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Laboratoire de Biologie CellulaireINRA

Plant Virus Evolution and Biosafety Group

Overall strategy:All biotechnology projects should include:

• Creation of the GMO and evaluation of its effectiveness

• Evaluation of the potential risks associated with the GMO

• Evaluation of the advantages and disadvantages of its deployment compared to other possible solutions (“precautionary approach”)

Viral diseases can have a major impact on plant productivity

Grapevine fanleaf virus on wine grapes

Plum pox virus infection of apricot

Strategies for creating virus-resistant transgenic plants that have been tested in Versailles

•Satellite RNA (CMV)

•3’ non-coding viral genomic RNA (TYMV)

•Coat protein gene (CMV, LMV, PVY, PFBV)

•Mammalian 2’,5’-oligoadenylate synthase (PFBV)

•Schizosaccharomyces pombe Pac1 RNase (PFBV)

(specifically degrades dsRNAs)

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The concept of pathogen-derived resistance

It is possible to interfere with the development of a pathogen by causing the host to synthesize a pathogen gene product, RNA or protein:

- at an inappropriate time- in an inappropriate amount- in modified form

Sanford and Johnston (1985)

Squash and pepper infected by CMV

Tomato infected by a necrogenic strain of CMVPepper infected by CMV

Maize infected by CMV Electronmicroscopic view of CMV virions

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helicaseRNA 1

RNA 2 polymerase

2bRNA 4a

movementRNA 3

coatRNA 4

Structure of the cucumovirus genome Expression of a CMV coat protein gene in tobaccoconfers resistance to CMV

Expression of the LMV coat protein in tobacco confers resistance to PVY

Expression of the LMV coat protein in oilseed rape confers resistance to TuMV

Expression of the PFBV coat protein in geranium confers resistance to PFBV

TABLE 2a. Authorizations of field tests of virus-resistant transgenic plants in the USthrough July 2001, except for plants expressing a CP gene1

Type of gene Plant Target number oftransformed virus authorizations

Nonstructural viral genesTYLCV replicase tomato TYLCV 2PLRV replicase potato PLRV 89PRSV replicase papaya PRSV 2CMV replicase tomato CMV 1PVY replicase potato PVY 1PVY protease potato PVY 2RBDV MP raspberry RBDV 2TMV MP tomato TMV 1CyMV MP dendrobium CyMV 1PVY NIa potato PVY 4PRSV NIa squash PRSV 3PRSV NIa melon PRSV 3PRSV NIa cucumber PRSV 4PRSV NIb squash PRSV 3WSMV NIb wheat WSMV 1PVY NIb potato PVY 7PRSV NIb melon PRSV 3PRSV NIb cucumber PRSV 4PVY VPg potato PVY 5BYDV ORF 1+2 oat BYDV 1PYDV 17k potato PYDV 6PLRV 17k potato PLRV 3TRV 60k potato TRV 3CMV satellite RNA tomato CMV 1TVMV 42k potato PVY? 2TVMV HC potato PVY? 2TVMV HC tobacco TVMV 1TVMV protease potato PVY? 2TVMV CI tobacco TVMV 1

1 Data extracted from http://www.nbiap.vt.edu/cfdocs/fieldtests3.cfm

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TABLE 2b. Authorizations of field tests of virus-resistant transgenic plants in the USthrough July 2001, except for plants expressing a CP gene1

Type of gene Plant Target number oftransformed virus authorizations

Nonviral genesS. pombe dsRNase wheat several 5S. pombe dsRNase potato several 9S. pombe dsRNase pea several 5tobacco cyt P450 tobacco aphids 1S. pombe dsRNA binding pea PEMV, PYMV 1tobacco N tobacco TMV 3mouse protein kinase potato PLRV, PVY, TRV 1potato pseudoubiquitin potato PLRV, PVY, TRV 2tobacco SAR 8.2 walnut CLRV 11 Data extracted from http://www.nbiap.vt.edu/cfdocs/fieldtests3.cfm

TABLE 1. Virus-resistant transgenic plants authorized for unrestricted use in the USthrough July 2001a

Plant species Gene expressed target virus

squash WMV2 CP WMV2ZYMV CP ZYMV

squash WMV2 CP WMV2ZYMV CP ZYMVCMV CP CMV

potatob PLRV replicase PLRV

potatob PVY CP PVY

papaya PRSV CP PRSVa Data extracted from http://www.nbiap.vt.edu/cfdocs/fieldtests3.cfmb These lines also express a Bacillus thuringiensis endotoxin gene conferring resistance tocolorado potato beetle.

Overall strategy:All biotechnology projects should include:• Creation of the GMO and evaluation of its

effectiveness• Evaluation of the potential risks associated with

the GMO• risk assessment• risk reduction• risk management

• Evaluation of the advantages and disadvantages of its deployment compared to other possible solutions (“precautionary approach”)

VRTP IMPACT

Virus-resistant transgenic plants:

ecological impact of gene flow

Cell factory area 3.2.4

Project QLRT-1999-30361

Dec. 1, 2000 - March 31, 2004

Participants in VRTP IMPACT• Mark Tepfer, INRA-Versailles, France• Mireille Jacquemond, INRA-Avignon, France• René Delon, Institut du Tabac, Bergerac, France• Ervin Balázs, Agricultural Biotechnology Center - Gödöllö, Hungary• Edgar Maiss, University of Hannover, Hannover, Germany• Detlef Bartsch, Aachen University of Technology, Aachen, Germany• Fernando García-Arenal, ETSI-Agronomos, Madrid, Spain• Peter Palukaitis, Scottish Crop Research Institute, Dundee, UK• Alan Raybould, Centre for Ecology & Hydrology, Dorchester, UK• Ian Cooper, Centre for Ecology & Hydrology, Oxford, UK• Erkki Truve, Talinn Technical University, Tallinn, Estonia

Participants in VRTP IMPACT

Gödöllö

Hannover

Madrid

Versailles

Dundee

Oxford

Aachen

Avignon

Bergerac

Talinn

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Basic premises of VRTP IMPACT

Potential durable effects would be mediated by changesin genotype, in particular two types of gene flow :

- plant-to-plant gene flow by sexual outcrossing- plant-to-virus gene flow by recombination

Assume these events will occur, focus on their impact

Compare what occurs in transgenic plantswith what occurs in non-transgenic plants

Plant to plant gene flow by sexual outcrossing

Could gene flow lead to creation of a virus-resistant "super-weed"?

Would a virus resistance gene confer a selective advantage on the plant?If so, what would be the impact of gene flow?

Increased frequency of the gene in the plant population?increased population size (increased weediness)?

But, an increase in fitness will result in an increase in population size only if virus infection contributes strongly to determining population size.

To what should we compare the use of a transgene (presence/absence of a natural resistance gene)?

Plant-to-plant gene flow by sexual outcrossing

From TuMV-resistant Brassica napus to B. rapa

Alan Raybould - NERC Dorchester, UKIan Cooper - Oxford, UK

From BNYVV-resistant sugar beet to hybrid weed beet

Detlef Bartsch - U. Aachen, Germany

Plant-to-virus gene flow by recombination

Cucumovirus (cucumber mosaic and tomato aspermy viruses)

Mark Tepfer - INRA Versailles, FranceMireille Jacquemond - INRA Avignon, FranceErvin Balázs - ABC Gödöllö, Hungary

Potyviruses (potato virus Y, plum pox, zucchini yellow mosaic viruses)

Edgar Maiss - U. Hannover, Hannover, GermanyErvin Balázs - ABC Gödöllö, HungaryPeter Palukaitis - SCRI Dundee, UK

Sobemoviruses and luteovirusesErkki Truve - Talinn Technical University, Tallinn, Estonia

Possible unwanted epidemiological effects of plant-to-virus gene flow by recombination

Recombination could create a virus with novel properties:• worsening of symptoms• changes in other properties, such as host range

But will the recombinant virus have increased fitness? If not, it will disappear, have no impact.

Proposed strategy for evaluation of potential risk associated with recombination

Under conditions of minimum selection pressure, examine thenature of the recombinants produced:

- in singly-infected transgenic plants- in doubly-infected non-transgenic plants

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Strategy applied to the cucumoviruses

Compare two situations:

Non-transgenic plants infected with two related viruses

Tobacco infected with two cucumoviruses:

either CMV and TAV or two strains of CMV

Transgenic plants expressing a CP infected with a related virus

Tobacco expressing a CP-CMV gene infected with TAV

or with another strain of CMV

Overall strategy:All biotechnology projects should include:

• Creation of the GMO and evaluation of its effectiveness

• Evaluation of the potential risks associated with the GMO

• Evaluation of the advantages and disadvantages of its deployment compared to other possible solutions (“precautionary approach”)

Integration of risk assessment in a broader cost-benefit analysis

Compare advantages and disadvantages of different solutions to a problem, with or without GM plants.

- Potential impact can be minimized, but there will always be residual impact (risk always non-zero). What other disadvantages?

- What are the advantages for the environment, farmers, consumers?

Grapevine fanleaf virus : no source of natural resistance.Solutions : nematicides (toxic), bad wine, or transgenics?

Overall strategy:All biotechnology projects should include:

• Creation of the GMO and evaluation of its effectiveness

• Evaluation of the potential risks associated with the GMO

• Evaluation of the advantages and disadvantages of its deployment compared to other possible solutions (“precautionary approach”)

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GMO RES COM

A European initiative to enhance communication

regarding GMO biosafety research

Cell factory area 3.2.5

Project QLAM-2001-00037

Starting date: 01 March 2002

Activities supported by GMO RES COM

• Creation of a database of European GMO biosafety research

• Editorial office of Environmental Biosafety Research

• Support for the European delegation attending the 7th ISBR Symposium in 2002 in China

• Support for hosting the 8th ISBR Symposium in 2004 in France

Official journal of the International Society for Biosafety Research

Dedicated to publishing peer-reviewed research and review articles pertinent to science-based risk assessment of all types of GMOs that are intended for release into the environment.

Activities supported by GMO RES COMSupport for hosting the 8th ISBR Symposium in 2004 in France

• A typical research-oriented Symposium• (6th symposium: http://www.ag.usask.ca/isbr/Symposium/biosafety.html)• (7th symposium: http://www.worldbiosafety.net/index.html)

• An event for contact between researchers and the public

• A North-South workshop

• Invite researchers involved in using GMOs in developing countries

• Identifybiosafety research needs for developing countries

• Favor collaboration with labs with experience in biosafety research

8th International Symposium on theBiosafety of Genetically Modified Organisms

The 8th Symposium is planned to include two novel elements:

A special event for better contact between scientists and the public, which could be in the form of a public workshop.

A special North-South workshop, whose aim will be to mobilize the GMO biosafety research community to identify the GMO-related biosafety research that countries in the South will need in order to develop GMOs for their own uses. The workshop will also help foster better contacts between researchers in North and South.

International Organizing CommitteeChair:Mark Tepfer (ISBR Vice-President), France

Members:Alan McHughen (ISBR President), USAAllison Snow (ISBR Secretary-Treasurer), USAAriel Alvarez-Morales, MexicoKlaus Ammann, SwitzerlandErvin Balázs, HungaryZhangliang Chen, ChinaIoannis Economidis, Belgium (EC)Philippe Feldmann, FranceKen-Ichi Hayashi, JapanJulian Kinderlerer, UKSally McCammon, USATerry Medley, USAThomas Nickson, USAJoachim Schiemann, GermanyKornelia Smalla, Germany

To find out more about the symposium, visit our web site athttp://www.inra.fr/gmobiosafety

Or contact us:E-mail: isbgmo@versailles.inra.fr

Phone:(+33) 1 30 83 37 30 / Fax: (+33) 1 30 87 37 28Post: Sophie Masliah Plant Cell and Molecular Biology, INRA-Versailles

78026 Versailles Cedex, France

An International Symposium on The Biosafety of Genetically Modified Organisms (GMOs) has been held biennially, to address the scientific basis for biosafety associated with GMOs. The Symposium series is designed for senior scientists, policy makers, regulators, environmentalists and industry representatives involved in GMO field releases.

International Society for Biosafety Research

September 26 - 30, 2004, Montpellier, France

Conclusions

The potential positive impact of biotechnology is immense

Biosafety research is an integral part of developing GMOs

Biosafety research can be extremely time consuming and expensive

But, a community of GMO biosafety research exists

Developing contries should take full advantage of existing biosafety results

Concentrate their own efforts on the few areas of specific research

Participate in joint research efforts wherever possible