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