P
P.s. tabaci
Null
P.s. syringae Hrp- (TTSS) mutant
HR
P.s. syringae
>50 pathovars based on host specificityTobacco Bean Tomato
P.s. pv. tabaci P HR HRP.s. pv. syringae HR P HR P.s. pv. tomato HR HR P
30 m
P. s. tomato DC3000 model pathogen
•hosts tomato and Arabidopsis•representative stealth parasite•Hrp type III secretion system (TTSS)
Pseudomonas syringae
Tobacco leaves
ROBIN BUELL - TIGR
ALAN COLLMER Cornell
JIM ALFANO - Nebraska
XIAOYAN TANG - Kansas
ARUN CHATTERJEE - Missouri
GREG MARTIN - BTI
SANDY LAZAROWITZ - Cornell
TERRY DELANEY - Cornell
SAM CARTINHOUR
DAVID SCHNEIDER
CHRIS MEYERS
Modeling of virulence gene regulation networks in P. syringae
Molecular/cellular determinants of plant- bacterium interactions
USDA/ARSCenter for Agricultural Bioinformatics
Cornell Theory Center
NSF PGRP DBI-0077622
Experimental biology
Computational biology
Functional Genomics of the Interactions of Tomato and Pseudomonas syringae pv tomato DC3000
http://pseudomonas-syringae.org
http://monod.cornell.edu
ORF BLAST P value
HMM E value
Microarray signal ratio1
Comment
SyrEPto 0 9.8e-7 NT Toxin (syringomycin) biosynthesis
AvrPphFPto 3e-36 1.7e-6 3±2 Effector
HrpW-related 2e-4 1.9e-6 14±7 Candidate harpin
AvrPphD1Pto 0 1.9e-6 30±17 Effector
AvrXv3Pto 7e-12 3.4e-6 ND Effector
AvrPpiB11Pto 1e-152 7.8e-6 11±9 Effector
AvrPpiB12Pto 1e-150 7.8e-6 10±6 Effector
AvrPphD2Pto 2e-44 3.0e-5 27±11 Effector
HopPtoA2 1e-177 2.8e-4 ND Effector
HopPtoB2 2e-16 2.6e-3 ND Effector
AvrRps4Pto 2e-44 2.5e-2 ND Effector
CorRPto 2e-72 6.6e-2 ND Coronatine biosynthesis regulator 1Relative to 16S and 23S rRNA genes
Virulence-related ORFs newly found by Hidden Markov Model search of P.s. tomato DC3000 genome
48<1e-4
78<1e-3
212<1e-2
Fouts, Abramovitch, Alfano, Baldo, Buell, Cartinhour, Chatterjee, D'Ascenzo, Gwinn, Lazarowitz, Lin, Martin, Rehm, Schneider, van Dijk, Tang, and Collmer. 2002. Proc. Natl. Acad. Sci. USA 99:2275-2280.
Genome of P. s. tomato DC3000
Buell et al. 2003. PNAS 100:10181-10186
6.5 Mb
5,763 ORFs
3,797 ORFs also in P.aeruginosa and P. putida
811 unknown ORFs not in P.a. or P.p.
7% of genome mobile genetic elements
298 ORFs implicated in virulence, including
•38 confirmed TTSS substrates
•19 strong candidates
pDC3000A carries at least 4 avr/hop genes
The problem of genomewide identification of Hrp effector genes in P. syringae
HR
avr
hrp
hrp
HR
hrp
R
avr
R
R
HR
hrp
R
hrp
Effector candidate "Hop"
Effector candidate "Avr"
-10
-35 GGAACT GGCACC GAAACT GAAACC GGAACC
TCACNNA CCACNNA ACACNNA CTACNNA
# of occurrences
# of occurrences
13 1 1 1 45
1 58 1 1
-35
NNN GGAACC NNNNNNNNNNNNNNNN CCACNNA NNN
-10
Most effectors found by avirulence phenotype
•All known avr genes preceded by "Hrp box" promoters
Mutant phenotypes typically weak or lacking
Secretion/injection "Hops" testable, but slow
No common motifs reported in proteins Disease
EEL CEL
tRN
Ale
uor
f4or
f3or
f2
tnpA
orf1 orf8
orf1
avrE
avrF
orf3
orf4
hrpW
orf5
orf6
orf7
avrP
to
avrP
toB
hrp gene cluster
mini-Tn5gus tagging of genes activated by HrpL alternative sigma factor
hrp
hrpL
tRNAleu
Fouts, Abramovitch, Alfano, Baldo, Buell, Cartinhour, Chatterjee, D'Ascenzo, Gwinn, Lazarowitz, Lin, Martin, Rehm, Schneider, van Dijk, Tang, and Collmer. 2002. Proc. Natl. Acad. Sci. USA 99:2275-2280.
EEL CEL
Hrp Pathogenicity Island
ORF1-avrPphF M K N A F D L L V E G L A K D Y N M P P L P D K K H I D E V Y C F E F Q S G M N
ORF2-avrPphF M G N I C G T S G S R H V Y S P S H T Q R I T S A P S T S T H V G G D T L T S I
a. Position 3 or 4 is I, V, or L, and between this residue and the starting M is a polar, positively charged or P residue
b. No MIVLFYW residues appear in position 5.
c. No D or E in first 12 residues.
d. The first 50 residues are amphipathic, rich in polar amino acids, and never have more than 3 of the MIVLFYW group in a row.
e. No C between positions 5 and 50
a c edbViolations:
Violations: none
ORF1-'AvrRpt2
ORF2-'AvrRpt2
Tsiamis et al. 2000. EMBO J. 19:3204
The rules successfully predict which unknown ORFs encode effectors
Petnicki-Ocwieja, Schneider, Tam, Chancey, Shan, Jamir, Schechter, Buell, Tang, Collmer, and Alfano. 2002. Proc. Natl. Acad. Sci. USA 99:7652-7657.