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