

Trangenic M and N lines were transformed with the full-length translatable CP
gene using the transformation vector pGAWS4. Transgenic S line was transformed with
the antisense full-length CCMV CP gene using transformation vector pGAMDlOl(-).
Transgenic X and Y lines were transformed with the antisense 3’ 2/3 of the CCMV CP
gene using transformation vector pGAMD202(-). For each plasmid construct, fifty

transgenic plants were regenerated from the callus tissue and transferred to soil for

92

 

XI
X1
X3
X4
X5
X6 #
X7
X9 #
Xll #
Y1 #
Y2
Y3 #
Y4
Y5
Y6
Y7
Y9
Y12
Sl #
$2 #
$6 #
S9 #
810 #
$12 #
516 #
Sl7 #

 

Figure 2. Northern blot analysis reflecting viral transgene transcription in transgenic
plants. (A) Transgenic M and N lines were transformed with a full-length translatable CP
gene using transformation vector pGAWS4. (B) Transgenic X and Y lines were
transformed with an antisense constnrct of the 3’ 2/3 of the CCMV CP gene using
transformation vector pGAMD202(-); transgenic S lines were transformed with an
antisense construct of the lull-length CCMV CP gene using transformation vector
pGAMD101(-).Total RNA from an equal amount of transgenic plant leaf tissue with high
anti-NPT H ELISA readings was loaded in each lane. Dig-labeled probe RA518(+),
which is specific for the 3’ UTR of the positive sense CCMV RNAs, was used to detect
the viral transgene in M and N lines. Dig-labeled probe RA518(-), which was specific for
the 3’ UTR of the negative sense CCMV RNAs, was used to detect the antisense viral
transgene in X, Y, and S lines. Transgenic plant lines marked with a “#” were used to test

virus resistance.

93

further examination. Anti-NPT H ELISA tests were used to screen for expression of the
NPT II marker gene which was located adjacent to the viral gene in the transformation
vector. A total of thirty-six transgenic lines were examined for viral transgene
accumulation in the total RNA. As shown in Fig. 2, a band at the expected transgene size
was detected in most of the plants. For transgenic lines M and N, 29% of the transgenic
plants regenerated from tissue culture were sterile. Although all of these plants had

flowers, no seeds were produced.

Viral protein detection

For transgenic lines expressing the full-length CCMV CP, Western blot analysis
was used to analyze the translation of viral protein. As discussed in Chapter 3, the
detection limit of the Western blot was approximately 1 ng wild type CCMV virion. A
small amount of viral protein (~20 KDa) was detected in transgenic lines M7 and N1
when lOOug of the total protein was loaded in each lane. These two bands are less strong
than the control lane with 1 ng CCMV virion, thus the concentration of the viral protein
in lines M7 and N] was less than 1 ng/lOOug total protein. No band was observed in the

Western blot analysis of the other transgenic lines (data not shown).

Test of virus resistance in transgenic plants

Transgenic plants with a range of viral transgene accumulation (indicated with
“#” in Fig. 2) were selected for the resistance test. These included: five lines transformed
with antisense 3’ 2/3 of the CCMV CP, eight lines transformed with antisense full-length

CCMV CP, and five lines transformed with messenger sense full-length CCMV CP.

94

Three micrograms of each wild type CCMV RNA transcripts (C1, C2, and C3) were
applied to each transgenic plant at the concentration of 120ug/ml. Systemic CCMV
infections were analyzed by dot blot at 14 and 30 dpi. The dot blots shown in Fig. 3 were
carried out at 14 dpi, and similar viral RNA accumulation patterns were observed

at 30 dpi. The results of this resistance test were compared with the results of previous

studies (Chapters 2 and 3; Greene and Allison, 1994; 1996) and are described in Table 1.

Translatable CP transgene and virus resistance

Transgenic plants expressing full-length translatable CP gene displayed two types
of virus resistance, and this resistance was not correlated with the expression of the coat
protein. Viral coat protein expression was observed only in lines M7 and N1. Both
showed reduced level of viral RNA accumulation. However, for several other transgenic
lines such as M2 and N2, no viral protein expression was detected, but both plant lines
showed a reasonable level of resistance (Fig. 3A). For most of the M and N lines of
transgenic plants, levels of viral transgenic mRNA correlated with levels of virus
resistance (see Fig. 2 and 3, transgenic lines M2, M3, M7, M9, N1, N3, N4, and N5). One
exception is transgenic line N2, which displayed a high level of resistance despite its low

accumulation of transgenic transcripts (Fig. 2A and 3A).
In the experiments testing virus resistance in transgenic plants expressing a

truncated CCMV CP gene (Chapter 3, G and H lines), a level of resistance similar to that

in transgenic plant M2, M7, and N2, was observed (data not shown). Thus, a truncated

95

M2 M3 M7 M9 N2 N3
N4 N5 S1 82 S6 S9
S 10 S 12 S 16 S 17 X6 X9

x11 Y1 Y3 (+) (-) (-)

M1 M1 N1 (+)

   

A B

Figure 3. Dot blot analysis of virus resistance in transgenic plants expressing viral genes.
(A) & (B) Clonally propagated cuttings of transgenic plants were inoculated at the 5-leaf
stage with 3 ug each of CCMV RNA transcripts at the concentration of 120 ug/ml. At 14
dpi, viral RNA accumulation was detected by dot blot analysis with the dig-labeled
RA518(+) probe. In Fig. 3A, the order of the samples in the dot blot is shown in the grid
above the blot, and in Fig. 38, each sample number is above the blot. Transgenic M and
N lines were transformed with a full-length translatable CP gene. Transgenic S line was
transformed with an antisense construct of the full-length CCMV CP gene. Transgenic X
and Y lines were transformed with an antisense construct of the 3’ 2/3 of the CCMV CP
gene. The same patterns of viral RNA accumulation were observed at 30 dpi. (+)
designates RNA fi'om CCMV infected nontransgenic plants. (-) designates RNA from

non-inoculated N. benthamiana.

96

Table 1: Test of Virus Resistance. Transgenic plants were challenged with either BMV
C“) or CCMV and analyzed by dot blots. Five “+” indicate full resistance, one “+”
indicate low level of resistance. Minus “-“ indicates fully susceptible plants.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Transgene Transgenic Lines Transgene Resistance
Transcription
3’ 1.6 Kb BMV polymerase 3-5“ H .-
gene. . 4-1“ H- +++-H-
Potential translation could start 6-9“ ++ -
at the beginning of the 7-1 * +++ -
transgene where an inframe 7.2!!R ++ -
start codon is present 7.3:: +1- _
(nucleotide 1259). 94: +++ -
3’ 2/3 CCMV CP gene with no 3-57 ++ -
start codon and TEV leader A69 ++ -
sequence added (G&A, 94;96) A83 ++ _
3’ 2/3 CCMV CP gene with a A7 H- +
stop codon following the 82 +++ ++
added start codon. TEV leader B6 +++ ++-
sequence was added at the 5’ 133 +++ .H.
of the transgene.Untranslatable 139 +++ ++
3’ 2/3 CCMV CP gene with G1 +++ +++
inframe start codon. TEV G18 +++ +++
leader sequence was added at H] +++ +++
the 5’ end of the transgene. H3 +++ +++
Translatable. H4 +++ +++
Full-length translatable CCMV Ml - +
CP gene, with TEV leader M2 ++ ++
sequence added at the 5’ end M3 - +-
of the transgene. Messenger M7# H H
sense. M9 ++ -
N l # +++—l- ++
# - coat protein detected in N2 4., +++
Western blots N3 +_ _
N4 +- +-
N5 -H- +
F ull-length antisense CCMV Sl - +
CP gene. 82 + +++-
S6 +—+ +++
No TEV leader sequence 39 +++ +++
added. S 10 +++ ..
S 12 ++ -
S 16 ++ -
S 17 + ++
3’ 2/3 CCMV CP gene. X6 +++- +-
Antisense. X9 +++ +-
X11 +++ -
No TEV leader sequence Y1 +++ +-
added. Y3 +++ +-

 

 

 

 

 

 

97

CCMV CP gene with added AUG start codon consistently provided CCMV resistance

while the selected full-length CCMV CP lines provided various levels of resistance.

Antisense transgene and virus resistance

CCMV resistance in transgenic plants expressing full-length antisense CP gene
provided variable results. For transgenic lines S2, S6, S9, and 817, high viral transgene
accumulation seemed to correlate with high level of virus resistance because the
accumulation level of viral RNA was highly reduced (Fig. ZB, Fig 3A). However, this
correlation was not observed in transgenic lines 810, $12 and 816 where a highlevel of
viral transgene accumulation resulted in low virus resistance (Fig. 3A). For transgenic
line 81, low viral transgene accumulation resulted in an intermediate level of resistance.
Transgenic lines transformed with tmncated antisense CP gene, lines X and Y, had
slightly reduced viral RNA accumulation and displayed low virus resistance (Fig. 3A).
Compared to the antisense lull-length CP gene (S lines), antisense truncated CP gene (X

and Y lines) provided lower resistance.

DISCUSSION

Data provided in this experiment indicate that the CP gene of CCMV provides
virus resistance to transgenic plants. Several transgenic plants expressing the translatable
full-length CP also had a reduced level of viral RNA accumulation (Fig. 3A). This
resistance was not correlated with the translation of the CP protein. Although the 3’ 2/3
of the CCMV CP gene did not provide any resistance to transgenic plants (Greene and

Allison, 1994; 1996), data in Chapter 3 indicated that the addition of an AUG start codon

98

resulted in a lower infection rate (50% success) and lower viral RNA accumulation
levels. This resistance was observed in both translatable (G and H lines) and
untranslatable transgenic lines (A and B lines), although viruses accumulated even lower
in translatable lines (BO-90% reduction in translatable lines, 50% reduction in
untranslatable lines). Several transgenic plants expressing the full-length CP gene (M and
N lines) exhibited a similar level of resistance as the plants expressing the truncated
translatable CP gene (G and H lines). However, when antisense CP gene was used, full-

length CP gene provided better resistance than truncated CP gene.

The data provided in this study and previous studies (Greene and Allison, 1994;
Chapter 3) suggested that the mechanisms of CPMR in CCMV are complex. Although
the addition of a start codon at the 5’ end of the truncated CCMV CP gene provided virus
resistance, truncated CP transgene expression did not provide a detectable amount of
protein. Therefore, the presence of the viral protein may not be required for virus
resistance. This is especially true in transgenic plants expressing the full-length CP gene,
where several transgenic lines lacking detectable coat protein expression had high virus
resistance (Fig. 3A). Using transgenic rice expressing the fiill-length BMV CP gene,
Huntley and Hall (1996) found that a viral protein product was not likely to be the
bioactive agent conferring resistance to BMV because transgenic lines with 80 to 90%
reduction in progeny virus accumulation did not have a detectable amount of coat protein.
Huntley and Hall (1996) concluded that virus resistance in transgenic plants expressing
BMV CP gene was more likely mediated through viral RNA rather than through the coat

protein. However, results in this study indicated that, for the two constructs with a

99

translatable 3’ 2/3 CP transgene, translatable lines showed a higher level of resistance
than untranslatable lines because the viral RNA accumulated at a lower level in
translatable lines (Chapter 3; Table 1, this chapter). Thus, we are unable to rule out the
possibility that trace amounts of coat protein could also play some role in virus resistance

in CCMV.

Substantial evidence indicates that segments of the BMV genome regulate plus-
strand (Pogue et al., 1990), minus-strand (Dreher and Hall, 1988; Dreher et al., 1989),
and subgenomic RNA synthesis (Marsh et al., 1988). A strong interference of transgenic
BMV mRNA on BMV viral RNA replication was observed in transgenic rice protoplasts
expressing the BMV polymerase gene and the antisense BMV RNA3 (Huntley and Hall,
1996). In each case, viral RNA accumulation was decreased to 5 to 30% of that in wild
type (Huntley and Hall, 1996). In another study, sense and antisense transcripts of the (-)
strand promoter of BMV interfered with progeny RNA accumulation in a concentration-
dependent manner (Huntley and Hall, l993). These observations implied that the
interference of transgenic mRNA on viral RNA replication could play a major role in the
resistance observed in transgenic plant 4-1 (Chapter 2) and transgenic S lines (this
chapter). Transgenic plant 4-1 which was transformed with a truncated BMV 2a gene was
completely resistant to BMV in that no viral RNA accumulation was observed in
repeated experiments (Chapter 2). For transgenic S lines which was transformed with an
antisense CCMV CP gene, high levels of viral transgene accumulation were associated

with high levels of virus resistance.

100

In plants expressing firll-length or truncated CP genes, the viral RNA
accumulation was generally reduced, but not completely eliminated. This was different
from the resistance provided by the truncated BMV polymerase gene in transgenic plant
4-1, where the viral RNA accumulation was completely eliminated (Chapter 2). This data
agrees with the general observations that a polymerase transgene can provide stronger
resistance than a CP transgene. It is also another example that a truncated polymerase
transgene is sufficient to provide strong virus resistance to transgenic plants. These above
observations did not give direct evidence that gene silencing was involved in and was
responsible for the resistance provided by the viral transgene, although we could not rule
out the possibility that gene silencing played some role in virus resistance observed in

this study.

In conclusion, our data indicates that CCMV CP gene can provide virus resistance
to transgenic plants. Although high levels of viral transgene accumulation is generally
correlated with high levels of virus resistance, one exception was found in line N2, where
a low level of transgene expression resulted in a high level of resistance. While full-
length polymerase and CP genes were used in many other studies to generate VRTPs,
truncated polymerase and CP transgenes were shown in this study to provide strong
resistance to transgenic plants. Thus, our data indicates that a smaller viral transgene
fragment and a lower level of viral transgene expression may provide useful viral
resistance. Viral transgenes with both properties would be less likely involved in RNA

recombination with infecting viruses.

lOl

SUMMARY AND CONCLUSIONS

The development of Agrobacterium-mediated transformation techniques has
enabled plant breeders to generate virus resistance transgenic crops (VRTPs) ' by
introducing viral genes into plants. These viral genes are usually cDNA fragments of
plant RNA viruses, and transcription of these viral transgenes driven by constitutive
promoter leads to accumulation of the viral transgene transcripts in the cytoplasm. RNA
recombination is a natural process that occurs during viral RNA replication and has the
capacity to unite unrelated RNA fi'agments. Upon viral infections of transgenic plants,
RNA recombination between the viral transgene and an infecting virus could generate
chimeric viruses with distinct properties. Using a bromovirus model system, Greene and
Allison (1994) demonstrated RNA recombination between a bromovirus capsid transgene
with a complete 3’ untranslated region (UTR) and an infecting virus. Three percent of the
plants produced recombinant virus, but a decrease in recombinant recovery rate was
found when the 3’ UTR was deleted (Greene and Allison, 1996). Experiments in this
study were based upon these two previous studies and attempted to expand our
knowledge of RNA recombination in plants with viral transgenes. An additional goal was
to understand and predict the risks of releasing virus resistant transgenic plants to the

field.

In Chapter 1, a full-length minus strand copy of the viral transgene was detected
in virus infected transgenic plants transcribing the CCMV CP gene with a complete 3’

UTR. This data provides an explanation for the difference in recovery of recombinant

102

virus in transgenic plants with a complete or deleted 3’ UTRs (Greene and Allison, 1994;
1996). The presence of both plus and minus strand copies of the viral transgene in a
single infected cell would increase the incidence of interactions between the viral
transgene and an infecting virus. Additionally, the presence of both the transgene and its
complement would enable recombination to occur during either plus or minus strand

RNA synthesis.

In Chapter 2, a bioassay experiment similar to Greene and Allison (1994) was
designed to assess the involvement of a viral polymerase transgene in RNA
recombination. Nicotiana benthamiana plants were transformed with a segment of the
BMV replicase gene which bore a silent marker mutation that provided an Eco47III
restriction site. The challenging virus, PT50, included a 307-nucleotide deletion near the
3’ end of the polymerase gene’open reading frame (ORF) in BMV RNA2. While PT50
remained capable of replication in N. benthamiana, it was unable to move systemically.
Transgenic plant lines contained the segment of the BMV polymerase gene that was
absent in PT50 mutant. Thus, systemic movement of the virus in PT50 infected
transgenic plants signaled the restoration of the polymerase gene through RNA
recombination. Eight of 202 challenged transgenic plants yielded systemic infection.
Sequence analysis of cDNA clones of the RNA2 components from these infections
revealed molecular marker Eco47III site which was originally present only in the viral
transgene. Thus, these viruses were generated through recombination between the viral
transgene and the infected PT50. The recovery rate of recombinant was 4%, which is

comparable to the 3% recovery rate observed with the capsid transgene.

103

Experiments in Chapter 3 were designed to determine if a translatable transgenic
mRN A is more or less likely to be involved in RNA recombination than a nontranslatable
version of the same transgene. The viral gene fragment used for transformation by
Greene and Allison (1994) was excised directly from the CCMV RNA3 cDNA clone and
no AUG start codon was added to encourage translation of the transcript. Thus,
translation of the viral gene was unlikely. In this study, N. benthamiana plants were
transformed with two modified constructs of the CCMV CP gene used by Greene and
Allison (1994). One construct had an inframe start codon added at the 5’ end of the
truncated CP gene and theoretically, this viral transgene was translatable. The other
construct had an out-of-frame start codon followed by a stop codon, and this viral
transgene was untranslatable. For both constructs, three single nucleotide substitutions
near the 3’ end of the CCMV CP gene characterized the transgene, and the translational
enhancer TEV leader sequence was added at the 5’ end of both transgenes. The infecting
virus was the AG3 mutant used by Greene and Allison (1994), which lacks the 3’ 1/3 of
the CCMV CP ORF. Only two recombinant viruses were recovered from 232 clonally
propagated plants from untranslatable lines, and none was recovered from 221
translatable plant lines. This gave a 0.85% recombinant recovery rate for untranslatable
lines, and 0% for translatable lines. Thus, there was an overall decrease in the
recombinant recovery rate compared to the 3% rate of Greene and Allison (1994), with a

lower recovery in translatable lines.

104

It is complicated to explain the data because both translatable and untranslatable
lines were resistant to CCMV viral RNA inoculation and a 50% infection rate was
observed for both lines. This could be one reason of the overall decrease in recombinant
recovery. The translatable lines showed higher resistance because the viral RNA
accumulation was reduced to 10-20% of that in nontransgenic N. benthamiana plants. For
untranslatable lines, a 50% reduction in viral RNA accumulation was observed. Thus, it
is unclear whether the lower recombinant recovery in translatable lines was due to the
association the transgenes with ribosomes during translation, or due to reduced viral
RNA replication. Additionally, this study indicated that RNA recombination is less likely
when the transgene provides resistance to the infecting virus, especially if this viral

transgene could interfere with the replication of that virus.

Chapter 4 experiments established that CCMV CP gene could provide virus
resistance to transgenic plants. N. benthamiana plants were transformed with either a full-
length CP gene, an antisense lull-length CP gene, or an antisense truncated CP gene.
Twenty three transgenic lines were tested for CCMV resistance. Resistance test results in
Chapters 2 and 3 were also analyzed and compared with the new data described in
Chapter 4. While full-length messenger sense and antisense CP genes provided virus
resistance, truncated capsid and polymerase genes also provided good resistance.

Variations in resistance were observed in different transgenic lines.

Based on this thesis research data and previous results obtained in our lab (Greene

and Allison, 1994; 1996), several conclusions are drawn below:

105

1)

2)

3)

4)

A complete 3’ UTR which bears the replication initiation site allows the
synthesis of minus strand copies of the viral transgene, and thus permit RNA
recombination to occur at an easily detectable frequency, presumably during
both plus and minus strand synthesis. Thus, exclusion of the regions required
for replication initiation is a must for generating VRTPs with a low risk for
generating recombinant viruses.

Both capsid and polymerase viral transgenes can be involved in RNA
recombination with infecting viruses and their recombinant recovery rates are
comparable. Thus, cautions should be taken in generating VRTPs using either
viral gene.

A translatable transgene is less likely to be involved in RNA recombination,
especially if the transgene provides resistance to the infecting virus.

Truncated bromovirus capsid and polymerase transgenes provided virus
resistance to transgenic plants. Such smaller viral transgenes may be more
desirable than complete genes as they should less likely contribute to viral

evolution through RNA recombination.

106

APPENDIX

107

Appendix

A Recombinant Virus Isolated from Transgenic Plants Expressing CCMV Coat

Protein Gene with a Deleted 3’ UTR

The 3’ UTR of bromoviruses bear the replication recognition and initiation site.
We have demonstrated that when the 3’ UTR is included in the viral transgene, it may be
recognized by the replication complex of infecting viruses, and thus a negative-sense
copy of the transgene may be synthesized (Chapter 1). When the 3’ UTR of the viral
transgene was deleted, a decrease in the rate of RNA recombination was observed
(Greene and Allison, 1994). However, the exclusion of the 3’ UTR from the viral
transgene does not guarantee that the transgenic transcripts will not be involved in RNA
recombination. Sequence analysis of several recombinant viruses generated in our
laboratory indicated that cellular mRNA, which does not have any sequence homology
with the 3’ UTR of bromoviruses, could be incorporated into the genome of recombinant

viruses through RNA recombination (Greene, 1995).

Transgenic plant A69 transcribes the 3’ 2/3 of the CCMV CP gene.
Characteristics of the A69 transgene are the deletion of the 3’ 69 nucleotides of the 3’
UTR. While I was working on the project for detecting minus-sense copies of the viral
transgene (Chapter 1), a transgenic A69 plant infected with CCMV looked sick (probably
because of senesce). Assumed that a recombinant virus which made the plant sick might

have been generated through RNA recombination between the viral transgene and

108

CCMV, I transferred the virus to both nontransgenic N. benthamiana and cowpea. While
no symptom was observed in N. benthamiana, the inoculated cowpea plants showed
severe chlorotic symptoms which were different from wild type CCMV at 10 days post
inoculation (dpi). This virus was named A69-1. At 14 dpi, viral RNA was extracted from
virus A69-1 and wt CCMV infected cowpea. Viral RNA from 0.1g of the plant tissue was
loaded in each lane. Northern blot analysis using dig-labeled probe RA518(+) indicated
that virus A69-l had all four RNAs of CCMV and thus was a CCMV variant. Virus A69-
1 also accumulated around three to five- fold higher than wt CCMV in cowpea (Fig. 2).
RT-PCR was used to amplify the RNA3 of virus A69-1. Primer CCMV 3’ (5’
CAGTCTAGATGGTCTCCTTAGAGAT 3’), which anneals at the 3’ end of CCMV
RN A3, was used for first strand cDNA synthesis (Boehringer-Mannheim). Primer 5’ CC-
D/3 (5’ CGCTGCAGTAATACGACTCACTATAGTAATCTTTACCTAAC 3’) which
anneals to the 5’ end of CCMV RNA3, was added for PCR amplification. Primers
CCMV 3’ and 5’ CC-D/3 add Xbal and P311 sites at the ends of the cDNA, respectively.
A 2.1 Kb cDNA was amplified and cloned into PstI-Xbal digested pBS/KS+. The
complete coat protein gene was sequenced and only one single nucleotide substitution
(G—9C at 1926) was found in the CP ORF. This mutation was one of the three mutations
introduced into the transgene of A69 plants (Greene and Allison, 1996). The other two

mutations in the transgene of A69 plants were absent in virus A69-l.

The presence of the single nucleotide substitution indicated that virus A69-1 could
be a recombinant virus which was generated through RNA recombination between the

viral transgene of A69 plants and the wild type CCMV. To acquire a marker mutation

109

which is only present near the 3’ end of the CP transgene, two crossovers have to occur
between the transgene and CCMV RNA3, and this can only happen during the minus-
strand synthesis (see Fig. 3 for the model). However, we could not rule out the possibility
that this single G—+C substitution was introduced through polymerase errors during the

process of RNA replication.

This single G—>C substitution at 1926 is a silent mutation which does not cause
any amino acid change in the CCMV coat protein. This mutation is unlikely the genetic
determinant of the severe chlorotic symptom, because AGl mutant which bears this
mutation causes symptoms similar to wild type CCMV in cowpea (Greene and Allison,
1994). Previous studies indicate that the movement protein of BMV influences virus-host
interactions that are crucial to systemic infection. A single Ser 180—)Arg mutation in the
3a cell-to-cell movement protein of a CCMV hybrid conferred compatibility for systemic
infection of a new host, and also affected the symptom development (Fujita et al., 1996).
In another study, a single amino acid change (Val 266—)Ile) in the 3a movement protein
of BMV altered symptom expression in N. benthamiana (Rao and Grantham, 1995). To
search for the genetic determinant of the severe chlorotic phenotype of this CCMV
variant A69-l, the movement protein gene of the cDNA clone was sequenced and the
data is presented in Fig. 3. Two G to A substitutions were found in the 3a gene ORF.

Both nucleotide substitutions resulted in amino acid changes, one is Val 17——)Ile, and the

other one is Glu 41—)Lys.

110

 

 

 

Figure 1. Infection of cowpea with virus A69-1. Trifoliate leaves fi'om cowpea inoculated
with wild type CCMV show typical chlorotic mottling symptom (right). Leaves from

cowpea inoculated with virus A69-l produce severe chlorotic symptoms (left).

111

WT CCMV RNA3

 

 

I 3a I I

 

 

 

 

A69 Transgene

 

 

Figure 2. Model for RNA recombination to generate recombinant A69-1.
The transgenic mRNA in A69 plants has a 69-nucleotide deletion at the
3’ terminus of the UTR. Thus, initiation of negative-strand synthesis is
unlikely. To generate a recombinant virus with a marker mutation which
originally exists only in the transgenic mRNA of A69 plants, RNA
recombination has to happen during the negative-strand RNA synthesis
through two template switches. Filled triangles, A and v, indicate the
G—)C marker mutation within the CP transgene (nucleotide 1926). The
other two introduced mutations in the transgene of A69 plants create a
NotI restriction site near the 3’ end of the CCMV CP gene in the 3’ UTR.

These two mutations were not present in the RNA3 of virus A69-1.

112

To determine the amino acid(s) responsible for the severe chlorotic phenotype,
experiments were designed to introduce the mutations into wild type CCMV RNA3 clone
pCC3TP4 by replacing the restriction fragment of wild type RNA3 with the same
fragment bearing the mutations. Plasmid pCC3MD6901 contains both 3a gene mutations
and was made by replacing the 1054-nucleotide EcoRV-Sunl fragment of pCC3TP4 with
the same fragment of the A69-1 clone. Plasmid pCC3MD6902 has the G to A mutation at
position 287 and was made by replacing the EcoRV-Narl fragment of pCC3TP4 with that
of the A69-1 clone. Plasmid pCC3MD6903 has the G to A mutation at position 359 and
was made by replacing the 950-nucleotide NarI-Sunl fragment of pCC3TP4 with that of

the A69-l clone.

Following dideoxy sequence analysis to confirm the correct orientation and
joining of the fragments, infectious RNA transcripts will be made from the Xbal
linearized clones. The mutant RNA3 transcripts (MD6901, MD6902 or MD6903) will be
applied to cowpea together with wild type CCMV RNA] and RN A2 transcripts (C1 and
C2). Comparing the development of the symptoms of the three mutants with that of virus
A69-l, we will establish which amino acid is responsible for the symptom change of

CCMV.

If the above mentioned experiments fail to determine the genetic determinant of

the symptom alteration of A69-1, alternative experiments are:

113

WT CCMV RNA3

F San I

 

 

 

A69 transgenic plant

 

 

 

 

 

A69-l l 3a I
l l 1
CR1 G/A G/A
27 287 359
A
17 41
1 . l . . . 50
WT MSNTTFRPFTGSSRTVVEGEQAGAQDDMSLLQSLFSDKSREEFAKECKLG
A69—1 ................ I ....................... K .........
B

Figure 3. Nucleotide and amino acid changes in virus A69-1 compared with wild
type CCMV. (A) Nucleotide changes in RNA3 of virus A69-l compared with wild
type CCMV. Only one single nucleotide mutation (G—)C at 1926, indicated by
triangle v) was found in the CP ORF of A69-1. This was one of the three marker
mutation present originally in the transgene. Two single nucleotide substitutions
were present in the 3a gene ORF, and one single nucleotide substitution was found in
the 5’ UTR. (B) Deduced amino acid changes in the 3a movement protein of A69-1.
Two amino acid changes were deduced according to the nucleotide changes in the

RNA3 of A69-1, Val 17—>Ile and Glu 41—+Lys.

114

pCC3TP4

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

202 306 1255
EcoRV NarI SunI
II 3a I
A69“ 202 306. 1255
EcoRV NarI SunI v
i 3. l I
1
cm G/A G/A
27 287 359
pCC3MD6901
3a I
I I
G/A G/A
287 359
pCC3MD6902
l 3a l
1
CIA
237
pCC3MD6903

 

 

 

G/A
359

115

Figure 4. Plasmid construction for making infectious transcripts bearing the mutations of
virus A69-1. Plasmid pCC3TP4 is the lull-length clone of CCMV RNA3. Plasmid A69-l
is the full-length clone the virus A69-1 RNA3. Two G to A substitutions were present in
the 3a gene of A69-l virus, and these two nucleotide substitutions result in two amino
acid substitutions. To determine the mutations responsible for the altered symptoms in
cowpea, three clones were created by replacing the restriction fragment of pCC3TP4 with
the same fragment of A69-1. Plasmid pCC3MD690l bears both mutations, and plasmids

pCC3MD6902 and pCC3MD6903 bear one of the two mutations.

116

1) Transfer virus A69-1 to local lesion host Chenopodium quinoa and transfer
viruses from several local lesions to individual cowpea plants. This allows the
isolation of a single virus which contributes to the symptom alteration in
cowpea.

2) Clone the three genomic RNAs of the virus (A69-l). By exchanging the RNAs
of virus A69-1 with wild type CCMV, we would identify the RNA responsible
for the altered symptoms.

3) Sequence analysis would firrther locate the mutation(s) contributing to the
virus-host interaction.

4) Inoculate cowpea plants with infectious transcripts containing the mutations
and identify the mutation(s) responsible for the symptom development.

5) Host range tests of virus A69-l

Previous studies indicate that bromovirus RNA3 is involved in virus-host
interactions and contributes to symptom development (Fujita et al., 1996; Rao and
Grantham, 1995). BMV RNA2 was found to play a role in host specificity and systemic

infection (Traynor et al., 1991). Thus, experiments should be concentrated on the RNA3.

117

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