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

EU-US ‘HoneySweet’ working group

• 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
• 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
• f developing HoneySweet PPV resistant plum
• Our experiences with the regulatory

approval process in the U.S.

• Regulatory approval in the EU?
• Public acceptance

PPV Symptoms

• Fruit deformation

and reduced quality

• Premature fruit drop
• Leaf chlorosis
• Tree decline in severe

infections

The need for PPV resistance

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%

• f 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.

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

PPV infected buds Transgenic plum plant

Greenhouse tests

• ver 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 A natural virus resistance mechanism

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

1 2 3

Vaccine - a portion of the pathogen's structure that upon administration stimulates immunity against the pathogen but is incapable of causing infection.

Pathogen-derived resistance in animal systems is represented by familiar and highly effective vaccines

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

Effectiveness of HoneySweet

EXAMPLE: Temporal spread of Plum pox virus - Romania 10 20 30 40 50 60 1998 2000 2004 2005 2006

years

% PPV

conventional C5

Additional tests: Graft inoculation of ‘HoneySweet’ with PPV

PPV infected plum tree HoneySweet tree PPV infected bud

HoneySweet graft inoculum (PPV-Rec) Year 2 Year 5

Polak et al., 2008

Czech Republic Field Test - Graft inoculation

PPV inoculum outgrowth

‘HoneySweet’ tree

 - 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’ is well-known in Europe and has been included in the following EU – funded projects:

‘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.

Destroy prunings

APHIS Field test regulations

Harvest and destroy all fruit not analyzed in the lab

Monitoring pollen flow from GE plum trees 1999-2010 (>12,000 seeds)

1060 m 510 m

N

• 390 m

600 m 73 m 450 m 880 m 740 m

GE plum block

Plum trees planted at various distances to monitor gene flow

Gene Flow - Co-existence

HoneySweet flowers Non-GE plum

The PPV-CP gene can only enter the embryo

• f the seed.

Non-GE plum flowers

50% of the ‘HoneySweet’ Pollen will carry the inserted PPV-CP gene

GE Non-GE

50% chance

• Seeds are usually destroyed
• Plums are not planted by seed
• Seedlings are killed by herbicides

and cultivation

• If used as rootstocks trees do not

produce fruit

2003 2004 2005 2006 2007 2008 2009 2010 2011

APHIS FDA EPA

Oct Aug Sept

submitted

Feb Nov Mar April Oct

U.S. Regulatory Time-Line

June

submitted resubmitted submitted

June

deregulated

Jan

cleared

August Registered

Oct

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)

1000 2000 3000 4000 5000 6000 7000 8000 mg GAE/Kg dry weight

Average Phenolics

Stanley Jojo HoneySweet Others 10 20 30 40 50 60 70 % by dry weight

Average Total Sugars

Stanley JoJo HoneySweet Others 10 20 30 40 50 60 mg per 100 dry grams

Average Vitamin C

Stanley JoJo HoneySweet Others

Fruit Compositional Studies

Food Safety and Healthfulness of HoneySweet

A total of ~8.02 x 1010 bases. Coverage is ~120X.

Sequencing of the ‘HoneySweet’ genome

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!

Leaf HoneySweet ‘Stanley’ Fruit

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).

Does the insert interrupt genes or affect flanking genes?

Average mRNA Sequencing Reads per 1,000,000 Reads (RPM) of mRNAs Fruit Stanley Infected Stanley HoneySweet PPV 4.1 20,882 15 PPV-CP 0.75 5,812 11 UidA 0.25 0.22 174 NPTII 0.01 3.2 PBR322 0.01 0.45 Are virus genes are being expressed in HoneySweet?

sRNA production in HoneySweet

HoneySweet hairpin Infected Stanley

Results of Environmental Risk Studies

Zagrai et al. 2011 Transgenic plums expressing Plum pox virus coat protein gene do not assist the development of virus recombinants under field conditions. Journal of Plant Pathology No virus recombinants Capote et al. 2007 Risk assessment of the field release of transgenic European plums susceptible and resistant to Plum pox virus ITEA

No effects on non-target insects

Capote et al. 2008 Assessment of the diversity and dynamics of Plum pox virus and aphid populations in transgenic European Plums under Mediterranean conditions Transgenic Research

No effects on aphid populations No effects on virus diversity

Zagrai et al. 2008 Plum pox virus silencing of C5 transgenic plums is stable under challenge inoculation with heterologous viruses. Journal of Plant Pathology

No breakdown of resistance in presence of other Prunus viruses Gene flow is low (Scorza et al PLOS ONE in press)

Sum Summar mary

None of the analyzed features of ‘HoneySweet’ suggest safety issues for cultivation or consumption.

This work would be impossible without the work of European collaborators

Shouldn’t European growers and consumers also benefit from ‘HoneySweet’? ‘HoneySweet’ International Working Group

Those outside the U.S. wishing to exploit ‘HoneySweet’ will be responsible for obtaining the required regulatory clearances.

Qualities- Sweet (21% sugar), large size (60 g). A good variety for the fresh market. Useful for breeders to develop new PPV resistant varieties

Czech Republic 2002- present Spain 1996-present Poland 1996-2007 Romania 1996-2006 , 2012 -

Based on over 15 years of study in Europe and the U.S. the ‘HoneySweet’ working group, headed by scientists at the Crop Research Institute, Czech Republic, is developing a dossier to submit to EFSA

Public Acceptance

• 2007 APHIS received 1,725 comments (1,708

negative). Most negative comments were received as cut and paste comments from a single anti-GMO website.

• 2010 EPA received 78 comments of which 76

were positive.

An opportunity for public research

Public institution development favored: “Please allow this genetically modified product to be marketed freely. The public will accept this disease resistant product and it will promote public discussion of genetically modified products.” “My primary concern with GM food lies with the industry behind their

production……….. “

“Good work on coming up with new virus resistant fruit! Definitely going to make it easy for my family to keep eating healthy (and at a reasonable price!)” “As long as the new, resistant plum trees will be available to growers without

financial strings attached …..then I welcome this development.”

PUBLIC ACCEPTANCE - excerpts of comments received

Population growth and food supply is a concern: “Am in favor of this genetically-engineered strain. All our foods are “engineered” by humans.

Modern techniques are merely more efficient, and it’s this efficiency which is needed more than ever, given our population pressures.”

“I am a private citizen who is for using science to improve crops to feed the

large amount of people in the world. Please plant the resistant plum.”

“I would gladly devour a plum that produced a coat protein that protects from the plum pox

• virus. Please keep the public informed of further positive research into genetic

engineering being used to help mankind.”

More Comments

The technology has a good safety record: “This shouldn’t be controversial. Approve the genetically engineered plum plant. No ill effects have been seen in other genetically engineered crops.” “Do it. Genetic engineering may sound scary, but a lot of very smart

People have spent much of their time making it work safely.”

Recognized as an update of breeding technology:

“All of agriculture and husbandry since we came out of the fertile crescent has been “genetic engineering”. “Genetic modification of food is simply a more efficient method of what plant breeders have done for millennia.”

More Comments

GMO technology can help solve the problems that will be facing Europe, the U.S. and the rest of the world including climate change, the spread of invasive species, and population pressures for increased food production. Wisely used, genetic engineering can be an important tool to help meet these challenges.

‘HoneySweet’ International Working Group

Academy of Medicine, France American Medical Association American Society for Plant Biology Brazilian Academy of Science Chinese Academy of Science Food and Agriculture Organization Indian National Science Academy Mexican Academy of Science Pontifical Academy of Sciences Romanian Academy Romanian Academy of Agricultural and Forestry Sciences Royal Society of London Third World Academy of Science U.S. Department of Agriculture U.S. National Academy of Sciences U.S. National Research Council World Health Organization and others…….

Scientific Institutions expressing positive positions on GMO Crops