What Makes Xylella fastidiosa Virulent?

1. What Makes Xylella fastidiosa Virulent?

2. Identity of Xf virulence gene >50 pil , fim Associated Genes What Makes Xylella fastidiosa Virulent?

3. Xf Strains Gene Mutant ORF Gene Product Presence in Other WT (Temecula isolate) pilB 1A2 PD1927 Type IV pili assembly protein PilB yes pilQ 5A7 PD1691 Type IV pili assembly protein PilQ yes fimA 6E11 PD0062 Type I pili (shaft) subunit yes pilX1 20D10 PD0022 Type IV pili assembly protein PilX yes pilO TM5 PD1693 Type IV pili assembly protein PilO yes pilC TM6 PD1923 Type IV pili assembly protein PilC yes pilR TM7 PD1928 Type IV pili expression regulatory protein yes pilY1 TM14 PD0023 Type IV pili tip-associated protein PilY yes pilL TM25 PD0848 Type IV pili sensor histidine kinase regulator/ yes chemotaxis-related protein kinase chpY 3E10 PD1671 Type IV pili response regulator PilH yes fimA/ DM12 PD0062/ Fimbial subunit precursor/ pilO PD1693 Fimbrial assembly membrane protein fimA/ DM16 PD0062/ Fimbrial subunit precursor/ pilX PD0022 PilX protein Identity of Xf virulence gene >92% identity with Almond, Oleander, CVC 9a5c

4. Type I and type IV pili, and associated proteins, glycoproteins Localization of gene product Type IV pili Type I pili (fimbriae)  Type IV pili Type I pili

5. Gene Mutant WT (Temecula isolate) pilB 1A2 pilQ 5A7 fimA 6E11 pilX1 20D10 pilO TM5 pilC TM6 pilR TM7 pilY1 TM14 pilL TM25 chpY 3E10 fimA/ DM12 pilO fimA/ DM16 pilX Localization of gene product WT type I and IV pili pilQ type I pili only fimA type IV pili only fimA, pilQ no pili

6. Localization of gene product and function Prepilin leader sequences cleaved by PilD. Processed PilA assembled on a base of minor pilins (PilE, V, W, X, and FimU) by action of cytoplasmic membrane protein PilC and the NTP-binding protein PilB. Pilus extruded through outer membrane via pore composed of multimeric PilQ, stabilized by the lipoprotein PilP. Pili are retracted by action of PilT (aidedby PilU). JS Mattick. Annu. Rev. Microbiol. 2002. 56:289 Type IV pili assembly and retraction Pili labeled with amino-reactive Cy3 Skerker and Berg, 2001 PNAS 98:6901

7. Localization of gene product and function Type IV pili - Motility (twitching, rafts, migration) - Adhesion - Cell aggregration Type I pili - Adhesion - Cell aggregration - Biofilm formation - Biofilm formation No type IV pili Pili poles

8. Localization of gene product and function Type IV pili - Motility (twitching, rafts, migration in vitro ) - Adhesion - Cell aggregration Type I pili - Adhesion - Cell aggregration - Biofilm formation - Biofilm formation 20  m motility Media flow

9. Localization of gene product and function Type IV pili - Motility (twitching, rafts, migration in vitro ) - Adhesion Twitching motility—rates - Cell aggregration Type I pili - Adhesion - Cell aggregration - Biofilm formation - Biofilm formation Media flow 0.7 μm min -1 0.2 μm min -1 4.4 μm min -1

10. Localization of gene product and function Type IV pili - Adhesion - Motility (twitching, rafts, migration in planta ) - Cell aggregration Type I pili - Adhesion - Cell aggregration - Biofilm formation - Biofilm formation

11. Type IV pili - Adhesion - Motility (twitching, rafts, migration) Adhesion force—pN - Cell aggregration Type I pili - Adhesion - Cell aggregration - Biofilm formation - Biofilm formation Localization of gene product and function Media flow 147 pN 204 pN 119 pN pilB fimA

12. Type IV pili Type I pili - Adhesion - Adhesion - Cell aggregration - Biofilm formation - Motility (twitching, rafts, migration) - Cell aggregration - Biofilm formation Phenotype of mutants in vitro pilB fimA, pilO WT & f imA WT & f imA

13. Type IV pili Type I pili - Adhesion - Adhesion - Cell aggregration - Biofilm formation - Motility (twitching, rafts, migration) - Cell aggregration - Biofilm formation Phenotype of mutants in vitro pilB fimA, pilO f imA WT

14. Gene Mutant WT (Temecula isolate) pilB 1A2 pilQ 5A7 fimA 6E11 pilX1 20D10 pilO TM5 pilC TM6 pilR TM7 pilY1 TM14 pilL TM25 chpY 3E10 fimA/ DM12 pilO fimA/ DM16 pilX Phenotype of mutants in planta WT fimA fimA/pilO 3 months post inoc

15. Gene Mutant WT (Temecula isolate) pilB 1A2 pilQ 5A7 fimA 6E11 pilX1 20D10 pilO TM5 pilC TM6 pilR TM7 pilY1 TM14 pilL TM25 chpY 3E10 fimA/ DM12 pilO fimA/ DM16 pilX Phenotype of mutants in planta WT fimA fimA/pilO 7 months post inoc

16. Potential of the gene(s) as target for mitigation of PD fim / pil genes... DSF 1 citrus sap 2 fimT fimA pilA2 pilF pilM pilI pilT pilU pilY1 … decreased twitching motility Expression of 1 Lindow lab; 2 Cooksey lab