1. Julian Avila PLANT PATHOGEN INTERACTIONS IN TODAYS LECTURE: Effector-triggered Immunity Perceiving PAMPs Effectors AvrPto, mechanism of action

2. Apoplast Cell NUCLEUS PAMPs MAPKKK MAPKK4/ MAPKK5 P P MAPK3/ MAPK6 P PAMP Receptors FLS2 PRR DETECTION SYSTEMS Stomatal Closure ROS Production DEFFENSE GENES SA signaling Nitric Oxide ABA Systemic Acquired Resistance

3. 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!

4. Mutagenesis screen of Flg22 insensitive plants EMS-Generated

5. PATTERN RECOGNITION RECEPTORS - PRRs FLS2 TRANSMEMBRANE DOMAIN KINASE DOMAIN ECTODOMAIN RADARS FLS2 FLAG BAK1 PP SIGNAL TRANSMISSION

6. Apoplast Cell NUCLEUS Stomatal Closure Type Three Secretion System AvrPto Pathogenic EFFECTOR PROTEINS PAMPs MAPKKK MAPKK4/ MAPKK5 P ROS Production P MAPK3/ MAPK6 P PREVIOUSLY ON PTI CORONATINE (looks like JA) DEFFENSE GENES SA signaling Nitric Oxide ABA Systemic Acquired Resistance FLS2 PRR CHEMICAL WEAPONS PATHOGEN TRIGGERED IMMUNITY INNATE IMMUNITY BASAL IMMUNITY SABOTAGE

7. 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

8. SECRETION SYSTEMS ARE COMPLEX MACHINES Agrobacterium Pseudomonas

9. 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.

10. AvrPto, mechanism of action - Questions 1 - 4

11. KINASE DOMAIN HIS Plasmid AvrPto E. coli AvrPto GST Plasmid + Lysis Beads will bind: A) AvrPto? B) Kinase domain? Glutatione S-transferase Glutathione GST KINASE DOMAIN SDS PAGE Separate proteins bound to the beads & Proteins that bound the proteins bound to the beads WESTERN BLOT Detect what bound the beads Antibody against GST (Anti-GST) or Anti-AvrPto Antibody 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

12. AvrPto, mechanism of action - Questions 5 - 7

13. Kinase assays KINASE DOMAIN HIS Plasmid E. coli KINASE DOMAIN His Affinity Purification of Recombinant protein AvrPto GST Plasmid AvrPto γ- 32 P- ATP KINASE DOMAIN His In vitro kinase assay P Autphosphorylation Autoradiography (Radioactive) BSA (Gel Protein Stain)(Phosporimage) FLS2

14. Question 8

15. AvrPto Binds PRR In vivo FULL LENGTH GENE HA Plasmid AvrPto Protoplasts AvrPto Plasmid + Lysis AntiFLAG 3xFLAG FULL LENGTH His Total (INPUT) IMMUNOPRECIPITATION 3xFLAG SDS PAGE WESTERN BLOT

16. AvrPto Binds PRR In vivo

17. Pst PLANT R- GENE AvrPto, Pto, and Effector Triggered Immunity Apoplast Cell NUCLEUS Type Three Secretion System Pathogenic PAMPs MAPKKK MAPKK4/ MAPKK5 P P MAPK3/ MAPK6 P DEFFENSE GENES FLS2 PRR 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.

18. AvrPto, Pto, and Effector Triggered Immunity Apoplast Cell NUCLEUS Type Three Secretion System 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 INFECTION RESISTANCE DISEASE AvrPto + Pto

19. AvrPto, Pto, and Effector Triggered Immunity Apoplast Cell NUCLEUS Type Three Secretion System 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

20. AvrPto, Pto, and Effector Triggered Immunity Apoplast Cell NUCLEUS Type Three Secretion System 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