Genetic Engineering of Plum Pox Virus Resistance - Honeysweet Plum

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 C. Dardick, USA EU-US ‘HoneySweet’ working group

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 PPV Symptoms The need for PPV resistance -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- mechanism mediated insertion nucleus PPV-CP PPV-CP

cytoplasm

2 1 ds RNA Double stranded RNA is naturally destroyed by being cut into small PPV 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 degrades the virus PPV degradation 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

0 1998 2000 2004 2005 2006

conventional years C5 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)

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

HoneySweet flowers Non-GE plum flowers 50% of the ‘HoneySweet’ Non-GE plum Pollen will carry the inserted The PPV-CP gene can only enter the embryo PPV-CP gene of the seed.

- Seeds are usually destroyed 50% - Plums are not planted by seed chance

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

Oct Aug Feb Nov Mar June Sept April deregulated resubmitted submitted

FDA

Oct submitted Jan cleared

EPA

June Oct August submitted revised Registered 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

70 Average Total Sugars 60

Stanley 50 JoJo 40 HoneySweet 30 Others

20 % by dry weight dry by % 10 0 Average Phenolics

8000

7000 Stanley 6000 Jojo 5000 HoneySweet 4000 3000 Others Average Vitamin C

2000 60

mg GAE/Kg dry weightGAE/Kgdry mg 1000 50 Stanley 0 40 JoJo

30 HoneySweet

20 Others

mg per 100 dry grams dry 100 per mg 10

0 Sequencing of the ‘HoneySweet’ genome

 A total of ~8.02 x 1010 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). Are virus genes are being expressed in HoneySweet?

Average mRNA Sequencing Reads per 1,000,000 Reads (RPM) of mRNAs

Fruit Infected Stanley HoneySweet Stanley PPV 4.1 20,882 15

PPV-CP 0.75 5,812 11

UidA 0.25 0.22 174

NPTII 0 0.01 3.2

PBR322 0 0.01 0.45 sRNA production in HoneySweet

HoneySweet hairpin

Infected Stanley

Fig. Mapping of sRNA reads from sN047-050 F St ES I 2.3 to full genomes (9876 nt) of PPV-D and PPV-M. 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) Summary

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 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

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

Spain 1996-present 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 ACCEPTANCE - excerpts of comments received 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.”

More Comments 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.”

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.” More Comments

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.” 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 Scientific Institutions expressing positive positions on GMO Crops

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…….