Pathogen confusion as a strategy forcontrolling diseases caused by Xylella fastidosa

Steven Lindow

Department of Plant and Microbial BiologyUniversity of California, Berkeley

X. fastidiosa Decreases Virulence by Coordinating Virulence Genes in Cell Density-Dependent Fashion

Wild type rpfF mutant

Key Virulence Genes Controlled by DSF

Downregulated: Type IV piliPolyglacturonaseCellulase

Meng et al. 2005

Upregulated:Hemagglutinin adhesinsExtracellular polysaccharides (EPS): gumType I pili

Type IV pili

Wild Type - gfp rpfF - gfp

20µM

The RpfF- mutant of Xylella fastidiosa colonizes many more xylem vessels than the wild-type strain

Wild type RpfF mutant

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

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

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RpfC mutant of X. fastidiosa over-produce DSF andare less virulent since it does not move well within grape

DSF Abundance

Gum Production

Stickiness to SurfacesExpression of adhesins

Twitching MotilityType IV pili

Pit Membrane DegradationPgl and Eng expression

Plant colonization phase Insect acquisition phaseExtensive vessel colonization Low cell numbers in most vesselsDisease symptoms may not be present

Some vessels have high cell numbersDisease symptoms may be presentFurther multiplication in crowded vessels slows

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 cellularlocations and with different accessory proteins

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

35S SSU rpfF

C-DNA encoding the RUBISCO small subunit N-terminal was isolated from Arabidopsis leaf RNAIn-frame fusion with rpfF gene from Xylella fastidiosa

Strategy 2: Expression of chloroplast-targeted RpfF

Results:Enhanced DSF production in Arabidopsis, tobacco and TomatoTransformation of grape underway

MASSMLSSATMVASPAQATMVAPFNGLKSSAAFPATRKANNDITSITSNGGRVNCMQVWPPIGKKKFETLSYLPDLTDSELAE F MSAVQPFIRTNIGSTLRIIEEPQRDVYWIHMHADLAINPGRACFSTRLVDDITGYQTNLGQRLNTAGVLAPHVVLASDSDVFNLGGDLALFCQLIREGDRARLLDYAQRCVRGVHAFHVGLGARAHSIALVQGSALGGGFEAALSCHTIIAEEGVMMGLPEVLFXLFPG

SSU-rpfF WT-tomato Xf-DSF

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 constructTransformation of grape is underway

Phenotype of transformed, uninoculated plants: Normal

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 unfeasiblesince transgenic plants have required insecticide sprays to defend against pests in greenhouse

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Grape transformed with rpfF gene from Xylella fastidiosa and producing DSF are much more resistant to Pierce’s Disease compared to wild-type grape

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 ++++ ++

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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 ofDSF-overproducing Xylella fastidiosa mutants

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

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 ResearchMitigate Pierce’s Disease?

Panel:, David Gilchrist, Abhaya Dandekar, John Labavich, Steven Lindow