PLANT PATHOGEN
INTERACTIONS
IN TODAYS LECTURE:
Perceiving PAMPs
Effectors
AvrPto, mechanism of action
Effector-triggered Immunity
Julian Avila
PAMP Receptors
DETECTION SYSTEMS
PRR
PAMPs
Apoplast
Nitric Oxide
FLS2
ABA
Cell
Stomatal
Closure
P
ROS
Production
MAPKKK
P
MAPKK4/
MAPKK5
P
MAPK3/
MAPK6
SA signaling
Systemic
Acquired
Resistance
DEFFENSE GENES
NUCLEUS
HOW DO YOU THINK
FLS2 WAS FOUND?
From what you have learned so far, you should be
able to provide an experimental approach in order to
answer this question.
Hint: Mutants!
Mutagenesis screen
of Flg22 insensitive
plants
EMS-Generated
PATTERN RECOGNITION
RECEPTORS - PRRs
FLS2
ECTODOMAIN
P
P
TRANSMEMBRANE DOMAIN
KINASE DOMAIN
BAK1
FLS2
SIGNAL TRANSMISSION
RADARS
PREVIOUSLY ON PTI
CHEMICAL WEAPONS
CORONATINE
(looks like JA)
PRR
SABOTAGE
PAMPs
Apoplast
Pathogenic
Type
Three
Secretion
System
Nitric Oxide
FLS2
ABA
Cell
Stomatal
Closure
P
ROS
Production
AvrPto
PATHOGEN
TRIGGERED
IMMUNITY
MAPKKK
P
MAPKK4/
MAPKK5
EFFECTOR
PROTEINS
INNATE
IMMUNITY
P
MAPK3/
MAPK6
SA signaling
BASAL
IMMUNITY
Systemic
Acquired
Resistance
DEFFENSE GENES
NUCLEUS
SECRETION SYSTEMS ARE COMPLEX MACHINES
Protein delivery into eukaryotic cells by type III secretion
machines
Jorge E. Galán and Hans Wolf-Watz
Nature 444, 567-573(30 November 2006)
Cryo-EM reconstruction of the quaternary structure of a TTSS
SECRETION SYSTEMS ARE COMPLEX MACHINES
Agrobacterium
Pseudomonas
IS CORONATINE TTSS-Secreted?
We will talk
about this
next week
TTSSTrue or False:
This figure suggests that:
- In addition to coronatine, TTSSsecreted factors are required
for stomatal opening.
- Coronatine is likely a small
hydrophillic molecule.
The stomatal manipulation doesn’t depend
on the delivery of Pst molecules into the
host’s cytosol.
AvrPto, mechanism of action - Questions 1 - 4
AvrPto Binds PRR In vitro
Glutatione S-transferase
GST
AvrPto
SDS PAGE
Plasmid
Separate proteins bound to the beads &
Proteins that bound the proteins bound to the
beads
+
KINASE
DOMAIN
HIS
Plasmid
WESTERN BLOT
Detect what bound the beads
Antibody against GST (Anti-GST) or Anti-AvrPto
Antibody against 6xHIS (Anti-HIS)
E. coli
His
Lysis
KINASE
DOMAIN
AvrPto
GST
Glutathione
Anti-6xHis?
Anti-AvrPto?
Total
(INPUT)
PULL-DOWN
Beads will bind:
A) AvrPto?
B) Kinase domain?
Would you trust this if the Total for
FLS2 looked like this?
AvrPto, mechanism of action - Questions 5 - 7
Kinase assays
HIS
KINASE
DOMAIN
AvrPto
GST
Plasmid
Plasmid
E. coli
Affinity Purification of
Recombinant protein
His
KINASE
DOMAIN
AvrPto
In vitro kinase assay
BSA
His
FLS2
KINASE
DOMAIN
Autoradiography
P
(Phosporimage)
(Gel Protein Stain)
Autphosphorylation
γ-32P- ATP
(Radioactive)
Question 8
AvrPto Binds PRR In vivo
AvrPto
3xFLAG
SDS PAGE
Plasmid
+
FULL LENGTH
GENE
HA
Plasmid
WESTERN BLOT
Protoplasts
His
FULL LENGTH
Lysis
AvrPto
3xFLAG
AntiFLAG
Total
(INPUT)
IMMUNOPRECIPITATION
AvrPto Binds PRR In vivo
PRR
AvrPto
PAMPs
Apoplast
No AvrPto
Pst
AvrPto, Pto, and Effector Triggered Immunity
Pathogenic
FLS2
Pto
Cell
Type
Three
Secretion
System
Pto
P
MAPKKK
P
MAPKK4/
MAPKK5
pto
P
MAPK3/
MAPK6
DEFFENSE GENES
NUCLEUS
PLANT
RGENE
Anytime AvrPto and Pto are present,
tomatoes are resistant (R). Any other time
they are susceptible (S).
This type of resistance has been
termed gene-for-gene resistance.
AvrPto, Pto, and Effector Triggered Immunity
PRR
PAMPs
Apoplast
Pathogenic
Type
Three
Secretion
System
FLS2
Pto
Cell
HYPERSENSITIVE RESPONSE
• KILLS CELLS
AROUND
INFECTION SITE
• CONTAINS
INFECTION
P
MAPKKK
P
RESISTANCE
MAPKK4/
MAPKK5
P
MAPK3/
MAPK6
CELL DEATH
DEFFENSE GENES
NUCLEUS
DISEASE
AvrPto + Pto
AvrPto, Pto, and Effector Triggered Immunity
PRR
PAMPs
Apoplast
Pathogenic
Type
Three
Secretion
System
FLS2
Pto
Cell
Gene-for-gene resistance systems fit
into the Red Queen Hypothesis
P
MAPKKK
P
MAPKK4/
MAPKK5
P
MAPK3/
MAPK6
“It takes all the running you can do, to keep
in the same place,” the Red Queen told Alice
(illustration by Sir John Tenniel)
DEFFENSE GENES
NUCLEUS
AvrPto, Pto, and Effector Triggered Immunity
PRR
PAMPs
Apoplast
Pathogenic
FLS2
Pto
Cell
Type
Three
Secretion
System
P
MAPKKK
P
This type of resistance has been
termed gene-for-gene resistance...
...but recent findings present a
hypothesis in which Pto acts as a
decoy that protects pathogen
recognition receptors.
- This alleviates the selection
pressure on PRRs.
MAPKK4/
MAPKK5
P
MAPK3/
MAPK6
DEFFENSE GENES
NUCLEUS
FLS2 Pto
DECOYS