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Final Report Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic Plants A Consultancy Report by CSIRO for Environment Australia June 2002

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Final Report

Environmental Risks Associated with Viral Recombination in Virus

Resistant Transgenic Plants

A Consultancy Report by CSIRO for Environment Australia

June 2002

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This is a report of research that was funded by Environment Australia as one project

in the CSIRO program: ‘Ecological Risk Assessment for GMOs’. This program aims

to develop research tools, assess potential risks, and help regulators develop

guidelines and policy on the use of GMOs in Australia. Further information on this

program can be obtained by accessing the following web-link:

http://www.ento.csiro.au/GMO-impact/index.html The authors of this report were Andy Richards (CSIRO Entomology) and Janelle

Scown (CSIRO Entomology).

Disclaimer The views and opinions in this report do not necessarily reflect those of the

Commonwealth Government or the Minister for the Environment and Heritage.

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Table of Contents ABBREVIATIONS...................................................................................................... 5 EXECUTIVE SUMMARY............................................................................................ 7 1. INTRODUCTION .................................................................................................. 10

PROJECT BACKGROUND ........................................................................................... 10 PROJECT AIMS ........................................................................................................ 11

2. DEVELOPMENT OF AN EXPERIMENTAL SYSTEM FOR MEASURING VIRUS RECOMBINATION FREQUENCY ........................................................................... 12

INTRODUCTION ........................................................................................................ 12 EXPERIMENTAL DESIGN............................................................................................ 13

Advantages of an experimental system based on potato virus y (PVY) and tobacco.............................................................................................................. 13 Development of an RT-PCR diagnostic assay for detecting and specifically identifying parental and recombinant PVY RNAs .............................................. 13 Evaluating primer specificity and sensitivity....................................................... 16

DEVELOPMENT OF AN INOCULATION STRATEGY TO MAXIMISE THE PROBABILITY OF DETECTING RECOMBINANT PVY STRAINS................................................................... 18

Assessing the virus inoculation strategy............................................................ 19 RESULTS ................................................................................................................ 20 CONCLUSIONS......................................................................................................... 25

3. LITERATURE REVIEW........................................................................................ 27 SCOPE OF THE REVIEW ............................................................................................ 27 I. EVOLUTIONARY SIGNIFICANCE OF RECOMBINATION IN PLANT RNA VIRUSES............. 30

Nomenclature .................................................................................................... 30 The recombination mechanism ......................................................................... 30 Evolutionary advantages of recombination........................................................ 32 Evolutionary constraints on virus recombination ............................................... 33

II. MECHANISMS OF PATHOGEN-DERIVED RESISTANCE (PDR) IN TRANSGENIC PLANTS 37 Protein mediated resistance .............................................................................. 37

Coat protein (CP) mediated resistance .......................................................... 37 Replicase - mediated resistance.................................................................... 38 Movement protein - mediated resistance ...................................................... 39

RNA-mediated resistance.................................................................................. 39 Suppression of RNA silencing ........................................................................... 40

III. EVIDENCE OF RECOMBINATION AMONG PLANT RNA VIRUSES AND TRANSGENIC PLANTS .................................................................................................................. 42

Detecting recombination among RNA viruses ................................................... 42 Recombination in VRT plants ............................................................................ 44

IV. PERCEIVED RISKS ASSOCIATED WITH VIRUS RECOMBINATION AND TRANSGENIC PLANTS .................................................................................................................. 47

VRT plants introduce no new or increased risk: supporting and opposing arguments ......................................................................................................... 47 Intra- and inter-group recombination among RNA viruses................................. 50 Evaluating frequency and hazard in risk assessment........................................ 51 Mitigating risk .................................................................................................... 53 Ecological risk experiments ............................................................................... 54

V. REFERENCES CITED............................................................................................. 57

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4. APPENDIX I ..................................................................................................... 65

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ABBREVIATIONS

AMV Alfalfa mosaic virus

BBTV Banana bunchy top virus

BMV Brome mosaic virus

BSB Banana streak badnavirus

BWYV Beet western yellows virus

CPMR Coat protein mediated resistance

CaMV Cauliflower mosaic virus

CCMV Cowpea chlorotic mottle bromovirus

CNV Cucumber necrosis virus

DI RNA Defective interfering RNA

HDGS Homology dependent gene silencing

HR Homologous recombination

MPMR Movement protein mediated resistance

NHR Non-homologous recombination

nt nucleotides

PLRV Potato leaf roll virus

PVY Potato virus Y

PVX Potato virus X

PDR Pathogen-derived resistance

PTGS Post-transcriptional gene silencing

RdRp RNA dependent RNA polymerase

RMR Replicase mediated resistance

SCSV Sub-clover stunt virus

SMV Soybean mosaic virus

TMV Tobacco mosaic virus

TAV Tomato aspermy virus

TGS Transcriptional gene silencing

TBSV Tomato bushy stunt virus

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TuMV Turnip mosaic virus

TYMV Turnip yellow mosaic virus

VRTP Virus resistant transgenic plant

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EXECUTIVE SUMMARY

In Australia, the commercialisation of transgenic plants containing viral derived

sequences for disease resistance promises both economic and environmental

benefits through improvements in crop productivity and quality concurrent with a

reduction in the use of chemical pesticides and other agricultural inputs. The mass

cultivation of these crops, however, also poses potential risks.

One of the more controversial of these risks is the potential for environmental

impacts arising as a result of genetic exchange between naturally occurring plant

viruses and virus-derived sequences deployed in some GM crop plants. This is

because there is a finite probability that virus-virus recombination and virus-

transgene recombination could give rise to a new (chimeric) virus capable of

spreading as a virulent disease in the Australian environment.

While virus recombination is a natural phenomenon, and has occurred many times in

the past, the use of virus transgenes in plants is a very recent development and

many people have voiced significant concern about the potential problems these may

pose. In April 2001, as part of a new CSIRO program on the scientific assessment of

GM technologies in Australia, Environment Australia commissioned a twelve-month

consultancy to conduct research and review current scientific understanding on the

risk associated with virus recombination as this relates to transgenic plants. This

report describes the progress in three parts.

A ‘Hazard Assessment’ workshop to identify and prioritise the risks associated

with RNA recombination and transgenic plants.

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Design and development of an experimental system to measure the relative

frequency of virus recombination in transgenic and non-transgenic plants.

A review of the scientific literature concerning virus recombination and its

relevance to transgenic plants.

The workshop was held in October, 2001. It involved a number of leading Australian

scientists in this field, environmental managers and gene technology regulators in

discussions on the need for and nature of future research effort in this country. The

workshop report is provided in Appendix I. Discussions concluded that while it was

not practicable to assess the probability and consequences of virus recombination for

all virus-plant-transgene combinations, several key points could be made:

1/ Very similar genetic sequences are much more likely to recombine than dissimilar

sequences to form a viable new virus. The likelihood of successful recombination

diminishes rapidly as sequence similarity declines.

2/ Risk is a product of two components: hazard x frequency. There is no scientific

reason to suspect that the nature of the hazard associated with virus recombination

(i.e. formation of a new virus) will differ for transgenic and non-transgenic plants. The

transgenic plant carrying a virus-derived sequence presents an increase in risk

compared to the non-transgenic plant only if the frequency with which viable

recombinants are generated in the former is significantly greater.

The workshop recommended that priority in research should be to identify an

experimental system (year 1) in order to measure the relative frequency of virus

recombination in a transgenic and non-transgenic plant (years 2-3). Two types of

experimental system were suggested:

a transgenic plant containing any one of several virus-derived ‘promoter’

sequences used for controlling the action of a variety of transgenes;

a transgenic plant containing a virus-derived transgene for conferring disease

resistance (a virus resistant transgenic plant or ‘VRTP’).

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An experimental system based on the second type (i.e. a VRTP) was chosen for

development. The plant component was tobacco and the virus component was

represented by several closely related strains of potato virus Y (PVY). Potato virus Y

is an economically important viral disease in Australian crops. To detect genetic

exchange a highly sensitive diagnostic procedure was designed. It was based on a

procedure known as the ‘polymerase chain reaction’ (PCR). In experiments, tobacco

plants were deliberately infected with two PVY strains and the PCR diagnostic tool

was used to search for chimeric PVY strains (recombinants). In one of these tests a

virus recombinant was detected.

This result represents a highly significant achievement and a very successful

conclusion to the project aims in year 1:

the research has demonstrated a system for measuring virus recombination

frequency in transgenic plants carrying virus-derived sequences. Notably

recombination was detected in the first experiment in the development of this

experimental system, in non-transgenic tobacco after only two virus strains

had been sequentially passed through five host plant generations. This is

equivalent to many millions of virus generations; and

the project generated valuable baseline data on virus recombination

frequency.

However, it cannot yet be concluded from this result that recombination between a

transgenic plant and a single virus strain occurs at a lower rate than recombination

between two naturally infecting strains. Experiments involving the inoculation, with a

single virus strain, of a transgenic tobacco carrying a PVY transgene were completed

for two generations of plants, with no recombinants detected. Further tests on

transgenic and non-transgenic plants (years 2-3) need to be conducted for a reliable

estimation of relative recombination frequency in this system.

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1. INTRODUCTION

Project background Recombination among co-infecting plant RNA viruses is a natural phenomenon that

appears to have played a significant role in the speciation and evolution of many

strains and has particular significance for risk assessment of plants that have been

genetically modified for disease resistance by incorporating virus sequences into

plant genomes. Virus recombination also has relevance to other types of transgenic

plant that use virus-derived sequences for driving the expression of non-viral

transgenes.

The incorporation of virus-derived sequences into plants for disease resistance has

provided the means of combating economically significant crop viruses for which

there are no readily accessible sources of natural resistance. Both coding and non-

coding viral gene sequences can be used for this purpose. In the former category,

the virus coat protein gene of the targeted virus has generally been used for

conferring disease resistance in virus resistant transgenic (VRT) plants, although the

mechanism by which this occurs is not known. For non-expressing viral sequences,

the mechanism of disease resistance appears to have arisen as part of an adaptive

defence strategy against invading viruses through sequence specific enzymatic

degradation. This mechanism is commonly referred to as post-transcriptional gene

silencing (PTGS) or RNA interference (RNAi).

Where an invading virus has the opportunity to replicate in a transgenic plant, the

transgenic sequence may be available for recombination with the invader if the two

sequences are sufficiently related. By a process termed ‘template switching’, the viral

replicase may switch from its viral RNA template to the transgenic mRNA and give

rise to a recombinant RNA molecule. This mirrors the natural phenomenon of RNA

cross-over between two co-replicating viruses that is known to have played an

important role in the evolution of some plant virus families e.g. the Luteoviridae.

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Project aims Phase 1: Problem definition and model development (year 1)

Conduct a hazard assessment workshop to identify the environmental

risks arising from viral recombination in transgenic crops in Australia. Develop and validate an experimental system for generating key risk

assessment data on virus recombination frequency in transgenic and non-

transgenic plant models. Review the current scientific literature on virus recombination particularly

as this relates to environmental risk associated with virus resistant

transgenic plants.

Phase 2: Empirical Evaluation (years 2-3)

Measure and compare RNA recombination frequency in a co-infected

wild-type host and a singly infected VRT plant using the experimental

system developed in Phase 1.

Environment Australia funding was only for Phase 1 (year 1) of this project.

A report of the development of the experimental system for measuring recombination

frequency, described on pages 11-25, is the central part of Phase 1 of this project

and this has been reflected in the position of this section within this report. However,

readers may benefit by first reading the report of the conclusions and

recommendations from the ‘Hazard Assessment’ workshop (Appendix I; page 64)

and the literature review (pages 26-63).

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2. DEVELOPMENT OF AN EXPERIMENTAL SYSTEM FOR MEASURING VIRUS RECOMBINATION FREQUENCY

Introduction

The key aim of Phase 1 of this project (year 1) was to identify, develop and validate

an experimental system in order to measure and compare virus recombination

frequency in a transgenic and a non-transgenic plant in Phase 2 (years 2-3). To

achieve this a collaborative arrangement was established with Dr Peter Waterhouse

(CSIRO Plant Industry).

Dr Waterhouse leads a research group in the area of plant-virus interactions, the

molecular biology of gene silencing and the application of this knowledge to the

development of virus resistant transgenic (VRT) plants. The Waterhouse group was

the first to describe in detail the underlying mechanism responsible for post-

transcriptional gene silencing (PTGS) in plants, showing that it almost certainly

evolved as an adaptive defence strategy to minimise the fitness costs associated

with virus disease.

The application of PTGS to the development of VRT plants has considerable and

growing importance to Australian agribusiness. A technical officer from CSIRO

Entomology, Ms Janelle Scown, was appointed to work with Dr Waterhouse and his

group to assist with the development and validation of the experimental model for

assessing recombination risk.

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Experimental design

The experimental system that has been developed is based on tobacco and potato

virus Y (PVY). The transgenic component of this system is tobacco (Nicotiana

tabaccum) containing a PVY protease (Pro) sense gene and was regarded as being

the optimal system for this work for the reasons outlined below.

Advantages of an experimental system based on potato virus y (PVY) and tobacco

Potato virus Y (PVY) belongs to the largest group of plant viruses, the potyviruses,

and along with potato leaf-roll virus (PLRV) it causes significant commercial losses in

Australian potato crops. CSIRO Plant Industry is presently engaged in developing

commercial transgenic cultivars with immunity to both these viruses based on PTGS

approaches.

A key benefit in using PVY and tobacco in this project was that experimental plants

could be inoculated manually. In contrast, many other plant viruses including PLRV

have to be inoculated by an insect vector for successful establishment. In addition,

several naturally occurring PVY strains were available for experimentation, and RNA

sequence data from each of these strains was available for use in the design of a

diagnostic tool based on a standard molecular method called the polymerase chain

reaction (PCR). The approach was to design the PCR detection tool to identify

recombination events between co-infecting PVY strains in wild-type tobacco plants

and recombination events between a singly infecting PVY strain and the PVY (Pro)

sense transgene in the transgenic tobacco plants.

Development of an RT-PCR diagnostic assay for detecting and specifically identifying parental and recombinant PVY RNAs

Three PVY strains were available in this study. These had sufficient sequence

diversity to allow PCR primers to be designed for strain-specific amplification of a

cDNA product from each strain (Table 1). The three strains are herein referred to as

‘D’, ‘N’ and ‘F’ strains. For example, as Figure 1 illustrates, the D5/ D3 primers were

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designed to specifically amplify a 700 base-pair PCR product from the ‘D’ strain only,

while the N5/ N3 primers were designed to specifically amplify a 700 base pair PCR

product from the ‘N’ strain only. A third set of PCR primers (F5/F3) was designed to

specifically generate an amplification product from the ‘F’ strain only.

Virus Primer1 Primer 2 Expected size

Recombinant 3

Recombinant 2

Recombinant 1Only Recombinant 2 will be detected

Wt PVY-N

Wt PVY-D

PVY-N D5 N3 X

Recombinant D5 N3 700

PVY-D D5 N3 X

PVY-N N5 N3 700

PVY-D D5 D3 700

b.

a. Figure 1. (a) The PCR detection of a recombinant RNA molecule relies on the fact that only certain combinations of PCR primers (here, either D5/N3 or N5/D3) will recognise the hybrid RNA sequence to produce a PCR product. (b) a recombination event outside of the targeted amplification region (i.e. situated outside the 5’ and 3’ primer annealing sites) will not be detected using this approach.

Co-infection experiments (described in later sections) were designed to utilise two

combinations of PVY strain: PVY-D + PVY-F and PVY-D + PVY-N. The PCR

detection assay was therefore designed so that the primer combinations, e.g. D5/ N3

and N5/ D3, would only amplify a cDNA PCR product transcribed from a hybrid RNA

generated following a cross-over event in the region situated between the 5' and 3'

primers (Figure 1). The full nucleotide sequence of each diagnostic PCR primer pair

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used in the study is given in Table 1. The relative location of the ‘D’ and ‘F’ primer

pairs is illustrated in Figure 2.

PVY-F

Transgene derived from PVY-D

PVY-D

Figure 2. Relative locations of ‘D’ and ‘F’ priming sites in PVY-D derived transgene, and the ‘D’ and ‘F’ virus strains. To detect a double recombination event i.e. a ‘D’ sequence containing an internal region of the ‘F’ sequence, a fourth set of primers called mD5/mD3 (represented by green arrows) were designed.

Table 1. Primer sequences

Primer Sequence (5’ to 3’) pvy D 5 cgacgtgaaggacataccagc pvy D 3 ccaccactacatcatgatcgatt pvy mid D 5 tttccctgtctttccacagaaattgca pvy mid D 3 tgttggagctcggaaatgcaat pvy F 5 tttgtgaaggacataccaaa pvy F 3 acaactacatcatgatctgcc pvy N 5 gatgtaagggacataccaaa pvy N 3* acaactacatcatgatctgcc

* same sequence as pvy F 3

Since a double recombination event in the targeted PCR region is also possible (e.g.

producing a virus that is PVY-D at its 5’ and 3' ends but containing an internal region

of PVY F) a third set of primers (‘Mid D’) were designed (Table 1; Figure 2). This was

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necessary because a double cross-over event would not be detected by the D5/F3

primer combination or by the F5/D3 combination.

Evaluating primer specificity and sensitivity

The specificity of the PCR primers was tested using PVY-D and PVY-F template

RNA (Table 2). Both the D5/D3 and F5/F3 primer combinations generated a PCR

product from RNA samples taken from plants infected with PVY-D and PVY-F

respectively. The D5/mD3 and mD5/D3 combinations were also tested and

successfully generated a PCR product from samples taken from plants infected with

PVY-D only. Importantly, primer combinations comprising: F5/D3, D5/F3, and F5/Mid

D3 did not generate PCR products from samples taken from plants infected with

PVY-D and PVY-F strains alone - confirming that these primer combinations were

likely to be useful in selectively detecting recombinant RNA only (see Figure 1).

One primer combination (Mid D5/F3) did, however, unexpectedly produce an

amplification product in an RNA sample taken from a PVY-D infected plant (Table 2).

While this PCR product was not of a size expected for a recombinant viral RNA, this

primer combination was discarded and not used in recombination experiments.

Table 2. Results of primer specificity.

Template Primer 5’ Primer 3’ Product F F F D D D F F D D D F F D F D F D D Mid D D D D Mid D D Mid D F D F Mid D F Mid D F F F Mid D

Products of wrong size, so excluded from further work

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The sensitivity of the RT-PCR reaction was examined by diluting RNA samples from

PVY-F infected plants using first water and then using extracts from PVY-D infected

plants as the diluent. The latter treatment was included in order to test primer

specificity when the target RNA was present against a background of non-target

RNA.

The results show that extracts from 100mg of leaf tissue could be diluted circa

80,000 fold and sufficient PCR product could still be generated for visual confirmation

of the presence of target PVY-F RNA (Figure 3 below). From the specificity and

sensitivity results it was clear that the RT-PCR diagnostic assay had the potential to

detect PVY RNA when present at very low titre in infected tobacco leaves and

therefore, by selecting the appropriate primer combinations, this diagnostic system

has the capability to detect recombinant PVY strains.

a b

Figure 3. Primer sensitivity tests. The F5/ F3 primers were used to generate PCR products from RNA extracts from PVY-F infected plant tissue using as a diluent: (a) PVY-D infected plant extract, and (b) water. PVY-F template RNA was diluted 10-fold to a final concentration of approx. 1:80,000 (lanes 4 and lane 9). Lane 5 is PVY-D RNA only as a negative control. PCR products are represented in the 650-800 nucleotide size range.

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Development of an inoculation strategy to maximise the probability of detecting recombinant PVY strains

The key objective in the design of the experimental strategy was to maximise the

probability of generating viral recombinants under quarantine conditions where the

limited availability of space imposes a severe constraint on the number of plants that

can be grown. This is important because if, as we expect, the recombination

frequency is very low then large numbers of plants would need to be inoculated in

order to detect and measure the frequency. The second objective of the experimental

strategy was to provide a convenient means of assessing the relative fitness of

parental and recombinant PVY strains in order to determine whether or not any

recombinants generated were capable of persisting in the environment. This was a

key recommendation of the Hazard Assessment Workshop (see Appendix I).

To meet these objectives a strategy was devised based on an idea originally

proposed at the Hazard Workshop by Dr Ali Rezaian. The procedure involved the

serial inoculation of experimental tobacco plants through several generations

(Figures 4 and 5). The concept was that since RNA viruses go through many millions

of rounds of replication in a single plant, by inoculating a subsequent generation of

plants using homogenised leaf extract from the plants originally inoculated, and

repeating this for subsequent generations, this procedure would maximise the

probability of detecting inter-viral recombination. In addition, if a recombinant virus

was generated, its fitness relative to the parental viruses could also be assessed by

monitoring its persistence through each generation because samples of infected

plant tissue from each inoculation round could be retained for future PCR analysis.

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Tested by PCR for Recombination

PVY-D+N

PVY-D PVY-N

G1

G2

G3

G4

G5

G6

Tested by PCR for Recombination

PVY-D+N

PVY-D PVY-N

G1

G2

G3

G4

G5

G6

Tested by PCR for Recombination

PVY-D+F

PVY-D PVY-F

G1

G2

G3

G4

G5

G6

Tested by PCR for Recombination

PVY-D+F

PVY-D PVY-F

G1

G2

G3

G4

G5

G6

Figure 4. The experimental strategy for serial co-inoculation of wild-type tobacco plants (a) PVY-D and PVY-F passed through 6 generations of plants for 5 independent lines (replicates) (generation 6 (G6) has been analysed for the presence of recombinants) (b) PVY-D and PVY-N passed through 6 generations of tobacco plants for 5 independent lines (all the G6 plants and the G2-G5 line 5 plants were assayed for virus recombinants). Assessing the virus inoculation strategy Single local lesions of PVY-D and PVY-F were separately inoculated onto healthy

tobacco plants. Leaf tissue extracts were taken from these plants two weeks later

when viral symptoms had become apparent (Figure 5). Extracts from PVY-D and

PVY-F infected plants were then mixed and inoculated onto each of 5 healthy

tobacco plants (Figure 4a). Extracts from each of these plants were subsequently

taken and inoculated onto another 5 healthy tobacco plants. This procedure was

repeated until 6 generations of plants co-infected with PVY-D and PVY-F had been

established.

An additional inoculation lineage was included in which a third PVY strain (PVY-N)

was used in the place of PVY-F (Figure 4b). This lineage was included both as an

additional treatment and to allow easier monitoring of the infection status of doubly

infected plants since the N strain gives a clear necrotic phenotype (Figure 5) that is

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easily distinguished from both the PVY-D and PVY-F strains (indistinguishable

symptoms).

Results

Application of the PCR detection assay to G6 plants from the PVY-D/F experiment

(Figure 4a) resulted in no recombinants being detected (Table 3). Using the mD5/N3

primer combination on RNA extracts taken from plant 5 of generation 6 of the PVY-

D/N co-infection experiment (Figure 4b), an amplification product was generated that

was indicative of the molecular weight of a putative recombinant molecule of 450

nucleotides (nt) (Table 4; Figure 6). The G2-G5 plants of this line (Figure 4b) were

also analysed using the PCR detection assay and while some spurious high

molecular weight PCR products were observed, no bands within the expected size

range of 450 nucleotides were observed (Figure 6).

Automated nucleotide sequence analysis was used to confirm that the 450nt

amplification product was a putative recombinant RNA formed by genetic exchange

between the co-infecting PVY-D and PVY-N strains (Figure 7). These are impressive

data given the limited time available for this work and we conclude that this

experimental system provides an excellent opportunity to measure and compare

virus recombination frequency in a transgenic and non-transgenic plant model.

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Table 3. Application of PCR to determine the presence of virus recombinants in experimental tobacco plants (G6) co-inoculated with PVY D and PVY F strains. No recombinants were detected.

D 5’

F 5’

F 5’

F 5’

Mid D 5’ D 3’ F 3’ D 3’ Mid D 3’ F 3’

plant 1

plant 2

plant 3

*

plant 4

*

plant 5

* one or more bands of wrong size Table 4. PCR detection of virus recombinants in experimental tobacco plants (G6) co-inoculated with PVY D and PVY N strains. Ticks in the midD5/N3 column indicate a recombinant.

D 5’

N 5’

N 5’

N 5’

Mid D 5’

D 3’ N 3’ D 3’ Mid D 3’ N 3’

plant 1

*

plant 2

plant 3

*

plant 4

*

plant 5

*

* one or more bands of wrong size

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PVY-D

PVY-N

PVY-F

PVY-D+N

PVY-D+F

Figure 5. PVY disease symptoms in tobacco. PVY-D, PVY-F and PVY-D + PVY-F co-infections produce similar, relatively mild, symptoms of vein clearing and tissue blistering. PVY-N produces a vein necrosis (visibly darkened) and growth is significantly retarded and similar but less pronounced symptoms are apparent for PVY-D + PVY-N co-infected tissue.

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Figure 6. Detection of a 450nt recombinant RNA molecule in plant tissue co-infected with PVY-D + PVY-N (lane 5). The PCR detection assay was carried out using mD5/N3 primers on extracts from plants representing generation 6, 5, 4, 3 and 2 (lanes 5-9 respectively) of line 5. Sterile water controls (x2) (lanes 2 and 3) and RNA extracts from uninfected tobacco (lane 4) did not produce an amplification product. A PCR product from PVY-N amplified using N5/ N3 primers produced a band of 770bp (lane 1). Lane M = 1Kb Plus DNA ladder (Invitrogen Life Technologies).

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PVY-D TGTCTAGTCGGAATCCACAGTTTGGCAAACAACAGACACACCACGAACTACTACTCAGCCTTCGATGAAGATTTTGAAAGCAAGTATCTCCGAACCA PCR product TGTCTAGTCGGAATCCACAGTTTGGCAAACAACAGACACACCACGAACTACTACTCAGCCTTTGATGAAGATTTCGAGAGCAAATATCTCCGAACTA PVY-N TGTCTACTTGGAATACATAGTTTGGCAAACAATGCGCAGTCCACGAACTACTACTCAGCCTTTGATGAAGATTTCGAGAGCAAATATCTCCGAACTA

Recombination occurred

in this region Figure 7. Comparison of the nucleotide sequence of the PCR product of the putative recombinant RNA and parental co-infecting PVY-D and PVY-N strains in experimental tobacco. The nucleotides shown in red mismatch with the sequence of the PCR product. Blue boxes show regions of perfect sequence homology.

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Conclusions

From these experiments the following conclusions can be drawn:

• The PVY-D/F and PVY-D/N stains have sufficient sequence diversity to allow

straightforward detection using an optimised RT-PCR diagnostic assay.

• The virus combinations will co-replicate for multiple generations and are

sufficiently closely related to maximise the likelihood of inter-viral recombination

• The serial inoculation procedure appears to provide a convenient method for

maximising recombination between two co-infecting PVY strains under

quarantine conditions without needing to process many hundreds of plants.

• An RT-PCR band indicative of a recombination event was found in one

generation of one virus combination line.

• Nucleotide sequence analysis of the RT-PCR band indicated that this

amplification product was almost certainly a recombinant RNA formed by genetic

exchange between co-infecting PVY-D and PVY-N strains.

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Possible future directions • The amplified DNA indicative of a recombination event was sequenced and the

result is strongly supportive of the view that a recombination event has been

detected. However, further analyses of replicate PCR products are needed to

confirm this very exciting result.

• The mixed infection of G6 line 5 of PVY-D/N should be passaged for a number of

generations to see whether the recombinant virus would survive competition with

its parental viruses, i.e. is it "fit"?

• Samples of total RNA have been retained from each plant of the inoculation

experiment and this material is available for RT-PCR analysis. This material

should be analysed for recombination events that have not persisted to G6.

• For the transgenic tobacco containing the PVY-D transgene, plants singly

infected with both PVY-N and PVY-F strains should be inoculated for a total of 6

generations and RNA samples assayed for the presence of parental and

recombinant RNAs. This has been done for G1 plants for parental RNA but not

for recombinant RNA due to lack of time.

• The PVY-D/N experiment should be repeated several times, with greater

numbers of plants and generations, to determine recombination frequency since

one recombinant detected out of 30 plants tested is insufficient to know whether

a very infrequent event had simply occurred very early on in the tests.

• If the repeat experiments show that one cannot expect to routinely detect

recombination using the system developed here, there are still several other

approaches that could be attempted.

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3. LITERATURE REVIEW

Scope of the review The risks associated with the introduction of transgenic plants carrying virus-derived

sequences include heterologous encapsidation, synergism and recombination.

Heterologous encapsidation (‘transencapsidation’) describes the complete or partial

encapsidation of the genome of one virus with the coat protein (CP) of another virus.

Synergism describes a phenomenon in which one virus may complement the effects

of other viruses resulting in more severe disease (Maki-Valkama and Valkonen

1999). Virus recombination describes the exchange of genetic material between two

co-infecting viruses or between a single infecting virus and a virus-derived transgene

with the potential for generating a chimeric virus with a novel pathology. Of these

three, only recombination has the potential to persist in the environment in the

absence of the transgenic plant and this review deals exclusively with this issue.

The intention in writing this review was to provide the reader with an informative

digest of our present understanding of the nature and significance of virus

recombination in transgenic plants containing virus-derived sequences. To this end,

the review is set-out as follows:

1. Evolutionary significance of recombination in plant viruses.

2. Mechanisms of pathogen-derived resistance (PDR) in transgenic plants.

3. Evidence of recombination among plant viruses and in plants.

4. Risks associated with recombination and transgenic plants.

5. References cited.

The review focuses almost entirely on RNA recombination. This is because the vast

majority of plant viruses and virus-derived sequences (promoters and transgenes)

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used in transgenic plants have an RNA origin. While there is presently little published

information on other types of genetic exchange involving plant viruses the following

generalities can be stated:

• recombination among RNA plant viruses appears to be relatively frequent.

• recombination among DNA plant viruses appears to be relatively frequent.

• recombination between RNA and DNA plant viruses appears to be relatively

infrequent.

• recombination between RNA or DNA viruses and host plant DNAs appears to

be relatively infrequent.

For a review of recombination involving plant DNA viruses the reader is directed to a

very recent publication by Frischmuth (2002) (unavailable to the authors at the time

of writing) and for a wider treatment of virus recombination and transgenic plants, to

the following web-links:

http://www.aibs.org/biosciencelibrary/vol46/transgenic/virus.html

http://www.aphis.usda.gov/biotech/virus/virussum.html

Two types of transgenic plant are relevant to discussions on recombination (i) GM

plants in which virus-derived promoters are used to control the expression of non-

viral transgenes, and (ii) GM plants in which virus-derived transgenes are used to

confer disease resistance in virus resistant transgenic plants (VRTPs). Virus-derived

promoters are used in a wide range of plants that are not necessarily hosts of the

virus (or its relatives) from which the promoter sequence was originally sourced.

Since the key constraint on genetic exchange is the degree of homology

(relatedness) shared by donor and recipient sequences (next section) recombination

involving virus promoters will only be an issue for relatively few of the plant species in

which the promoter is deployed.

Of the reference material surveyed, several publications were extensively used in the

preparation of this review. These were: Lai (1992), Baulcombe (1996); Gibbs et. al.

(1997); Kasschau and Carrington (1998); Aaziz and Tepfer (1999); Rubio et. al.

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(1999a,b); Zaitlin and Paulkaitis (2000) and Waterhouse (2001). Of these, the reader

is directed to the paper by Gibbs et. al. (1997) which was of particular value in the

final part of this review.

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i. Evolutionary Significance of Recombination in Plant RNA Viruses Questions concerning whether novel viruses with altered virulence, host range, and

vector specificity might evolve through recombination between RNA viruses and viral

derived transgenes used in genetically modified plants arose out of findings

implicating genetic exchange in the evolution of a large number of RNA viruses. Two

distinct types of genetic exchange operate in RNA viruses. The first, re-assortment,

occurs only in segmented viruses and involves the exchange of one or more discrete

RNA molecules that comprise the multipartite virus genome. The second type of

genetic exchange involves the introduction of a donor nucleotide sequence into a

single contiguous acceptor molecule to form a novel recombinant RNA molecule and

can occur in segmented as well as unsegmented viruses.

Nomenclature

Based on the similarity of the parental RNA molecules, two types of RNA

recombination have been defined: homologous recombination and non-homologous

recombination (Lai 1992). Homologous recombination (HR) occurs between two

similar or identical RNAs involving precisely matched (precise HR) or divergent

(aberrant HR) RNA sequences. Non-homologus recombination (NHR) involves the

exchange between identical viruses in which strict alignment is not maintained or

involves exchange of unrelated sequences. This nomenclature is not entirely

satisfactory since the term ‘homologous’ implies common ancestry which need not

necessarily be the case (Aaziz and Tepfer 1999), nevertheless, it remains in common

usage and is therefore adopted in this review.

The recombination mechanism

Two mechanistic models have been proposed to account for RNA recombination: 1)

a breakage-rejoining model which supposes the cleavage at two distinct sites of the

same or two different precursor RNAs followed by ligation of the two cleaved

molecules into one recombinant RNA and 2) an RNA dependent RNA polymerase

(RdRp) mediated copy-choice model, also known as ‘template switching’, in which

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the viral RdRp complex switches from one RNA template to another during

replication. Where replication of the new strand continues precisely where it left the

first template RNA the recombinant virus is homologous and non-homologous when

it does not.

The only experimental evidence in support of the breakage-rejoining model for RNA

recombination has come from Chetverin et. al. (1997) from in vitro studies of the Qβ

bacteriophage in which only non-homologous recombinants were observed.

Nevertheless, almost all the other evidence supports the RdRp-mediated copy-

choice model (e.g. Kirkegaard and Baltimore 1986; Nagy et. al. 1995; Nagy and

Simon 1997; Figlerowicz et. al. 1997 and 1998) although it should also be noted that

the template-switching mechanism has yet to be formally demonstrated.

Since template switching occurs during RNA synthesis several physical requirements

need to be met for successful cross-over to occur. The first of these is that the RNA

polymerase, which is responsible for catalysing the synthesis of the new RNA

molecule, must first pause and then dissociate from the template viral RNA (or DNA)

molecule. The second condition is that an alternative template must be sufficiently

close for the dissociated polymerase to bind to. The third condition is that the new

template has a polymerase binding site to facilitate cross-over and continued

synthesis on the new RNA molecule (Lai 1992).

Evidence indicates that these conditions are generally met. Transcriptional pausing

has been observed in a range of RNA viruses and RNA polymerase binds only

weakly to RNA, which increases its potential for dissociation. Also, in most RNA

viruses, RNA synthesis occurs in membrane bound cellular compartments and this

may promote the local concentration of viral RNAs making template switching by

polymerase more likely. The tendency for some regions of an RNA molecule to form

double stranded ‘heteroduplexes’ may also serve to promote both transcriptional

pausing and physical bridging of the template RNAs by the polymerase. The

mechanism by which polymerase recognises binding sites on an alternative RNA

template probably involves either sequence complementarity (where homologous

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recombination occurs), or recognition of common RNA secondary structure where

non-homologous recombination occurs, or both (Lai 1992; Aaziz and Tepfer 1999).

Evolutionary advantages of recombination Evidence derived in the main from nucleotide sequence data has been used to

compile a large and rapidly growing list of RNA viruses that have evolved as a result

of genetic recombination (Nagy and Simon 1997; Gibbs et. al. 1997). It is now

unequivocal that recombination has played a central role in the evolution of many

RNA viruses. Theoretical explanations of the evolution of sexual reproduction tend to

emphasise the spread of advantageous genes and the removal of deleterious genes

as the main drivers. The second of these derives from ‘Mueller’s ratchet’ theory

which predicts the gradual build-up of deleterious alleles in finite asexual populations.

RNA viruses represent one of only a few examples that have been used to

empirically test this prediction and results have generally indicated agreement i.e.

decreased fitness in populations in the absence of reassortment (Chao et. al. 1992

and Chao et. al. 1997).

Direct experimental evidence supporting a similar role for recombination has not

been provided to date, but expectations are that genetic exchange through

recombination provides for a similar function to reassortment by fixing advantageous

alleles in the population and removing disadvantageous alleles by combining

mutation free parts of different genomes. Although it has been proposed that

recombination in monopartite RNA viruses and reassortment in segmented RNA

viruses represent alternative evolutionary pathways in these two groups (Chao et. al.

1992) the weight of evidence for plant viruses, as well as other RNA viruses, indicate

that these two mechanisms are not necessarily exclusive and in some cases may

even operate simultaneously (Masuta et. al. 1998).

The evidence that has been presented in favour of the theory that recombination can

eliminate deleterious alleles has come from studies that show that weakly replicative

mutant tombusviruses, for example, can be reconstituted through recombination with

intact wild-type homologues (White and Morris 1994). Similar evidence has also been

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generated for the bromoviruses (Rao and Hall 1993). Several landmark studies have

also shown that mutant viruses can repair their genomes through genetic

recombination with virus transcripts in VRT plants carrying viral derived transgenes

(pages 38-42). Recombination between viruses and host transgenes under

conditions of strong selection has been shown not only to produce viruses that cause

disease symptoms quite distinct from the parental strain (Green and Allison 1994),

but also hybrid viruses more pathogenic than the parental strains (Jacquemond et al.

1997). Two important studies have shown that RNA recombination in plant viruses

may even provide a telomerase-like function by repair to the 3’ ends of satellite

RNAs, for example, of turnip crinkle virus (Burgyan and Garcia-Arenal 1998) and

cucumber mosaic virus (Simon and Nagy 1996).

In addition, a growing body of information indicates that recombination between viral

and host genomes has occurred (as illustrated by sequence comparison) between a

luteovirus and a plant chloroplast exon (Mayo and Jolly 1991). Closteroviruses have

also contributed supporting evidence with sequence data indicating that this group

has evolved in part by acquiring various host protein-coding genes (Dolja et. al.

1994). Evidence for the uptake of viral sequences by plant hosts is also beginning to

emerge with a report of the suspected integration of Banana Streak Badnavirus

(BSB) into the Musa genome (Harper et. al. 1999). Presumably, the selective

advantages to host and pathogen are resistance to or amelioration of disease and in

maximising disease transmission, respectively.

Evolutionary constraints on virus recombination Recombination appears to play a central role in the evolution of many RNA viruses

although it is now clear that RNA viruses are not equally prone to recombination.

Even for those that do show evidence of recombination, the frequency of

recombinants both in nature and in experimental studies appears to vary

considerably depending on the virus family, genus and strain (Worobey & Holmes

1999).). Therefore, given the likely advantages of recombination, a crucial question

is why does recombination frequency vary so considerably across the RNA virus

group?

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To be able to answer this question would put us in a position to predict the likelihood

that a given virus and viral transgene in a specified plant would recombine and may

even point to how the risks arising from such an event could be mitigated or

managed. Clearly, our present knowledge falls a considerable way short of this.

Nevertheless, it is possible to outline the main steps involved in the process of

recombination and, in so doing, identify some of the constraints that appear to limit

the potential for recombinants to arise and persist (Figure 10).

For successful genetic exchange between two plant RNA viruses the first condition is

that the host plant must be permissive to both strains (step 1). The only exception to

this is recombination between viruses of which at least one has diverged following an

initial clonal infection of the host. Host co-infection may never be possible for many

combinations of viruses simply because their spatial and/or temporal distributions do

not overlap. Most plant viruses require an insect vector for transmission and vector

specificity is likely to be a key determinant of whether the condition for host co-

infection can be satisfied for any two viruses. If the two potential recombining viruses

are not transmitted simultaneously host defence response is likely to play a critical

role either in rapidly clearing the primary infection or by preventing or limiting the

potential for superinfection.

The second condition is co-infection of the same host cell (Step 2). Barriers to the

simultaneous infection of a host cell are likely to be even more severe than co-

infection at the level of the whole organism and are likely to be mediated by host as

well as virus factors. Recent evidence indicates that intra-cellular competition for host

cell resources can be costly to virus reproduction (Turner and Chao 1998), and as

with other parasites, many RNA viruses appear to have evolved mechanisms to

promote the selfish acquisition of host resources by reducing the potential for

competition (Simon et. al. 1990; Singh et. al. 1997). Predicting the efficacy of virus

induced constraints to cellular co-infection will require an analysis of the likely

advantages arising from recombination balanced against the likely costs to the two

pathogens of sharing a limiting resource.

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The third condition is replication of at least one of the co-infecting viruses in proximity

to the RNA of the other (Step 3). Where the sites of replication for two potential

recombinants are not the same this condition is unlikely to be met. However, the

available evidence indicates that RNA synthesis for many RNA viruses occurs in

membrane bound cellular compartments - a situation that is likely to promote the

local concentration of RNAs (Lai 1992). The most important factor constraining the

probability that a viral polymerase will successfully switch from one RNA template to

another is the degree of sequence similarity of the co-infecting RNAs. In addition, the

capacity of viral polymerases to undergo template switching (Bujarski and Nagy

1996) will also be a central determinant of the likelihood of recombination across

RNA virus taxa.

While Steps 1-4 are necessary for a hybrid RNA virus to arise, Step 5 must be

satisfied if the recombinant is to persist in the environment. Although recombination

may offer a way to eliminate disadvantageous traits and fix useful traits within the

population, it is also the case that most new combinations of genes invariably prove

lethal and will be rapidly selected against. Studies of both plant and animal RNA

viruses indicate that recombination occurs randomly throughout the genome.

Recombination ‘hot-spots’ are simply the cross-over points remaining after limited

selection rather than regions of elevated genetic exchange, because even these

crossover points nearly always disappear after sufficient passages (Banner and Lai

1991; Jarvis and Kirkegaard 1992; Desport et. al. 1998).

Recombination among RNA viruses is probably relatively common but only hybrids

for which the benefits of exchange outweigh the costs and for which the hybrid is

able to compete with parental strains are likely to persist. As with mutation, genetic

exchange is a random process and the vast majority of new combinations will either

be non-functional or suffer decreased fitness compared to the parental viruses. As

such, the vast majority of recombinants are likely to be rapidly eliminated from the

virus gene pool.

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Figure 10. The constraints operating against recombination between two plant RNA

viruses. Virus or host constraints can block or limit any of the five steps that lead first

to the formation of a recombinant and subsequently (step 5) to persistence in the

environment (modified from Worobey and Holmes 1999).

STEPS CONSTRAINTS

t Environmental distribution of viruses Host plant defence responses to limit

primary infection

1. Co-infection of hos

l Virus-mediated exclusion of

competitor strains Host plant defence response to

2. Co-infection of cel

n

avoid superinfection

Lack of physical proximity of replicating RNA viruses limits opportunity for template switching

3. Replicatio

Insufficient sequence similarity

between co-infecting viruses

4. Template switch

5. Selection

RECOMBINANT

severely limits the potential for effective template switching

Purifying selection of hybrid RNA sequences and viruses in plant hosts and transmission vectors

VIABLE RECOMBINANT

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ii. Mechanisms of Pathogen-Derived Resistance (PDR) in Transgenic Plants

The incorporation of viral-derived sequences into plants has provided the means of

combating economically significant crop viruses for which there are no readily

accessible sources of natural resistance. Only two plant genes demonstrating

resistance to viruses have been isolated to date. These include the N gene from

tobacco (Erikson et. al. 1999) and the Rx gene from potato (Bendahmane et. al.

1995) which encodes a hypersensitive response to tobacco mosaic virus (TMV) and

an extreme resistance potato virus X (PVX) respectively. However, the mechanism

by which each of these responses is induced is not known and while the N gene has

been successfully transferred to tomato, the wider applicability of these transgenes to

other crop plants has not been demonstrated.

Strategies for pathogen-derived resistance (PDR) are divided into those that require

the production of proteins and those that require only the presence of RNA

sequences. Generally speaking, protein-mediated strategies provide incomplete

resistance to a wide range of RNA viruses, whereas RNA-mediated resistance

provides very high levels of resistance to a narrower range of viruses (Beachy 1997).

Protein mediated resistance Coat protein (CP) mediated resistance

Following empirical foundations laid by Sherwood and Fulton (1982) and Bevan et al.

(1985), the concept of pathogen-derived resistance (PDR) (Sanford and Johnston

1985) was demonstrated experimentally for the first time in transgenic plants by

Powell-Abel et al. (1986). This study showed that expression of the TMV coat protein

gene in tobacco delayed the onset of disease. Similar experiments quickly followed

and the same phenomenon was demonstrated for a variety of other viruses including,

for example, alfalfa mosaic virus (AMV), PVX, and cauliflower mosaic virus (CMV)

(Baulcombe 1996; Lomonossoff 1995).

Coat protein mediated resistance (CPMR) can provide either broad or narrow

resistance to RNA viruses. The coat protein of TMV for instance is effective against

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closely related strains, but resistance decreases with diminishing sequence similarity

(Nejidat and Beachy 1990). In contrast, the CP gene of soybean mosaic virus (SMV)

which is not capable of infecting tobacco, confers resistance in tobacco to two

unrelated potyviruses, potato virus Y (PVY) and tobacco etch virus (TEV) (Stark and

Beachy 1989). It is not known why some coat proteins afford high levels of protection

and why others provide broader or lower levels of resistance, though this issue will

almost certainly have direct relevance to the risks of virus recombination where

transgenes from several virus strains are required to deliver sufficiently broad

resistance.

The underlying mechanisms involved in CP mediated resistance are not well

understood although inhibition of virion assembly and reduced spread of the virus

appear to play central roles (Chapman et al. 1992; Clark et. al. 1995; Taschner et. al.

1994). Nevertheless, based on the success achieved using CP mediated resistance

against TMV (for which the three dimensional structural of the virus CP is now

known), the design of mutant coat proteins with improved efficacy and host range are

anticipated in the future.

Replicase - mediated resistance

Genes encoding viral replicase proteins can confer near immunity to infection. All

plant viruses have replicase genes and transformation of plants with both defective

and non-defective replicase genes can provide very strong resistance - though

usually only to the donor virus or very closely related strains (Palukaitis and Zaitlin

1997). The mechanisms involved in replicase-MR are not known but are likely to

involve the suppression of virus replication and gene expression.

While a feature of replicase-MR is that it is generally quite specific, some interesting

exceptions have been observed. In one case, transgenic resistance to potato leaf roll

virus (PLRV) turned out to be effective against a wide range of PLRV strains. In

another example, the TMV replicase gene containing an Agrobacterium sequence

insertion was found to confer high level resistance in tobacco plants to a wide range

of tobamoviruses (Donson et. al. 1993).

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Movement protein - mediated resistance

Movement protein mediated resistance (MPMR) is thought to arise through

competition between the dysfunctional introduced MP and the invading virus for

binding sites on host plasmodesmata resulting in reduced local and systemic spread

of infection in MPMR transgenic plants. In contrast to replicase-MR, the expression of

dysfunctional MP genes facilitates resistance to a much wider range of viruses

(Malyshenko et. al. 1993). This approach has not seen wide application due to the

scale of effort necessary to make and screen banks of mutant MP genes to identify

effective candidate mutants.

The expression of functional MPs appears either to have no effect or actually

increases susceptibility of plants to virus disease (Ziegler-Graff et. al. 1991). It has

been anticipated that improved knowledge of MP structure and function will lead to

the development of mutant MPs that can act as highly effective inhibitors of many

different types of virus (Beachy 1997).

RNA-mediated resistance

Several PDR strategies involve the expression of genes that do not encode proteins.

One of the first was expression of antisense RNA sequences to inhibit virus

replication (Hammond and Kamo 1995) although some doubt exists as to whether

this effect was actually due to RNA suppression (below). Other mechanisms may

also be responsible including interruption of template selection by viral replicase

and/or the formation and subsequent degradation of double-stranded RNA.

Antisense mediated suppression is generally very narrow in terms of the range of

viruses that it targets.

RNA mediated resistance in transgenic plants has also been shown to operate

through competitive inhibition of the invading virus with the transgene or its RNA

transcript acting as a decoy for proteins needed for virus replication and movement.

This process is known to operate in instances where the transgene specifies a

‘defective-interfering’ (DI) viral RNA as, for example, in transgenically expressed

turnip yellow mosaic virus (TYMV) 3’ DI RNA which competes with the invading

TYMV genome (Zaccomer et.al. 1993). Satellite RNAs are similar to DI molecules but

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do not depend on sequence similarity with the donor virus. Transgenic plants

encoding DI RNAs (or DNAs) and satellite RNAs have been tested for their capacity

to reduce replication and ameliorate disease with some degree of success (Stanley

et. al. 1990 and Kollar et. al. 1993).

To date, the most well known type of RNA mediated resistance is RNA suppression -

a process that describes the post-transcriptional destruction of viral RNA also known

as “gene silencing” (Baulcombe 1996). Virus gene silencing was suspected early on

in the development of VRT plants from observations that in plants designed to be

resistant through CP expression, protection was maintained even in the absence of

the transprotein (Lindbo et. al. 1993). Gene silencing in plants derives either from a

block on the initiation of viral transcription or from degradation of viral RNA after

transcription and is described as transcriptional gene silencing (TGS) and post-

transcriptional gene silencing (PTGS) respectively. In either case, the host cell

activates a destruction mechanism upon detecting aberrant RNA sequences and

both viral and transgene RNA is destroyed.

Our present understanding of how PTGS works involves the cleavage of the targeted

RNA following recognition by, and sequence-specific hybridisation to, dicer-like

protein complexes. This highly elaborate mechanism is thought to represent a highly

evolved defence strategy on the part of the plant to nullify invading viruses and

transposable elements (Waterhouse et. al. 2001). The PTGS signal is capable of

systemic spread within and between host plant cells through plasmodesmata and

diffusion through the vascular system. Since signal specificity is conserved the

systemic spread is thought to comprise, at least in part, the transgene RNA and may

be facilitated by movement proteins or factors. Although levels of gene silencing can

be extremely high, resistance is usually only effective against viruses that are very

similar or identical to the transgene.

Suppression of RNA silencing

A key finding in PTGS research was the discovery in 1998 that some plant viruses

encode proteins capable of suppressing RNA silencing (Kasschau and Carrington

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1998). Suppression of RNA silencing probably represents an evolutionary adaptation

by plant viruses to a generic host antiviral defence strategy since many of the

suppressor proteins have previously been shown to be important determinants of

virulence. In fact, the ability to suppress PTGS may be crucial for many viruses to be

able to infect their plant hosts. It seems that suppression occurs in a general manner,

independent of the gene or sequence responsible for inducing the silencing

mechanism that, by contrast, can only act against near homologous sequences.

Transgenic plants designed to work by gene silencing are only effective because the

PGTS mechanism is activated prior to infection and is therefore able to degrade the

target RNA before viral proteins can be translated from it. The implication, if any,

regarding the potential for recombination should suppression of the PTGS

mechanism in VRT plants by naturally occurring viruses be possible, does not appear

to have received much, if any, empirical attention.

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iii. Evidence of Recombination among Plant RNA Viruses and Transgenic Plants Mutation, reassortment and recombination are the drivers for evolution in RNA

viruses. Recombination probably plays an additional role in rescuing viruses by

repairing mutations in virus genomes that have occurred as a result of proof-reading

errors during replication (Carpenter and Simon 1996). The realisation that

recombination has a central role in the evolution of many RNA viruses began with

experiments on poliovirus by Hirst (1962) and Ledinko (1963). Since that time

recombination has been documented for many animal and plant RNA viruses. The

rapid accumulation of evidence for recombination is all the more impressive when

one realises that prior to these formative studies, recombination was not thought to

be a property of RNA genomes at all.

Recombination in plant RNA viruses was first demonstrated for brome mosaic virus

(BMV) (Bujarski and Kaesberg 1986) and there is now an extensive literature

documenting recombination among both plant and animal RNA viruses (Aaziz and

Tepfer 1999) as well as in bacteriophages (Chetverin 1997) and double-stranded and

negative sense RNA viruses (Suzuki et. al. 1998 and Sibold et. al. 1999). The genetic

engineering revolution, which has brought with it the development of the polymerase

chain reaction (PCR), automated nucleotide sequencing techniques and phylogenetic

analysis, has transformed the study of virus recombination by providing the means of

detecting and characterising recombination events. Using these techniques it has

been possible not only to detect rare recombination events and exchanges that have

occurred long ago (e.g. Weaver et. al. 1997) but also to identify the precise location

of the cross-over point (Olsthoorn and Duin 1996). Two types of data have been

used to provide evidence of RNA recombination, one based on phylogenetic analysis

and the other based on empirical observation.

Detecting recombination among RNA viruses Methods for analysing putative recombination events are often graphical in nature

and exploit the fact that many recombinant sequences are mosaics of virus

phylogenies. For example, ‘Split Decomposition’ analysis (Huson 1998) employs a

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split graph highlighting transition from a dichotomously branching tree form indicative

of sequence relationships to a complicated network indicative of a history of genetic

exchange. Other graphical programmes look for discordant nucleotide sequence

relationships suggestive of recombination, examples of which include ‘boot scanning’

(Salminen et. al. 1995), ‘PhylPro’ (Weiller 1998), ‘TOPAL’ (McGuire and Wright 1998)

and ‘DIVERT’ (Gao et. al. 1998).

Statistical approaches employ procedures based on maximum-likelihood

computation of goodness-of-fit estimates by comparing the frequency distribution of

polymorphisms in putative parental and recombinant strains (Maynard-Smith 1992;

Holmes et. al. 1999). Some statistical approaches attempt to quantify the amount of

recombination rather than document specific recombination events and do this by

calculating an ‘Index of Association’ which measures the degree of linkage between

alleles at different loci (Maynard-Smith et. al. 1993). A further development on this

approach is the ‘Homoplasy Test’ (Maynard-Smith and Smith 1998) which compares

the number of convergent and parallel base changes (homoplasies) in a maximum

parsimony tree to the number expected by chance alone where excessive homoplasy

indicates a history of recombination.

There is also an abundance of direct experimental data demonstrating homologous

RNA recombination using movement-defective viruses in which functional

recombinants have been restored through genetic exchange. These are summarised

below in Table 5.

Non-homologous recombination (NHR) between plant viruses has been

demonstrated in two instances. The first involved co-inoculation of protoplasts and

plants with defective cucumber necrosis virus (CNV) and tomato bushy stunt virus

(TBSV) RNAs which produced recombinant defective interfering (DI) strains as well

as fully infectious derivatives (White and Morris 1994). The second case involved the

co-inoculation of tomato aspermy virus (TAV) RNAs 1 and 2 and cucumber mosaic

virus (CaMV) RNAs 2 and 3, resulting in a novel cucumovirus encoding a novel

replicase comprising TAV helicase and CaMV polymerase subunits (Masuta et. al.

1998).

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Table 5. Direct experimental evidence of homologous recombination in plant RNA

viruses – first reference cited only (from Aaziz and Tepfer 1999).

Virus family

Species Reference

Bromovirus Brome mosaic virus

Cowpea chlorotic mottle virus

Bujarski and Kaesberg

(1986)

Allison et. al. (1990)

Carmovirus Turnip crinkle virus Cascone et. al. (1990)

Tobamovirus Tobacco mosaic virus Beck and Dawson (1990)

Alfamovirus Alfalfa mosaic virus Van der Kuyl et. al. (1991)

Tombusvirus Cucumber necrotic virus, tomato

bushy stunt virus DI-RNA

White and Morris (1994)

Cucumovirus Cucumber mosaic virus, tomato

aspermy virus

Fernandez-Cuartero et. al.

(1994)

Potyvirus Zucchini yellow mosaic virus Gal-On et. al. (1998)

The phylogenetic evidence as well as the empirical evidence indicates that not only

do different RNA viruses not recombine at the same frequency, but also that some

strains are highly recombinative while others show no evidence of recombination at

all. Phylogenetic analyses of potyvirus strains, for example, indicate numerous

recombination events, whereas analyses of cucumovirus genomes have yielded no

evidence at all of past recombination events (Revers et. al. 1996; Candresse et. al.

1997). While these findings may be explained to some extent by differences in the

detection limits of the different virus systems, it is also likely to be due, in part at

least, to the inherent characteristics of the virus-host interaction. This last point, in

particular, is significant for VRT plant risk assessment since almost nothing is known

about the role of host factors in viral RNA recombination.

Recombination in VRT plants

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Since the first empirical demonstration of PDR in transgenic plants in the mid 1980s,

numerous transgenic crops have been developed for tolerance or complete

resistance to a range of economically important virus diseases. Recombination in

transgenic plants has been investigated in systems in which movement defective

viruses are inoculated into plants expressing the functional homologous movement

protein gene, and then tested for the presence of systemic (recombinant) viruses.

This phenomenon has been reported in three experimental systems: cauliflower

mosaic virus (CaMV), cowpea chlorotic mottle bromovirus (CCMV) and tomato bushy

stunt tombusvirus (TBSV).

The first extensively documented case of recombination involving a transgene was in

Brassica napus expressing the cauliflower mosaic virus (CaMV) translational

transactivator gene ORF VI (Gal et. al. 1992). Experimental plants were inoculated

with a mutant CaMV strain lacking the ORF VI gene and recombination with the

transgene restored systemic movement to the inoculated strain. In an important

related study a CaMV strain unable to infect solanaceous plants acquired ORF VI

from transgenic tobacco, Nicotiana bigelovii, generating recombinants with altered

symptoms and extended host range (Schoelz and Wintermantel 1993). It should be

noted, however, that while both of these examples provide strong evidence that

recombination arose by two template-switching events between viral RNA and the

transgene transcripts, DNA-DNA recombination cannot be ruled out since this has

previously been shown to occur in CaMV (Gal et. al. 1991). Moreover, the copy-

choice mechanism in CaMV is due to viral reverse transcriptase and therefore this

system is not directly comparable to recombination involving RNA dependent RNA

polymerase (RdRp).

A strain of cowpea chlorotic mottle bromovirus (CCMV) that had been crippled by

deletion of part of the coat protein gene was used to elegantly demonstrate

recombination in transgenic N. benthamiana containing the missing CP sequence

(Green and Allison 1994, 1996). In this example, mutation markers unequivocally

confirmed a single recombination event between the transgene mRNA and the

movement-defective CCMV to generate fully restored systemic hybrids containing the

functional CP gene. Sequence analysis of the recombinant strains revealed

numerous modifications flanking the putative cross-over site, and when tested on a

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range of host species, four out of seven recombinants displayed novel symptoms,

although none was more fit than the wild-type virus (Allison et. al. 1997 and 1999). In

these experiments, recombinant viruses were recovered under conditions of strong

selection pressure in that the challenging virus was movement defective and had to

recombine with the transgene in order to regain function. Searching for recombinant

viruses generated under conditions of little or no selection pressure is generally

regarded as necessary for realistic risk assessment.

In experiments by Borja et. al. (1999), recombination events between a tomato bushy

stunt tombusvirus (TBSV) mutant containing a deletion in the coat protein transgene

were readily detected. In this example, the TBSV mutants were systemic but at a

clear disadvantage to the recombinants that must have been generated by at least

two cross-over events and as a result of precise homologous recombination. There

are, as yet, no published experimental data where fully intact inoculating strains have

been tested in the absence of experimentally induced selection pressure. The closest

so far has been a published study involving isolation of recombinant CaMV from

transgenic N. bigelovii plants under moderate selection pressure (Wintermantel and

Schoelz 1996).

Taken as a whole the evidence points to the conclusion that evolution of new virus

genotypes, possibly with altered pathology, can occur through a large array of

recombination events that could include exchange between a naturally occurring

virus and a viral transgene employed in a GM plant. Nevertheless, since all of these

experiments have been performed under conditions of either strong or moderate

selection pressure the results from these studies in themselves afford little insight

into the likelihood that viruses will successfully recombine with viral transgenes in

transgenic plants under field conditions and become successfully established in the

environment.

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iv. Perceived Risks Associated with Virus Recombination and Transgenic Plants

The key characteristic that distinguishes genetically modified organisms (GMOs)

from most other technologies (e.g. chemical pesticides) is that GMOs can replicate

and, consequently, once established in the environment a GMO may be able to

persist indefinitely and prove difficult and costly to remove if this becomes necessary.

Persistence does not just imply temporal continuity but also indicates spatial spread

since not only could the entire GMO disperse beyond the boundary of the intended

release site but the transgene itself may be able to disperse into the wider

environment as a result of out-crossing between the GM plant and compatible wild

relatives.

Uptake of the transgene by wild-type organisms provides the potential for unique and

unintended combinations of genes and for this reason considerable attention is now

being directed at understanding the risks associated with gene introgression (see

e.g. Sindel 1997). In addition to out-crossing, VRT plants present an additional

concern because of the possibility that the viral derived transgenes used to confer

disease resistance in GM crops may recombine with the genomes of naturally

occurring plant viruses. The key risk associated with recombination between a viral

derived sequence and a plant virus is that this may result in the generation of a

unique virus with a novel pathology.

VRT plants introduce no new or increased risk: supporting and opposing arguments

In contemplating the risks associated with recombination in VRT plants the central

point has tended to be that their use will not introduce any additional risk to that

already present in nature when two or more co-infecting viruses recombine to form a

hybrid. Gibbs et. al. (1997) have outlined the key points used both in support and in

opposition to this view (Table 6).

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Table 6. Supporting arguments (A) and opposing arguments (B) to the view that the

cultivation of VRT plants in the environment introduces no additional risk in terms of

recombination to that already present when two or more co-infecting viruses

recombine (modified from Gibbs et. al. 1997).

A.

Supporting arguments:

1.

Viral recombination is a natural phenomenon and will continue to contribute

to the evolution of RNA viruses irrespective of whether transgenic plants are

used.

2.

Several viruses are frequently detected infecting the same host plant

providing ample opportunity for recombination. Nevertheless, new

recombinants are rarely detected and this provides strong evidence that

successful new recombinants are actually very rare. Therefore, it is likely

that the key limiting factor influencing the generation of a recombinant virus

is not the opportunity for genetic exchange but the purifying effect of natural

selection which acts to eliminate the vast majority of hybrids.

3.

Because the efficacy of the virus transgene in the VRT plant depends on the

degree of sequence similarity to the challenging virus, the capture of part or

all of the transgene to form a recombinant would likely result in it being

targeted by the PDR mechanism.

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B.

Opposing arguments:

1.

Too little is known about recombination in natural systems and there may

be critical barriers to natural recombination for many potential

combinations of RNA viruses. These could include, for example, sub-

cellular compartmentalisation and tissue tropism that leads to a physical

separation of co-replicating viruses as well as differences in geographic

distribution and epidemiology. The mass cultivation of VRTPs across

geographic zones may mitigate or entirely overcome some or all of these

barriers resulting in an increase in recombination frequency.

2.

Too little is known about virus fitness and virus-virus competitive

interactions to be able to predict with confidence the potential for

recombinant viruses to establish in the population (Fernandez-Cuartero

et. al. 1994). In natural systems it may be the case, for example, that

recombinants are displaced by one or more parental strains. It may be

possible that in a transgenic plant that expresses only partial resistance to

a given virus, a fitness cost imposed on the parent strain may be sufficient

to advantage the recombinant virus which may subsequently become

established.

3.

Changes over time in the incidence and severity of viral diseases in crops

have been recorded. The reasons for these changes are not well

understood and it is not known whether or not recombination plays a role

in this process (Revers 1996).

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Intra- and inter-group recombination among RNA viruses The potential for a recombinant virus with a new pathology to arise following genetic

exchange between two or more parental virus strains will be significantly influenced

by the degree of sequence homology shared by the two RNA viruses. Gibbs et. al.

(1997) have regarded two types of non-homologous recombination as being pertinent

to risk assessment: (a) intra-group exchange in which the contributing genomes are

taxonomically very closely related (i.e. strains of the same ‘species’) and (b) inter-

group exchange involving more distantly related viruses that would be regarded as

being from different ‘species’. The problem though is distinguishing between these

classes in practice, as the concept of a virus ‘species’ is not particularly well defined.

Nevertheless, the general point can be made that the experimental evidence to date

indicates that intra-group recombination occurs at a measurable frequency (it is

relatively common) whereas inter-group recombination has yet to be experimentally

detected even though much effort has been focussed on this class and data from

sequence analysis shows that it has occurred many times in the past. Given the

absence of experimental evidence, therefore, inter-group recombination is

considered to be relatively rare.

The consequences arising from intra- versus inter-group recombination are likely to

differ markedly. While both classes of recombination may increase the rate of virus

evolution, inter-group recombination is more likely to lead to radically new

combinations of genes and over time result in the generation of entirely novel genera

and families (Gibbs 1994). For example, sequence analysis data has indicated that

inter-group exchange was probably responsible for the evolution of the potyviruses

(Goldbach 1992) and the subsequent diversification of this group into the largest

family of RNA virus species (Revers et. al. 1996). It is noteworthy that several

potyvirus strains have achieved status as economically important diseases of

agricultural crops.

An interesting point in VRT plant risk assessment is that many viruses considered as

nonpathogens are capable of replicating in host cells but are limited in their ability to

move systemically within the plant. The evidence for this comes from experiments

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showing that some viruses can replicate in non-host protoplasts and therefore there

is a possibility that in VRT plants, viruses capable of replication may have

considerable opportunity for recombination with the transgene, particularly where the

transgene is involved in the systemic spread of virus through the plant. One

immediate possibility is virus recombination involving a movement protein (MP)

transgene, although viral MPs have not proved particularly useful for conferring

disease resistance. Similarly, virus coat protein (CP) transgenes may present similar

opportunities, as these genes are known to contribute not only to the systemic

movement of virus in the host plant but also play a role in defining vector specificity

(Allison et. al. 1997).

Evaluating frequency and hazard in risk assessment

Two distinct though inter-related elements are central to considerations of risk

assessment: frequency and hazard (Hull 1994). The frequency component of risk for

VRT plants is evaluated in relative terms and as stated above, intra-group

recombination among co-infecting viruses is measurable and appears to be relatively

frequent and is therefore also likely to be the case for recombination between an

RNA virus and a homologous host transgene (Schoelz and Wintermantel 1993;

Green and Allison 1994). Empirical detection of inter-group recombination among co-

infecting viruses has proved far more difficult with only two published examples

available to date (White and Morris 1994; Masuta et. al. 1998). Similarly, inter-group

recombination between a virus and a host transgene has not been demonstrated and

is therefore not readily measurable.

As discussed previously, detection of mixed infections in plants does not necessarily

mean that the RNA of one virus is available to the other’s replication machinery.

Recombination requires two viruses to replicate in the same place even down to the

level of the same sub-cellular locality at the same time. Evidence indicates that

infections proceed as a wave, initiating and completing viral replication progressively,

and leaving encapsidated virions in its wake (Wang and Maule 1995). The RNAs of

two viruses may thus have limited opportunity for contact (depending on the temporal

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dynamics of the infection waves of the two strains) even if they share the same

cellular compartment.

Physical and temporal proximity as a key constraint to recombination between two

co-infecting viruses may not be as critical in transgenic plants, however, where the

transgene is constitutively expressed in all host cells. Under these conditions,

simultaneous replication of the virus and transgene mRNA may increase the

probability (and hence frequency) of recombination. Even so, there appears to be no

data either to support or refute this hypothesis under the conditions of little or no

selection pressure (Aziz and Tepfer 1999). Perhaps one reason for the lack of

progress on this question has been that mixed infections have not been extensively

studied because successful co-infection of plant cells in most experimental systems

(necessary for comparison with singly infected transgenic plants) has been difficult to

achieve as one strain usually succeeds in excluding the other.

It is likely therefore that success in this area will come from studies of intra-group

recombination using experimental methods and systems in which co-infecting strains

can be readily distinguished. Concurrent with this last point is the issue that

experimental assessment of RNA recombination is dependent on the availability of

sufficiently sensitive techniques for the detection and unequivocal identification of

chimeric RNA molecules. The reverse transcriptase-polymerase chain reaction (RT-

PCR) provides the diagnostic capability to detect sequence specific RNA when

present at extremely low titre (in theory a single molecule). The reaction conditions

and pairs of oligonucleotide primers designed specifically to detect hybrid RNAs,

require thorough empirical evaluation and optimisation to reach the required level of

sensitivity and specificity.

Several workers have attempted to address the hazard component of risk

assessment by creating recombinant strains in vitro then determining qualitative

effects such as changes to host range and disease severity (Ding et. al. 1996; Salanki et. al. 1997, Carrere et. al. 1999). The main criticisms of this approach are

first, that selection pressure in vitro will be quite different to that encountered by the

virus in vivo and second, the relative fitness of recombinant and parental strains has

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not been determined and in the absence of this data, little can be concluded as to the

realistic hazards posed by these strains.

Comparison of recombination frequency in singly infected transgenic versus doubly

infected non-transgenic plants, is generally considered as providing the most useful

experimental data for risk assessment regarding VRT plants (Aaziz and Tepfer

1999). A suitably amenable experimental system is obviously key since quantitative

comparison lacks confidence where both treatments have no detectable

recombinants. If such a system is available, however, one can then contemplate

testing for changes to disease pathology and fitness of any recombinants relative to

parental strains can also ascertained i.e. evaluating the hazard component of risk.

Mitigating risk

Considering the enormous scale of potential future commercial releases of VRT

plants, the implications for both the hazard and frequency components of risk

becomes a non-trivial issue - particularly if the geographic deployment of VRT crops

present opportunities for new virus associations. Clearly, genetic engineering

strategies that reduce the potential for genetic exchange between viral RNAs in VRT

plants would be valuable in mitigating the risks associated with this phenomenon.

Genetic engineering of crops based on homology dependent gene silencing may

present certain advantages from a biosafety perspective - since the mechanism

quickly degrades the mRNAs produced by both the transgene and the targeted viral

RNAs. Under these circumstances the expectation is that the opportunity for

recombination will be removed. However, suppression of PTGS by certain viruses

has been recently demonstrated (Kasschau and Carrington 1998; Vaucheret et. al.

2001) and this may have ramifications for virus recombination in VRT plants. For

example, Jakab et. al. (1997) demonstrated with the inoculation of experimental

plants with two potato virus Y strains, in which one was targeted by RNA-mediated

resistance and the other was not, that selection pressure favoured recombination

between the viral genomes resulting in the replacement of the targeted sequence.

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Experimental work with the cowpea chlorotic mottle bromovirus (CCMV) coat protein

gene has shown that, during virus replication, the replication complex initiates

synthesis of the complementary strand at the 3’ end of the genomic RNA.

Nucleotides responsible for directing this are located at the terminus of the 3’

untranslated region (UTR) of the CP gene. Consequently, a CP transgene in which

the terminal nucleotides have been removed may carry significantly less chance of

recombination with a challenging RNA virus. Experiments appear to confirm this as a

potential mitigation strategy for CP-mediated transgenic plants (Green and Allison

1996). Various CP mutants have now been designed to eliminate vector transmission

of potential recombinants expressing heterologous CP sequences (Jacquet et. al.

1998).

Finally, Rubio et. al. (1999a) have made the case that the use of viral derived

transgenes may actually constrain rather than increase the frequency of

recombination by reducing the background level of virus (sources of recombination)

that exist in susceptible crops. These include not only complex mixtures of

autonomously replicating viruses but also a large number of defective virus-like

agents that may become capable of assuming autonomy by acquisition of a single

critical gene. These authors contend that a major ancillary benefit of widespread use

of transgenic PDR would be to reduce the proportion of multiple and co-dependent

infections in cultivated crops. Since replication is normally several times lower in

highly resistant compared to fully susceptible plants (Lomonossoff 1995), these

authors conclude that this should provide downward pressure on recombination

frequency.

Ecological risk experiments

It is probably fair to say that the majority view among scientists expert in the art is

that, in terms of the potential range of viral phenotypes arising from recombination,

VRT crops pose no additional per capita risk over and above that which already

exists from recombination among naturally occurring co-infecting strains in non-VRT

crops. That is not to say, however, that the mass cultivation of VRT crops will not

increase the overall frequency with which chimeric viruses appear.

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There are few published ecological studies assessing the potential for environmental

impact from the cultivation of virus recombination in VRT plants. One exception is a

study by Raybould et. al. (1999) that has focused on generating baseline data on the

distribution of plant viruses in natural populations of oilseed rape, Brassica oleracea,

in the United Kingdom and evaluating their impact on host plant fitness.

Oilseed rape crops are widely grown in the UK and genetically modified rape has

been developed for both insect and virus disease resistance. Five feral populations of

B. oleracea in the Dorset region of southern England were monitored using ELISA for

the presence of four virus pathotypes. These were beet western yellows virus

(BWYV), cauliflower mosaic virus (CaMV), turnip mosaic virus (TuMV), and turnip

yellow mosaic virus (TYMV). The distribution of all four viruses was found to be

highly over-dispersed. For example, 84% of TYMV infected plants were confined to

only one of the five populations and associations of viruses were also found to be

significantly non-random.

Genetic differences in host response to virus infection did not appear to be driving

this pattern, however, since results from controlled field trials showed that plants

sourced from these populations could become infected with viruses in equal

proportions regardless of their origin. Tests of the effect of viruses on host fitness

were then conducted, in which 574 seedlings from the 5 study populations were

grown in insect-screened glasshouses and inoculated with either TuMV, TYMV or

sterile water. At 3 months, data revealed that not only was disease mortality highly

significantly different between treatments (P<0.001) but that in survivors, total seed

production per plant was significantly impacted in virus treated hosts.

These data indicate that agriculturally important plant viruses are capable of

impacting on natural host plant populations and that impacts are unlikely to be

uniformly distributed among host populations. This was almost certainly as a result of

differences in the distribution and behaviour of the aphid, weevil and flea vectors

responsible for transmitting these virus pathotypes. Since evidence from the

controlled trials discounted the possibility that vector species had imposed differential

selection for virus resistance among the study populations, the conclusion was that

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the potential for virus induced impacts on wild B. oleracea populations will be highly

dependant on the distribution and behaviour of the vector species involved.

Nevertheless, Raybould et. al. (1999) also emphasise that it remains to be shown

whether these plant viruses actually play any significant role in determining the

persistence or abundance of natural or feral Brassica populations. The doubt arises

because B. oleracea has a very high seed output compared to seedling recruitment

and because B. oleracea seed disperses only short distances. The overall

conclusion, therefore, was that both inter- and intra-specific competition are likely to

have a much greater effect on the likelihood of B. oleracea seedlings establishing

than by impacts from insect predators or virus pathogens.

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v. References cited

Aaziz R and Tepfer M (1999) Recombination in RNA viruses and in virus-resistant transgenic plants. J. Gen. Virol. 80: 1339-46.

Abdel PP, Nelson RS, De B, Hoffman N, Rogers SG, Fraley RT and Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232: 738-743.

Allison R, Thompson C, and Ahlquist P (1990) Regeneration of a functional RNA virus genome by recombination between deletion mutants and requirement for cowpea chlorotic mottle virus 3a and coat genes for systemic infection. Proc. Nat. Acad. Sci. USA. Vol. 87, pp. 1820-1824

Allison RF, Greene AE and Schneider WL (1997) Significance of RNA recombination in capsid-protein-mediated virus-resistant transgenic plants. In Virus-Resistant Transgenic Plants: Potential Ecological Impact. Edited by M. Tepfer and E. Balazs. Versailles and Heidelberg: INRA and Springer-Verlag, pp. 40-44.

Allison RF, Schneider WL and Deng M (1999) Risk assessment of virus resistant transgenic plants. In Proceedings of the 5th International Symposium on Biosafety Results of Field Tests of Genetically Modified Plants and Microorganisms. (Ed. J. Schiemann and R Casper).

Banner LR and Lai MMC (1991) Random nature of coronovirus RNA recombination in the absence of selection pressure. Virology 185: 441-445.

Baulcombe DC (1996) Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8:1833-44.

Beachy RN (1997) Mechanisms and applications of pathogen-derived resistance in transgenic plants. Curr. Opin. Biotechnol. 8:215-220.

Becher P, Meyers G, Shannon A and Thiel HJ (1996) Cytopathogenicity of border disease is correlated with integration of cellular sequences in the viral genome. J. Virology 70: 2992-98.

Beck DL and Dawson WO (1990) Deletion of repeated sequences from tobacco mosaic virus mutants with two coat protein genes. Virology 177, 462-469.

Bendahmane A, Kohm BA, Dedi C and Baulcombe DC (1995) The coat protein of potato virus X is a strain specific elicitor of Rx-1-mediated virus resistance in potato. Plant J. 8: 933-41.

Bergelson J (1994) Changes in fecundity do not predict invasiveness: a model study of transgenic plants. Ecology 75: 249-252.

Bevan MW, Mason SE, Goelet P (1985) Expression of tobacco mosaic virus coat protein by a cauliflower mosaic virus promoter in plants transformed by Agrobacterium. EMBO J 4: 1921-26

Borja M, Rubio T, Scholthof HB, and Jackson A (1999) Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene. Mol. Plant-Microbe Interact. 12:153-162.

Broer I (1996) Stress inactivation of foreign genes in transgenic plants. Field Crops Res. 45: 19-25.

Bujarski JJ and Kaesberg P (1986) Genetic recombination between RNA components of a multipartite plant virus. Nature (London) 321: 528-531.

57

Page 58: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Bujarski JJ and Nagy PD (1996) Different mechanisms of homologous and non-homologous recombination in brome mosaic virus: role of RNA sequences and replicase proteins. Semin. Virol. 7: 363-372.

Burgyan J and Garcia-Arenal F (1998) Template-independent repair of the 3’ end of cucumber mosaic virus satellite RNA controlled by RNAs 1 and 2 of helper virus. J. Virol. 72: 5061-5066.

Candelier-Harvey P and Hull R (1993) Cucumber mosaic virus genome is encapsulated in alfalfa mosaic virus coat protein expressed in transgenic tobacco plants. Trans. Res. 2: 277-285.

Candresse T, Revers F, Le Gall O, Kofalvi S, Marcos J, and Pallas V (1997) Systematic search for recombination events in plant viruses and viroids. In Tepfler M and Balazs E (eds), Virus resistant transgeneic plants: potential ecological impact, pp 20-25. Springer, Berlin, Germany

Carpenter CD and Simon AE (1996) In vivo restoration of biologically active 3’ ends of virus-associated RNAs by non-homologous RNA recombination and replacement of a terminal motif. J. Virol. 70: 478-486.

Carrere I, Tepfer M and Jacquemond (1999) Recombinants of cucumber mosaic virus (CMV): determinants of host range and symptomatology. Arch. Virol. 144: 365-379.

Cascone PJ, Carpenter CD, Li XH, and Simon AE (1990). Recombination between satellite RNAs of turnip crinkle virus. EMBO J. 9: 1709-1715

Chapman S, Hills GJ, Watts J, and Baulcombe DC (1992) Mutational analysis of the coat protein gene in potato virus X: effects on virion morphology and viral pathogenicity. Virology 191, 223-230.

Chao L, Tran TR and Matthews C (1992) Mueller’s ratchet and the advantage of sex in the RNA virus Ф6. Evolution 46: 289-299.

Chao L, Tran TT and Tran TT (1997) The advantage of sex in the RNA virus Ф 6. Genetics 147: 953-959.

Chetverin AB (1997) Recombination in bacteriophage Qβ its satellite RNAs: the in vivo and in vitro studies. Semin. Virol. 8: 121-129.

Clark WG, Fitchen JH and Beachy RN (1995) Studies of coat protein-mediated resistance to TMV. Virology 206, 307-313.

Cooper PD, Steiner-Pryor A, Scotti PD and Delong D (1974) On the nature of poliovirus genetic recombinants. J. Gen. Virol. 23: 41-49.

Desport M, Collins ME and Brownlie J (1998) Genome instability in BYDV: an examination of the sequence and structural influences on RNA recombination. Virology 246: 352-361.

Ding SW, Shi BJ, Li WX, and Symons RH (1996) An interspecies hybrid RNA virus is significantly more virulent than either parental virus. Proceedings of the National Academy of Sciences, USA 93: 7470-7474.

Dolja VV, Karasev AV, and Koonin EV (1994) Molecular biology and evolution of closteroviruses: sophisticated build-up of large RNA genomes. Annual review of Phytopathology 32: 261-285.

Donson J, Kearney CM, Turpen TH, Khan IA, Kurath G, Turpen AM, Jones GE, Dawson WO and Lewandowski DJ (1993) Broad resistance to tobamoviruses is mediated by a modified tobacco mosaic virus replicase transgene. Molecular Plant Microbe Interactions 6:635-642.

58

Page 59: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Edwards ML and Cooper JI (1985) Plant virus detection using a new form of indirect ELISA. J. Virol. Methods 11: 309-319.

Erikson FL, Dinesh-Kumar SP, Holzberg S, Ustach CV, Dutton M, Handley V, Corr C and Baker BJ (1999) Interactions between tobacco mosaic virus and the tobacco N gene. Philos. Trans. R. Soc. London Ser. B 354: 653-58.

Fagard M and Vaucheret H (2000) Systemic silencing signal(s). Plant Mol. Biol. 43: 285-293. Falk BW and Bruening G (1994) Will transgenic crops generate new viruses and new

diseases? Science 263: 1395-1396. Fernandez-Cuartero B, Burgyan J, Aranda MA, Salanki K, Moriones E and Garcia-Arenal F

(1994) Increase in the relative fitness of a plant virus RNA associated with its recombinant nature. Virology 203: 373-377.

Figlerowicz M, Nagy PD and Bujarski JJ (1997) A mutation in the putative RNA polymerase gene inhibits non-homologous, but not homologous genetic recombination in an RNA virus. Proc. Nat. Acad. Sci. USA 94: 2073-2078.

Figlerowicz M, Nagy PD, Tang N, Kao CC, and Bujarski JJ (1998) Mutations in the N terminus of the brome mosaic virus polymerase affect genetic RNA-RNA recombination. J. Virol. 72: 9192-9200.

Frischmuth T (2002) Recombination in plant DNA viruses. Plant viruses as molecular pathogens. vol. (). pp. 339-363.

Fuchs M, Gal-On A, Raccah B and Gonsalves D (1999) Epidemiology of an aphid non-transmissible potyvirus in field of non-transgenic and coat protein transgenic squash. Trans. Res. 8: 429-439.

Fuh-Jyh J, Fagoaga C, Sheng-Zhi P and Gonsalves D (2000) A single chimeric transgene from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing. J. Gen. Virol. 81: 2103-09.

Gal S, Pisan B, Hohn T, Grimsley N, and Hohn B (1991) Genomic homlogous recombination in planta. EMBO Journal 10: 1571-1578.

Gal S, Pisan B, Hohn T, Grimsley N, and Hohn B (1992) Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination. Virology 187: 525-533.

Gal-On A, Meiri E, Raccah B and Gaba V (1998) Recombination of engineered defective RNA species produces infective potyvirus in planta. J. Virol. 72: 5268-5270.

Gao F, Robertson DL, Carruthers CD, Morrison SG, Jian BX, Chen YL, Barre -Sinoussi F, Girard M, Srinivasan A, Abimiku AG, Shaw GM, Sharp PM and Hahn BH (1998) A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. Journal of Virology 72, 5680-5698.

Gibbs MJ (1994) Risks in using transgenic plants? Science 264: 1650-1651. Gibbs MJ, Waterhouse PM and Weiller GF (1997) Analysis of natural viral recombination

may assist the design of new virus resistance traits. In: Commercialisation of transgenic crops: Risk, benefit and Trade Considerations. Proceedings of a workshop held in Canberra 11-13 March, 1997 (edited by G.D. McLean, P.M. Waterhouse, G. Evans and M.J. Gibbs). Cooperative Research Centre for Plant Science and Bureau of Resource Sciences, Canberra. pp. 159-172.

Goldbach R (1992) The recombinative nature of potyviruses: Implications for setting up a true phylogenetic taxonomy. Arch. Virol. 299-304.

Gray AJ and Raybould AF (1998) Reducing transgene escape routes. Nature 392: 653-654.

59

Page 60: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Greene A E and Allison RF (1994) Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-25.

Greene AE, and Allison RF (1996) Depletion in the 3' untranslated region of cowpea chlorotic mottle virus transgene reduce recovery of recombinant viruses in transgenic plants. Virology 225: 231-234

Hammond J and Kamo KK (1995) Effective resistance to potyvirus infection conferred by expression of antisense RNA in transgenic plants. Molecular Plant Microbe Inter. 5: 67-682.

Harper G, Osuji JO, Heslop-Harrison JS and Hull R (1999) Integration of Banana Streak Badnavirus into the Musa genome: Molecular and cytogenetic evidence. Virology 255: 207-213.

Hilder VA and Gatehouse AMR (1991) The phenotypic costs to plants of an extra gene. Trans. Res. 1: 54-60.

Hirst GK (1962). Genetic recombination with Newcastle disease virus, poliovirus and infuenza. Cold Spring Harbor Symposia on Quantitative Biology 27, 303-308.

Holmes EC, Worobey M and Rambau A (1999) Phylogenetic evidence for recombination in dengue virus. Mol. Biol. Evol. 16: 405-409.

Hoyle R (1994) Let’s finally get the threat of virus resistant plants straight. Bio/Technology 12: 662-663.

Hull R (1994) Risks in using transgenic plants? Science 264: 1649-1650. Huson DH (1998) SplitsTree: a program for analyzing and visualizing evolutionary data.

Bioinformatics 14:68–73. Jacquemond M, Salanki K, Carrere E, Balazs E and Tepfer M (1997) Behaviour of

cucumovirus pseudorecombinant and recombinant strains in solanaceous hosts. In: Virus-resistant transgenic plants: potential ecological impact (M Tepfer and E. Balazs eds.). pp.52-64. Berlin; New York: Springer; Paris: INRA Editions: OECD/OCDE.

Jacquet C, Decolle B, Raccah B, Lecoq H, Dunez, J, and Ravelonandro M (1998) Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants. J. Gen. Virol. 79:1509-1517

Jakab G, Vaistij FE, Droz E and Malnoe P (1997). Transgenic plants expressing viral sequences create a favourable environment for recombination between viral sequences. In Virus-Resistant Transgenic Plants : Potential Ecological Impact, pp. 45±51. Edited by M. Tepfer & E. Balazs. Versailles & Heidelberg : INRA & Springer-Verlag. 2547-2556. 67

Jarvis TC and Kirkegaard K (1992) Poliovirus RNA recombination: mechanistic Studies in the absence of selection. EMBO journal 11: 3135-3145.

Kasschau KD and Carrington JC (1998) A counter-defensive strategy of plant viruses: suppression of post-transcriptional gene silencing. Cell 95:461-70.

Kawchuk LM, Martin RR and McPherson J (1990) Resistance in transgenic potato expressing the leafroll virus coat protein gene. Mol. Plant Microbe Int. 3: 301-307.

Kirkegaard K and Baltimore D (1986) The mechanism of RNA recombination in poliovirus. Cell 47: 433-443.

Kollar A, Dalmay T, and Burgyan J (1993) Defective interfering RNA mediated resistance against cymbidium ringspot tombusvirus in transgenic plants. Virology 193: 313-318.

Lai MMC (1992) RNA recombination in animal and plant viruses. Microbiol. Rev. 56: 61-79.

60

Page 61: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Ledinko N (1963). Genetic recombination with poliovirus type 1: studies of crosses between a normal horse serum-resistant mutant and several guanidine-resistant mutants of the same strain. Virology 20, 107-119.

Leqoc H, Ravelonandro M, Wipf-Scheibel C, Monison M, Raccah B and Dunez J (1993) Aphis transmission of a non-aphid transmissible strain of zucchini yellow mosaic potyvirus from transgenic plants expressing the capsid protein gene of plum pox potyvirus. Molecular Plant-Microbe Interactions 6: 403-406.

Lindbo JA, Silva-Rosales L, Proebsting WM and Dougherty WG (1993) Induction of a highly specific antiviral state in transgenic plants: implications for regulation of gene expression and virus resistance. Plant Cell 5, 1749-1759.

Lomonossoff GP (1995) Pathogen-derived resistance to plant viruses. Annu. Rev. Phytopathol 33: 323-343.

Mäki-Valkama T and Valkonen JPT (1999) Pathogen derived resistance to Potato virus Y: mechanisms and risks. Agricultural and Food Science in Finland 8: 493-513.

Malyshenko SI, Kondakova OA, Nazarova JV, Kaplan IB, Taliansky ME, and Atabekov JG (1993) Reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. J. Gen. Virol. 74, 1149-56.

Marathe R, Anandalakshmi R, Smith TH, Pruss GJ and Vance VB (2000) RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing. Plant Mol. Biol. 43: 203-220.

Masuta C, Ueda S, Suzuki M and Uyeda I (1998) Evolution of a quadripartite hybrid virus by interspecific exchange and recombination between replicase components of two related tripartite RNA viruses. Proc. Nat. Acad. Sci. USA 95:10487-92.

Maynard-Smith J (1992) Analyzing the mosaic structure of genes. J. Mol. Evol. 34: 126-129. Maynard-Smith J and Smith NH (1998) Detecting recombination from gene trees. Mol. Biol.

Evol. 15: 590-599. Maynard-Smith J, Smith NH, O’Rourke M and Spratt BG (1993) How clonal are bacteria?

Proc. Nat. Acad. Sci. USA 90: 4384-4388. Mayo MA and Jolly CA (1991) The 5’ terminal sequence of potato leafroll virus RNA:

evidence of recombination between virus and host RNA. J. Gen. Virol. 72: 2591-95 McGuire G and Wright F (1998) TOPAL: recombination detection in DNA and protein

sequences. Bioinformatics 14, 219-220. Miller WA, Koev G, and Mohan BR (1997) Are there risks associated with transgenic

resistance to luteoviruses? Plant Dis. 81:700-710. Mueller E, Gilbert JE, Davenport G, Brigneti G and Baulcombe DC (1995) Homology-

dependent resistance: Transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J. 7: 1001-13.

Nejidat A and Beachy RN (1990) Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses. Mol. Plant Microbe Int. 3: 247-251.

Nagy PD and Simon AE (1997) New insights into the mechanisms of RNA recombination. Virology 235: 1-9.

Nagy PD, Dzianott A, Ahlquist P and Bujarski JJ (1995) Mutations in the helicase-like domain of protein 1a alter the sites of RNA-RNA recombination in brome mosaic virus. Journal of Virology 69: 2547-2556.

61

Page 62: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Olsthoorn RCL and van Duin J (1996) Evolutionary reconstruction of a hairpin deleted from the genome of an RNA virus. Proc. Nat. Acad. Sci. USA 93: 12256-61.

Palukaitis P and Zaitlin M (1997) Replicase-mediated resistance to plant virus disease. Adv. Virus. Res. 48:349-77

Powell-Abel PP, Nelson RS, De B, Hoffmann N, Rogers SG, Fraley RT and Beachy RN (1986) Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743.

Rao ALN and Hall TC (1993) Recombination and polymerase error facilitate restoration of infectivity in brome mosaic virus. J. Virol. 67: 969-979.

Raybould AF, Moyes CL, Maskell LC, Mogg RJ, Warman EA, Wardlaw JC, Elmes GW, Edwards ML, Cooper JI, Clarke RT and Gray AJ (1999) Predicting the ecological impacts of transgenes for insect and virus resistance in natural and feral populations of Brassica species. In: Methods for Risk Assessment of Transgenic Plants. III. Ecological risks and prospects of transgenic plants, where do we go from here? A dialogue between biotech industry and science (Ammann K, Jacot Y, Kjellsson G, and Simonsen V, Eds.). 1999. Birkhauser Verlag. pp. 3-15.

Revers F, Le Gall O, Candresse T, Le Pomancer M, and Dunez J (1996) Frequent occurrence of recombinant potyvirus isolates. J. Gen. Virol. 776: 1953-65.

Rubio T, Borja M, Scholthof HB and Jackson AO (1999a) Recombination with host transgenes and effects on virus evolution: an overview and opinion. Mol. Plant-Microbe Int. 12: 87-92.

Rubio T, Borja M, Scholthof HB, Feldstein PA, Morros TJ and Jackson AO (1999b) Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants. J. Gen. Virol. 73:5070-78.

Salanki K, Carrere I, Jacquemond M, Balazs E and Tepfer M (1997) Biological properties of pseudorecombinant and recombinant strains created with cucumber mosaic virus and tomato aspermy virus. J. Virol. 71: 3597-3602.

Salminen MO, Carr JK, Burke DS and McCutchan FE (1995). Identifcation of breakpoints in intergenotypic recombinants of HIV type-1 by bootscanning. AIDS Research and Human Retroviruses 11, 1423-1425.

Sanford JC and Johnston SA (1985) The concept of pathogen derived resistance: deriving resistance from the parasite’s own genome. J. Theor. Biol. 113: 395-405.

Schoelz JE and Wintermantel WM (1993) Expansion of viral host range through complementation and recombination in transgenic plants. Plant Cell 5: 1669-1679.

Sherwood JL and Fulton RW (1982) The specific involvement of coat protein in tobacco mosaic virus cross protection. Virology 119:10-158.

Sibold C, Meisel H, Kruger DH, Labuda M, Lysy J, Kozuch O, Pjcoch M, Vaheri A and Plyusnin A (1999) Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia. J. Virol. 73: 667-675.

Simon AE and Bujarski JJ (1994) RNA-RNA recombination and evolution in virus-infected plants. Ann. Rev. Phytopathol. 33:337-362.

Simon AE and Nagy PD (1996) RNA-RNA recombination and evolution in virus-infected plants. Annu. Rev. Phytopathol. 32: 337-362.

Simon KO, Cardamone JJ, Whitaker-Dowling PA, Youngner J and Widnell CC (1990) Cellular mechanisms in the superinfection exclusion of vesicular stomatitis virus. Virology 177: 375-379.

62

Page 63: Environmental Risks Associated with Viral Recombination in Virus Resistant Transgenic ... · Environmental Risks Associated with Viral Recombination in Virus ... is an economically

Sindel BM (1997) Outcrossing of transgenes to weedy relatves. In: Commercialisation of Transgenic Crops: Risk, Benefit and Trade Considerations. Edited by GD McLean, PM Waterhouse, G Evans and MJ Gibbs. Proceedings of a workshop held in Canberra 11-13 March, pp. 43-81.

Singh I, Suomalainen M, Varadarajan S, Garoff H and Helenius A (1997) Multiple mechanisms of entry and uncoating of superinfecting Semliki Forest virus. Virology 231: 59-71.

Stanley J, Frischmuth T, and Ellwood S (1990) Defective viral DNA ameliorates symptoms of geminivirus infection in transgenic plants. Proc. Nat. Acad. Sci. USA 87: 6291-6295.

Stark DM and Beachy RN (1989) Protection against potyvirus infection in transgenic plants: evidence for broad spectrum resistance. Biotechnology 7:1257-62.

Suzuki Y, Gojobori T and Nakagomi O (1998) Intragenic recombinations in rotavirses. FEBS Letters. 427: 183-187.

Taschner PEM, Van Marle G, Brederode FT, Tumer NE, and Bol JF (1994) Plants transformed with a mutant alfalfa mosaic virus coat protein gene are resistant to the mutant but not to wild-type virus. Virology 203, 269-276.

Tepfer M (1993) Viral genes and transgenic plants: what are the potential environmental risks? Bio/Technology 11: 1125-1132.

Turner PE and Chao L (1998) Sex and the evolution of intrahost competition in RNA virus θ6. Genetics 150, 523-532.

van der Kuyl AC, Neeleman L, Bol JF (1991) Complementation and recombination between alfalfa mosaic virus RNA 3 mutants in tobacco plants Virology 183: 731-738

Vaucheret H, Beclin C and Fagard M (2001) Post-transcriptional gene silencing in plants. J. Cell Sci. 114 (17): 3083-3091.

Wang D and Maule AJ (1995) Inhibition of host gene expression associated with plant virus replication. Science. 267: 229-231.

Waterhouse PM, Wang M-B, and Lough T (2001) Gene silencing as an adaptive defence against viruses. Nature. 411: 834-841.

Weaver SC, Kang WL, Shirako Y, Rumenap T, Strauss EG and Strauss JH (1997) Recombinational history and molecular evolution of western equine encephalomyelitis complex alphaviruses. J. Virol. 1: 613-623.

Weiller GF (1998). Phylogenetic Profiles : a graphical method for detecting genetic recombinations in homologous sequences. Molecular Biology and Evolution 15, 326-335.

Wintermantel WM and Schoelz JE (1996) Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure. Virology 223, 156-164.

White DO and Morris TJ (1994) Recombination between defective tombusvirus RNAs generates functional hybrid genomes. Proc. Nat. Acad. Sci. USA 91:3642-46.

Worobey M and Holmes EC. (1999). Evolutionary aspects of recombination in RNA viruses. J.Gen.Virol. 80, 2535-2544

Zaccomer B, Cellier F, Boyer JC, Haenni AL, and Tepfer M (1993) Transgenic plants that express genes including the 3’ untranslated region of the turnip yellow mosaic virus (TYMV) genome are partially protected against TYMV infection. Gene 136: 87-94.

Zaitlin M and Palukaitis P (2000) Advances in understanding plant viruses and plant virus diseases Annu. Rev. Phytopathol. 38: 117-43.

63

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Ziegler-Graff V, Guilford PJ and Baulcombe DC (1991) Tobacco rattle virus RNA-1 29K gene product potentiates viral movement and also affects symptom induction in tobacco. Virology 182: 145-55.

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4. APPENDIX I

Report to Environment Australia on a hazard assessment workshop on viral recombination and transgenic plants held at CSIRO Entomology in Canberra, October 23-24, 2001 Introduction In Australia, the commercialisation of transgenic plants containing viral derived sequences for disease resistance and for driving the expression of other types of transgene promises economic as well as environmental benefit through improvements in productivity and crop quality concurrent with a reduction in the use chemical pesticides and other inputs. The mass cultivation of these crops, however, also poses novel risks. One of the more controversial of these is the potential for environmental impact arising from recombination between plant viruses and viral-derived sequences. This is because there is a finite probability that a recombination event will result in a chimeric virus capable of establishing as a severe disease in species hitherto unaffected. Agencies involved in the regulation of GM technologies in Australia have been concerned that there is insufficient information to adequately inform risk assessment in this country. To improve on this position Environment Australia recently commissioned a twelve-month project to review current scientific understanding of the risks associated with viral recombination and transgenic plants in Australia. As part of this review a workshop was organised by the project lead organization, CSIRO Entomology, in collaboration with Environment Australia, to bring together relevant scientists and regulators to discuss the subject of virus recombination and its associated risk. Whilst it is recognised that genetic recombination may have significance to a broad range of transgenic organisms, the focus for this workshop was recombination between plant viruses and viral-derived sequences deployed in transgenic plants of particular significance to Australia. Background The incorporation of viral-derived sequences into plants has provided the means of combating economically significant crop viruses for which there are no readily accessible sources of natural resistance. Both coding and non-coding viral gene sequences can be used for this purpose. In the former category the virus coat protein gene of the targeted virus has generally been used for conferring disease resistance in virus resistant transgenic plants (VRTPs) although the mechanism by which this occurs is not known.

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For non-expressing viral sequences the mechanism of disease resistance appears to have arisen as part of an adaptive defence strategy against invading viruses through sequence specific enzymatic degradation. This mechanism is commonly referred to as post-transcriptional gene silencing (PTGS) or RNA interference (RNAi). Where an invading virus has the opportunity to replicate in a transgenic plant, the transgenic sequence may be available for recombination with the invader if the two sequences are sufficiently homologous. By a process, termed ‘template switching’, the viral replicase may occasionally switch from its viral RNA template to the transgenic mRNA template, resulting in the formation of a recombinant RNA molecule. This mirrors the natural phenomenon of occasional template switching between two co-replicating viruses, which is known to have played an important role in the evolution of some plant virus families e.g. the Luteoviridae. Objectives The objectives for the workshop were three-fold: • to promote discussion across relevant scientific disciplines and between scientists

and regulators on viral recombination and risk in Australia, • to gauge a range of scientific opinion on the significance of viral recombination,

and • to identify key areas of scientific uncertainty and determine the need for and

nature of future research effort into viral recombination in this country

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List of participants Dr Paul Chu CSIRO Plant Industry

Dr Louise Morin CSIRO Entomology

Prof. James Dale Queensland University of Technology

Mr Declan O'Connor-Cox Office of the Gene Technology Regulator

Dr Owain Edwards CSIRO Entomology

Dr Ali Rezaian CSIRO Plant Industry

Prof. Adrian Gibbs Australian National University

Dr Andy Richards CSIRO Entomology

Dr Bob Godfree CSIRO Plant Industry

Dr Peter Stoutjesdijk Environment Australia

Dr Paul Hattersley Environment Australia

Dr Peter Waterhouse CSIRO Plant Industry

Dr Mark Lonsdale CSIRO Entomology

Dr Kent Williams CSIRO Sustainable Ecosystems

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Composition of the workshop The workshop was attended by 14 individuals representing five organizations involved in the scientific research and development, and regulatory oversight of transgenic technologies in Australia. The majority of the scientists present were from a virology background. Other disciplines represented included plant ecology, weed pathology, both vertebrate and invertebrate virus ecology, and insect molecular ecology. The collective virological expertise of the workshop was considerable and afforded both a depth of insight and a wide range of opinion on the significance of virus recombination in regard to the risk assessment of transgenic plants. Approach As a means of structuring the workshop discussion sessions a recombination “scenario” was proposed describing a sequence of events beginning with recombination between a plant virus and a viral-derived sequence in an unspecified transgenic plant. The acquisition, dispersal and transmission of the recombinant virus by an insect vector eventuated in the establishment of a novel, highly pathogenic disease in a new host plant species in the Australian environment (see Annex). The participants were asked to critically evaluate this scenario and to identify key areas of scientific uncertainty. Over the course of the two-day workshop this event sequence was re-defined and the steps in the sequence mapped against a priority list of research topics and questions as a means of addressing the key areas of scientific uncertainty associated with viral recombination and transgenic plants (Figure 1 below). The list of priority research questions and topics at each step in this sequence is given in Table 1. The scenario presented to participants (Annex) proposed a specific ecological impact or ‘end point’ as a basis for beginning the process of identifying potential ecological hazards. The overriding opinion of the participants was that such an outcome was extremely improbable and no more likely as a result of the deployment of transgenic plants than through recombination among naturally occurring viruses. The rationale behind these conclusions is outlined in the ‘summary of key points’ below. From a methodological standpoint, participants concurred that proper evaluation of risks associated with viral recombination and transgenic plants should proceed by systematic progression down the event sequence defined in Figure 1.

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Figure 1. Flow diagram of key events in the scientific evaluation of the risks associated with recombination between plant viruses and viral-derived sequences deployed in virus resistant transgenic plants (VRTPs).

Survi

Re No recombinant

Uptakeve

Transhost ospecie

tra

No change in phenotype

No survival

Infection of a virus resistant transgenic plant (VRTP) by a

plant virus

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STEP 1 combination

STEP 2 val in cell/ plant

STEP 3 by same or new ctor species

STEP 4 mission to new f same/ different s; effect on seed nsmissibility

Change in phenotype

STEP 5

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Table 1. Key areas of scientific uncertainty (priority research questions and experimental variables) associated with risk, viral recombination and transgenic plants Event Priority research questions and experimental

variables STEPS 1 and 2 What is the relative frequency of viral recombination and/or

probability of survival of viral recombinants in vivo arising from plant-virus versus virus-virus interactions?

Experimental variables: RNA versus DNA viruses

Sequence homology of viral derived sequence in the plant and the virus challenger (strain vs species vs genus vs family) Host species Host plant quality Disease status and virus competition Transmission mode (important to replicate, as far as possible, the natural route of infection in experimental systems) Transgene length and function

STEPS 3-4 Effect of recombination on host plant range – issue of which test

species to select? Effect on virus-vector relations – e.g. specificity and transmission?

Vector ecology – e.g. dispersal and host plant selection. STEP 5 Virus characterisation and diagnostic assays for identifying

progeny recombinant viruses and the relative proportion of progeny and parental viruses. Biological studies to evaluate effect on virulence, pathogenicity and transmissibility (virus fitness).

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Summary of workshop discussions and key points Workshop participants concluded that risk assessment effort at this stage ought to be directed at determining the relative frequency of recombinant survivors arising from virus-VRTP versus virus-virus interactions. The rationale behind this conclusion was that the potential range of genetic variants (and therefore the potential for unique virus phenotypes) arising from recombination is unlikely to differ between these two categories. In other words it was generally held that the introduction of genetically modified plants containing viral derived sequences did not introduce any additional qualitative risk, in terms of the potential range of viral phenotypes, over and above that which presently exists in Australia from recombination among naturally occurring co-infecting viruses. In terms of frequency of recombination three important points were emphasised. The first was that successful recombination requires the two parental strains to be very closely related. The second was that the longer the transgenic viral sequence, and therefore the more recombination sites and functional units it contained, the greater the probability that it would successfully recombine with a virus. The third was that transgenic viral sequences expressing functional proteins in transgenic plants are more likely to generate a chimeric virus with phenotypic consequences than are non-coding RNA sequences. During the course of the discussions the workshop participants identified several other key points presented in summary form below. These are listed in no particular order of priority and are presented to provide a general guide to the discussion surrounding the key research topics and questions and indicate the basis for the recommendations given at the end of this report. • Examination of genome sequences of a large range of viruses shows that

recombination has been a driving force in the evolution of some virus groups. • Under experimental conditions recombination has proved difficult to

demonstrate. This apparent contradiction maybe explained as follows: while virus recombination may not be infrequent the probability of survival of recombinants is likely to be very low and hence the frequency with which recombinants are detected is correspondingly low.

• The lack of empirical data and uncertainty relating to most areas of virus

recombination means that few conclusions as to its risk can be made with confidence. ‘Absence of evidence is not evidence of absence’.

• The issue of heterologous versus homologous recombination is a grey area.

The term ‘homology’ does not necessarily imply 100% sequence similarity but is generally taken to indicate intra-specific relatedness (ie. among viral strains of the same species). By analogy the term ‘heterology’ is generally taken as indicating inter-specific sequence divergence. Virus recombination has been demonstrated between different strains of the same virus species as well as between different species from the same genus. Nevertheless, the probability of a recombinant surviving decreases with increasing genetic distance between the recombining sequences.

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• The phenotypic consequences of a virus recombination event, even between

“homologous” isolates, cannot be predicted. There are potentially many outcomes. However, the most likely outcome is the failure of a recombinant to survive in the host plant.

• There is no evidence for plant DNA being taken up by RNA viruses although

there is some evidence that viral DNA has been taken up by plant genomes (cf banana streak virus sequences in banana).

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Identifying experimental systems for risk research A great deal of consideration by participants was given to identifying experimental systems available in Australia to address the research questions raised. Three types of viral-derived sequence were considered: viral-derived gene promoter sequences, expressing viral transgenes (e.g. virus capsid protein genes) and non-expressing viral transgenes developed on gene silencing approaches. Experimental systems appropriate to each of these three categories were scored against various criteria as a means of assessing their relative suitability for addressing the research topics outlined in Table 1. The experimental models and their scores are shown in Tables 2 and 3 for viral promoter sequences and expressing/ non-expressing viral transgene sequences respectively. The term ‘virus challenger’ refers to a virus capable of infecting the transgenic plant. The key point is that it must have sufficient homology to the viral promoter/ transgene sequence. Table 2. Viral-derived transgene promoter sequences, transgenic plants and appropriate virus challengers available in Australia for risk research. CaMV= cauliflower mosaic virus; BSV= banana streak virus; SCSV= sub-clover stunt virus, and BBTV= banana bunchy top virus Promoter

Transgene(s)

Target plants

Plants/ promoters available

Virus challenger

CaMV 35s

many types

many types

Many types

BSV

BSV

GUS, GFP, Invertase

Banana, sugarcane, tobacco

BSV in sugarcane

CaMV

SCSV

Virus resistance genes, GUS, GFP, HPT, BAR, Bt, SSA

cotton, tobacco, white clover, sugercane, rice, potato, lupins, lucerne

SCSV in banana

BBTV

BBTV

Virus res genes, GUS, GFP, NPT2, “novel constructs”

Bananas, sugarcane,sorghum, tobacco, papaya

BBTV in subclover

SCSV

GFP = green fluorescent protein (report gene); GUS = β-glucuronidase (reporter gene); BAR = BASTA herbicide resistance; HPT = hygromycin resistance; Bt = Bacillus thuringiensis (bacterial gene encoding an insecticidal protein); SSA = sunflower albumin

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Table 3. Suitability of VRTP experimental systems based on expressed and non-expressed viral-derived transgenes available in Australia for risk research. Ratings (1=low suitability) are shown for the level of virus resistance achieved, the relative potential for genetic recombination and the degree of experimental flexibility afforded by the VRTP-virus system.

Virus origin

Construct

Host plant

VRTP resistance level (1-5)

Recomb’n potential (1-5)

Exp’tal flexibility (1-3)

Release stage

PVY (potyviridae)

Hairpin (sense X anti-sense)

Tobacco/ potato

5 2 3 Glasshouse,Field

PRSV (potyviridae)

Hairpin (sense)

Papaya 5 2 1.5 G and F

BYDV (luteoviridae)

Hairpin Barley 5 3 1.5 G and F

AMV/ WCMV (tricornoviridae)

Coat protein (expressed)

Alfalfa/ White clover

5 5 3 G and F

CYVV (potyviridae)

Coat protein (expressed) and Hairpin (sense)

White clover 5 2 3 G

BBTV (Babuviridae)

Mutant Banana 2 ? 1 G

SCMV (potyviridae)

Hairpin (sense)

Sugarcane 5 2 2 G and F

FDV (reoviridae)

Hairpin (sense)

Sugarcane 2 ? 0.5 G

CMV (tricornoviridae)

Hairpin (sense)

Lupin 5 4 1.5 G and F

Abbreviations used: PVY=potato virus Y; PRSV=papaya ringspot virus; BYDV=barley yellow dwarf virus; AMV=alfalfa mosaic virus; WCMV=white clover mosaic virus; CYVV=clover yellow vein virus; BBTV=banana bunchy top virus; SCMV=sub-clover mosaic virus; FDV=Fiji disease virus; CMV=cucumber mosaic virus; CCMV=cowpea chlorotic mottle virus

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RECOMMENDATIONS

In the final discussion session the workshop participants agreed on the following recommendations:

1. The priority question to address is “Does the presence of viral-derived sequences in transgenic plants lead to an increase in the frequency of virus recombinants compared to that arising from virus-virus interaction in non-transgenic plants?” The design of experiments to address this question should reflect the important distinction between frequency of recombination and probability of survival (detection) of recombinants.

2. This question should be addressed for two functional classes of viral

sequence used in transgenic plants: viral-derived promoters used for driving the expression of transgenes in GM plants and viral-derived transgenes used specifically for conferring disease resistance in virus resistant transgenic plants (VRTPs).

3. For VRTPs two types of transgenic plant should be assessed: those

expressing functional proteins e.g. the capsid protein gene, and those transformed with non-coding RNA viral sequences developed on gene silencing approaches.

4. The choice of experimental system for each of these categories was

determined using several criteria (Tables 2 and 3) including: relevance to Australia, potential for recombination based on sequence homology of virus challenger and the viral transgene, virus challengers available for use in Australia, and the overall experimental flexibility afforded by the experimental system. The following systems were identified as the best candidates for each category of viral transgene.

a. PROMOTERS: The banana streak virus promoter (BSV) in sugarcane

challenged with the cauliflower mosaic virus (CaMV) or any one of several plants containing either the sub-clover stunt virus promoter (SCSV) or the banana bunchy top virus promoter (BBTV) and challenged with the reciprocal virus i.e. the BBTV promoter challenged with SCS virus/ the SCSV promoter challenged with the BBT virus.

b. EXPRESSING TRANSGENES: white clover expressing the alfalfa

mosaic virus (AMV) and clover yellow vein virus (CYVV) coat protein genes.

c. NON-EXPRESSING TRANSGENES: potato virus Y ‘hairpin’ sequences

either in potato or tobacco.

5. The design and execution of risk assessment studies should be based on a collective approach involving a “multi-node” arrangement whereby

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experiments are repeated across several laboratories and organizations1 (though not necessarily simultaneously). The rationale for this is that given the sensitive nature of this area results generated from experiments conducted by only one organization is likely to be regarded with scepticism particularly if that organization had commercial interests in the technology under assessment. Repeating key risk experiments across unrelated organizations was regarded as a sensible approach for this work.

6. Risk assessment studies should be costed according to the experimental

design collectively deemed to be the minimum necessary to adequately address this question.

1 The identity of organizations has not been confirmed.

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Annex The event sequence below was presented to workshop participants on day 1. The

scenario begins with a viral recombination event and finishes with a specified

ecological impact or ‘end-point’. Workshop participants were asked to critically

evaluate and redefine this event sequence. The result of this process is presented in

Figure 1.

tr

Replication of rVirus and acquisition by an insect

vector

Infection by a virus that recombines with the viral

transgene

Virus resistant transgenic plant (VRTP) with a viral-derived transgene insert

Vector disperses: ansmission of rVirus to non-target plant

species

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END-POINT: The rVirus establishes as a highly pathogendisease in one or more environmental species

ic

A recombinant virus with a novel pathology

(highly pathogenic/ wide host range)