Download - Andres Alvarez Dr. Jeff Chang IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS
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Andres AlvarezDr. Jeff Chang
IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS
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Plants are susceptible to pathogens
Bacterial speck disease: Pseudomonas syringae
Pictures courtesy of www.apsnet.org/education/IntroPlantPath
Bacterial soft rot: Erwinia carotovora
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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
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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
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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
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My project•Use a yeast two-hybrid screen to identify candidate targets of TTEs.
•Hypothesize targets are involved in host defense.
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Yeast two-hybrid overview
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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
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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
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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
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Protein-protein interaction in selective media• Takes about 4 days to get to this. Now ready for phenotype screening
-leu –trp selection
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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
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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
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Confirmation of transformed yeast w/ effectors genes• PCR screen of transformed Gal4 BD yeast cells with
effector genes
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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.
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Acknowledgements • Howard Hughes Medical Institute• URISC program• Cripps funds• Dr. Jeff Chang and lab members especially Cait Thireault and Allison Creason• Dr. Kevin Ahern