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P P.s. tabaci Null P.s. syringae Hrp - (TTSS) mutant HR P.s. syringae >50 pathovars based on host specificity Tobacco Bean Tomato P.s. pv. tabaci P HR

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Text of P P.s. tabaci Null P.s. syringae Hrp - (TTSS) mutant HR P.s. syringae >50 pathovars based on host...

Tobacco Bean Tomato
hosts tomato and Arabidopsis
Hrp type III secretion system (TTSS)
This is a slide depicting the different macro and microscopic reactions that Pseudomonas has in plants.
Sepcifically, the tobacco plant.
On the left most panel, macroscopically, we see disease, and this is Pathovar tabaci infecting its natural host tobacco.
Microscopically the bacteria thriving,as you see in this coinfocal image taken by Wen-Ling Deng. – The bacteria are labelled with GFP, and the red is autofluorescent. AS you can see the bacteria are extracellular – they thrive in the intercellular space of plant cells known as the apoplast.
In the middle, we have infiltration of P. syringae, a bean pathogen on a nonhost, tobacco. Macroscopically we see the hypersensitive response, a localized programmed cell death that restricts the pathogen from infecting the whole plant. The orange in this panel are phenolic compounds which make up part of a chemical burst the plant induces when it recognizes a pathogen. this HR occurs because the bacteria is delivering proteins into the plant cell that the plant recognizes through its defense system
Finally we have a bean pathogen mutant that does not cause HR on tobacco, and this is because the mechanism by which it delivers proteins into the plant cell is not functional dues to a mutation, hrcC, in the delivery apparatus. The plant does not recognize this bacteria, and the bacteria cannot thrive in this environment becaseu it cannot put viruelnce proteins into the plant.
The system that delivers proteins is encoded by genes in the hrp/hrc cluster.
-starting from the left: tabaci causes disease on tobacc; the plant does not recognize the pathogen until it is too late.
-Pseduomonas syrngae can cause two types of reactions on plants: a hypersensitive response (HR) or disease
-in the HR, the plant immediately recognizes the pathogen, and induces a. --secretion mutant cannot cause HR
-Red is autofluorescnence of chlorplasts
-P the bacteria is huge!!
ROBIN BUELL - TIGR
ALAN COLLMER Cornell
JIM ALFANO - Nebraska
XIAOYAN TANG - Kansas
ARUN CHATTERJEE - Missouri
GREG MARTIN - BTI
SANDY LAZAROWITZ - Cornell
TERRY DELANEY - Cornell
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
Molecular/cellular determinants of plant- bacterium interactions
complementary approaches to total biology
use red and blue arrows
2e
Virulence-related ORFs newly found by Hidden Markov Model search of
P.s. tomato DC3000 genome
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.
ORF
BLAST
found effectors but note:
6.5 Mb
5,763 ORFs
7% of genome mobile genetic elements
298 ORFs implicated in virulence, including
38 confirmed TTSS substrates
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
Disease
-35
-10
All known avr genes preceded by "Hrp box" promoters
Secretion/injection "Hops" testable, but slow
classic method that keen and staskawicz pioneered in 1984
but not useful for genomewide, systematic effector mining
explain hop and effector (copy animal workers)
so that is where our effector mining began
EEL
CEL
tRNAleu
orf4
orf3
orf2
tnpA
orf1
orf8
orf1
avrE
avrF
orf3
orf4
hrpW
orf5
orf6
orf7
avrPto
avrPtoB
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
here is hrp pai
a
c
e
d
b
Violations:
Violations:
none
ORF1-'AvrRpt2
ORF2-'AvrRpt2
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.
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

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