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