Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

What Makes Xylella fastidiosa Virulent? What Makes Xylella fastidiosa Virulent?

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Identity of Xf virulence gene Identity of Xf virulence gene What Makes Xylella fastidiosa Virulent? What Makes Xylella fastidiosa Virulent?

>50 pil, fim Associated Genes

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Xf StrainsGene Mutant ORF Gene Product Presence in Other

WT (Temecula isolate)pilB 1A2 PD1927 Type IV pili assembly protein PilB yespilQ 5A7 PD1691 Type IV pili assembly protein PilQ yesfimA 6E11 PD0062 Type I pili (shaft) subunit yespilX1 20D10 PD0022 Type IV pili assembly protein PilX yespilO TM5 PD1693 Type IV pili assembly protein PilO yespilC TM6 PD1923 Type IV pili assembly protein PilC yespilR TM7 PD1928 Type IV pili expression regulatory protein yespilY1 TM14 PD0023 Type IV pili tip-associated protein PilY yespilL TM25 PD0848 Type IV pili sensor histidine kinase regulator/ yes

chemotaxis-related protein kinasechpY 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 Identity of Xf virulence gene

>92% identity with Almond, Oleander,

CVC 9a5c

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Type IV pili

Type I pili (fimbriae)

Type I and type IV pili, and associated proteins, glycoproteins

Localization of gene product Localization of gene product

Type IV piliType I pili

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Gene Mutant

WT (Temecula isolate)pilB 1A2pilQ 5A7fimA 6E11pilX1 20D10pilO TM5pilC TM6pilR TM7pilY1 TM14pilL TM25

chpY 3E10

fimA/ DM12 pilO

fimA/ DM16 pilX

Localization of gene product Localization of gene productWT type I and IV pili

pilQ type I pili only

fimA type IV pili only

fimA, pilQ no pili

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Localization of gene product and function 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

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

No type IV pili

No type IV pili

Localization of gene product and function Localization of gene product and function

Type IV pili- Motility (twitching, rafts, migration)- Motility (twitching, rafts, migration)

- Adhesion

Pili poles

- Cell aggregration

Type I pili- Adhesion

- Cell aggregration

- Biofilm formation

- Biofilm formation

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Localization of gene product and function Localization of gene product and function

Type IV pili- Motility (twitching, rafts, migration in vitro)- Motility (twitching, rafts, migration in vitro)

- Adhesion

20 m

motility

Media flowMedia flow

- Cell aggregration

Type I pili- Adhesion

- Cell aggregration

- Biofilm formation

- Biofilm formation

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Localization of gene product and function Localization of gene product and function

Type IV pili- Motility (twitching, rafts, migration in vitro)- Motility (twitching, rafts, migration in vitro)

- Adhesion

Twitching motility—ratesTwitching motility—rates

Media flowMedia flow

- Cell aggregration

Type I pili- Adhesion

- Cell aggregration

- Biofilm formation

- Biofilm formation

0.7 μm min-1

0.2 μm min-1

4.4 μm min-1

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Localization of gene product and function Localization of gene product and function

Type IV pili

- Adhesion

- Motility (twitching, rafts, migration in planta)- Motility (twitching, rafts, migration in planta)

- Cell aggregration

Type I pili- Adhesion

- Cell aggregration

- Biofilm formation

- Biofilm formation

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Type IV pili

- Adhesion- Adhesion

- Motility (twitching, rafts, migration)

Adhesion force—pNAdhesion force—pN

Media flowMedia flow

- Cell aggregration

Type I pili- Adhesion- Adhesion

- Cell aggregration

- Biofilm formation

- Biofilm formation

147 pN

204 pN

119 pN

pilB

fimA

Localization of gene product and function Localization of gene product and function

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Type IV pili

Type I pili

- Adhesion

- Adhesion

- Cell aggregration- Cell aggregration

- Biofilm formation

- Motility (twitching, rafts, migration)

- Cell aggregration- Cell aggregration- Biofilm formation

pilB

fimA, pilO

WT & fimA

WT & fimA

Phenotype of mutants in vitro Phenotype of mutants in vitro

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Type IV pili

Type I pili

- Adhesion

- Adhesion

- Cell aggregration

- Biofilm formation- Biofilm formation

- Motility (twitching, rafts, migration)

- Cell aggregration- Biofilm formation- Biofilm formation

pilB fimA, pilOfimA WT

Phenotype of mutants in vitro Phenotype of mutants in vitro

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Gene Mutant

WT (Temecula isolate)pilB 1A2pilQ 5A7fimA 6E11pilX1 20D10pilO TM5pilC TM6pilR TM7pilY1 TM14pilL TM25

chpY 3E10

fimA/ DM12 pilO

fimA/ DM16 pilX

WT fimA fimA/pilO

3 months post inoc

Phenotype of mutants in planta Phenotype of mutants in planta

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes

Gene Mutant

WT (Temecula isolate)pilB 1A2pilQ 5A7fimA 6E11pilX1 20D10pilO TM5pilC TM6pilR TM7pilY1 TM14pilL TM25

chpY 3E10

fimA/ DM12 pilO

fimA/ DM16 pilX

Phenotype of mutants in planta Phenotype of mutants in planta

WT fimA fimA/pilOfimA/pilO

7 months post inoc

Fimbrial (fim, pil) Associated GenesFimbrial (fim, pil) Associated Genes Potential of the gene(s) as target for mitigation of PD Potential of the gene(s) as target for mitigation of PD

fim/pil genes... DSF1 citrus sap2

fimTfimApilA2pilFpilMpilIpilTpilUpilY1

…decreased twitching motility

Expression of

1Lindow lab; 2Cooksey lab