1. Andres Alvarez Dr. Jeff Chang IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS

2. Plants are susceptible to pathogens Bacterial speck disease: Pseudomonas syringae Pictures courtesy of www.apsnet.org/education/IntroPlantPath Bacterial soft rot: Erwinia carotovora Erwinia carotovora

3. Why should we care about plants’ health? Agriculture is essential for food production In the U.S. 10-20% of crops are lost to disease annually Billions of dollars each year Threat to food availability

4. How do plants defend themselves? Two branches of immunity First branch: PAMP – Triggered Immunity (PTI) PAMP = Pathogen Associated Molecular Pattern Pattern recognition receptors (PRRs) detect PAMPs on bacteria PTI response: e.g., strengthen cell wall

5. How are bacteria able to infect a plant? Many host-assoc., Gram- negative bacteria use a type III secretion system (TTSS) Molecular syringe Injected proteins are known as type III effectors (TTE) Effectors target defense- assoc. proteins inside the host cell Marlovits et al

6. My project Use a yeast two-hybrid screen to identify candidate targets of TTEs. Hypothesize targets are involved in host defense.

7. Yeast two-hybrid overview

8. Bait & Prey cDNA library derived from a plant that was infected – BAIT Target proteins are produced while plant is infected Effectors (HopW and HopAY) – PREY Proteins that could potentially interact with a protein within the cDNA library

9. Confirmation of transformed yeast DNA transformation into yeast Confirmation via PCR Prey: control protein (Krev1) Baits: control protein interactors (WT, M1, M2) Krev1 clones C 1 2 3 C 1 2 3 C 1 2 3 C 1 2 3 1 2 3 WT M1 M2

10. Protein-protein interaction in yeast Day 1: plate both strains on selection Day 2: replica plate to mate yeast strains, plate on non selective media Day 3: replica plate on to selective media, let grow 1 day YEP-Leu -Trp -Trp -Leu

11. Protein-protein interaction in selective media Takes about 4 days to get to this. Now ready for phenotype screening -leu –trp selection

12. Confirmation of protein-protein interaction Grow one day then replica plate Immediately replica clean Culture is replica plated from –leu, -trp plate to a –leu, -trp, -his + 3AT plate

13. Protein- protein interaction results Expected Conclusions: Krev1 + WT = Strong Krev1 + M1 = Weak Krev1 + M2 = None Experimental Conclusions Krev1 + WT = Interaction Krev1 + M1 = Interaction Krev1 + M2 = None -trp –leu – ura plate

14. Confirmation of transformed yeast w/ effectors genes PCR screen of transformed Gal4 BD yeast cells with effector genes

15. Future Work Mate yeast cells containing cDNA library and yeast cells with effectors Confirm mating results through 3 reporter genes PCR screen and sequence positive interactions to determine candidate plant protein sequence.

16. Acknowledgements Howard Hughes Medical Institute URISC program Cripps funds Dr. Jeff Chang and lab members especially Cait Thireault and Allison Creason Dr. Kevin Ahern