Role of Type I Secretion in Pierce’s Disease

Project Leader: Dean W. GabrielPlant Pathology DepartmentUniversity of FloridaGainesville, FL 32611

Cooperator: Donald HopkinsMid Florida Res. Edu. CenterUniversity of FloridaApopka, FL

Postdoctoral Associate:Shujian ZhangPlant Pathology DepartmentUniversity of FloridaGainesville, FL 32611

Funding: U.C. Davis subaward from USDA Cooperative Agreement SA6477

Research Associate:Stephanie ReddyPlant Pathology DepartmentUniversity of FloridaGainesville, FL 32611

Working hypothesis:

• Leaf scorch (PD) and necrotic leaf lesions (CVC) not typical of vascular blockage alone

• Two Type I secretion systems (hemolysin & colicin V) available.

• 10 PFTs: 6 hemolysins, 3 colicinVs, & 1bacteriocin

Multiple RTX effectorsfound in Temecula genome(equivalents in Dixon, Ann1 & 9a5c):

Hemolysins:PD0143 PD1506PD0282 PD2094PD0305 PD536

Colicin V precursors:PD0215PD0216(PD0217)

Bacteriocin:PD1427

Mechanism/s of the disease: Type I secreted RTXEffectors (PFTs)?

O.M.

Peptidoglycan

I.M.

Periplasm

Secretion of proteins produced by the bacterium

Efflux of toxicsubstances entering the bacterium

ATPADP

Type I secretion machine has 2 roles

Type I secretion

(PD1964)

Figure from Koronakis et al. 2004 Ann. Rev. Biochem. 73:467

(PD0783)

(PD1404)

AcrF

AcrD

Only one (1) TolC (PD1964) in X. fastidiosa

Figure from Koronakis et al. 2004 Ann. Rev. Biochem. 73:467

Type I secretion: PD1964 (TolC) is common component.

(PD1964)

(PD1413)

(PD1412)

Two potential toxin secretion systems: HlyD (PD1413)/ HlyB (PD1412) & CvaA (PD0496)/ CvaB (PD0499)

Reddy et al. 2007. MPMI 20:403-10.nptII

4033 bp

FE DA B C

2019 bp

2000 bp 1454 bp

1kb wt M1 M2 wt M1 M2 wt M1 M2 wt M1 M2 wt M1 M2 100 bp

A & D B & C A & E F & D RST31&33

PD1964 (tolC)

1019 bp

3033 bp

A DCB

pJR6.1

nptII

PD1964 (tolC)

23

9

Wt M1

Marker exchange of tolC in Temecula

PD-T Wild Type 64 dpi Type I secretion/efflux tolC¯

tolC ::nptII mutants are completely nonpathogenic...

Reddy et al. 2007. MPMI 20:403-10.

var. Carignane

Protein Secretion (hlyBD ::nptII) (cvaA ::nptII) (cvaB ::nptII)

Drug efflux (acrF ::nptII) (acrD ::nptII)

...and knockouts of all other Type I system components exhibit reduced pathogenicity.

Xf Temecula (tolC)

PD-T Wild Type 64 dpi Type I MDR efflux acrD¯

PD-T Wild Type 64 dpi Type I hemolysin secretion hlyBD¯

PD-T Wild Type 64 dpi Type I colicin secretion cvaA¯

PD-T Wild Type 64 dpi Type I colicin secretion cvaB¯

Expt #1 1-29-0764 days post inoculation

0

10

20

30

40

50

60

Strain inoculated

Qu

anti

ty o

f sy

mp

tom

s

# total leaves produced

# total symptomatic leaves

WT acrD- acrF- hlyB-D-

# total leaves produced = # asymptomatic (healthy) leaves, # symptomatic (diseased) leaves, # bare petioles, # bare nodes

# total symptomatic leaves = # symptomatic leaves, # bare petioles, # bare nodes

MDR efflux: acrD, acrF, tolC

Type I effector secretion: hlyB, hlyD, cvaA, cvaB, tolC

Type I mutations

0

0.2

0.4

0.6

0.8

1

1.2

0 dpi 40 dpi 88 dpi

Days Post Inoculation (dpi)

% D

isea

sed

Leav

es

WT

tolC

acrD

acrF

hlyBD

cvaA

cvaB

colicins

TolC is externally exposed and might be blocked by chemicals or engineered plants.

(PD1964)

Conclusions

• X. fastidiosa absolutely requires tolC for in planta survival; MDR efflux is critical.

• Knockouts of all other Xf Type I machine components exhibit reduced pathogenicity, including those used for PFT (hemolysin & colicin V) secretion.

• Knockouts of all 3 colicin V effectors exhibit reduced pathogenicity, possibly indicating a direct role in symptom elicitation (apoptotic?).

• TolC should have an exposed region and present a potential target for drugs/antibody strategy to control this pathogen

Funding: U.C. Davis subaward from USDA Cooperative Agreement SA6477