Rootstock based management of disease in vineyard crops

1. Plant Science Department Rootstock based management of disease in vineyard crops “Using Biotechnology to extend the rootstock concept” Eric Stafne ICABR-AAWE Pre-Conference Workshop: Technology and Innovation in the Grape and Wine Industries Feudi di San Gregorio – Localita’ Cerza Grossa 83050 June 24, 2012, 9:30 am Abhaya M. Dandekar

2. DHS ‘Most Destructive’ List of Plant Pathogens • How do we generate resistance to destructive diseases when none exists in the germplasm? • How do we manage the disease while maintaining production and the livelihood of growers?

3. Key Problem: • Pest and Pathogen Populations and their ability to cause disease • Lower Productivity • Lower Quality • Chemical pesticides control the insect vector but do not target the pathogen directly. • Once the pathogen infects the plants, no treatments are available to eliminate it except vine/tree removal 1900ft radius citrus canker eradication scars

4. Early Disease Detection: • Eradication • Replant with clean stock Disease Triangle for Orchard/Vineyard Crops • Disease/Pest Resistance: • Genetic • Epigenetic HOST Disease/Pest Complex Vector & Environment Pathogen • Chemical Warfare: • Eradicate target pest • Pheromone confusion Disease is the exception rather than the rule!

5. “Cloning” • Varietal vines are clones • Vegetative propagation • Genetic uniformity • All vines are composite genetic systems • Genetically distinct scion • Unique wine quality • Genetically distinct rootstock • Pest/disease resistant • Nutrient or abiotic stress tolerant Varietal vine Eric Stafne Locally Adapted Rootstock “Grafting”

6. American Rootstocks fight the Phylloxera Plague in Europe • Phylloxera is native to N America • Damages the very young roots • Resistance to phylloxera evolved/selected in the natural environment among the wild grapevine relatives Phylloxera feasting on the best wines Europe had to offer

7. Anatomy of the “Graft-Union” • Adhesion of rootstock and scion tissues • Proliferation of a bridge callus at the graft interface • Vascular differentiation across the graft interface allowing the flow of water, nutrients and growth regulators including pathogens • Can we move proteins and siRNA? Rootstock Callus Scion Callus Rootstock

8. Deliver Therapeutics via Rootstocks Strategy: Genetic resistance mediated by transgenic expression of therapeutic proteins and/or siRNA to control the multiplication of pests and pathogens and to counteract their virulence Applications: “Transgenic Rootstocks”: To limit pests and pathogens in grafted scions. “Transgenic trap crop”: Trap crops to reduce pathogen inoculums and to attract the insect vector thus reducing pesticide applications. Therapeutic Proteins

9. Xylella fastidosa (Xf) causes serious diseases Dixon M12 M23 Xf strain Temecula 9a5c Ann-1 Grape: Pierce’s Disease Citrus: Variegated Chlorosis Almond: Leaf Scorch Oleander: Leaf Scorch In many economically important plants

10. Colonization of Xf in the plant xylem and the clogging of the xylem is the primary cause of the disease Plant Vascular System

11. Complex lifestyle of Xylella fastidiosa the causative agent of Pierce’s Disease Symptomatic Asymptomatic Long distance movement Type 4 pili Degradation of pit pore membranes Polygalacturonase (PG) Surface Adhesion/attachment Biofilm formation Vessel clogging Disease symptoms Chatterjee et al., Ann Rev Phytopathol. (2008) 46: 243-271.

12. Testing two Promising Protein-Based Therapeutics • Expression of PGIP to counteract the virulence and movement of the pathogen Xylella fastidiosa • Expression of (Chimeric Antimicrobial Proteins) to improve pathogen destruction and inoculum reduction blocking transmission of PD in the field PGIP Xf PG CAP binds to mopB on Xf surface Goutam Goupta Surface binding Pore formation

13. Interfering with Xylella movement • Pit-pore membranes connect one xylem vessel to another • Pit membranes are made of pectin • Xylella expresses a polygalacturonase (PG) to degrade the pectin • PGIP (polygalacturonase inhibitory protein) inhibits the activity of PG Cook Lindow

14. Leaf symptoms and stem MRI of PGIP expressing Grapevines 45-77 TS Round 3 chi-PGIP 45-77 TS Round 3 chi-PGIP Ana Maria Ibanez, Hossein Gouran

15. Degrading the inoculum: • Express a chimeric antimicrobial protein (CAP) in xylem tissues. CAP binds to bacteria surface then destroys the bacteria

16. Construction and testing of binary vectors for Agrobacterium-mediated transformation expressing a synthetic gene encoding chimeric antimicrobial protein 1 month 2 months 2 months 2 months 2 months Uninfected Control Infected Control 051096-004 051096-003

17. Reduced Leaf Scorching in Transgenic Grapevines expressing CAP Leaf number 8 above the point of inoculation was harvested 10 weeks post infection with 20 million Xf cells

18. Measuring xylem blockage using MRI Transgenic Wild Type Transgenic expressing CAP show less xylem blockage as compared to the wild type controls 10 cm above the site of inoculation with 20 million Xf cells

19. Riverside Yolo Human Sharpshooters Riverside and Armstrong Vineyards Yolo Human Sharpshooters Yolo

20. Xylem Sap from HNE-CecB transgenic lines display antimicrobial activity against Xf

21. Strategies to Control Crown Gall • Contaminated Nursery Stock • Infection of Crown and roots • Young trees do not establish • Tree decline • Loss of productivity • Paradox leading rootstock is very susceptible • Contaminated orchards cannot be replanted Escobar and Dandekar, TRENDS in Plt Sci. 8(8): 380-386 (2003)

22. Wild type T-DNA iaaH ipt iaaM EXPRESSION pDE00.0201 ipt iaaM Maai tpi 35S 35S Dicer siRNA Vctr. 01/6 Vctr. 01/6 Wt Wt 41bp 21bp 01/6 01/6 iaaM ipt WT 01/6 Inhibiting Genetic Colonization of Agrobacterium with RNAi Induced Silencing Crown Gall Cells AUXIN CYTOKININ

23. Walnut: shoot inoculation Arabidopsis: root bundle assay Tomato: stem inoculation Wild-Type Wild-Type Wild-Type Line 01/6 Line 01/33 Line 01/258 Crown Gall Resistance by Silencing Gall Formation Model Systems Escobar et al., PNAS 98: 13437-13442 (2001) Escobar et al., Plant Sci 163: 591-597 (2002)

24. Transgenic Paradox Rootstocks Resistant to Crown Gall Site of Infection Charles Leslie

25. Stacking Resistance to Crown Gall and Nematodes in Walnut Rootstocks -Strong vigor -High yield efficiency -Resistance to pests and disease -Graft compatibility -Transplantability CA Walnut orchards –Nematodes- 90% Crown gall - 85%

26. Pratylenchusvulnus Life cycle of lesion nematode (Dr. G .N. Agrios).

27. Binary vectors used for co-transformation • GFP • noselectable marker gene Binary vector in Agrobacterium rhizogenes used for P. vulnus gene silencing • GUS • nptII selectable marker gene Binary vector in Agrobacteriumtumefaciensused for induction of oncogene silencing Monica Britton

28. Transformed plantlets of J1 genotype with Pv010 gene High PV010 expression Very low PV010 expression Transgenic-nematode damage is significant Transgenic-nematode damage is minimal Lalani Walawage

29. Inhibition of root lesion nematodes in transformed walnut plantlets • Pv010 gene expression level in different J1 transgenic lines in co-transformation Lalani Walawage

30. Why Transgenic Rootstocks? Conventional Transgenic • Disease resistance • No linkage drag • Rapid genotype development • Pest resistance • Secretion of therapeutic proteins • Destroy pathogens • Control their virulence • Synthesis of sRNA • Control Pathogens • Control Pests • No Gene Flow • No Pollen • No Seeds 1) Promote Vigor 2) Stress tolerance -Biotic -Abiotic 3) Mineral nutrition 4) Precocity 5) Dwarfing Can Save an Industry French wine industry was saved from phylloxera with American native rootstocks Rangpur lime rootstocks saved citrus in Brazil from CTV decline on sour orange

31. Benefit to the Industry • Rootstocks in new orchards • Inarched grafts within existing orchards • Trap crops around existing orchards • No transgenic fruit • Reduce pesticide use • Compatible with IPM • Destroys pathogen • Easy to stack resistances In-arch grafting of ‘Rangpur Lime Rootstock’ to save CTV infect trees on ‘Sour Orange Rootstocks’ from ‘quick decline’ in Brazil

32. Acknowledgments Gale McGranahan Chuck Leslie Cristina Davis Weixiang Zhao Alexander Aksenov William Cheung Goutam Gupta LANL Anu Choudhry Paige Pardington Cecilia Aguero George Bruening UCD Luiz Goulart Rodrigo Almeida Raissa D’Souza UCD Soumen Roy Elizabeth Leicht Tim Spann UFL Kim BowmanUSDA-ARS Ute Albrecht Oliver Feihn Kirsten Skogerson Dandekar Lab UCD Sandra Uratsu Matthew Escobar Federico Martinelli Ana Maria Ibanez Hossein Sadeghi-Gouran My Phu Russell Reagan Rafael Nascimento Sarah McFarland Lalani Walawage Mary Lou Mendum Kevin Quach David Dolan Special Services Transformation David Tricoli Metabolomics Valdimir Tolstikov DNA Sequencing Charles Nicolet Bioinformatics Core Dawei Lin Monica Britton Joseph Fass Vince Buffalo Funding Sources Citrus Research Board California Walnut Board UC Discovery FCPRAC/CDRF CDFA-PD UC-PD