A1258689937BvxQg

1

• "The pathogen mechanisms of attack and novel
  targets
• Bacterial pathogenicity
• Structure and function of T3SS
• 2. Effector proteins with virulence functions
• 3. AvrPtoB as a suppressor of basal defence
• 4. Hormonal targets?

John Mansfield, Marta de Torres, Ian Brown,
Richard Thwaites, Mark Bennett and Murray
Grant Division of Biology, Imperial College
2
P. syringae pathovar phaseolicola

• Causal agent of halo-blight disease in bean

GENE-FOR-GENE INTERACTIONS Nine races of P. s.
phaseolicola, differentiated by their ability to
infect different cultivars of bean
3
Formation of the T3SS pilus The appendage grows
rapidly from the bacterial surface and is
coated with HrpZ (small gold label). The pilus
subunit is HrpA (large gold label).
Immunocytochemistry identifies protein
components
4
Secretion of effector protein through the pilus
5
Plant cell
Bacterium
HrpA
Effector protein
HrpZ
TTSS
6
Beware of the plague!
Yersinia type III secretion system
Animal and plant pathogens use the same secretion
system to deliver proteins to the host cell
7
P. syringae pv. phaseolicola hrp cluster

?
avrPphE
hrcQa
hrcQb
hrcN
hrpS
hrpQ
hrpV
hrpT
hrcJ
hrpB
hrpA
hrpO
hrcT
hrpG
hrpD
hrpY
hrpJ
hrcV
hrcR
hrcS
hrcU
hrcC
hrpF
hrpE
hrpZ
hrpR
hrpL
hrpP
Harp box
? 54 promoter
?
HrpV is a negative regulator HrpA is also
required for HrpL function
8
Taqman PCR analysis of the expression of core
T3SS components eg hrpA, hrpP and effectors
egvirPphA
9
hrpA
hrpP
virPphA
8
7
6
mRNA (relative to 16S)
5
mRNA (relative to 16S)
mRNA (relative to 16S)
4
3
2
1
0
0
0
Induction time (min)
Rapid induction and different levels, but not
sequential as in flagellum production
9
Type 3 puzzles..

• How is construction co-ordinated?
• What is the regulatory role of HrpA?
• Is there ordered secretion?
• Is there a role for wall degradation to allow
  delivery?
• How do effectors travel through the pilus?

10
How do proteins move in the pilus lumen?
11
Evolution of plant /pathogen interactions a
simple three step hypothesis

• Multiple effector proteins evolve to suppress
  basal defences induced by common microbial
  molecules (PAMPS)
• R gene-based recognition of effectors triggers
  the HR to give gene-for-gene interactions
• Loss of the recognised effector breaks
  gene-for-gene mediated resistance

Effector proteins are delivered through the type
three secretion system TTSS
12
Nd
Ws
Basal resistance - cell wall alterations
including callose deposition
13
Virulent bacteria suppress basal defences in
Arabidopsis
14
The hypersensitive reaction cell death
15
Bacterial effector proteins Identified
by Function (many as Avr proteins) Secretion
through T3SS Bioinformatics Testing effectors
by pseudomonas delivery using the attenuated
bean pathogen strain RW60 or expression in the
plant
16
Effectors on the plasmid identification of
virulence functions
Race 6 has lost avrPphF but gained a second
potential effector
17
The HopAB family includes effectors from several
pathovars of P.syringae
Names have changed! The first member to be
identified was VirPphA
18
Use of RW60 to examine the properties of virPphA
homologues the reaction matrix
Homologue Reaction on plant if delivered by
RW60 Bean Soybean Tomato
Arabidopsis virPphA Vir Avr Avr
- virPphAPgy Vir - Avr
- virPphAPsv Vir Avr Avr
- avrPtoB Vir Avr Avr
Vir hopPmaL - - - - holPmaN
- - - - virPphAPsi -
- - - avrPto -
- Avr -
19
Effects of avrPtoB in ecotypes of Arabidopsis
after 5 days
Columbia - 5
Wassilewskija 3
avrPtoB 0.2 0.4 OD
avrPtoB 0.2 0.4 OD
RW60 0.4 OD
RW60 0.4 OD
Ecotype and concentration influence the symptoms
produced by RW60
20
(No Transcript)
21
Mapping basal resistance genes
CAPS markers position the locus on chromosome
5 Co-segregation with FLS2 !!!!!
Ws lacks a functional the FLS2 receptor
22
FLS2 detects flagellin (from Gomez Gomez and
Boller)
23
Onset of wilting syptom in Ws with a high level
of AvrPtoB expression after Dex-mediated induction
24h after Dex treatment
24
In planta expression of AvrPtoB promotes growth
of hrp mutant
25
Papilla deposition in response to DC3000 hrpA
mutant
26
Suppression of cell wall responses after in
planta expression of AvrPtoB
27
Callose deposition detected by aniline blue
fluorescence staining in a leaf of Nd
infiltrated with flagellin suspension
28
Callose deposition detected by aniline blue
fluorescence staining in a leaf of Nd
infiltrated with flagellin suspension
The edge of the infiltrated zone is shown. Note
that trichomes contain callose and fluoresce
without elicitation
29
AvrPtoB suppresses the induction of callose if
the effector is induced 6h before challenge with
flagellin
Wound induced callose is also suppressed
30
Transgenic Nd plants expressing avrPtoB under
control of the Dex-inducible promoter were used
to examine effect on the callose deposition
response to flagellin peptide
Callose deposition 18h after flagellin injection
into leaves treated with Dex 6, -3, 0 or 2h
before or after flg22 elicitation -6 -3 0
2 No Dex control No callose

Expression of avrPtoB does suppress the callose
response but only if the effector is there
before flagellin How does AvrPtoB work?
31
Suppression of specific transcripts by AvrPtoB
AvrPtoB was expressed from a Dex inducible
promoter in leaves before challenge with the
hrpA mutant of DC3000
32
Transcription profiling shows up regulation of
ABA responsive genes during disease
i, ii and iii induced by TT3Es and osmotic
stress iv down reg by both and v differential
More than 50 of disease induced genes Contain
ABA associated response elements
33
Increased ABA during disease development after
challenge with DC3000
Not just ABA changes but other hormones too
34
In planta expression of AvrPtoB causes ABA
synthesis
35
AvrPtoB induces NCED3 expression for ABA
biosynthesis but suppresses defences
Defence response markers
(NCED) 9-cis-epoxycarotenoid dioxygenase
36
Ler mutants with enhanced sensitivity to ABA
show enhanced
symptoms to DC3000
37
ABA status influences callose deposition after
DC3000 challenge in Ler mutants and treatment
with ABA in Col backgrounds
ABA insensitive
Wild-type Ler
abi1-sup7 35S HAB-1
Wild-type Col
ABA sensitive
ABA insensitive
abi1-sup7
abi1-sup7 35S HAB-1
Col ABA
abi-sup7 is hypersensitive to ABA, whereas
expression of HAB-1 renders the mutant
insensitive to ABA. Insensitivity to ABA was
reflected by increased callose deposition (a-d).
ABA treatment suppresses callose deposition in
Col (e and f)
38

• Conclusions.
• A single bacterial effector protein delivered by
  the T3SS
• suppresses defence
• 2. Primary target of AvrPtoB may be regulation of
  ABA and other
• plant hormones
• 3. Link between ABA and defence responses
• Wood RKS 1967 in Physiological Plant Pathology
• Changes in hormone levels in diseased plants may
  simply be
• side-effects of successful parasitism
• In fact hormonal homeostasis may be a target for
  the
• establishment of disease

39
Collaboration.
HOME
AWAY

• George Tsiamis
• Alec Forsyth
• Hassan Ammouneh
• Guillaume Lafforgue
• Nina Grabov

• Laszlo Bogre
• Suvi Taira
• Jens Boch
• Silke Robatsek