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IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS Andres Alvarez Dr. Jeff Chang Plants are susceptible to pathogens Bacterial speck disease: Pseudomonas syringae Bacterial soft rot: Erwinia carotovora Pictures courtesy of www.apsnet.org/education/IntroPlantPath 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 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 How are bacteria able to infect a plant? • Many host-assoc., Gramnegative bacteria use a type III secretion system (TTSS) • Molecular syringe • Injected proteins are known as type III effectors (TTE) • Effectors target defenseassoc. proteins inside the host cell Marlovits et al My project •Use a yeast two-hybrid screen to identify candidate targets of TTEs. •Hypothesize targets are involved in host defense. Yeast two-hybrid overview 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 Confirmation of transformed yeast WT • DNA transformation into yeast M1 M2 C 1 2 3 C 1 2 3 C 1 2 3 • Confirmation via PCR • Prey: control protein (Krev1) • Baits: control protein interactors (WT, M1, M2) Krev1 clones C 1 2 3 1 2 3 Protein-protein interaction in yeast -Trp YEP -Leu -Trp -Leu 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 Protein-protein interaction in selective media • Takes about 4 days to get to this. Now ready for phenotype screening -leu –trp selection Confirmation of protein-protein interaction Immediately replica clean Culture is replica plated from –leu, -trp plate to a –leu, -trp, -his + 3AT plate Grow one day then replica plate 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 Confirmation of transformed yeast w/ effectors genes • PCR screen of transformed Gal4 BD yeast cells with effector genes 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. Acknowledgements • Howard Hughes Medical Institute • URISC program • Cripps funds • Dr. Jeff Chang and lab members especially Cait Thireault and Allison Creason • Dr. Kevin Ahern
