function of zar1 resistance protein · shalom r. jamena1,2, jana a. hassan2, and jennifer d....
TRANSCRIPT
HopZ1a
myc
CC +NBS
CC NBS
LRR
My special thanks go to Allison Schwartz for making some of the
constructs for me to work on, to Jane Zhou for being a great lab
partner, and to the ELP program for providing this wonderful
opportunity to experience scientific research firsthand.
Katagiri, F., Thilmony, R., & He, S.Y. (2002). The Arabidopsis Thaliana-Pseudomonas Syringae
Interaction
Lewis, J.D., Wu, R., Guttman, D.S., & Desveauz, D. (2010). Allele-Specific Virulence Attenuation of
the Pseudomonas syringae HopZ1a Type III Effector via the Arabidopsis ZAR1 Resistance Protein
Belkhadir, Y.,Subramaniam, R., and Dangl, J.L. (2004). Plant disease resistance protein signaling:
NBS–LRR proteins and their partners
Swiderski, M. R. , Birker, D., Jones, J.D.G. (2009). The TIR domain of TIR-NB-LRR resistance
proteins is a signaling domain involved in cell death induction .
FUTURE DIRECTIONS
Cloning Strategy- ZAR1 gene RESULTS
Nicotiana benthamiana growth
Sterilize seeds
Sow on soil
Transplant
in 1-2 weeks
Grow until 6-7 weeks
post- germination
Transient expression using Agrobacterium
Grow culture with appropriate
construct in induction media
Measure the optical density (OD)
Dilute to OD600=0.3
Infiltrate
N. benthamiana plants
Spray with DEX
to induce expression
Observe phenotypes
• Successfully cloned and sequence confirmed CCNBS, or ∆LRR
domains, and the full length ZAR1 into pMAC15 vector with myc
epitope tag.
• Transformed pMAC15 containing CC, NBS, or LRR into
Agrobacterium
• Infiltrated Nicotiana benthamiana with Agrobacterium carrying
pMAC15-CC, pMAC15-NBS, or pMAC15-LRR to observe the
HR.
• Also infiltrated HopZ1a, positive control that
induces the HR and myc, negative control.
• HopZ1a showed the HR on 4 out of 14 leaves
• No phenotype was observed with the ZAR1
domains.
Shalom R. Jamena1,2, Jana A. Hassan2, and Jennifer D. Lewis2,3
1Ohlone College, 2University of California- Berkeley, 3United States Department of Agriculture
Plate on selective media
Colony PCR to screen for the clone of interest
Grow culture and prepare stock
1kb
PCR amplification using
gene-specific primers and
the colony as template. The
PCR products were
separated by size on an
agarose gel. Clones 1 and 3
contain gene of interest.
PCR to add XhoI restriction site
Restriction digest
Ligate the vector and inserts
Transform E.coli with ligation
Plate on selective media
Colony PCR to screen for positive clone
Culture bacteria with positive clone
Extract plasmid
Sequence to confirm clone
Chromatogram of sequenced clone with each peak
representing a specific base of DNA
E. coli colonies are
KAN-resistant
Digested PCR products
(CCNBS,LRR, ZAR1,
or, vector [pMAC15-
myc]) separated by
size on an agarose gel
PCR amplification using gene
specific primers and colony
as template. PCR products
separated by size on an
agarose gel. Colonies 3, 6, and
7 contain the gene of interest.
Colonies 1, 2, 4, and 5 do not
have the gene of interest.
ladder 1 2 3 4 5 6 control 7
1kb
To determine whether specific ZAR1 domains can induce the
HR in Nicotiana benthamiana.
• HopZ1a had an inconsistent phenotype and more
experiments are required.
• A phenotype was not observed from the ZAR1 domains.
• HopZ1a should have the HR on every leaf
• Try combination of ZAR1 domains
• Western blot to confirm protein expression
• If a specific domain causes the HR:
• Delineate minimal region for the phenotype
• Look at role of specific amino acids in
contributing to the phenotype
+
pathogen
effector
CC CC
NBS NBS LRR
LRR
NO PATHOGEN
WHEN
PATHOGEN
EFFECTOR IS
RECOGNIZED
R protein unfolds
and downstream
defense signaling
is induced
R protein R protein
• Host Resistance (R) genes play a significant role in plant
defenses against pathogens by recognizing specific pathogen
effectors. Recognition triggers a rapid, cell death response
called the hypersensitive response (HR).
• The ZAR1 R protein recognizes the P. syringae Type III
Effector protein HopZ1a.
• ZAR1, like many R proteins, contain a coiled-coil (CC)
domain, a nucleotide-binding site (NBS) domain, and a
leucine-rich repeat (LRR) domain.
• The CC domain is involved in protein-protein interactions.
• The NBS domain is believed to induce downstream signaling.
• The LRR domain appears to maintain the resistance protein
in a non-signaling state.
• When the R protein detects an effector, the R protein unfolds,
reveals the NBS domain, and induces defense signaling.
OBJECTIVE
INTRODUCTION
Kanr
XhoI
XhoI StuI
StuI StuI
XhoI
Kanr Kanr
ACKNOWLEDGEMENT
REFERENCES
The ZAR1 truncations were designed to test the role of each individual domain in defense
induction. The DEX promoter allows dexamethasone-inducible gene expression.
The myc tag is an epitope tag, which allows protein to be detected in Western blot.
Agro-infiltrated leaf
with ZAR1
domains, HopZ1a
(positive control),
and myc (negative
control). HR was
observed on
HopZ1a 24 hours
post-Dex spray. No
phenotype was
observed with the
ZAR1 domains.
Xho1
Transform the clone into A. tumefaciens
Function of ZAR1 resistance protein
domains in defense induction in
Nicotiana benthamiana