Andres Alvarez Dr. Jeff Chang IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS.
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Transcript of Andres Alvarez Dr. Jeff Chang IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS.
Andres AlvarezDr. Jeff Chang
IDENTIFICATION OF CANDIDATE TARGET PROTEINS OF TYPE III EFFECTORS
Plants are susceptible to pathogens
Bacterial speck disease: Pseudomonas syringae
Pictures courtesy of www.apsnet.org/education/IntroPlantPath
Bacterial soft rot: Erwinia carotovora
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., 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
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
• 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
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
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
Grow one day then replica plate
Immediately replica clean
Culture is replica plated from –leu, -
trp plate to a –leu, -trp, -his + 3AT 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