From concept to product - Co-developed by U.S. and European partners - PowerPoint PPT Presentation

Genetic engineering of plum pox virus resistance - HoneySweet Plum – From concept to product - Co-developed by U.S. and European partners - Field tested in Europe for over 15 years - Solely the work of publicly-supported scientists, at public research institutions – For the benefit of growers and consumers Ralph Scorza, Ann Callahan, Chris Dardick USDA-ARS Michel Ravelonandro INRA Jaroslav Polak, Czech Republic Tadeusz Malinowski, Poland Ioan Zagrai, Romania Mariano Cambra, Spain

‘HoneySweet’ R & D collaborators M. Ravelonandro, France J.M. Hily, France J. Polak, Czech Republic* J. Kundu, Czech Republic M. Cambra, Spain I. Zagrai, Romania T. Malinowski, Poland A. Atanassov, Bulgaria I. Kamenova, Bulgaria S. Paunovic, Serbia S. Dolgov, Russia H. Prieto Chile R. Scorza, USA A. Callahan, USA EU- US ‘HoneySweet’ working group C. Dardick, USA Black Sea Biotechnology Association and others………. *blue type indicate scientists who have conducted ‘HoneySweet’ field tests

To be covered: - An overview of the 20 year EU-US process of developing HoneySweet PPV resistant plum - Our experiences with the regulatory approval process in the U.S. - Regulatory approval in the EU? - Public acceptance

The need for PPV resistance PPV Symptoms -Fruit deformation and reduced quality -Premature fruit drop -Leaf chlorosis -Tree decline in severe infections

PPV infection in selected European countries: Serbia - 42 million bearing plum trees; 58% are infected with PPV Croatia - 2004 survey found 51% of sampled plum trees infected Bosnia-Herzegovina - up to 41% of plum trees infected. Bulgaria - infection in plums 62% Romania – infection in plums 70% Moldova – yield loses in plum 16-48% Czech Republic - in the last 30 years, the average fruit yield has reduced by 80% and the number of plum trees reduced from 18 million to 4 million. Greece – processed apricot production decreased from 35% to 13% of world production due to PPV Spain - 2.3 million PPV-infected trees removed between 1989 and 2006 at a cost of over 63 M Euros 2006 OEPP/EPPO 36 (2) Zagrai et al UASMV 67 2010

Few sources of readily usable highly resistant Prunus germplasm are available. Resistance multigenic, strain specific. In collaboration with European partners, beginning with Dr. Michel Ravelonandro at INRA, France in 1989 we Initiated a project to produce PPV resistant stone fruits, beginning with plum.

The strategy of pathogen- derived resistance was selected as the most promising for resistance development. The PPV coat protein (CP) gene had been cloned by M. Ravelonandro (INRA).

The gene insert contained the PPV-CP gene genes for selection (NPTII and GUS) This genetic insert is the same used to transform papaya (approved in the US, Canada, Japan) using a the PPV coat protein gene in place of the PRSV gene. EFSA classifies NPTII for unlimited use, can continue being used in GM plants.

Transgenic plum plant PPV infected buds The transgenic shoots were rooted, planted in a greenhouse, then inoculated with buds from P. tomentosa infected with PPV.

Greenhouse tests over several years both in the U.S. and in Bordeaux, France indicated that one clone, C5, was highly resistant to plum pox virus.

Pathogen derived resistance through RNA silencing An inverted repeat of the PPV- CP gene (hairpin) formed A natural virus resistance naturally, likely during Agro- mediated insertion mechanism PPV-CP PPV-CP nucleus cytoplasm 1 2 ds RNA Double stranded RNA is naturally PPV destroyed by being cut into small pieces call siRNA RISC complex ds RNase DICER nuclease siRNA with PPV-CP sequences attaches 3 to infecting plum pox virus and the natural defense system PPV degradation degrades the virus

Pathogen-derived resistance in animal systems is represented by familiar and highly effective vaccines Vaccine - a portion of the pathogen's structure that upon administration stimulates immunity against the pathogen but is incapable of causing infection.

Effectiveness of HoneySweet In over 15 years of field testing in Europe no ‘HoneySweet’ trees have been naturally Infected with PPV by aphids

EXAMPLE: Temporal spread of Plum pox virus - Romania % PPV 60 50 40 30 20 10 0 1998 2000 2004 2005 2006 conventional C5 years

Additional tests: Graft inoculation of ‘HoneySweet’ with PPV HoneySweet tree PPV infected plum tree PPV infected bud

Czech Republic HoneySweet graft inoculum Field Test - (PPV-Rec) Graft inoculation ‘HoneySweet’ tree Year 2 Year 5 PPV inoculum outgrowth Polak et al., 2008

‘HoneySweet’ is well -known in Europe and has been included in the following EU – funded projects:  - FP4-Bio4CT-960773: Risk assessment of genetic engineering woody plants expressing virus coat protein gene (1996-1999)  - FP5-QLK3-2002-024:Environnmental impact assessment of transgenic grapevines and plums on the diversity and dynamics of virus population (Transvir: 2003-2006)  - FP7-204429: Sharka containment in view of EU expansion (Sharco: 2008-2012)  -FP7-269292: Intercontinental and temporal research studies on transgene engineered plants (Interest: 2011-2014) FP7-PEOPLE- 2010- IRSES ‘INTEREST’ (2012 -2015)  Spain Ministerio de Educación y Ciencia grant no. INIA RTA03-099, RTA05-00190 and AGL05-01546  Czech Ministry of Agriculture, grants No. QI101A123  No. 0002700604 and Romanian Research Ministry contract 37/2003

‘HoneySweet’ It is highly resistant to PPV - no trees were ever infected by aphids. Resistant to all PPV strains tested The mechanism of resistance RNA silencing is a resistance natural to plants No PPV-CP produced, eliminating the risk of virus transcapsidation Resistance can easily be transferred to progeny through traditional breeding Fruit quality is excellent – 21-22% sugar, large size (60 g), productivity very good.

U.S. Regulatory Approval Three agencies: Jurisdiction __________________ Animal and Plant Health Inspection Service (APHIS) Safety for Agriculture Food and Drug Administration (FDA) Safety for Food Environmental Protection Agency Safety for Environment

APHIS regulated the field tests of ‘HoneySweet’ in the U.S. All permit documents were routinely checked by APHIS inspectors. All field operations were verified, and the field tests were regularly inspected.

APHIS Field test regulations Destroy prunings Harvest and destroy all fruit not analyzed in the lab

Monitoring pollen flow from GE plum trees 1999-2010 (>12,000 seeds) N GE plum block 73 m 600 m 450 m 390 m 510 m 1060 m 740 m 880 m - - Plum trees planted at various distances to monitor gene flow -

Gene Flow - Co-existence Non-GE HoneySweet flowers plum flowers Non-GE plum 50% of the ‘HoneySweet’ The PPV-CP gene can only enter the embryo Pollen will carry the inserted of the seed. PPV-CP gene - Seeds are usually destroyed 50% chance - Plums are not planted by seed Non-GE GE - Seedlings are killed by herbicides and cultivation - If used as rootstocks trees do not produce fruit

U.S. Regulatory Time-Line 2003 2004 2005 2006 2007 2008 2009 2010 2011 APHIS Feb Nov Mar June Oct Aug April deregulated Sept resubmitted submitted FDA Oct Jan submitted cleared EPA August June Oct Registered submitted revised

Examples of some of the information provided to U.S. regulators: Transformation system Donor genes and sequences Molecular characterization (DNA, RNA, protein) Mechanism of resistance Stability of resistance Inheritance of resistance Gene flow Fruit compositional analyses Allergenicity potential Environmental consequences (including non-target effects)

Food Safety and Healthfulness of HoneySweet Fruit Compositional Studies Average Total Sugars 70 60 Stanley % by dry weight 50 JoJo HoneySweet 40 Others 30 20 10 0 Average Phenolics 8000 mg GAE/Kg dry weight 7000 Stanley 6000 Jojo 5000 HoneySweet 4000 Average Vitamin C Others 3000 2000 60 1000 mg per 100 dry grams 50 Stanley 0 40 JoJo HoneySweet 30 Others 20 10 0

Sequencing of the ‘HoneySweet’ genome  A total of ~ 8.02 x 10 10 bases.  Coverage is ~ 120X .

What We Know from DNA sequencing The location, size, and organization of the inserts. Sequence of the regions flanking the inserts. No DNA from outside the T-DNA borders was inserted No unexpected proteins are produced by the inserts.

What We Know from RNA sequencing PPV-CP RNAs are produced by HoneySweet PPV infected Stanley produce 500-1,000 x more PPV-CP specific sRNA than HoneySweet PPV infected Stanley produces sRNA from the entire PPV genome The sequences of PPV-CP sRNAs are the same. Some, of low abundance, are different in length Humans have been ingesting PPV-CP RNAs as long as PPV has been infecting stone fruits!

Does the insert interrupt genes or affect flanking genes? HoneySweet ‘Stanley’ Fruit Leaf mRNAs sequenced were mapped to the genes flanking insertion event. The genes are indicated by blue arrows at the top of each map. The location and abundance of reads that mapped to C5 Fruit (top left) and C5 Leaf (bottom left) were nearly identical to those of ‘Stanley’ fruit (top right) and ‘Stanley’ leaf (bottom right).