julian avila plant pathogen interactions in todays lecture: effector-triggered immunity perceiving...
TRANSCRIPT
Julian Avila
PLANT PATHOGEN INTERACTIONS
IN TODAYS LECTURE:
Effector-triggered Immunity
Perceiving PAMPs
EffectorsAvrPto, mechanism of action
Apoplast
Cell
NUCLEUS
PAMPs
MAPKKK
MAPKK4/MAPKK5
P
P
MAPK3/MAPK6
P
PAMP Receptors
FLS2
PRRDETECTION SYSTEMS
Stomatal Closure
ROSProduction
DEFFENSE GENES
SA signaling
Nitric Oxide
ABA
Systemic Acquired Resistance
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
TRANSMEMBRANE DOMAIN
KINASE DOMAIN
ECTODOMAIN
RADARS
FLS2
FLAG
BAK1
PP
SIGNAL TRANSMISSION
Apoplast
Cell
NUCLEUS
Stomatal Closure
TypeThreeSecretionSystem
AvrPto
Pathogenic
EFFECTOR PROTEINS
PAMPs
MAPKKK
MAPKK4/MAPKK5
PROSProduction
P
MAPK3/MAPK6
P
PREVIOUSLY ON PTICORONATINE(looks like JA)
DEFFENSE GENES
SA signaling
Nitric Oxide
ABA
Systemic Acquired Resistance
FLS2
PRRCHEMICAL WEAPONS
PATHOGEN TRIGGERED IMMUNITY
INNATE IMMUNITY
BASAL IMMUNITY
SABOTAGE
SECRETION SYSTEMS ARE COMPLEX MACHINES
Protein delivery into eukaryotic cells by type III secretion machinesJorge E. Galán and Hans Wolf-WatzNature 444, 567-573(30 November 2006)
Cryo-EM reconstruction of the quaternary structure of a TTSS
SECRETION SYSTEMS ARE COMPLEX MACHINES
Agrobacterium Pseudomonas
TTSS-We will talk about this next week
The stomatal manipulation doesn’t depend on the delivery of Pst molecules into the host’s cytosol.
IS CORONATINE TTSS-Secreted?
True or False:
This figure suggests that:
- In addition to coronatine, TTSS-secreted factors are required for stomatal opening.
- Coronatine is likely a small hydrophillic molecule.
AvrPto, mechanism of action - Questions 1 - 4
KINASE DOMAINHIS
Plasmid
AvrPto
AvrPto
AvrP
to
E. coli
AvrPtoGST
Plasmid
+
Lysis
Beads will bind: A) AvrPto?B) Kinase domain?
Glutatione S-transferase
GlutathioneGST
KINASE DOMAIN
SDS PAGE Separate proteins bound to the beads &Proteins that bound the proteins bound to the beads
WESTERN BLOTDetect what bound the beadsAntibody against GST (Anti-GST) or Anti-AvrPtoAntibody against 6xHIS (Anti-HIS)
Anti-6xHis?Anti-AvrPto?
His
Total(INPUT)
PULL-DOWN
Would you trust this if the Total for FLS2 looked like this?
AvrPto Binds PRR In vitro
AvrPto, mechanism of action - Questions 5 - 7
Kinase assaysKINASE
DOMAINHIS
Plasmid
E. coli
KINASE DOMAIN
His
Affinity Purification of Recombinant
protein
AvrPtoGSTPlasmid
AvrPto
γ-32P- ATP
KINASE DOMAIN
His
In vitro kinase assay
P
Autphosphorylation
Autoradiography
(Radioactive)
BSA
(Gel Protein Stain)(Phosporimage)
FLS2
Question 8
AvrPto Binds PRR In vivo
FULL LENGTH GENE HA
Plasmid
AvrPto
AvrPto
AvrP
to
Protoplasts
AvrPto
Plasmid
+
Lysis
AntiFLAG3xFLAG
FULL LENGTH
His
Total(INPUT)
IMMUNOPRECIPITATION
3xFLAG
SDS PAGE
WESTERN BLOT
AvrPto Binds PRR In vivo
Pst
PLANTR-
GENE
AvrPto, Pto, and Effector Triggered Immunity
Apoplast
Cell
NUCLEUS
TypeThreeSecretionSystem
Pathogenic
PAMPs
MAPKKK
MAPKK4/MAPKK5
P
P
MAPK3/MAPK6
P
DEFFENSE GENES
FLS2
PRR
AvrPto
AvrPto No AvrPto
Pto
pto
Pto
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
Apoplast
Cell
NUCLEUS
TypeThreeSecretionSystem
Pathogenic
PAMPs
MAPKKK
MAPKK4/MAPKK5
P
P
MAPK3/MAPK6
P
DEFFENSE GENES
FLS2
PRR
AvrPto
Pto
HYPERSENSITIVE RESPONSE
CELL DEATH
• KILLS CELLS AROUND INFECTION SITE
• CONTAINS INFECTIONRESISTANCE
DISEASE
AvrPto + Pto
AvrPto, Pto, and Effector Triggered Immunity
Apoplast
Cell
NUCLEUS
TypeThreeSecretionSystem
Pathogenic
PAMPs
MAPKKK
MAPKK4/MAPKK5
P
P
MAPK3/MAPK6
P
DEFFENSE GENES
FLS2
PRR
AvrPto
Pto
“It takes all the running you can do, to keep in the same place,” the Red Queen told Alice (illustration by Sir John Tenniel)
Gene-for-gene resistance systems fit into the Red
Queen Hypothesis
AvrPto, Pto, and Effector Triggered Immunity
Apoplast
Cell
NUCLEUS
TypeThreeSecretionSystem
Pathogenic
PAMPs
MAPKKK
MAPKK4/MAPKK5
P
P
MAPK3/MAPK6
P
DEFFENSE GENES
FLS2
PRR
AvrPto
Pto
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.
DECOYS
FLS2 Pto