1. The document describes a strategy to control the plant pathogen Xylella fastidiosa using pathogen confusion by expressing its DSF synthase gene rpfF in transgenic grape plants. 2. Transgenic grape plants expressing rpfF were more resistant to Pierce's disease compared to wild-type grape when challenged with X. fastidiosa. 3. Expression of rpfF and the accessory gene rpfB together in Arabidopsis enhanced DSF production and reduced symptoms caused by a DSF-overproducing strain of Xanthomonas campestris more than rpfF alone.

1. Pathogen confusion as a strategy for controlling diseases caused by Xylella fastidosa Steven Lindow Department of Plant and Microbial Biology University of California, Berkeley

4. X. fastidiosa Decreases Virulence by Coordinating Virulence Genes in Cell Density-Dependent Fashion Wild type rpfF mutant

5. Key Virulence Genes Controlled by DSF Downregulated: Type IV pili Polyglacturonase Cellulase Meng et al. 2005 Upregulated: Hemagglutinin adhesins Extracellular polysaccharides ( EPS): gum Type I pili Type IV pili

6. Wild Type - gfp rpfF - gfp 20 µM The RpfF- mutant of Xylella fastidiosa colonizes many more xylem vessels than the wild-type strain

7. Wild type RpfF mutant RpfF- mutant tends to fill xyllem vessels more frequently than wild-type strain of Xylella fastidiosa

8. Over-expression of RpfF in Xylella fastidiosa reduces the movement of the pathogen in the plant and limits disease to site of inoculation

9. Distance of migration (cm) Log cells/g Weeks after inoculation Symptomatic leaves/plant RpfC mutant of X. fastidiosa over-produce DSF and are less virulent since it does not move well within grape

10. Gum Production Plant colonization phase Insect acquisition phase Extensive vessel colonization Low cell numbers in most vessels Disease symptoms may not be present Some vessels have high cell numbers Disease symptoms may be present Further multiplication in crowded vessels slows DSF Abundance Stickiness to Surfaces Expression of adhesins Twitching Motility Type IV pili Pit Membrane Degradation Pgl and Eng expression

11. The goal : Production of DSF in transgenic grape The approach : Expression of rpfF encoding DSF synthase from Xylella fastididosa The strategy : Express RpfF in different cellular locations and with different accessory proteins

12. Strategy 1: Express un-targeted Rpf 35S rpfF pCAMBIA 1390 Results: Very modest production of DSF At threshold of detection in grape, Arabidopsis, and tomato

13. 35S SSU rpfF C-DNA encoding the RUBISCO small subunit N-terminal was isolated from Arabidopsis leaf RNA In-frame fusion with rpfF gene from Xylella fastidiosa Strategy 2: Expression of chloroplast-targeted RpfF Results: Enhanced DSF production in Arabidopsis, tobacco and Tomato Transformation of grape underway MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQ VWPPIGKKKFETLSYLPDLTDSELAE F MSAVQPFIRTNIGSTLRIIEEPQRDVYWIHMHA DLAINPGRACFSTRLVDDITGYQTNLGQRLNTAGVLAPHVVLASDSDVFNLGGDLALFC QLIREGDRARLLDYAQRCVRGVHAFHVGLGARAHSIALVQGSALGGGFEAALSCHTIIA EEGVMMGLPEVLFXLFPG SSU-rpfF WT-tomato Xf-DSF

14. Strategy 3: C0-expression of chloroplast-targeted RpfF and RpfB Logic: The role of RpfB in DSF synthesis is not clear but it seems to be an “accessory” protein that may help supply needed substrate for RpfF 35S SSU-rpfF nos 35S SSU-rpfB nos 35S gus nos Results: Somewhat higher expression of DSF in Arabidopsis compared to SSU- rpfF construct Transformation of grape is underway

15. Phenotype of transformed, uninoculated plants: Normal

16. Method for challenging with Xylella fastidiosa Stem droplet-needle puncture Vector inoculation may be superior in that it delivers fewer cells to fewer vessels, but it has been unfeasible since transgenic plants have required insecticide sprays to defend against pests in greenhouse

17. Plant Genotype Symptomatic leaves/plant Grape transformed with rpfF gene from Xylella fastidiosa and producing DSF are much more resistant to Pierce’s Disease compared to wild-type grape

18. Disease severity from topical application of Xanthomonas campestris strains varying in DSF production to Arabidopsis transformed with rpfF or with both rpfB and rpfF Arabidopsis genotype Xcc strains Wild type rpfF- Col (WT) ++++ - rpfF transformed ++++ + rpfF & rpfB transformed ++++ ++

19. Distance of migration (cm) Log cells/g Studies of the movement and titer of Xylella fastidiosa in transgenic grape is underway, but we expect both to be reduced based on the observation of disease symptoms that are limited to near the point of inoculation in RpfF- expressing plants and the reduced growth/movement of DSF-overproducing Xylella fastidiosa mutants

20. Susceptible Cabernet sauvignon have been grafted onto transgenic rpfF-expressing rootstocks to test for mobility of DSF within plants

21. 1. Rationale for achieving transgenic protection against Xf 2. Introduced DNA construction, its construction, and its expected affects 3. Observed phenotype of the transformed, uninoculated grapevine 4. Xf challenge method described and compared to vector inoculation 5. Comparison of challenged transgenic and non-transgenic grapevine 6. Xf accumulation (titer) and localization in transgenic vs non-transgenic grapevine 7. Assessment of the value and applicability of the transgenic approach taken Session 4 Panel Discussion Can Transgenic Research Mitigate Pierce’s Disease? Panel:, David Gilchrist, Abhaya Dandekar, John Labavich, Steven Lindow