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