Download Identification of candidate target proteins of type III effectors

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