CHARACTERIZATION OF THE PATHOGEN-REGULATED …

The Pennsylvania State University

The Graduate School

Huck Institute of Life Sciences

CHARACTERIZATION OF THE PATHOGEN-REGULATED ARABIDOPSIS

BONZAI1/COPINE1 PROTEIN AND ITS ROLE IN CALCIUM SIGNALING

A Dissertation in

Integrative Biosciences

by

Tzuu-fen Lee

Submitted in Partial Fulfillment

of the Requirements

for the Degree of

Doctor of Philosophy

August 2008

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The dissertation of Tzuu-fen Lee was reviewed and approved* by the following:

Timothy W. McNellis

Associate Professor of Plant Pathology

Thesis Advisor

Chair of Committee

Surinder Chopra

Associate Professor of Crop & Soil Sciences

Seogchan Kang

Associate Professor of Plant Pathology

Paula McSteen

Assistant Professor of Biology

Andrew Stephenson

Professor of Biology

Peter Hudson

Director,

Integrative Biosciences Program

Huck Institutes of the Life Sciences

*Signatures are on file in the Graduate School

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ABSTRACT

The Arabidopsis BONZAI1/COPINE1 (BON1/CPN1) gene is a suppressor of

defense responses controlled by the disease resistance gene homolog SNC1. The

BON1/CPN1 null mutant cpn1-1 has a recessive, temperature- and humidity-dependent

lesion mimic phenotype that includes enhanced disease resistance and activation of

Pathogenesis Related (PR) gene expression. This study shows that calcium perturbation

activated defense responses in the absence of BON1/CPN1. Leaf infiltration with the

calcium ionophore A23187 triggered strong PR gene expression specifically in cpn1-1

mutant plants grown under permissive conditions while co-infiltration of the calcium

chelator EGTA attenuated this effect. This suggests that BON1/CPN1 is required for

normal responses to calcium fluxes. Using a polyclonal anti-BON1/CPN1 antibody, the

accumulation of BON1/CPN1 protein was shown to be up-regulated by the activation of

defense signaling responses controlled by two Resistance (R) genes, SNC1 and RPS2.

Promoter deletion analysis identified a 280 bp portion of the BON1/CPN1 promoter

required for pathogen- and A23187-induced GUS expression, which occurred in a

punctate pattern in treated leaves. Finally, two BON1/CPN1 promoter T-DNA mutants

over-accumulated BON1/CPN1 despite T-DNA inserted extremely close to the

BON1/CPN1 transcriptional start site. Our study explains the conditional nature of the

cpn1-1 phenotype. These results are consistent with BON1/CPN1 being a calcium- and

pathogen-responsive plant defense suppressor protein.

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TABLE OF CONTENTS

List of figures...............................................................................................................vii

Chapter 1 The Arabidopsis copine BONZAI1/COPINE1 protein and its role in

calcium signaling ..................................................................................................1

1.1 Plant-pathogen interaction ..............................................................................1

1.2 Calcium signals and signaling components ....................................................2

1.3 Biochemical properties and functions of copines...........................................3

1.4 Arabidopsis BON1/CPN1 as a suppressor of defense responses ...................6

1.5 Objectives of my thesis research ....................................................................8

Chapter 2 The role of BON1/CPN1 in calcium signaling specificity.........................10

2.1 Summary.........................................................................................................10

2.2 Background information.................................................................................10

2.3 Materials and methods....................................................................................11

2.3.1 Plant materials and growth conditions .................................................11

2.3.2 Chemicals and cold stimuli treatments.................................................12

2.3.3 RNA preparation and northern analysis ...............................................13

2.4 Results.............................................................................................................13

2.4.1 Calcium ionophore triggered cell death in cpn1-1 mutant plants.........13

2.4.2 Calcium ionophore triggered accumulation of PR gene transcripts

in cpn1-1 mutants ...................................................................................16

2.4.3 Ca2+ chelator EGTA suppressed A23187-induced PR1 gene

expression in cpn1-1 plants ....................................................................18

2.4.4 Cold-induced PR gene expression in cpn1-1 plants .............................18

2.5 Discussion.......................................................................................................20

Chapter 3 Generation of anti-BON1/CPN1 antisera and troubleshooting for

artifact signals in western blot analysis ................................................................23

3.1 Summary.........................................................................................................23




3.3.2 Bacterial expression of BON1/CPN1 VWA domain and antibody

production...............................................................................................26

3.3.3 Protein extraction and western blot analysis ........................................27

3.4 Results.............................................................................................................29

3.4.1 GST-VWABON1/CPN1

protein purification and antigen preparation.......29

3.4.2 Anti-BON1/CPN1 antisera produced high levels of background

signals in western blots ..........................................................................31

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3.4.3 β-mercaptoethanol in the loading buffer caused the background

problems .................................................................................................34

3.4.4 Lower concentrations of reducing agent in the loading buffer

eliminated the background signals .........................................................36

3.5 Discussion.......................................................................................................38

Chapter 4 Pathogen regulation of BON1/CPN1 protein level ....................................41

4.1 Summary.........................................................................................................41


4.3 Materials and methods...................................................................................43


4.3.2 Bacterial inoculations and bacterial growth analysis ...........................43

4.3.3 Plant protein extraction and western analysis ......................................44

4.4 Results.............................................................................................................45

4.4.1 Tissue-specific BON1/CPN1 protein accumulation patterns ...............45

4.4.2 BON1/CPN1 protein accumulation was induced by both avirulent

and virulent Pseudomonas syringae pv tomato......................................46

4.4.3 BON1/CPN1 protein accumulation was induced in the snc1 gain-

of-function mutant..................................................................................49

4.4.4 Induction of BON1/CPN1 protein accumulation by avirulent P. s. t.

DC3000 (avrRpt2) depended on NDR1 but not EDR1 ..........................50

4.5 Discussion.......................................................................................................52

Chapter 5 Pathogen- and calcium-responsive BON1/CPN1 promoter activity ..........56

5.1 Summary.........................................................................................................56



5.3.1 BON1/CPN1 promoter-GUS fusion constructs and plant

transformation ........................................................................................60

5.3.2 Bacterial and chemical treatments........................................................60

5.3.3 Histochemical staining for GUS activity..............................................61

5.3.4 Genomic Southern analysis ..................................................................62

5.3.5 Total RNA extraction and semi-quantitative RT-PCR.........................62

5.3.6 In planta bacterial growth analysis......................................................63

5.4 Results.............................................................................................................63

5.4.1 A 280 bp region of the BON1/CPN1 promoter is required for

pathogen-induced GUS expression ........................................................63

5.4.2 A 280 bp region of the BON1/CPN1 promoter is required for

calcium ionophore-induced GUS expression .........................................68

5.4.3 Over-accumulation of BON1/CPN1 in two BON1/CPN1 promoter

T-DNA insertion mutants.......................................................................68

5.5 Discussion.......................................................................................................73

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Chapter 6 Conclusions and future directions ..............................................................76

Bibliography ................................................................................................................81

Appendix A Changes in BON1/CPN1 protein level in response to abiotic stimuli ...90

A.1 Background information ................................................................................90

A.2 Materials and methods ...................................................................................91

A.2.1 Plant materials and growth conditions.................................................91

A.2.2 Chemical, cold treatments and western analysis .................................91

A.3 Results............................................................................................................92

A.3.1 BON1/CPN1 protein level in response to low temperature and low

humidity .................................................................................................92

A.3.2 BON1/CPN1 protein level in response to calcium ionophore

treatment.................................................................................................93

A.4 Dicussion .......................................................................................................95

Appendix B Subcellular redistribution of YFP::BON1/CPN1 protein in response

to calcium ionophore and cold stimuli..................................................................97

B.1 Background information ................................................................................97

B.2 Materials and methods ...................................................................................98

B.2.1 Plant materials and growth conditions.................................................98

B.2.2 Chemical and cold treatments and confocal microscopy.....................98

B.3 Results ............................................................................................................99

B.4 Discussion ......................................................................................................102

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LIST OF FIGURES

Figure 1: The calcium ionophore A23187 triggered cell death in HH/HT-grown

cpn1-1 mutant plants.............................................................................................15

Figure 2: A23187 induced PR gene expression in HH/HT-grown cpn1-1 mutant

plants.....................................................................................................................17

Figure 3: 1h cold treatment induced PR gene expression in HH/HT cpn1-1

mutant plants.........................................................................................................19

Figure 4: A coomassie-stained SDS-PAGE gel showing bacterial-expressed

GST-VWA BON1/CPN1

fusion protein and protease-cleaved products after

electrophoresis. .....................................................................................................30

Figure 5: A coomassie-stained SDS-PAGE gel showing the purified

VWABON1/CPN1

protein (indicated by asterisk in lane 1).. .....................................31

Figure 6: Western blot background problems encountered using two anti-

BON1/CPN1 antisera to detect BON1/CPN1 in Arabidopsis plant protein

extracts. .................................................................................................................33

Figure 7: β-mercaptoethanol in the loading buffer caused the background signals

in western blotting.. ..............................................................................................35

Figure 8: Replacing 286 mM β-mercaptoethanol with 5mM DTT in the 2×SDS

loading buffer eliminated the background signal and allowed specific

detection of BON1/CPN1. ....................................................................................37

Figure 9: Lower concentrations of reducing agent eliminated the background

signals in western blotting. ...................................................................................38

Figure 10: BON1/CPN1 protein level in different Arabidopsis tissues......................45

Figure 11: BON1/CPN1 protein level in LH/LT-grown plants after bacterial

pathogen challenge. ..............................................................................................48

Figure 12: BON1/CPN1 protein accumulation in snc1 mutant plants........................50

Figure 13: Pathogen-induced BON1/CPN1 protein accumulation in edr1 and

ndr1 mutant plants. ...............................................................................................52

Figure 14: A model for BON1/CPN1 regulation by RPS2 and SNC1 defense

pathways. ..............................................................................................................53

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Figure 15: Some predicted cis-acting elements in the BON1/CPN1 promoter

region. ...................................................................................................................59

Figure 16: Expression of BON1/CPN1 promoter-GUS fusion construct in

transgenic Arabidopsis plants. ..............................................................................65

Figure 17: BON1/CPN1 promoter deletion analysis in response to pathogen and

calcium ionophore stimuli.. ..................................................................................67

Figure 18: Phenotypic effects of two T-DNA insertions in the BON1/CPN1

promoter................................................................................................................70

Figure 19: Overexpression of BON1/CPN1 in cpn1-2 and cpn1-3 mutants. .............72

Figure 20: A proposed model for the function of BON1/CPN1.. ...............................77

Figure A1: BON1/CPN1 protein level in LH/LT or HH/HT-grown Col-0 plants. ....92

Figure A2: BON1/CPN1 protein level in Col-0 plants after 4°C cold treatment. ......93

Figure A3: BON1/CPN1 protein level in wild-type Col-0 plants after A23187

treatment. ..............................................................................................................94

Figure B1: Subcellular localization of YFP::BON1/CPN1 in response to bacterial

pathogen, calcium ionophore, and cold stimuli in transgenic plant leaves. .........101

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ACKNOWLEDGEMENTS

First and foremost, I would like to express my greatest gratitude to my thesis advisor, Dr.

Timothy W. McNellis, for his full support and guidance throughout my doctoral study. I

would also like to thank my thesis committee members, Dr. Surinder Chopra, Dr. Paula

McSteen, Dr. Seogchan Kang, and Dr. Andrew Stephenson for their helpful suggestions

and discussions regarding my research projects. Furthermore, I would like to thank my

previous and current lab members, Dr. Niran Jambunathan, Dr. Jianxin Liu, Justin Dillon,

Dr. Philip Jensen, Judy Sinn, Steven Lee, and Dharmendra Singh for all their help,

discussion, and encouragement during my time in the lab. Finally, my deepest

appreciation and gratitude goes to my family in Taiwan and my fiancé Yilun Zhao. I

would have been able to complete this long journey without their love and support.

This thesis is dedicated to my dear late father Guang-Jie Lee, whose unconditional love

lives on forever in our memory

.

Chapter 1

The Arabidopsis copine BONZAI1/COPINE1 protein and its role in calcium

signaling

1.1 Plant-pathogen interaction

Plants frequently encounter a range of abiotic and biotic challenges during their

lifetime. Therefore, complex non-self recognition and defense mechanisms are deployed

to protect against microbial attack. The genetic basis of plant-pathogen recognition can

be explained by the “gene-for-gene” concept (Flor, 1971). In an incompatible plant-

pathogen interaction, a bacterial effector protein encoded by an avirulence gene (Avr) can

be specifically recognized by a corresponding protein encoded by a resistance (R) gene in

the plants (Flor, 1971; Staskawicz et al., 1995; Dangl and Jones, 2001; Nimchuk et al.,

2003). The recognition event then triggers a sequence of signaling events that leads to

defense activation and renders the plants resistant to pathogen invasion. On the other

hand, a compatible interaction occurs when either determinant from the plants or the

pathogens is absent, which leads to the breakdown of resistance and renders the plants

susceptible to the pathogen attack (Dangl and Jones, 2001; Nurnberger and Scheel, 2001;

Nimchuk et al., 2003).

The earliest steps in defense are changes in ion fluxes such as calcium influx

(Nurnberger and Scheel, 2001; Hetherington and Brownlee, 2004; Garcia-Brugger et al.,

2006). Other defense responses include production of reactive oxygen species (ROS),

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production of nitric oxide (NO), cell wall reinforcement, and production of antimicrobial

compounds such as phytoalexins (Beynon and Dickinson, 2000; Dangl and Jones, 2001).

One of the most rapid defense responses is the hypersensitive response (HR), which

involves necrosis cell death at the infection sites to contain the pathogen progress. The

HR is thought to be a form of programmed cell death and has some similarity to

mammalian apoptosis (Greenberg, 1997). At the later stage of the defense activation, the

accumulation of the phenolic signaling molecule salicylic acid (SA) triggers a

heightened, systemic immune response throughout the plants by inducing several

Pathogenesis-Related (PR) defense genes and producing antimicrobial compounds

(Durrant and Dong, 2004). This phenomenon, termed systemic acquired resistance

(SAR), protects the host plants systemically against subsequent invasion of a broad range

of pathogens (Dong, 1998; Nurnberger and Scheel, 2001; Durrant and Dong, 2004).

1.2 Calcium signals and signaling components

In plants, a rise in cytosolic free Ca2+ concentration is a component of signal

transduction events in response to numerous developmental processes and environmental

stimuli (White and Broadley, 2003; Hepler, 2005; Lecourieux et al., 2006). For instance,

cold induces a rapid and transient rise in cytosolic Ca2+ level, whereas elicitors from

fungal pathogens trigger a sustained increase of cytosolic Ca2+ (Knight et al., 1991). The

transient changes in Ca2+ concentration serve as initial calcium signals which are then

perceived by various proteins that bind calcium ions. During calcium signaling, these

Ca2+-binding proteins may function as calcium sensors to detect changes in cytosolic

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Ca2+ level and transduce different stimuli into appropriate physiological responses

through interaction with downstream target proteins (Zielinski, 1998; Sanders et al.,

2002; Hetherington and Brownlee, 2004; Reddy and Reddy, 2004).

Plant calcium sensors can be divided into two major categories, which employ

different mechanisms to perceive and transduce calcium signals through downstream

signaling components. Sensor relays such as calmodulin (CaM) and calcineurin B-like

proteins undergo a calcium-induced conformational change, and subsequently change the

structure or enzyme activity of their targets proteins through protein-protein interaction

(Luan et al., 2002). Sensor responders such as Ca2+-dependent protein kinases (CDPK)

undergo a calcium-induced conformational change which affects the protein’s own

activity through intramolecular interaction (Harmon et al., 2000; Luan et al., 2002;

Harper et al., 2004). For calcium binding, many calcium sensors including CaMs and

CDPKs contain multiple “EF hand” motifs with a helix-loop-helix structure that binds a

single calcium ion (Zielinski, 1998). Calcium binding proteins without EF-hands, such

as copines and phospholipase D, contain C2 domains which bind Ca2+ ions and mediate

Ca2+-dependent phospholipid binding (Rizo and Sudhof, 1998). Together, the activation

of a specific set of calcium sensors and their downstream target proteins in a complex

network determines the specificity of calcium signaling.

1.3 Biochemical properties and functions of copines

Copines are a highly conserved protein family found in protozoa, nematodes,

animals, and plants (Tomsig and Creutz, 2002). The copines are defined as a protein

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class by the presence of two C2 domains in the N-terminal portion and a von Willebrand

A (VWA) domain in the C-terminal portion. In animals, C2 domain-containing proteins

such as protein kinase C and phospholipases are involved in processes such as lipid

metabolism, signal transduction, and membrane trafficking (Rizo and Sudhof, 1998). In

plants, C2 domain-containing proteins are considered potential Ca2+ signal transduction

proteins due to their Ca2+ binding ability (Tomsig and Creutz, 2002; Lecourieux et al.,

2006). On the other hand, VWA domain of copines have homology with the A domain

of integrin proteins and mediate protein-protein interactions (Creutz et al., 1998; Tomsig

et al., 2003). Proteins containing VWA domains are usually components of multi-protein

complexes (Whittaker and Hynes, 2002). The wide distribution of copines and their

conserved structure suggests that copines may play important roles in eukaryotic cells.

The biochemical activities of copines have been studied extensively. The C2

domains of copines exhibit Ca2+-dependent phospholipid binding activity with preference

for negatively charged phospholipids (Creutz et al., 1998; Hua et al., 2001; Tomsig and

Creutz, 2002). This characteristic could affect the subcellular localization of copine

proteins in response to Ca2+ stimuli. For example, Arabidopsis BONZAI1/COPINE1

(BON1/CPN1) protein is localized to the plasma membrane in Arabidopsis protoplasts

and intact plants (Hua et al., 2001). Dictyostelium discoideum CpnA binds membranes in

a Ca2+-dependent manner and transiently associates with the plasma membrane of some

starved cells (Damer et al., 2005). The Ca2+-dependent activity of the C2 domain may

also regulate the function of copines in coordination with the protein-protein interaction

mediated by the VWA domain. For instance, the interaction of human N-copine with its

interactor OS-9 is Ca2+ dependent (Nakayama et al., 1999). The VWA domains of

5

human copines have been shown to interact with different intracellular proteins such as

mitogen-activated protein kinase kinase (MEK1) and, protein phosphatase 5, and

ubiquitin C12 (UBC12) (Tomsig et al., 2003). Furthermore, human copine I regulates

the tumor necrosis factor-α (TNF-α) signaling pathway in a Ca2+-dependent manner,

possibly through copine I interaction with UBC12 to degrade a negative regulator

(Tomsig et al., 2004). Therefore, the basic function of copines may be to recruit target

proteins to membrane surfaces in response to calcium fluxes and subsequently affect the

activity of target proteins (Tomsig et al., 2003). This possible mode of action for copines

is further supported by studies of overexpressing VWA domains in Arabidopsis plants

and mammalian cells (Tomsig et al., 2004; Liu et al., 2005). Overexpression of the

VWA domain produces a dominant-negative effect on copine functions, possibly caused

by excess VWA domains competing with the endogenous copines for target protein

binding. Due to the lack of C2 domains, the VWA domain alone fails to recruit target

proteins to membranes in response to Ca2+ signals and the normal copine function is

disrupted.

Increasing studies of copines have revealed their diverse biological functions in

cellular processes including growth, development, cell death, stress and defense

responses, and neuronal signaling. In C. elegans, the copine protein GEM4 has been

shown to antagonize the function of GON2, a cation channel required for postembryonic

cell division (Church and Lambie, 2003). In addition, the silencing of copine NRA-1 by

RNA interference leads to nicotine resistance, probably caused by reduced expression of

synaptic nicotinic receptor in C. elegans (Gottschalk et al., 2005). The Dictyostelium

copine A is involved in cytokinesis, contractile vacuole function, and normal

6

development in the later stages prior to culmination (Damer et al., 2007). The human

copine I negatively regulates the TNF-α signaling in human embryonic kidney cells

(Tomsig et al., 2004). Furthermore, a recent study has shown that copine I blocks the

transcriptional activity of NF-κB, a downstream transcriptional factor regulating the

TNF-α-induced gene expression, through directing the proteolytic processing of p65

subunits in NF-κB complex (Ramsey et al., 2008).

In Arabidopsis, the BON1/CPN1 gene has been shown to negatively regulate cell

death and defense responses (Jambunathan et al., 2001). Loss of function of BON1 in

combination with BON2 or BON3 gene results in extensive cell death and lethality,

indicating that copine family may have overlapping functions in cell death suppression

(Yang et al., 2006b). In addition, the BON1 interactor BAP1 and its homolog BAP2 may

function together with BON/CPN family as the general inhibitors of cell death and

defense responses (Yang et al., 2006a; Yang et al., 2007). Interestingly, BAP1 and

BAP2 are small C2-domain containing proteins. Together, copines and associated C2-

domain proteins may function together in specific Ca2+ signaling that underlie processes

including cell death, defense responses, and growth regulation in Arabidopsis.

1.4 Arabidopsis BON1/CPN1 as a suppressor of defense responses

The phenotype of bon1/cpn1 knockout mutants indicates that BON1/CPN1 is a

suppressor of cell death and defense responses (Hua et al., 2001; Jambunathan et al.,

2001; Jambunathan and McNellis, 2003). The null mutant cpn1-1 exhibits a humidity-

and temperature-sensitive, lesion-mimic phenotype with an accelerated hypersensitive

7

response (HR), constitutive expression of PR genes and increased resistance to a bacterial

and an oomycete pathogen under nonpermissive growth conditions with low humidity

(LH, 35% RH) and low temperature (LT, 21°C) (Jambunathan et al., 2001). These

mutant phenotypes suggest that the BON1/CPN1 protein negatively regulates the plant

defense responses.

BON1/CPN1 has been shown to negatively regulate a disease resistance (R) gene

homolog, SUPPRESSOR OF npr1-1, CONSTITUTIVE 1(SNC1) (Yang and Hua, 2004).

SNC1 gene encodes an R protein homolog which belongs to the Toll/interleukin1

receptor (TIR)-nucleotide binding site (NBS)-leucine rich repeat (LRR) class of R

proteins (Dangl and Jones, 2001; Martin et al., 2003). Interestingly, SNC1 and six other

closely-related TIR-NBS-LRR R genes are located in the RPP5 (for recognition of

Peronospora parasitica 5) locus in the Arabidopsis thaliana Columbia ecotype (Noel et

al., 1999) and these genes appear to be regulated coordinately at the transcriptional level

(Yi and Richards, 2007). Among the R genes in this locus, RPP4 have been

demonstrated to confer resistance against fungal pathogens (Noel et al., 1999; van der

Biezen et al., 2002) and the activation of SNC1 results in resistance against bacterial and

fungal pathogens (Zhang et al., 2003; Yang and Hua, 2004). Furthermore, genetic

studies have shown that the loss-of-function mutation in bon1-1 mutant activates the

SNC1 gene and leads to constitutive defense activation and reduced cell growth.

However, the molecular mechanism of how SNC1 activity is regulated by BON1/CPN1 is

not fully understood. It has been suggested that BON1/CPN1 may be one of the factors

that regulates SNC1 gene at the transcript level (Li et al., 2007).

8

When the cpn1-1 mutant is grown under permissive conditions with high humidity

(HH, 75% RH) and high temperature (HT, 25°C), the lesion-mimic and enhanced disease

resistance phenotype is abolished and constitutive PR gene expression is suppressed

(Jambunathan et al., 2001; Jambunathan and McNellis, 2003). These results

demonstrated that the cpn1-1 mutant phenotype is sensitive to temperature and humidity.

Interestingly, BON1/CPN1 gene expression is up-regulated in response to non-

permissive, LH or LT growth conditions in wild-type Col-0 plants (Jambunathan and

McNellis, 2003). In addition, BON1/CPN1 expression is up-regulated by pathogen

inoculation in wild-type Col-0 plants. The accumulation of BON1/CPN1 transcript is

induced rapidly and transiently in Col-0 leaves inoculated with an avirulent strain of

Pseudomonas syringae pv. tomato (P. s. t.) carrying the avrRpt2 gene, which is

recognized by the cognate RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2)

gene in Col-0 plants (Jambunathan and McNellis, 2003). This result indicates that the

BON1/CPN1 transcript is up-regulated specifically by gene-for-gene recognition events

in the presence of corresponding R and Avr proteins (Jambunathan and McNellis, 2003)

1.5 Objectives of my thesis research

Based on the conditional phenotype of the cpn1-1 mutant, we speculated that the

activation of PR gene expression in response to low temperature and humidity in cpn1-1

mutant plants may reflect an inappropriate response to fluxes in intracellular Ca2+

concentration in the absence of BON1/CPN1. To test this hypothesis, we examined the

effect of the calcium ionophore A23187 on HH/HT-grown Col-0 and cpn1-1 plants.

9

Next, an anti-BON1/CPN1 antibody was generated to detect the BON1/CPN1 protein

level in wild-type Col-0 plants after bacterial challenge. To investigate how BON1/CPN1

gene expression is regulated, β-glucuronidase (GUS) reporter gene (Jefferson, 1989) was

used to study the spatial expression of BON1/CPN1 and its promoter activity in response

to calcium and pathogen stimuli. Finally, we characterized two BON1/CPN1 T-DNA

insertion mutants which over-accumulate the BON1/CPN1 protein. Our results help to

elucidate the involvement of Ca2+ in BON1/CPN1 function and the regulation of

BON1/CPN1 expression in response to abiotic and biotic stresses.

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Chapter 2

The role of BON1/CPN1 in calcium signaling specificity

2.1 Summary

We investigated whether perturbations of intracellular Ca2+ levels could play a

role in the development of the cpn1-1 conditional phenotype. Calcium ionophore and

cold treatments triggered strong PR gene expression in cpn1-1 mutant plants under the

permissive growth conditions, whereas Ca2+ chelator EGTA attenuated the calcium

ionophore-induced PR1 expression. Our results indicate that perturbation of intracellular

calcium level activates defense responses in the absence of BON1/CPN1. This suggests

that BON1/CPN1 is required for normal responses to calcium fluxes.

2.2 Background information

One intriguing characteristic of the bon1/cpn1 loss-of-function mutants is the

conditional mutant phenotype. The cpn1-1 mutant shows an aberrant, stunted

morphology with spontaneous cell death and constitutive defense activation under non-

permissive, low humidity and low temperature (LH/LT) growth conditions. When the

cpn1-1 mutant is grown under permissive, high humidity and high temperature (HH/HT)

conditions, the lesion-mimic and enhanced disease resistance phenotypes were abolished

and constitutive PR gene expression was suppressed (Jambunathan et al., 2001;

11

Jambunathan and McNellis, 2003). When considering copines as potential Ca2+ sensors,

we speculated that the cpn1-1 mutant phenotype including cell death activation and

constitutive PR gene expression is due to inappropriate response to calcium fluxes

triggered by low humidity and low temperature in the absence of BON1/CPN1. In this

study, we take advantage of the conditional phenotype of cpn1-1 mutant plants, which

behave like the wild-type Col-0 plants under permissive HH/HT conditions. The effect

of the calcium ionophore A23187 on HH/HT-grown Col-0 and cpn1-1 plants was

investigated by monitoring PR gene expression, which served as a marker for activated

defense responses. A23187 is commonly used to produce a cytosolic Ca2+ perturbation

by allowing extracellular calcium ions released from the cell wall to enter freely into the

cytosol and elevate the cytosolic Ca2+ level (Williams et al., 1990). In addition, several

studies have utilized A23187 to elevate the cytosolic Ca2+ level which in turn induces the

expression of Ca2+-dependent genes or physiological responses to ABA and chilling

temperature (Monroy and Dhindsa, 1995; Sheen, 1996; Sangwan et al., 2001). Our

results indicate that perturbations of intracellular Ca2+ level lead to defense activation in

the absence of BON1/CPN1.

2.3 Materials and methods

2.3.1 Plant materials and growth conditions

All plants were grown in soil-less medium (Redi-Earth Plug and Seedling Mix,

Sun Gro Horticulture) and irrigated with distilled water. For the permissive HH/HT

12

conditions, the Arabidopsis thaliana ecotype Columbia (Col-0) plants and cpn1-1 mutant

plants were grown at 25°C and 75% RH under an 8 h photoperiod with 100 µmol m-2 s

-1

light intensity. 5-week-old plants were used for all experiments.

2.3.2 Chemicals and cold stimuli treatments

For leaf infiltration, the underside of an Arabidopsis leaf was first scratched with

a razor blade, and then infiltrated with chemical solutions using a needle-less syringe

pushed against the leaf surface until the intracellular space of the whole leaf was filled

with the solution. For cell death observations and PR gene expression analysis, 50 µM or

100 µM of calcium ionophore A23187 (Sigma, St. Louis, MO, USA) were used along

with 0.25% or 0.5% DMSO (dimethyl sulfoxide; Sigma) as solvent controls, respectively.

Sterilized water was used as an infiltration control. 10 mM EGTA (Ethylene glycol-

bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; Sigma) was used alone or in

combinations with 50µM A23187. Cold treatments were administered by placing potted

plants at 4°C for 1 h and then returning them to the original growth chamber for periods

of time as described in Results. Untreated control plants were also included for chemical

and cold stimulus experiments. For cell death observations, results from four replicate

experiments were analyzed by Student’s t-test. For PR gene expression analysis, control

or chemical- or cold-treated leaves were collected at the indicated time points, flash-

frozen in liquid nitrogen, and stored at -80°C prior to RNA isolation.

13

2.3.3 RNA preparation and northern analysis

150mg of frozen Arabidopsis leaf tissues were grounded by pestle and mortar,

and the total RNA was isolated using the RNeasy plant mini kit (Qiagen, Valencia, Ca,

USA). For northern analysis, 5µg of total RNA was separated on 1% agarose gel,

transferred onto a Hybond-N nitrocellulose membrane (GE Healthcare), and probed with

PR1 or PR2 probes using the NorthernMax kit (GE Healthcare). PR1 and PR2 probe

fragments were prepared as described (Liu, 2007) and labeled using the Rediprime II kit

(GE Healthcare).

2.4 Results

2.4.1 Calcium ionophore triggered cell death in cpn1-1 mutant plants

The calcium ionophore A23187 was a potent trigger of cell death in cpn1-1

mutants; in contrast, A23187 triggered a weak cell death response in wild-type plants

(Figure 1 A, upper panel). Leaves of HH/HT-grown cpn1-1 plants usually showed a

visible cell death response with partially or totally collapsed tissues within 24h post

infiltration (hpi) with 50µM A23187 (Figure 1 A, lower panel). The same treatment

usually produced no cell death or only slight collapse in HH/HT-grown Col-0 plants

(Figure 1 A, upper panel). Tissue collapse in A23187-treated cpn1-1 leaves was initially

observed starting at 16 hpi and gradually progressed until around 24 hpi. Water and

0.25% DMSO did not trigger cell death in Col-0 or cpn1-1 plants (Figure 1 A). Both

HH/HT Col-0 and cpn1-1 plants seemed to be more sensitive to environmental stimuli

14

than LH/LT-grown plants, which may contribute to the batch-to-batch variation in

replicate experiments. In general, 50µM A23187 triggered much stronger cell death

response in cpn1-1 plants than in Col-0 plants under the permissive growth conditions.

Around 70% of A23187-treated cpn1-1 leaves displayed visible collapse, while fewer

than 20% of A23187-treated Col-0 leaves showed visible collapse (Figure 1 B). A23187-

induced collapse in wild-type plants, when it occurred, was less extensive than that

observed in cpn1-1 plants (Figure 1 and data not shown).

15

A B

Figure 1: The calcium ionophore A23187 triggered cell death in HH/HT-grown cpn1-1 mutant

plants. A, A wild-type plant grown under permissive conditions and infiltrated in one leaf with

water (infiltration control), a second leaf with 0.25% DMSO (solvent control), and a third leaf

with 50µM A23187 in 0.25% DMSO as indicated by the arrows (upper panel). A cpn1-1 mutant

plant with the same treatments is shown (lower panel). Photographs were taken at 26 hpi. B,

Bar graph comparing percentages of cpn1-1 (black bars) and wild-type (open bars) plant leaves

that showed visible cell death in response to A23187 infiltration. Percentage is the number of

the leaves that showed any visible collapse out of the total number of treated leaves. The results

represent the pooled average of four independent experiments.

16

2.4.2 Calcium ionophore triggered accumulation of PR gene transcripts in cpn1-1

mutants

PR1 and PR2 gene expression was monitored by northern blot analysis in wild-

type and cpn1-1 plant leaves infiltrated with calcium ionophore A23187. Total RNA was

isolated from leaves that showed no collapse or partial collapse in response to A23187.

50µM and 100µM A23187 treatment triggered high-level PR1 and PR2 transcript

accumulation in cpn1-1 plants but not in wild-type plants at 24 hpi (Figure 2 A). 100µM

A23187 triggered a low level of PR2 transcript accumulation and a very low level of PR1

transcript accumulation in wild-type plants. However, these transcript levels were much

lower than those observed in cpn1-1 plants after the same treatment. There was a clear

dosage effect of calcium ionophore: in HH/HT cpn1-1 plants, 100µM A23187 caused

higher accumulation of both PR1 and PR2 transcripts than 50µM A23187. Both the

water control and the solvent control consisting of 0.5% DMSO in water triggered low

but detectable PR gene transcript accumulation in HH/HT cpn1-1 plants (Figure 2 A).

PR transcript accumulation was slightly higher in the solvent control than in the water

control. These experiments were repeated four times with similar results.

17

Figure 2: A23187 induced PR gene expression in HH/HT-grown cpn1-1 mutant plants.

Northern blots showing the effect of calcium ionophore A23187 and calcium chelator EGTA on

PR gene transcript accumulation in Col-0 wild-type (WT) and cpn1-1 mutant (M) plants grown

under permissive conditions. A, Effect of A23187 on PR1 and PR2 gene transcript

accumulation. Treatments: water (W, infiltration control), 0.5% DMSO (D), 50 µM A23187 in

0.25% DMSO (50A), or 100µM A23187 in 0.5% DMSO (100A). B, Suppression of calcium

ionophore-induced PR1 gene expression by EGTA. Treatments: water (W, infiltration control),

0.25% DMSO (D, solvent control), 50 µM A23187 in 0.25% DMSO (50A), 10 mM EGTA

(10G), and 10 mM EGTA mixed with 50 µM A23187 (10G+50A). Time, time after treatment in

hours; Un, untreated plant; rRNA, ribosomal RNA stained with methylene blue to show the

relative amount of RNA in each lane. Five µg of total RNA were loaded in each lane.

18

2.4.3 Ca2+ chelator EGTA suppressed A23187-induced PR1 gene expression in cpn1-

1 plants

To determine whether calcium ionophore-induced responses were due to the

influx of extracellular Ca2+, we tested whether chelation of extracellular Ca

2+ could

attenuate the effect of A23187. The Ca2+ chelator EGTA was co-infiltrated with A23187

into leaves of Col-0 and cpn1-1 plants growing under permissive conditions, and the

effect on PR1 transcript accumulation was determined by northern blot. 10mM EGTA

strongly suppressed PR1 transcript accumulation when it was co-infiltrated with 50µM

A23187 (Figure 2 B, compare lanes 50A to 10G+50A in HH/HT cpn1-1). In this

experiment, 50µM A23187 triggered some PR1 transcript accumulation in wild-type

plants; however, the accumulation level was much lower than that observed in cpn1-1

plants receiving the same treatment (Figure 2 B). This result illustrates some of the

variability of our experimental system: A23187 sometimes induced PR gene expression

in wild-type plants as well as in cpn1-1 plants; however, in all cases, A23187-induced PR

gene expression was much higher in cpn1-1 plants than in wild-type plants. Also, 10mM

EGTA largely suppressed A23187-induced PR1 transcript accumulation in wild-type

plants, similar to the pattern observed in cpn1-1 plants. The solvent control (DMSO)

triggered slight PR1 gene transcript accumulation in cpn1-1 plants growing under

permissive conditions (Figure 2, A and B).

2.4.4 Cold-induced PR gene expression in cpn1-1 plants

19

Because the cpn1-1 mutant phenotypes are temperature-sensitive, we decided to

test whether transient cold treatment of HH/HT-grown cpn1-1 plants would also trigger

PR gene transcript accumulation. Wild-type and cpn1-1 plants growing under

permissive, HH/HT conditions were cold-treated at 4ºC for 1 h. PR gene expression was

monitored by northern blots either immediately after the 1h cold treatment period or after

the plants had been returned to 25°C for 23h (Figure 3). No visible cell death response

was observed in any Col-0 or cpn1-1 plants at any point in this experiment. However,

cold treatment specifically triggered the accumulation of PR1 and PR2 gene transcripts in

Figure 3: 1h cold treatment induced PR gene expression in HH/HT cpn1-1 mutant plants. Wild-

type Col-0 and cpn1-1 mutant plants grown under permissive conditions were subjected to 4ºC

for 1h and then returned to their original growth chamber at 25ºC for either 0 or 23 hours. PR

gene transcript levels were monitored by northern analyses. Transcript levels in untreated plants

were monitored at 0h and 24h. Dashed line indicates where two sections of the same RNA blot

were joined together with unnecessary middle lanes removed. rRNA, ribosomal RNA stained

with methylene blue to show the relative amount of RNA in each lane.

20

cpn1-1 plants at 23h after the end of the cold treatment period. No cold-induced PR

transcript accumulation was observed in wild-type plants. In addition, no PR transcript

accumulation was observed in cpn1-1 plants immediately after the end of the 1h cold

treatment.

2.5 Discussion

This study implies that BON1/CPN1 is required for normal responses to Ca2+

fluxes in Arabidopsis plants. In particular, our findings suggest that BON1/CPN1 plays a

role in maintaining the specificity of Ca2+

signaling by preventing the activation of plant

defenses by changes in intracellular Ca2+ level that are unrelated to pathogen attack, at

least in plants containing SNC1. We used the conditional nature of the cpn1-1 mutant

phenotype to explore the involvement of intracellular Ca2+ in the development of the

cpn1-1 mutant phenotype. The results presented here indicate that the cpn1-1 mutant is

especially sensitive to perturbations of intracellular Ca2+ level. The calcium ionophore

A23187 triggered a much stronger cell death response and higher PR gene transcript

accumulation in cpn1-1 plants than in wild-type plants growing under permissive

conditions. This indicates that a chemically-induced influx of Ca2+ into the cytoplasm

stimulates strong defense-related gene expression in cpn1-1 plants. Moreover, the Ca2+

chelator EGTA largely blocked the A23187-induced PR1 gene transcript accumulation,

indicating that when extracellular Ca2+ is chelated and unavailable for transport, the effect

of A23187 is reduced. This result shows that Ca2+ influx from the apoplast is the main

factor responsible for the physiological responses caused by A23187 in cpn1-1 mutants.

21

In addition, cold stimulus also triggered the PR gene induction specifically in cpn1-1

mutant plants. Cold treatments cause a transient rise in cytosolic Ca2+ level and changes

of membrane fluidity (Plieth et al., 1999; Orvar et al., 2000). Collectively, our data

showed that perturbation of the cytosolic Ca2+ level, either by cold or A23187, was able

to elicit defense responses in the cpn1-1 mutant.

It appears that BON1/CPN1 is required for normal responses to Ca2+ fluxes in

Arabidopsis plants. In particular, it appears that certain environmental stimuli, such as

LT or LH, may trigger intracellular Ca2+ perturbations that have the potential to activate

cell death and defense responses. Our findings suggest that BON1/CPN1 plays a role in

maintaining the specificity of Ca2+

signaling and preventing the activation of plant

defenses by changes in intracellular Ca2+ level that are unrelated to pathogen attack. Our

results suggest that the cpn1-1 mutant phenotype observed under the non-permissive

growth conditions results from inappropriate defense activation in responses to Ca2+

fluxes triggered by lower temperature and humidity.

It is interesting that DMSO treatment alone triggered slight PR gene transcript

accumulation in cpn1-1 plants but not in wild-type plants. DMSO is commonly used as a

membrane rigidifier that mimics the effects of cold on biological membranes (Orvar et

al., 2000; Sangwan et al., 2001). This result suggests that BON1/CPN1 is also required

for normal responses to changes in membrane fluidity, and that changes in membrane

fluidity may be able to trigger defense responses in the cpn1-1 mutant.

It is notable that 50µM A23187 triggered tissue collapse in wild-type plants

(Figure 1 A), although this response was much weaker than that observed in cpn1-1

mutant plants. The observation is not unexpected, since Ca2+ influx mediated by high

22

concentration of A23187 (15mM) caused cell death in soybean suspension cells (Levine

et al., 1996). Similarly, cytosolic Ca2+ overload can cause cytotoxicity and trigger cell

death in animals (Orrenius et al., 2003). The A23187 concentration we applied was

probably high enough to allow excessive amounts of apoplastic Ca2+ to enter the cytosol,

and therefore caused cell death due to cytosolic Ca2+ overload even in wild-type plants.

Both A23187 and cold induced PR gene expression in cpn1-1 plants, while cell

death was triggered only by A23187. The difference may be related to the degree and

type of Ca2+ perturbation elicited by the two treatments. A brief cold shock induces an

immediate and transient rise in cytosolic Ca2+ level (Knight et al., 1991; Plieth et al.,

1999). Intracellular Ca2+ levels return to baseline level when the temperature reaches a

steady value (Plieth et al., 1999). In contrast A23187 may cause a more sustained

increase in intracellular Ca2+ levels, thus creating a more extensive response.

The findings presented here explain the conditional nature of the cpn1-1 mutant

phenotypes. They are also consistent with the proposed function of copines as Ca2+-

sensitive membrane trafficking proteins and Ca2+-responsive signal transduction proteins.

Finally, the accelerated HR observed in cpn1-1 (Jambunathan et al., 2001) may also be

due to a greater sensitivity to pathogen-induced increases in intracellular Ca2+. Further

work will be required to elucidate the function of BON1/CPN1 in regulating responses to

cytosolic Ca2+ fluctuation.

23

Chapter 3

Generation of anti-BON1/CPN1 antisera and troubleshooting for artifact signals in

western blot analysis

3.1 Summary

Two polyclonal antisera were generated to detect BON1/CPN1 protein in

Arabidopsis. When these antisera were used in western blot analysis, we encountered

problems with artifact bands associated with the β-mercaptoethanol in the protein gel

loading buffer. Here, we demonstrated that lowering the concentration of reducing agent

in the loading buffer, either β-mercaptoethanol or dithiothreitol (DTT), eliminated the

artifact signals and allowed the specific detection of BON1/CPN1 band in plants extracts.

The possible cause of the artifact bands by keratin contamination of protein samples is

discussed.


Western blotting involves sodium dodecyl sulfate–polyacrylamide gel electrophoresis

(SDS-PAGE) of proteins (Laemmli, 1970) followed by electrophoretic transfer of the

protein to a solid membrane and detection with antisera (Renart et al., 1979; Towbin et

al., 1979; Burnette, 1981). Western blot analysis is one of the most common

immunoassays for antigen detection, and this technique requires an antibody that can

24

specifically recognize the antigen of interest (Kurien and Scofield, 2003). However, with

the increasing sensitivity of protein detection methods, problems with artifact bands

involved in SDS-PAGE analysis and immunoblotting have been reported (Ochs, 1983;

Tasheva and Dessev, 1983; Shapiro, 1987; Riches et al., 1988; Berube et al., 1994). The

presence of artifact bands between 50-68 kDa is frequently found and correlated with the

use of β-mercaptoethanol as reducing agent in sample preparation. Evidence suggests

that these artifact bands originate from the contamination of human skin protein keratin

in protein samples or SDS-PAGE electrophoresis process, which presents a common

problem for immunoblotting when polyclonal antibodies are used (Ochs, 1983; Berube et

al., 1994).

The Arabidopsis BONZAI1/COPINE1 (BON1/CPN1) protein is a suppressor of

plant cell death and defense responses (Hua et al., 2001; Jambunathan et al., 2001; Yang

et al., 2006b). It belongs to the ubiquitous, highly conserved copine protein family,

whose members appear to be involved in membrane trafficking and protein interactions

(Tomsig and Creutz, 2002). BON1/CPN1 contains two C2 domains at the N-terminus

with calcium-dependent, phospholipid-binding activity, and a von Willebrand A (VWA)

domain at the C-terminus that mediates protein-protein interactions. We set out to

produce polyclonal anti-BON1/CPN1 antibodies in order to monitor the protein presence

and amount in plant tissues during plant disease resistance responses. In this chapter, we

described a problem with artifact bands between 50-75kDa which we encountered in

western analysis using the anti-BON1/CPN1 antisera. Furthermore, we demonstrate that

lower concentrations of reducing agent in the SDS-PAGE loading buffer eliminated the

25

artifact bands possibly caused by keratin contamination, and allowed the specific

detection of BON1/CPN1 protein bands in plant extracts.



All plants were grown in soil-less medium (Reid-Earth Plug and Seedling Mix,

Sun Grow Horticulture) and irrigated with distilled water. Wild-type plants were of the

Arabidopsis thaliana ecotype Columbia-0 (Col-0). cpn1-1 mutant plants are BON1/CPN1

null mutants (Jambunathan et al., 2001). cpn1-2 and cpn1-3 are two mutants

(SAIL_865_A09 and SAIL_723_E11, respectively) with T-DNA inserted in the

BON1/CPN1 promoter region from the SAIL T-DNA insertion line collection

(http://www.tmri.org/en/partnership/sail_collection.aspx) (Sessions et al., 2002).

YFP::BON1/CPN1 plants are transgenic lines expressing yellow fluorescence protein

(YFP) fused to the N-terminus of BON1/CPN1 (Liu, 2007). BON1/CPN1::myc plants

are transgenic lines expressing BON1/CPN1 protein with a C-terminal c-myc epitope tag

(Liu, 2007). All plants were grown at 21°C and 35% RH under a 10 h photoperiod with

75 µmol m-2 s-1 light intensity. 5-week-old plants were used for all experiments.

26

3.3.2 Bacterial expression of BON1/CPN1 VWA domain and antibody production

The VWA domain of the Arabidopsis BON1/CPN1 protein (VWABON1/CPN1

) was

expressed in E. coli and purified using the glutathione-S-transferase (GST) expression

system (GE Healthcare, Piscataway, NJ, USA). Sequences encoding the VWA domain

(amino acids 291-578) of BON1/CPN1 were amplified with the following PCR primers:

5´-GAATTCCTTCATTTGGCGGGCCAAGGA-3´ (Forward) and 5´-

CTCGAGTCATGGAGGAATCGGTTTCAT-3´ (Reverse); introduced EcoRI and XhoI

restriction sites are shown in italics. The PCR product was subcloned into the pGEX-6P-

1 vector cut with EcoRI and XhoI so that the VWABON1/CPN1

domain was translationally

fused with the C-terminus of GST. The GST- VWABON1/CPN1

fusion construct was

introduced into protease-deficient E. coli stain BL21 (DE3) pLys (EMD Chemical, Inc.,

Gibbstown, NJ, USA). Purification steps for the bacterial-expressed VWABON1/CPN1

domain were modified from Tomsig and Creutz, 2000. A bacterial culture was seeded

from an overnight culture, grown for 1 h at 37ºC, and then induced with 50 µM IPTG

(Sigma) for 3 h at 30ºC. The cells were then lysed by sonication in ice-cold 1×PBS

containing 10 mg/ml lysozyme (Sigma) and centrifuged at 9,600×g for 10 min. The

pellet was resuspended in 1×PBS buffer containing 5 mM EGTA, 5 mM DTT, 10 mg/ml

PMSF (phenylmethanesulphonylfluoride, Sigma), and 1×protease inhibitor cocktail

(Sigma, catalog #P2714). Proteins in the extract were solubilized with sarkosyl (Sigma)

prepared in 1×PBS to a final concentration of 0.5%. After the mixture was centrifuged at

9,600×g for 15 min, the supernatant was saved, mixed with Triton X-100 (Sigma)

prepared in 1×PBS to a final concentration of 1%, and incubated with glutathione

27

Sepharose 4B beads (GE Healthcare) with gentle shaking at room temperature for 1.5 h.

The beads were then washed with ice-cold 1×PBS 6 times followed by 6 washes in

PreScission Cleavage buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM

DTT). VWABON1/CPN1

was then cleaved from the GST moiety by resuspending the beads

in 1×bead volume of PreScission Cleavage buffer, adding 6 units of PreScission protease

(GE Healthcare) per 200µl of beads, and incubating at 4ºC overnight. Cleaved

VWABON1/CPN1

protein was released from beads in 2×SDS loading buffer (126 mM Tris-

HCl pH 6.8, 20% glycerol, 4%SDS, 0.005% bromophenol blue, 2% β-mercaptoethanol)

at 100ºC for 10 min and separated on 10% SDS-PAGE gels. Protein gels were stained

with GelCode Blue stain (Pierce, Rockford, IL, USA), and the 31 kDa VWABON1/CPN1

band was excised. The VWABON1/CPN1

protein fragment was electroeluted into 1×SDS

running buffer (25mM Tris, 192mM glycine, 0.1% SDS) using ElutaTube (Fermentas,

Burlington, Ontario, Canada) according to the manufacturer’s instructions. Eluted

proteins were concentrated using Amicon Ultra-4 centrifugal filter units (molecular

weight cut-off 30 kDa) (Millipore, Billerica, MA, USA). Purified VWABON1/CPN1

protein

was mixed with Titermax adjuvant, and used to immunize two rats following standard

procedures for polyclonal antibody production (Cocalico Biologicals, Inc., Reamstown,

PA, USA).

3.3.3 Protein extraction and western blot analysis

Arabidopsis total soluble proteins were isolated by re-suspending frozen,

pulverized tissue in extraction buffer containing 12% sucrose, 100 mM Tris-HCl pH 7.5,

28

1 mM EDTA, 1% Triton X-100, 1% sarkosyl, 2 mM DTT, and 1×protease inhibitor

cocktail (GE Healthcare). Supernatants were collected after centrifugation of the samples

at 11,000×g at 4ºC for 5 min. This protocol was based on a previously published

protocol (Hua et al., 2001). Protein extracts were mixed with an equal volume of 2×SDS

loading buffer (126 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol

blue) containing 286 mM β-mercaptoethanol or 5 mM DTT as the reducing agent unless

indicated specifically. β-mercaptoethanol used in this study was from Sigma (Catalog #

M3148-25ml), Mallinckrodt Baker (J.T. Baker Catalog #4049, Phillipsburg, NJ, USA),

and AMRESCO (Catalog # 0482-100ml, Solon, OH, USA). Protein samples were

separated on 7.5% SDS-PAGE gels and electrotransferred onto Hybond ECL

nitrocellulose membranes (GE Healthcare). Membrane blocking and primary and

secondary antibody incubations were performed in 1×TBST buffer (20 mM Tris-HCl pH

7.6, 137 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk. A 1:10,000

dilution was used for both the rat anti- BON1/CPN1 antisera and the rabbit-anti-rat

antibody conjugated with horseradish peroxidase (HRP; Sigma). A 1:16,000 dilution was

used for the HRP-conjugated, goat anti-rat antibody (Abcam, Cambridge, MA, USA).

1:8,000 and 1:25,000 dilutions were used for the monoclonal mouse anti-c-myc antibody

(BD Biosciences, Palo Alto, CA, USA) and the HRP-conjugated, sheep-anti-mouse

antibody (GE healthcare), respectively. Proteins were detected using a

chemiluminescence detection kit (ECL Plus Western Blotting Detection System, GE

Healthcare) according to the manufacturer’s instructions.

29

3.4 Results

3.4.1 GST-VWABON1/CPN1

protein purification and antigen preparation

The GST expression system was employed to express the VWA domain of

BON1/CPN1 protein in E. coli after a failed attempt using the His-tag expression system

previously (data not shown). Under the inducing condition, we were able to express of

GST-VWABON1/CPN1

fusion protein in bacteria cultures (Figure 4, compare lane 2 and 3).

The GST protein alone was also induced by IPTG in bacteria containing the empty vector

pGEX-6P-1 (Figure 4, lane 1). Two induced protein bands had similar molecular weight

to the 57kDa expected size of GST-VWA BON1/CPN1

protein (Figure 4 , lane 3). The band

with smaller molecular weight may be a partial fusion protein due to proteolysis or

processing in E. coli. Therefore, the band with the bigger molecular weight was

considered as the complete GST-VWA BON1/CPN1

fusion protein and subjected to further

purification. The GST-VWA BON1/CPN1

protein was bound with the glutathione Sepharose

4B beads after incubation with soluble proteins from IPTG-induced bacterial lysate

(Figure 4, lane 5) but not from un-induced bacterial lysate (Figure 4, lane 4). However,

the association of GST-VWA BON1/CPN1

protein was so tight that the fusion protein could

not be eluted using the regular GST elution buffer according to the manufacturer’s

instructions (GE Healthcare). Therefore, we conducted on-matrix protease cleavage,

released the cleaved protein products from beads in 2×SDS loading buffer (Figure 4, lane

6-9), and separated the products on SDS-PAGE gels. Protein gels were stained with

GelCode Blue (Pierce, Rockford, IL, USA), and the 31 kDa VWA domain band was

excised, electroeluted into 1×SDS running buffer (25 mM Tris, 192 mM glycine, 0.1%

30

SDS) using ElutaTube (Fermentas, Burlington, Ontario, Canada), and concentrated using

Amicon Ultra-4 centrifugal filter units (molecular weight cut-off at 30 kDa) (Millipore,

Billerica, MA, USA),. Finally, the purified VWABON1/CPN1

protein band (Figure 5, lane

1) was used as antigen to immunize two rats for polyclonal antibody production

(Cocalico Biologicals, Inc., Reamstown, PA, USA).

Figure 4: A coomassie-stained SDS-PAGE gel showing bacterial-expressed GST-VWA

BON1/CPN1 fusion protein and protease-cleaved products after electrophoresis. Protein molecular

weight marker sizes are shown in kDa. Arrows indicated the position of protein bands of GST-

VWABON1/CPN1

fusion protein (57kDa), PreScission protease (46kDa), VWABON1/CPN1

portion

(31kDa), and GST protein (26kDa). Protein samples of total bacterial lysate were from bacteria

containing the empty pGEX-6P-1 vector with IPTG induction (lane 1), and bacteria containing

the GST-VWABON1/CPN1

fusion construct without IPTG induction (lane 2) or with IPTG

induction (lane 3). The GST-VWABON1/CPN1

protein associated with the glutathione Sepharose

4B beads after incubation with soluble proteins from IPTG-induced bacterial lysate (lane 5) but

not from un-induced bacterial lysate (lane 4). After 4h or 24h of protease cleavage, only a

small portion of the cleaved VWABON1/CPN1

protein products could be eluted according to the

manufacturer’s instruction (lane 6 and 8, respectively). All bound proteins including the

cleaved VWABON1/CPN1

were released from the matrix in 2×SDS loading buffer (lane 7 and 9).

The 24h of protease cleavage resulted in more complete cleavage of VWABON1/CPN1

from GST

moiety comparing to the 4h cleavage (lane 7 and 9, compare intensity of GST-VWA band).

31

3.4.2 Anti-BON1/CPN1 antisera produced high levels of background signals in

western blots

When the rat antisera were used for western blot analysis, we encountered

problems with high level background signals in the region between 50-75 kDa, as shown

in Figure 6. The same background signals occurred in all sample lanes, including protein

samples from wild-type Col-0 Arabidopsis plants, cpn1-1 mutant plants with a null

mutation in the BON1/CPN1 gene, and cpn1-2 and cpn1-3 mutant plants that are known

to over-accumulate the BON1/CPN1 transcript (Chapter 5). Only the empty lane was

without background signals. The strong background signals appeared to mask the

Figure 5: A coomassie-stained SDS-PAGE gel showing the purified VWABON1/CPN1

protein

(indicated by asterisk in lane 1). The matrix-bound proteins after protease cleavage are shown

in lane 2. Protein molecular weight marker sizes are shown in kDa. Arrows indicated the

position of protein bands of GST-VWABON1/CPN1

fusion protein (57kDa), PreScission protease

(46kDa), VWABON1/CPN1

portion (31kDa), and GST protein (26kDa).

32

expected 63 kDa BON1/CPN1 protein band. In contrast, a 90 kDa YFP::BON1/CPN1

fusion protein was detected in extracts from transgenic plants expressing high levels of

YFP::BON1/CPN1, although these lanes also contained the background signals. The

YFP::BON1/CPN1 bands were detectable because they did not overlap with the

background signals. The ability of the antisera to detect the YFP::BON1/CPN1 fusion

protein demonstrated that the rat antisera contained anti-BON1/CPN1 antibodies.

Antisera from both of the immunized rats produced similar background signal patterns

(compare Figure 6 A and B). The secondary antibody was not the source of the problem,

since using a different secondary antibody (goat anti-rat, Figure 6 B) instead of rabbit

anti-rat (Figure 6 A) did not eliminate the background signals. Western blots without the

background signals were obtained when similar blots containing protein from transgenic

Arabidopsis plants expressing c-myc epitope-tagged BON1/CPN1 protein (Liu, 2007)

were probed with an anti-c-myc monoclonal antibody (Figure 6 C), showing that the

background problems seen in the other blots were not related to some general aspect of

our western blotting technique. Finally, the rat pre-immune sera did not produce any

background signals (data not shown), indicating that the background signals produced by

the antisera were a result of the immunization process.

33

Figure 6: Western blot background problems encountered using two anti-BON1/CPN1 antisera

to detect BON1/CPN1 in Arabidopsis plant protein extracts. Each lane contains 25 µg of protein

except for the empty lane in panel A. Protein molecular weight marker sizes are shown in kDa.

A; The anti-BON1/CPN1 antiserum from the first immunized rat was used to probe a western

blot containing protein extracts from wild-type Col-0 (WT), cpn1-1 null mutant, cpn1-2 mutant,

cpn1-3 mutant, and two YFP::BON1/CPN1 transgenic plants. The secondary antibody used

was an HRP-conjugated, rabbit-anti-rat antibody. An empty lane was included to show the

absence of background signals when nothing was loaded in a lane. The arrow marks the

expected location of the 63 kDa BON1/CPN1 band; the asterisk indicates the expected location

of the 90 kDa YFP::BON1/CPN1 transgenic fusion protein band. B; The anti-BON1/CPN1

antiserum from the second immunized rat was used to probe a western blot containing protein

extracts from wild-type Col-0 (WT) and cpn1-1 null mutant plants. The secondary antibody

used was an HRP conjugated, goat-anti-rat antibody. The arrow marks the expected location of

the 63 kDa BON1/CPN1 band. C; A monoclonal mouse anti-c myc antibody was used to probe

a western blot containing protein extracts from two BON1/CPN1::myc transgenic plants. The

secondary antibody used was an HRP-conjugated, sheep-anti-mouse antibody. The double

asterisk indicates the location of the 63 kDa BON1/CPN1::myc transgenic fusion protein band.

34

3.4.3 ββββ-mercaptoethanol in the loading buffer caused the background problems

We speculated that the background problems might be due to the β-

mercaptoethanol in the loading buffer as suggested in previous study (Tasheva and

Dessev, 1983). To test whether the anti-BON1/CPN1 antibodies cross-reacted with

reagents in the protein gel loading buffer, a western blot was prepared using samples that

contained only loading buffers without protein extracts. One lane contained complete

SDS loading buffer, and the other lanes contained loading buffers with one of each of the

five components omitted. When this blot was probed with anti-BON1/CPN1 antisera, all

of the lanes showed the usual background problems except for the lane containing

loading buffer that was missing β-mercaptoethanol, as shown in Figure 7. This result

showed that β-mercaptoethanol in the loading buffer was associated with the background

signals in the western blots.

In order to rule out the possibility that our β-mercaptoethanol bottle was

contaminated with something that caused the background signals, SDS loading buffers

containing β-mercaptoethanol from five different sources were tested by western blotting.

All of the different sources of β-mercaptoethanol produced the same background patterns

after probing with the anti-BON1/CPN1 antisera (Figure 7 B). Background signals were

not observed in a lane loaded with 1-year-old SDS loading buffer, indicating that storage

of the loading buffer for 1 year at -20°C eliminated the background problems. It was

likely that this was the result of the loss of β-mercaptoethanol from the tube by

evaporation or by chemical breakdown. Finally, a lane containing SDS loading buffer

wherein the β- mercaptoethanol had been substituted with the alternative reducing agent

35

DTT (Cleland, 1964) did not have background signals (Figure 7 B, lane 7). These data

conclusively demonstrate that the background signals were specifically caused by β-

mercaptoethanol in the loading buffer.

Figure 7: β-mercaptoethanol in the loading buffer caused the background signals in western

blotting. Molecular weight marker sizes are shown in kDa. A; Western blot of a gel loaded with

complete 2×SDS loading buffer (lane 1) and 2×SDS loading buffers with one of each of the five

components omitted (lanes 2-6) probed with anti-BON1/CPN1 antisera. Check marks in the

table indicate the presence of individual ingredients. Omitting β-mercaptoethanol from the

loading buffer eliminated the background signal (lane 6). BPB, bromophenol blue; β-ΜΕ, β-

mercaptoethanol. B; Western blot of a gel loaded with a 1-year-old 2×SDS loading buffer made

with 286 mM β-mercaptoethanol (Sigma, Lot 035K0054, lane 1), 2×SDS loading buffers

containing 286 mM β-mercaptoethanol from various sources (lanes 2-6), and 2×SDS loading

buffer with 5 mM DTT instead of 286 mM β- mercaptoethanol (lane 7) probed with anti-

BON1/CPN1 antisera. β-mercaptoethanol was from Sigma (Lot 035K0054, lane 2; Lot

09729MH, lane 3; Lot 39F-060715, lane 4), Mallinckrodt Baker (Lot 1-3350, lane 5), and

AMRESCO (Lot 3078A68, lane 6).

36

3.4.4 Lower concentrations of reducing agent in the loading buffer eliminated the

background signals

Changing the reducing agent in the loading buffer from 286 mM β-

mercaptoethanol to 5mM DTT eliminated the background signals in western blots

(Figure 8 A) and allowed better detection of the 90 kDa YFP::BON1/CPN1 protein.

However, the 63 kDa native BON1/CPN1 protein was not detected in lanes containing

25µg of protein extract, possibly due to the low abundance nature of BON1/CPN1 and

the competitive binding of anti-BON1/CPN1 antibody to the much stronger

YFP::BON1/CPN1 signal on the same blot (Figure 8 A). Increasing the amount of plant

proteins to 50 µg per lane allowed specific detection of BON1/CPN1 in protein extracts

of wild-type Col-0 plants but not cpn1-1 null mutant plants, as expected (Figure 8 B).

Two other protein bands with higher molecular weights were also detected, which could

represent proteins similar to BON1/CPN1.

37

We speculated that the lower concentration of the reducing agent in the loading

buffer was the reason of background elimination rather than the change of different

reducing agents. Therefore, we tested a range of concentration of β-mercaptoethanol and

DTT in the loaded protein samples (Figure 9). The concentration of β-mercaptoethanol,

a monothiol, was doubled to match the reducing ability of the dithiol DTT. Our results

showed that final concentrations of 2.5 mM DTT and 5mM β-mercaptoethanol in the

loaded protein samples provided the optimal situation for BON1/CPN1 detection in plant

extracts using the anti-BON1/CPN1 antibody. On the other hand, high concentrations of

either β-mercaptoethanol or DTT caused the reoccurrence of background signals

A B

Figure 8: Replacing 286 mM β-mercaptoethanol with 5mM DTT in the 2×SDS loading buffer

eliminated the background signal and allowed specific detection of BON1/CPN1. Arrow, the

expected location of the 63 kDa BON1/CPN1 band. Molecular weight marker sizes are shown

in kDa. A; Substitution of 5 mM DTT for 286 mM β-mercaptoethanol in the 2×SDS loading

buffer eliminated the background signals. Each lane contains 25 µg of protein from wild-type

Col-0 (WT), cpn1-1 mutant, and two YFP::BON1/CPN1 transgenic plants. Two sections of the

same blot are shown with unnecessary middle lanes omitted. Asterisk, the 90 kDa

YFP::BON1/CPN1 protein band. B; Western blot containing 50 µg of protein per lane.

38

(Figure 9, lanes with 10mM DTT or 20mM β-ME) as seen in previous experiments

(Figure 6). Reducing agents appear to be essential for better resolution of BON1/CPN1

detection in western analysis, since omitting the reducing agent caused poor detection of

BON1/CPN1 band (Figure 9, no reducing agent lane).

Figure 9: Lower concentrations of reducing agent eliminated the background signals in western

blotting. A western blot was loaded with wild-type Col-0 protein sample prepared with a range

of final β-mercaptoethanol and DTT concentrations, or no reducing agent at all in the loading

buffer. Final concentrations of the reducing agents, the monothiol β-mercaptoethanol and the

dithiol DTT, were shown as pairs with the same reducing ability. The arrow indicates the

BON1/CPN1 band. Molecular weight marker sizes are shown in kDa. Each lane contains 75

µg of protein from the same wild-type plant extract.

3.5 Discussion

Our findings demonstrate that high concentrations of reducing agents in protein

samples can causes the background signals in western blots. The background signals we

39

encountered in western analysis using the polyclonal anti-BON1/CPN1 antibody were

very similar to the keratin contamination reported before (Ochs, 1983; Tasheva and

Dessev, 1983; Shapiro, 1987; Riches et al., 1988; Berube et al., 1994). The presence of

artifact bands between 50 and 68 kDa was first associated with the use of β-

mercaptoethanol for sample preparation under reduced conditions (Tasheva and Dessev,

1983). It was later demonstrated that these artifact bands were caused by the presence of

antibodies that react with the keratin contamination in protein samples or electrophoresis

buffers (Ochs, 1983). Furthermore, purification by pre-absorption of polyclonal

antibodies on keratin was able to improve the artifact problem, indicating the presence of

anti-keratin antibodies in the immunized animal serum (Berube et al., 1994). Along the

same line, there was a high likelihood that anti-keratin antibodies were present in the

anti-BON1/CPN1 sera due to keratin contamination in the gel-eluted antigen. After

being reduced by reducing agents under the electrophoresis conditions, the keratin

contaminant in protein samples was recognized by anti-keratin antibody and caused the

artifact bands in immunoblots.

Several solutions for the keratin artifact problems have been suggested (Shapiro, 1987;

Riches et al., 1988; Berube et al., 1994). The best way to eliminate these artifact bands is

taking extreme caution to avoid any keratin contamination during antigen preparation, sample

preparation, and SDS-PAGE electrophoresis. However, this is difficult to implement when

human handling is involved in the antibody production and immunodetection procedures.

Antibody purification by affinity chromatography also provides a feasible way to solve the

artifact problem. Most importantly, we demonstrated a simple and direct method to eliminate

the artifact background bands in western blotting. It will be useful for researchers to test a

40

range of reducing agent concentrations when trying to eliminate artifact bands from western

blots.

41

Chapter 4

Pathogen regulation of BON1/CPN1 protein level

4.1 Summary

The anti-BON1/CPN1 antisera were used to monitor the BON1/CPN1 protein

level in response to pathogen stimuli. The BON1/CPN1 protein accumulated to a high

level in roots of Col-0 plants. Furthermore, the timing of BON1/CPN1 protein

accumulation in response to avirulent and virulent pathogen challenge correlated with

previously determined BON1/CPN1 transcript accumulation patterns. Finally, the

accumulation of BON1/CPN1 protein was shown to be up-regulated by the activation of

defense signaling responses controlled by two Resistance (R) genes, SNC1 and RPS2.


Previous studies have shown that the expression of the Arabidopsis BON1/CPN1

gene is up-regulated by pathogen stimuli (Jambunathan and McNellis, 2003). Leaf

inoculation with avirulent Pseudomonas syringae pv tomato (P. s. t.) DC3000 (avrRpt2)

bacteria specifically triggers a rapid and transient accumulation of BON1/CPN1

transcripts, whereas virulent P. s. t. DC3000 bacterial inoculation triggers a much slower

BON1/CPN1 transcript accumulation in wild-type Col-0 plants (Jambunathan and

McNellis, 2003). Furthermore, in planta expression of the bacterial effector avrRpt2 in

42

the presence of the RPS2 disease resistance gene in the Col-0 background is sufficient to

induce the BON1/CPN1 transcript accumulation (Jambunathan and McNellis, 2003).

Together, these results indicate that stronger and more rapid accumulation of

BON1/CPN1 transcripts is triggered by the gene-for-gene recognition between the RPS2

gene carried by Col-0 plants and its cognate avrRpt2 gene carried by the avirulent strain

of P. s. t. DC3000 (avrRpt2) bacteria compared to that by a compatible reaction (Whalen

et al., 1991; Innes et al., 1993; Kunkel et al., 1993). In other words, rapid accumulation

of BON1/CPN1 transcripts is triggered by RPS2-mediated defense activation following

the RPS2-mediated recognition of avrRpt2.

Since BON1/CPN1 has been shown to negatively regulate the R gene homolog

SNC1 (Yang and Hua, 2004), we were interested in whether BON1/CPN1 transcript or

protein level is also regulated by the activation of SNC1-mediated defense signaling.

However, SNC1 is an orphan R gene due to the unknown cognate Avr-gene product

recognized by wild-type SNC1, which makes the study of wild-type SNC1-mediated

defense signaling not possible. In this study, we take advantage of the suppressor of n-

pr1-1, constitutive 1 (snc1) mutant, which has a dominant, missense mutation in SNC1

that activates the R gene constitutively, and results in constitutive PR gene expression and

enhanced disease resistance to bacterial and oomycete pathogens (Li et al., 2001). The

constitutive activation of SNC1 gene in the snc1 gain-of-function mutant allows us to

study the BON1/CPN1 regulation by SNC1-mediated defense signaling.

In this study, the anti-BON1/CPN1 antisera we generated in Chapter 3 were first

used to examine the tissue distribution of BON1/CPN1 protein. Next, to investigate

whether RPS2 or SNC1 genes affect the abundance of BON1/CPN1, we monitored

43

BON1/CPN1 levels in wild-type Col-0 plants after pathogen challenge and in gain-of-

function snc1 mutant plants. We also monitored BON1/CPN1 protein level in a mutant

with defective RPS2-mediated defense signaling and a mutant defective in a putative

BON1/CPN1 interacting protein. These results provide more information about the

protein and defense signaling pathways that are important for pathogen-regulated

BON1/CPN1 accumulation.



All plants were grown in soil-less medium (Reid-Earth Plug and Seedling Mix,

Sun Grow Horticulture) and irrigated with distilled water. Wild-type Col-0 plants and the

various mutant plants were grown at 21°C and 35% RH under a 10 h photoperiod with 75

µmol m-2 s

-1 light intensity. 5-week-old plants were used for all experiments unless

indicated otherwise.

4.3.2 Bacterial inoculations and bacterial growth analysis

Arabidopsis leaves were infiltrated with bacteria using needle-less 1 cc syringes

as described in Chapter 2. P. s. t. DC3000 and P. s. t. DC3000 (avrRpt2) strains of

bacteria were grown at 28°C on Pseudomonas Agar F (PA Sigma) containing only

100µg/mL rifampicin (Sigma) or both 100 µg/mL rifampicin and 25 µg/mL kanamycin

44

(Sigma), respectively. Bacterially-infiltrated plants were returned to their original growth

conditions and leaves were collected at indicated time points for protein extraction.

4.3.3 Plant protein extraction and western analysis

Arabidopsis total soluble proteins were isolated as described in Chapter 3. For

western blot analysis, 75 µg of protein was mixed with an equal volume of 2×SDS

loading buffer (126 mM Tris-HCl pH 6.8, 20% glycerol, 4% SDS, 0.005% bromophenol

blue, 5 mM DTT), separated a 7.5% SDS-PAGE gel, and electrotransferred onto Hybond

ECL nitrocellulose membranes (GE Healthcare) as described (Sambrook and Russell,

2001). Membrane blocking and primary and secondary antibody incubations were

performed in 1×TBST buffer (20 mM Tris-HCl pH 7.6, 137 mM NaCl, 0.1% Tween-20)

containing 5% non-fat dry milk. A 1:6,000 dilution and a 1:8,000 dilution were used for

the rat anti- BON1/CPN1 antisera and the rabbit-anti-rat antibody conjugated with

horseradish peroxidase (Sigma), respectively. Chemiluminescence detection was

performed according to the manufacturer’s instructions (ECL Plus western blotting

detection system, GE Healthcare).

45

4.4 Results

4.4.1 Tissue-specific BON1/CPN1 protein accumulation patterns

A polyclonal anti-BON1/CPN1 antibody was generated in rats using the C-

terminal VWA domain of BON1/CPN1 produced in E. coli as the antigen (Chapter 3).

The antibody detected a 63 kDa protein band in total soluble protein extracts from wild-

type Col-0 tissues (Figure 10), which agreed with the predicted BON1/CPN1 molecular

weight. The 63 kDa protein band was absent in total soluble protein extracts from tissues

of the cpn1-1 mutants (Figure 10), indicating that it represents BON1/CPN1.

BON1/CPN1 appeared to be a low-abundance protein.

Figure 10: BON1/CPN1 protein level in different Arabidopsis tissues. BON1/CPN1 protein

level was monitored by western analysis using an anti-BON1/CPN1 antibody. Total soluble

proteins were extracted from various samples of LH/LT-grown Col-0 and cpn1-1 plants

including roots (R), 3-week-old seedlings (Sd), mature leaves (L), florets (F), and green siliques

(Gs). Arrow indicates the position of the BON1/CPN1 band. Protein molecular weight marker

sizes are shown in kDa. 75 µg of total soluble protein were in each lane. Lower panel,

membrane stained with Ponceau S.

46

To investigate the tissue-specific distribution pattern of BON1/CPN1, we

analyzed BON1/CPN1 levels in different Arabidopsis tissues (Figure 10). Florets,

mature leaves, and the aerial portions of 3-week-old seedlings had low levels of

BON1/CPN1 accumulation. On the other hand, BON1/CPN1 accumulated to a very high

level in the roots. All tissues except for green siliques had detectable BON1/CPN1

protein. The sizes and intensities of the background bands varied among the different

samples.

4.4.2 BON1/CPN1 protein accumulation was induced by both avirulent and virulent

Pseudomonas syringae pv tomato

Previous studies have shown that BON1/CPN1 transcript accumulation was

induced by P. s. t. bacterial inoculation (Jambunathan and McNellis, 2003). To test if

activation of RPS2 defense signaling affected the abundance of BON1/CPN1, we

monitored BON1/CPN1 levels in Col-0 wild-type and cpn1-1 mutant plants inoculated

with avirulent P. s. t. DC3000 (avrRpt2) bacteria (Whalen et al., 1991; Innes et al., 1993;

Kunkel et al., 1993). In inoculated Col-0 wild-type leaves, BON1/CPN1 accumulation

was induced at 12 h and 24 h after infiltration with 106 cfu/ml of avirulent P. s. t.

(avrRpt2) bacteria relative to untreated plants (Figure 11 A). In contrast, the 63 kDa

BON1/CPN1 band was completely absent in total soluble protein extracts from

bacterially-inoculated leaves of cpn1-1 mutant plants, which further supported the

authenticity of the 63 kDa BON1/CPN1 band. Two other bands with higher molecular

weight than BON1/CPN1 were also detected in the western blot (Figure 11 A). They

47

might represent proteins with similarity to BON1/CPN1. However, accumulation levels

of these proteins were not pathogen regulated (Figure 11 A) in wild-type Col-0 plants.

We also tested the dosage effect of increasing bacterial inoculum concentrations

on the level of BON1/CPN1 accumulation. Leaves of Col-0 wild-type plants grown

under LH/LT conditions were infiltrated with concentrations of avirulent P. s .t. DC3000

(avrRpt2) bacteria ranging from 104 to 10

8 cfu/ml. The level of BON1/CPN1

accumulation showed a bacterial dose-dependent increase in Col-0 wild-type leaves

infiltrated with 104, 10

5, and 10

6 cfu/ml of bacteria at 12 hpi (Figure 11 B). Inoculation

with 107 and 10

8 cfu/ml of bacteria did not substantially increase BON1/CPN1

accumulation beyond that observed with 106 cfu/ml inoculum. Mock-inoculation did not

stimulate BON1/CPN1 accumulation, indicating that bacteria specifically stimulated

BON1/CPN1 accumulation.

48

Figure 11: BON1/CPN1 protein level in LH/LT-grown plants after bacterial pathogen

challenge. A; BON1/CPN1 levels in Col-0 wild-type and cpn1-1 mutant plants that were

uninoculated (Un) or at 12 and 24 hours after challenge (P) with 106 cfu/ml P. s. t. DC3000

(avrRpt2). Protein molecular weight marker sizes are shown in kDa; arrow indicates 63 kDa

BON1/CPN1 protein band location. B; Dose-dependence of bacterially-induced BON1/CPN1

accumulation in Col-0 wild-type plants at 12 h post-inoculation with avirulent P. s. t. DC3000

(avrRpt2) at the concentrations indicated. M, sample from leaf mock-inoculated with 10 mM

MgCl2. C; Time course of BON1/CPN1 accumulation in wild-type plants after inoculation

with 106 cfu/ml of avirulent P. s. t. DC3000 (avrRpt2) bacteria. D; Time course of

BON1/CPN1 accumulation after inoculation with 105 cfu/ml of virulent P. s. t. DC3000

bacteria.

49

Time course experiments were performed to determine the timing of BON1/CPN1

accumulation in Col-0 wild-type leaves after avirulent or virulent bacterial inoculation.

In Col-0 wild-type leaves inoculated with 106 cfu/ml of avirulent P. s. t. DC3000

(avrRpt2), the level of BON1/CPN1 increased detectably at 6 hpi and remained elevated

through the end of the experiment at 72 hpi compared to the levels in untreated plants

(Figure 11 C). Virulent P. s. t. DC3000 also induced BON1/CPN1 accumulation, but

more slowly than avirulent P. s. t. DC3000 (avrRpt2) (Figure 11 D). Increased

BON1/CPN1 accumulation was detected at 36 hpi in Col-0 wild-type leaves inoculated

with 105 cfu/ml of virulent P. s. t. DC3000 bacteria. The level of BON1/CPN1 remained

elevated until the end of the experiment at 72 hpi. In summary, the timing of

BON1/CPN1 protein accumulation in response to avirulent and virulent pathogen

challenge correlated with previously determined BON1/CPN1 transcript accumulation

patterns (Jambunathan and McNellis, 2003).

4.4.3 BON1/CPN1 protein accumulation was induced in the snc1 gain-of-function

mutant

To investigate whether activation of SNC1 defense signaling affects the

abundance of BON1/CPN1, we monitored BON1/CPN1 levels in the gain-of-function

mutant snc1 (Figure 12). The snc1 missense mutation produces a constitutively activated

R gene, resulting in highly disease-resistant plants. Western analysis revealed an

extremely high accumulation of BON1/CPN1 in untreated leaves of the snc1 mutant

relative to that observed in Col-0 wild-type plants.

50

4.4.4 Induction of BON1/CPN1 protein accumulation by avirulent P. s. t. DC3000

(avrRpt2) depended on NDR1 but not EDR1

We tested the pathogen-inducibility of BON1/CPN1 accumulation in the edr1 and

ndr1 mutants. The VWA domain of BON1/CPN1 has been shown to interact with the

ENHANCED DISEASE RESISTANCE 1 (EDR1) protein in a yeast-two-hybrid

experiment (Sinn and McNellis, unpublished data). EDR1 encodes a MAPKK kinase

which negatively regulates disease resistance and ethylene-induced senescence (Frye and

Innes, 1998; Frye et al., 2001; Tang et al., 2005). Moreover, it has been demonstrated

that EDR1 negatively regulates a conserved, basal defense pathway mediated by R genes

RPW8.1 and RPW8.2 (Xiao et al., 2005). After leaf infiltration with avirulent P. s. t.

DC3000 (avrRpt2) bacteria, the level of BON1/CPN1 accumulation in edr1 mutant

plants was similar compared to that in inoculated Col-0 wild-type leaves at both 12 hpi

Figure 12: BON1/CPN1 protein accumulation in snc1 mutant plants. BON1/CPN1 protein level

was monitored by western analysis using an anti-BON1/CPN1 antibody in untreated snc1 plants

grown under LH/LT conditions. 75 µg of total soluble protein were in each lane. Arrows

indicate the position of the BON1/CPN1 band.

51

and 24 hpi (Figure 13). This result suggested that induction of BON1/CPN1

accumulation by P. s. t. DC3000 (avrRpt2) was not dependent on the BON1/CPN1

interactor EDR1.

The NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1) gene is required

for RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) gene-mediated defense

signaling (Century et al., 1995; Century et al., 1997; Aarts et al., 1998), which is

triggered by the recognition of avrRpt2-derived signals by the RPS2 gene product

(Kunkel et al., 1993; Yu et al., 1993). In ndr1 leaves inoculated with avirulent P. s. t.

DC3000 (avrRpt2) bacteria, the level of BON1/CPN1 accumulation was reduced

compared to that in inoculated Col-0 wild-type leaves at both 12 hpi and 24 hpi

(Figure 13). This result suggested that induction of BON1/CPN1 accumulation by P. s. t.

DC3000 (avrRpt2) was partially dependent on NDR1.

52

4.5 Discussion

Root tissues appeared to have high levels of BON1/CPN1 protein accumulation

(Figure 10). Roots are likely to be in constant contact with soil-borne microorganisms. It

is possible that BON1/CPN1 accumulates in the roots due to constant interactions with

microbes. Alternatively, BON1/CPN1 may be acting as a negative regulator of defenses

in the roots: runaway defense responses or HR-induced necrosis near root tips may be

deleterious to the growth of root meristems (Hawes et al., 2000). Interestingly, Hua et al.

(2001) detected BON1/CPN1 promoter activity in root tips using GUS reporter gene.

Figure 13: Pathogen-induced BON1/CPN1 protein accumulation in edr1 and ndr1 mutant plants.

BON1/CPN1 protein level was monitored by western analysis using an anti-BON1/CPN1

antibody. 75 µg of total soluble protein were in each lane. Arrows indicate the position of the

BON1/CPN1 band. BON1/CPN1 protein accumulation was monitored in Col-0, edr1 and ndr1

mutant plants after avirulent P. s. t. inoculation. Leaves of Col-0 and ndr-1 plants grown under

LH/LT conditions were infiltrated with 106 cfu/ml of avirulent P. s. t. DC3000 (avrRpt2)

bacteria. Total soluble proteins were extracted from infiltrated leaves 12 and 24h post-

inoculation (P). Un, protein sample from untreated control plant.

53

Our data showed that activation of RPS2 and SNC1 resistance pathways, either by

recognition of avrRpt2 or by gain-of-function snc1 mutation, triggered the accumulation

of BON1/CPN1 protein as explained in a model (Figure 14). In addition, avirulent P. s.

t.-induced BON1/CPN1 accumulation is suppressed in the ndr1 mutant, indicating that

the pathogen-induced BON1/CPN1 accumulation depends on a functional NDR1 in

defense signaling. Since BON1/CPN1 transcripts are regulated by SA at the transcription

level (Jambunathan and McNellis, 2003), increased accumulation of BON1/CPN1 protein

may result from the up-regulation of BON1/CPN1 expression by SA accumulation

downstream of activated R defense signaling. SNC1 expression is also induced by SA,

indicating the presence of the SA feedback regulation of SNC1 and its negative regulator

BON1/CPN1 upstream of SNC1 defense signaling (Yang and Hua, 2004).

Figure 14: A model for BON1/CPN1 regulation by RPS2 and SNC1 defense pathways.

BON1/CPN1 negatively regulates the R gene SNC1. Recognition of the AvrRpt2 effector by

RPS2 activates the RPS2-mediated, SA-dependent resistance pathway which requires the NDR1

gene. In the snc1 gain-of-function mutant, SNC1 and the downstream SA-dependent resistance

pathway is constitutively activated in the absence of pathogen. Activation of RPS2- and SNC1-

mediated defense pathways up-regulates BON1/CPN1 accumulation.

54

It is interesting that the accumulation of BON1/CPN1, a negative regulator of

defense responses, is induced by defense activation in response to bacterial pathogen

attack. One possible explanation is that increased BON1/CPN1 accumulation is required

for the negative regulation of cell death and defense response to cope with diverse types

of pathogen challenge. In a recent study about tradeoffs between different defense

responses, plants have developed a tight control for cross-talk between SA- and jasmonic

acid (JA)-dependent defenses in a spatial and pathogen-specific fashion (Spoel et al.,

2007). For various types of pathogen attack, SA induces the defense response against

biotrophic pathogens that propagate on living cells, while JA induces the defense

response against necrotrophic pathogens that kill cells and feed on the nutrients of cells.

Their results demonstrates that infection with the biotroph Pseudomonas syringae, which

triggers SA-mediated defense, makes plants more susceptible to the necrotroph

Alternaria brassicicola by suppressing the JA-mediated defense responses. In addition,

the tradeoffs occur only at the adjacent tissues of the initial infection sites but not in a

systemic manner (Spoel et al., 2007). Along the same lines, BON1/CPN1 may

participate in a complex regulation mechanism for controlling programmed cell death

against biotrophic pathogen challenge, and preventing run-away cell death which would

be beneficial for necrotrophic pathogens. Previous study has demonstrated the

accumulation of BON1/CPN1 transcripts in the bacterially-infiltrated leaves and not in

distal leaves after avirulent and virulent pathogen inoculation (Jambunathan and

McNellis, 2003). This result suggests that the function of BON1/CPN1 as cell death

suppressor may be needed more locally near the pathogen infection site than

55

systemically, which agrees with the scenario that local activity of BON1/CPN1 is

important for spatial regulation of defense activation.

56

Chapter 5

Pathogen- and calcium-responsive BON1/CPN1 promoter activity

5.1 Summary

BON1/CPN1 promoter activity was analyzed using the β-glucuronidase (GUS)

reporter gene. A 280 bp portion of the BON1/CPN1 promoter was identified conferring

pathogen- and A23187-inducible GUS expression, which occurred in a localized,

punctate pattern in treated leaves. Characterization of two BON1/CPN1 T-DNA

insertion mutants with T-DNA inserted extremely close to the BON1/CPN1

transcriptional start site showed that they over-accumulated BON1/CPN1 in response to

pathogen challenge. The function of the pathogen-responsive region of the BON1/CPN1

promoter was discussed.


The regulation of BON1/CPN1 expression appears to be under complicated

control by multiple factors. The rapid and transient accumulation of BON1/CPN1

transcript as early as 4 h after avirulent P. s. t. challenge demonstrates the tightly

controlled regulation of BON1/CPN1 expression during the early stage of the

incompatible defense response (Jambunathan and McNellis, 2003). In addition, the

57

expression of SNC1 and its negative regulator BON1/CPN1 gene is subjected to feedback

regulation by SA, the signaling molecule for later stage of defense responses like

systemic acquired resistance (Durrant and Dong, 2004). It is not uncommon for R genes

being regulated by feedback amplification. The Arabidopsis RPW8.1 and RPW8.2 genes,

which mediate powdery mildew resistance by inducing hypersensitive response (HR), are

regulated transcriptionally by a SA-dependent feedback circuit (Xiao et al., 2003).

Interestingly, the accumulation of RPW8.1 and RPW8.2 transcript is also induced by

environmental conditions including low temperature and low humidity (Xiao et al.,

2003). These studies suggest that R genes and components in R-mediated defense

signaling may commonly be regulated by mechanisms involved in multiple factors and

feedback regulation at the transcriptional level.

Many genes encoding C2 domain-containing proteins are induced by abiotic and

biotic stresses (Kim et al., 2003; Ouelhadj et al., 2006; Kim et al., 2008). In addition,

their expression is induced by chemically applied Ca2+ stimulus. For example, rice

OsERG1 transcript accumulation was induced by fungal elicitors and A23187 treatment

in rice suspension cells (Kim et al., 2003). The expression of the barley HvC2d1 gene

was transiently induced by heavy metal, reactive oxygen species (ROS), and A23187 leaf

treatments (Ouelhadj et al., 2006). In pepper leaves, the CaSRC2-1 gene was up-

regulated by bacterial and viral pathogen infection, cold and CaCl2 treatments (Kim et al.,

2008). Similarly, BON1/CPN1 and BAP1, which encodes a small C2 domain-containing

protein that interacts with and is a functional partner of BON1/CPN1, are

transcriptionally up-regulated by pathogens, salicylic acid (SA), and low temperatures

(Hua et al., 2001; Jambunathan and McNellis, 2003; Yang et al., 2006a). Taken together,

58

these results suggest that many C2 domain-containing proteins are subject to

transcriptional regulation by Ca2+ stimuli and various abiotic and biotic stresses.

A search for regulatory motifs based on the BON1/CPN1 promoter sequence has

found different putative cis-acting element responsive to abiotic and defense-related

signals (Figure 15). However, experimental analysis is needed to identify whether these

putative regulatory elements are functional or not. To further investigate how

BON1/CPN1 expression is regulated by different stimuli, we conducted a BON1/CPN1

promoter activity analysis using the β-glucuronidase (GUS) reporter gene. Dissection of

the BON1/CPN1 promoter has allowed us to identify a 280 bp promoter region which is

responsive to pathogen and calcium stimuli. Furthermore, we characterized two

BON1/CPN1 T-DNA insertion mutants with altered BON1/CPN1 gene expression.

Although they had T-DNA inserted extremely closed to the BON1/CPN1 transcriptional

start site, these two T-DNA insertion mutants had high, induced levels of BON1/CPN1

transcript and protein accumulation in response to pathogen challenge. This result

suggested the pathogen-responsive region of the BON1/CPN1 promoter may still regulate

BON1/CPN1 expression in a distance across the T-DNA interruption in these two

mutants.

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Figure 15: Some predicted cis-acting elements in the BON1/CPN1 promoter region. The 1.9

kb upstream sequence of the BON1/CPN1 gene was analyzed using the PlantCARE program

for known cis-acting element motifs (Rombauts et al., 1999; Lescot et al., 2002). Selected cis-

acting elements involved in defense-related signals and abiotic stimuli are shown. W box, a

fungal elicitor-responsive element (Rushton et al., 1996); CGTCA motif, a methyl jasmonate-

responsive element (Rouster et al., 1997); ERE, ethylene-responsive element (Itzhaki and

Woodson, 1993); EIRE, elicitor-responsive element (Hennig et al., 1993); TCA element, SA-

responsive element (Pastuglia et al., 1997); LTR, low-temperature-responsive element (Brown

et al., 2001); MBS, MYB binding site involved in drought-inducibility (Yamaguchi-Shinozaki

and Shinozaki, 1993); ABRE, abscisic-acid-responsive element (Shen et al., 1993; Yamaguchi-

Shinozaki and Shinozaki, 1993) and putative Ca2+ responsive element (Kaplan et al., 2006).

60


5.3.1 BON1/CPN1 promoter-GUS fusion constructs and plant transformation

For promoter activity analysis, different lengths of promoter region and the

complete first exon, first intron, and partial second exon (36 bp) of BON1/CPN1 were

translationally fused with the GUS reporter gene. Promoter fragments starting at -1518, -

1118, -718, -318, and -38 relative to the BON1/CPN1 transcriptional start site were

amplified by PCR using following primer pairs. Introduced restriction sites are italicized.

Forward primers were: pF1-1518, 5´-AAGCTTGTTGCTACTCTGGTATGC-3´; pF2-

1118, 5´-AAGCTTCTCACTCTTTCCACCTAC-3´; pF3-718, 5´-

AAGCTTGTGAACCTTACGATTTGT-3´; pF4-318, 5´-

AAGCTTGCTGGAAAGATTATGTCA-3´; and pF5-38, 5´-

AAGCTTGTGGGTCCCATTTACTGC- 3´. The reverse primer used for all constructs

was 5´-TCTAGAGTCGCGGTCTCGCAAATT-3´. PCR fragments were subcloned into

the HindIII-XbaI sites of the binary pBI101 vector. The resulting promoter-GUS fusion

constructs were introduced into wild-type Col-0 plants by Agrobacterium-mediated

transformation using the floral dip method (Clough and Bent, 1998). T2 or T3 plants

from selected transgenic lines were used for histochemical GUS staining experiments.

5.3.2 Bacterial and chemical treatments

Leaf infiltration was performed as described in Chapter 2. For chemical

treatment, leaves of transgenic plants expressing BON1/CPN1 promoter-GUS fusion

61

constructs were infiltrated with 20 µM or 50 µM of calcium ionophore A23187 (Sigma,

St. Louis, MO, USA), and collected at 24 hpi for GUS staining. 0.25% DMSO (dimethyl

sulfoxide; Sigma) was used as the solvent control. For bacterial treatment, 106 cfu/ml of

P. s. t. DC3000 (avrRpt2) strain of bacteria was suspended in 10 mM MgCl2 and used for

leaf infiltration. Mock (10 mM MgCl2) or bacteria-infiltrated leaves were collected 6hpi

or 24 hpi for GUS staining.

5.3.3 Histochemical staining for GUS activity

Leaf samples were fixed in ice-cold 90% acetone on ice for 5 min, then incubated

in a rinse solution containing 50 mM sodium phosphate buffer pH 7.0, 0.5 mM

potassium ferricyanide (Sigma), and 0.5 mM potassium ferrocyanide (Sigma) for 20 min

with gentle shaking at room temperature. Samples were then incubated in 1.5 mM of 5-

bromo-4 chloro-3-indolyl-β-D-glucuronide cyclohexylammonium salt (X-GlcA CHA

salt, Sigma) and 0.05% Triton X-100 prepared in the rinse solution, vacuum infiltrated

for 10 min, and then incubated at 37ºC for 24 h. Stained leaves were washed with 30%

ethanol for 1 h, and then fixed for 1 h in solution containing 50% ethanol, 5% acetic acid,

and 3.7% formaldehyde. Chlorophyll was cleared by several changes of 70% ethanol

following the fixation step.

62

5.3.4 Genomic Southern analysis

Genomic Southern analysis was performed essentially as described previously

(Southern, 1975). Genomic DNA of Col-0, cpn1-2, cpn1-3 mutant plants were digested

with HindIII (New England Biolabs, Ipswich, MA, USA) and separated on a 0.7%

agarose gel. The insert of a plasmid containing the 1.15kb of BON1/CPN1 genomic

fragment starting at -718bp upstream of the BON1/CPN1 transcriptional start site ant

extended 36bp into exon 2 was cut with HindIII-XbaI and used as the probe fragment.

Probe labeling and detection were performed using Phototope-Star Chemiluminescent

Detection Kit (New England Biolabs) according to the manufacturer’s instruction.

5.3.5 Total RNA extraction and semi-quantitative RT-PCR

Total RNA was extracted from Arabidopsis leaf tissues using the RNeasy plant

mini kit (Qiagen, Valencia, Ca, USA). 2 µg of total RNA was used to generate the first-

strand cDNA using the RETROscript kit (Applied Biosystems, Foster City, CA, USA)

following the manufacturer’s instructions. To determine the BON1/CPN1 transcript

abundance in Col-0, cpn1-2, and cpn1-3 mutant plants, PCR primers (5'-

TCTAGAATTATGGGGAATTGTTGCTCCGAT-3' and 5'-

GAATTCTCAATGAAGTTTTTCTAAGTCTGA-3') were used to amplify the

BON1/CPN1 cDNA sequence encoding the C2 domains which produced an 880-bp

product as described (Liu et al., 2005). Introduced XbaI and EcoRI restriction sites are

shown in italics in primer sequences. 18S rRNA primers were used to amplify the 18S

ribosomal RNA as internal standard using the QuantumRNA™ Universal 18S Internal

63

Standard kit (Applied Biosystems). PCR reactions were performed using Advantage® 2

polymerase (Clontech, Mountain View, CA, USA) and the following PCR parameters: 2

min initial denaturation at 95 ºC, 29 cycles of 95 ºC for 30 sec, 58ºC for 30 sec, and 72ºC

for 75 sec. Final extension was at 72ºC for 5 min. RT-PCR products were analyzed by

agarose gel electrophoresis followed by ethidium bromide staining.

5.3.6 In planta bacterial growth analysis

Growth of bacterial culture and bacterial infiltration of Arabidopsis leaves were

conducted as described in Chapter 4. In planta bacterial growth analysis was performed

basically as described (Jambunathan et al., 2001). In short, 105 cfu/ml of P. s. t. DC3000

(avrRpt2) strain of bacteria was suspended in 10mM MgCl2 and used for leaf infiltration.

Bacterially-infiltrated plants were kept at 75% RH conditions for the duration after

bacterial infiltration. Bacteria population in infiltrated leaves was monitored at day 0 and

day 3 after inoculation.

5.4 Results

5.4.1 A 280 bp region of the BON1/CPN1 promoter is required for pathogen-induced

GUS expression

Previous studies have shown that BON1/CPN1 transcript accumulation is

regulated by pathogen stimulus (Jambunathan and McNellis, 2003). To investigate the

pathogen-inducible activity of BON1/CPN1 promoter region and the spatial expression

64

pattern of BON1/CPN1 following pathogen inoculation, we generated a series of

constructs for BON1/CPN1 promoter activity analysis using GUS reporter gene

(Figure 16 A). First of all , for BON1/CPN1 spatial expression analysis, 1.5 kb

BON1/CPN1 promoter fragment, plus the complete first exon, first intron, and partial

second exon (36 bp) of BON1/CPN1, was translationally fused with the GUS reporter

gene (Figure 16 A, construct pF1-1518). The 1.5 kb BON1/CPN1 promoter fragment

extends 177 bp into the 3´ untranslated region of neighboring gene At5g61910. Over 30

independent transgenic Arabidopsis lines carrying the pF1-1518 construct were generated

and analyzed. Of those, 14 lines that showed little or no GUS background activity after

mock inoculation were selected for further experiments. Leaves from these transgenic

plants were infiltrated with 106 cfu/ml of avirulent P. s. t. DC3000 (avrRpt2) bacteria and

histochemically stained for GUS activity at 6 hpi or 24 hpi. The expression of the pF1-

1518 construct was induced by avirulent P. s. t. DC3000 (avrRpt2) as early as 6 hpi in

inoculated leaves (Figure 16 B), which coincided with the timing of BON1/CPN1

transcript accumulation after avirulent bacterial inoculation in previous studies

(Jambunathan and McNellis, 2003). The expression of the pF1-1518 construct was

localized as punctate GUS staining on the inoculated leaves.

65

Figure 16: Expression of BON1/CPN1 promoter-GUS fusion construct in transgenic

Arabidopsis plants. A; Schematic diagram of the 5´ promoter deletion-GUS reporter constructs.

B; Leaves of transgenic plants with the pF1-1518 construct infiltrated with 106 cfu/ml of

avirulent P. s. t. DC3000 (avrRpt2) bacteria and histochemically stained for GUS activity

(P.s.t.). Control leaves were infiltrated with 10 mM MgCl2 (Mock). Infiltrated leaves were

stained for GUS activity at indicated time after inoculation (hpi). Representative leaves are

shown. Middle and right panels of P. s. t. 24 hpi represent higher magnifications of the boxed

areas in the left P. s. t. 24 hpi panel.

Next, a series of 5´ BON1/CPN1 promoter deletion GUS reporter fusion

constructs was used to further define the promoter region which was responsible for

pathogen-regulated BON1/CPN1 expression (Figure 16 A, construct pF2-1118, pF3-718,

pF4-318, and pF5-38). Various lengths of the BON1/CPN1 promoter were fused with the

GUS gene in the same way as in the pF1-1518 construct and introduced into wild-type

66

Col-0 background. Constructs pF2-1118, pF3-718, pF4-318, and pF5-38 contained 1118,

718, 318, and 38 bp of promoter upstream of the BON1/CPN1 transcriptional start site,

respectively (Figure 16 A). Independent transgenic lines carrying each construct were

analyzed, and lines with little or no background GUS expression were selected for further

experiments. To test for BON1/CPN1 promoter activity in response to pathogen

inoculation, leaves from the transgenic plants were infiltrated with 106 cfu/ml of avirulent

P. s. t. DC3000 (avrRpt2) bacteria and stained for GUS activity at 6 hpi (Figure 17 A). In

plants carrying the constructs pF2-1118, pF3-718, and pF4-318, bacterially-inoculated

leaves displayed a localized GUS staining pattern similar to that observed in plants

expressing the pF1-1518 construct. However, there was no detectable GUS staining in

plants of leaves carrying the pF5-38 construct after bacterial inoculation. This result

showed that the -318 to -38 bp region of the BON1/CPN1 promoter was required for

pathogen-regulated expression of the GUS reporter gene.

67

A

B

Figure 17: BON1/CPN1 promoter deletion analysis in response to pathogen and calcium

ionophore stimuli. A; Histochemical GUS staining of pF1-1518, pF2-1118, pF3-718, pF4-318,

and pF5-38 transgenic plant leaves either mock-inoculated with 10 mM MgCl2 (Mock) or

inoculated with 1x106 cfu/ml of avirulent P. s. t. DC3000 (avrRpt2) bacteria (P.s.t.) at 6 h post

inoculation. n. the number of independent transgenic lines examined. Representative leaves

are shown. B; Histochemical GUS staining of pF1-1518, pF2-1118, pF3-718, pF4-318, and

pF5-38 transgenic plant leaves infiltrated with 0.25% DMSO, 20 µM A23187 in 0.1% DMSO

or 50 µM A23187 in 0.25% DMSO at 24 h post infiltration. Representative leaves are shown.

Leaves in each column are from a single transgenic plant.

68

5.4.2 A 280 bp region of the BON1/CPN1 promoter is required for calcium

ionophore-induced GUS expression

The presence of putative Ca2+-responsive elements in the BON1/CPN1 promoter

region suggests that expression of BON1/CPN1 might be responsive to Ca2+

stimulus.

We speculated that BON1/CPN1 expression could be regulated by calcium influx. To

test our hypothesis, leaves from transgenic plants carrying the 5´ promoter deletion

reporter constructs described above were infiltrated with 20 µM or 50 µM of calcium

ionophore A23187 and stained for GUS activity at 24 hpi (Figure 17 B). Transgenic

plant leaves showed little or no GUS expression after infiltration with 0.25% DMSO,

which served as the solvent control and contained the same amount of DMSO as 50 µM

A23187. In plants carrying the constructs pF1-1518, pF2-1118, pF3-718, and pF4-318,

leaves showed GUS expression at 24 h after infiltration with A23187. However, there

was no detectable GUS staining in leaves carrying the pF5-38 construct after 20 µM or

50 µM of A23187 treatment. This result shows that BON1/CPN1 promoter-GUS fusion

gene expression was inducible by the calcium ionophore A23187. Furthermore, the -318

to -38 bp region of the BON1/CPN1 promoter was required for calcium ionophore-

induced expression of the GUS reporter gene.

5.4.3 Over-accumulation of BON1/CPN1 in two BON1/CPN1 promoter T-DNA

insertion mutants

In an attempt to isolate mutants with altered BON1/CPN1 gene expression, we

obtained two mutants with T-DNA inserted in the BON1/CPN1 promoter region from the

69

SAIL T-DNA insertion line collection

(http://www.tmri.org/en/partnership/sail_collection.aspx) (Sessions et al., 2002). The

mutants SAIL_865_A09 and SAIL_723_E11 had T-DNA insertions at 348 bp and 38 bp

upstream of the BON1/CPN1 transcriptional start site and were designated as cpn1-2 and

cpn1-3, respectively. Despite the large T-DNAs inserted extremely close to the

BON1/CPN1 transcriptional start site, cpn1-2 and cpn1-3 mutants grew like wild-type

plants under LH/LT conditions (Figure 18 A) and HH/HT conditions (data not shown).

Southern blotting confirmed the presence of T-DNA insertions in the BON1/CPN1

promoter regions of cpn1-2 and cpn1-3 (Figure 18 B and C). The probe hybridized to

different sized HindIII genomic fragments in Col-0 wild-type, cpn1-2 and cpn1-3 mutant

DNA samples due to the introduced internal HindIII site on the T-DNA (Figure 18 C).

These results confirmed the T-DNA locations in cpn1-2 and cpn1-3 relative to the

BON1/CPN1 transcriptional start site. Only one band was detected in the cpn1-2 and

cpn1-3 mutants on the genomic Southern blot, corresponding to the portion of the probe

hybridizing to the BON1/CPN1 transcribed region. No bands corresponding to the

portion of the BON1/CPN1 promoter on the far side of the T-DNA insertions were

observed.

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Figure 18: Phenotypic effects of two T-DNA insertions in the BON1/CPN1 promoter. A; Five-

week-old Col-0 wild-type, cpn1-1, cpn1-2, and cpn1-3 plants grown under LH/LT conditions.

B; HindIII restriction map of the genomic region of BON1/CPN1, including the location of the

BON1/CPN1 transcribed region (open box), the T-DNA insertion sites in cpn1-2 and cpn1-3,

and the probe segment used for Southern blotting (grey box). C; Confirmation of T-DNA

location in cpn1-2 and cpn1-3. The T-DNA has a HindIII site 1.4 kb away from the left border;

the cpn1-2 and cpn1-3 T-DNA left borders are 1.2 and 0.9 kb distant from the first internal

HindIII site in BON1/CPN1. A Southern blot of genomic DNA cut with HindIII has the

expected 3.6, 2.6, and 2.3 kb bands for Col-0, cpn1-2, and cpn1-3 plants, respectively. DNA

molecular weight marker sizes are shown in kb. Scale is the same as in B.

71

BON1/CPN1 was present at similar levels in untreated Col-0 wild-type, cpn1-2,

and cpn1-3 leaves (Figure 19 A). Surprisingly, BON1/CPN1 accumulation was rapidly

induced after inoculation with avirulent P. s. t. DC3000 (avrRpt2) bacteria in both cpn1-2

and cpn1-3 mutant plants (Figure 19 A). In fact, the level of BON1/CPN1 accumulation

was higher in inoculated cpn1-2 and cpn1-3 plants compared to inoculated Col-0 wild-

type plants. In addition, cpn1-2 and cpn1-3 plants mock-inoculated with 10 mM MgCl2

also exhibited elevated levels of BON1/CPN1 accumulation, while Col-0 wild-type

plants did not. The mock-inoculated cpn1-3 mutant showed particularly high

BON1/CPN1 accumulation at 24 hpi. These results indicate that infiltration alone was

enough to cause induction of BON1/CPN1 accumulation in cpn1-2 and cpn1-3. Semi-

quantitative RT-PCR analyses demonstrated parallel trends in BON1/CPN1 transcript

accumulation in cpn1-2 and cpn1-3 mutant plants in response to avirulent P. s. t. DC3000

(avrRpt2) inoculation (Figure 19 B).

72

A

B

C

Figure 19: Overexpression of BON1/CPN1 in cpn1-2 and cpn1-3 mutants. A; BON1/CPN1

accumulation in Col-0, cpn1-2, and cpn1-3 plants that were uninoculated (Un), mock-

inoculated with 10 mM MgCl2 (M), or after inoculation with 106 cfu/ml of avirulent P. s. t.

DC3000 (avrRpt2) bacteria (P), at 6 and 24 h after inoculation. Arrows indicate BON1/CPN1

position. B; Semi-quantitative RT-PCR detection of BON1/CPN1 transcript accumulation in

Col-0 wild-type, cpn1-2, and cpn1-3 plants at 6 and 24 h after infiltration with 10 mM MgCl2

(M) or 106 cfu/ml of avirulent P. s. t. DC3000 (avrRpt2) bacteria (P). 18S, 18S rRNA internal

control. C; Populations of virulent P. s. t. DC3000 bacteria in Col-0, cpn1-1, cpn1-2, and cpn1-

3 plants at 0 and 3 days after infiltration with 105 cfu/ml of virulent P. s. t. DC3000 bacteria.

73

Since BON1/CPN1 is postulated to be a suppressor of plant defense, we

hypothesized that the elevated levels of BON1/CPN1 in cpn1-2 and cpn1-3 mutant plants

might suppress plant disease resistance. The growth of virulent P. s. t. DC3000 bacteria

was monitored in leaves of LH/LT-grown Col-0, cpn1-1, cpn1-2, and cpn1-3 plants on

day 0 and day 3 after inoculation with 105 cfu/ml of bacteria (Figure 19 C). On day 3

post-inoculation, the growth of P. s. t. DC3000 bacteria was reduced by 10 fold in cpn1-1

mutant plants when compared with wild-type Col-0 plants as expected. In contrast, the

growth of P. s. t. DC3000 bacteria in cpn1-2 and cpn1-3 plants was similar to that in

wild-type Col-0 plants. This experiment was repeated several times with similar results,

and was repeated with virulent and avirulent strains of P. s. t. and P. s. pv. maculicola

with similar results.

5.5 Discussion

Our 5´ promoter deletion analysis of BON1/CPN1 revealed a 280 bp promoter

region (-318 bp to -38 bp upstream of the transcription start site) that was required for

pathogen- and calcium-responsive GUS expression. On the other hand, pathogen-

induced BON1/CPN1 protein accumulation was observed in the cpn1-3 mutant despite a

T-DNA inserted at -38 bp upstream of the transcription start site of BON1/CPN1. In

other words, while the 38 bp length of promoter region was not sufficient to direct

pathogen-inducible GUS expression, the T-DNA insertion at -38 did not abolish

pathogen-induced BON1/CPN1 accumulation in cpn1-3. This suggests that the 280 bp

pathogen-responsive portion of the BON1/CPN1 promoter may be able to direct

74

BON1/CPN1 transcript accumulation across the T-DNA insertion in cpn1-3. It is not

clear from Southern blotting whether the 280 bp pathogen-responsive portion of the

BON1/CPN1 promoter still exists in cpn1-3 (Figure 18 C). An alternative explanation is

that elements within the T-DNA may somehow regulate the pathogen-induced

BON1/CPN1 expression. Finally, the relatively high level of pathogen-induced

BON1/CPN1 protein and transcript accumulation in cpn1-2 and cpn1-3 suggests that

there are negative regulatory elements in the BON1/CPN1 promoter upstream of -318 bp

before the BON1/CPN1 transcriptional start site, and that these regulatory elements were

at least partially disabled by the T-DNA insertions in cpn1-2 and cpn1-3.

Although BON1/CPN1 is believed to be a suppressor of plant defense, resistance

to virulenct P. s. t. appeared to be normal in cpn1-2 and cpn1-3, which have high levels

of BON1/CPN1 proteins. One possibility is that the disease resistance assays used were

not sensitive enough to detect altered disease resistance in cpn1-2 and cpn1-3 plants.

Another possibility is that BON1/CPN1 functional partners are needed for effective

suppression of disease resistance. A study by Yang et al. (2007) has demonstrated that

transient overexpression of both BON1/CPN1 and its interacting partner BAP1

significantly suppressed the HR induced by avirulent P. s. t. bacteria and programmed

cell death caused by either an apoptotic gene or an ROS-producing chemical. Therefore,

the over-accumulation of BON1/CPN1 alone in cpn1-2 and cpn1-3 may not affect

resistance to bacterial pathogens in the absence of crucial BON1/CPN1 functional

partner(s).

A regulatory motif search within the 280 bp, pathogen- and calcium-responsive

BON1/CPN1 promoter region identified several putative cis-elements, including an SA-

75

responsive TCA element, an elicitor-responsive W box, and an ABRE element which

may be responsive to Ca2+

stimulus (Rushton et al., 1996; Pastuglia et al., 1997; Kaplan

et al., 2006). Our results are in accordance with studies of several plant C2 domain-

containing proteins, which are regulated transcriptionally by calcium signals and various

abiotic and biotic stresses (Kim et al., 2003; Ouelhadj et al., 2006; Kim et al., 2008). The

need for specific regulation of the expression of these C2-domain containing proteins by

stress or pathogen-related stimuli reflects their biological functions as calcium sensors,

which determine the specificity of calcium signaling by perceiving and relaying calcium

signals precisely for the right biological outcome. Certainly, more detailed promoter

analysis will be needed to verify the functional regulatory sequences within the 280 bp

pathogen- and calcium-responsive section of the BON1/CPN1 promoter.

76

Chapter 6

Conclusions and future directions

Based on currently available information about BON1/CPN1, I propose a working

model for BON1/CPN1 function (Figure 20). In wild-type Col-0 plants, BON1/CPN1

negatively regulates SNC1 (Yang and Hua, 2004) by preventing the activation of SNC1

by intracellular Ca2+ fluxes induced by low temperature, A23187, pathogens, and

possibly low humidity. In the absence of BON1/CPN1 (in cpn1-1 mutant plants),

cytosolic Ca2+ perturbations triggered by calcium ionophore A23187, low temperature, or

pathogen inoculation activate SNC1. This model explains the conditional nature of the

cpn1-1 mutant phenotype: the cpn1-1 mutant phenotype observed under non-permissive

growth conditions may result from inappropriate defense activation in response to Ca2+

fluxes triggered by low temperature and possibly by low humidity. This model explains

the ability of calcium ionophore A23187 to trigger PR gene expression in cpn1-1, and the

ability of avirulent pathogens, like P. s. t. DC3000 (avrRpt2) to trigger an accelerated HR

in cpn1-1 (Jambunathan et al., 2001). The model is also consistent with the proposed

function of copines as Ca2+-sensitive membrane trafficking and signal transduction

proteins.

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Figure 20: A proposed model for the function of BON1/CPN1. In wild-type Col-0 plants, SNC1

has the potential to activate defense in response to fluxes in intracellular calcium level triggered

by pathogens, low temperature, and A23187. However, BON1/CPN1 negatively regulates the

response of SNC1 to intracellular Ca2+. In cpn1-1 mutant plants, cytosolic Ca

2+ perturbations

lead to SNC1-mediated defense activation because BON1/CPN1 is absent.

It is yet to be determined how BON1/CPN1 exerts a negative regulatory function

on SNC1-mediated defense. In line with the guard hypothesis, it has been suggested that

BON1/CPN1 is guarded by R proteins including SNC1 (Yang and Hua, 2004; Yang et

al., 2006). Instead of directly interacting with the pathogen effector proteins, plant R

proteins act as surveillance proteins and detect pathogen invasion by monitoring changes

in host proteins targeted by pathogen effectors (Dangl and Jones, 2001; Martin et al.,

2003). It is conceivable that BON1/CPN1 is a favorable target of pathogen effectors due

78

to its crucial role in cell death regulation and Ca2+ signaling. Among many known host

target proteins, Arabidopsis RIN4 protein is the best characterized "guardee", which is

guarded by at least two R proteins RPM1 and RPS2 (Chisholm et al., 2006).

Phosphorylation or elimination of the RIN4 protein by avirulent (Avr) effectors activate

the RPS2- or RPM1-dependent defense responses (Mackey et al., 2002; Axtell and

Staskawicz, 2003; Mackey et al., 2003). Assuming BON1/CPN1 is a host protein

guarded by SNC1, it is possible that BON1/CPN1 is targeted and modified by pathogen

effector proteins via similar mechanisms such as proteolytic degradation and

phosphorylation. Another possible scenario is that SNC1 monitors the change in the

subcellular localization of BON1/CPN1 protein caused by pathogen effectors as

suggested by BON1/CPN1 localization studies (Appendix B and (Liu, 2007). However,

it will be challenging to investigate the mechanism of SNC1 surveillance of BON1/CPN1

because the cognate Avr protein recognized by SNC1 is not known. Future work to

identify the pathogen effector protein for SNC1 and how BON1/CPN1 is modified will

help to elucidate the functional relationship between BON1/CPN1 and SNC1.

We have identified a 280 bp promoter region that directs the pathogen- and

calcium-inducible BON1/CPN1 promoter activity. Detailed deletion analysis of this

specific region will help to identify the functional regulatory sequences that are responsive

to pathogen and/or calcium signals. On the other hand, our results have provided more

information regarding the transcriptional factors involved in BON1/CPN1 regulation

based on these cis-acting regulatory elements predicted within the 280bp promoter

region. For instance, the elicitor-responsive W box is known to be the binding site for

members of the WRKY family of transcriptional factors (Rushton et al., 1996).

79

Moreover, the ABRE motif has shown to be the binding site of CAMTAs factors, a

newly identified family of calcium-dependent, calmodulin-binding transcriptial factors

(Bouche et al., 2002; Yang and Poovaiah, 2002; Mitsuda et al., 2003; Choi et al., 2005).

A sensible step after defining the pathogen- and calcium-responsive elements in

the BON1/CPN1 promoter region is to identify the transcription factors that specifically

bind with these regulatory sequences. By using yeast one-hybrid system, an in vivo

genetic assay designed for isolating DNA-binding proteins (Li and Herskowitz, 1993),

we will be able to determine the identity of the transcriptional factors that interact with

the pathogen-reponsive cis-acting element on the BON1/CPN1 promoter. Since the

detection of the DNA-protein interaction occurs in vivo while proteins are in their native

configurations, the one-hybrid assay offers high sensitivity to detect protein-DNA

association. Moreover, the genes encoding the DNA-binding proteins can be obtained

quickly after library screening. These results will provide important insights about the

transcriptional regulation of BON1/CPN1 and roles of the transcriptional factors in

defense and Ca2+ signaling.

Another important task in the future will be the identification of the BON1/CPN1

downstream target proteins in Ca2+ signaling. Our data support the proposed function of

Arabidopsis copines as Ca2+-responsive signal transduction proteins that perceive and

relay Ca2+ signals in response to abiotic and biotic stimuli. Since perturbation of

intracellular Ca2+ levels by cold or calcium ionophore leads to defense activation in the

absence of BON1/CPN1, BON1/CPN1 is essential for normal Ca2+ responses and the

maintenance of Ca2+ signaling specificity. Subsequently, the specificity of Ca

2+ signaling

also depends on a specific set of Ca2+ sensors and their target proteins that coordinate and

80

function together in an orderly and timely fashion in the cells.

In order to identify potential interactors of BON1/CPN1, a yeast two-hybrid assay

has been employed in previous studies and the C2-domain containing protein BAP1 was

characterized as interacting with and possibly functioning together with BON1/CPN1

(Hua et al., 2001; Yang et al., 2006a). However, the design of yeast two-hybrid

screening can only detect the interaction between two proteins, hence limiting the ability

to isolate interacting proteins that require the formation of a multi-subunit protein

complex in actual cellular situations. In this case, a pull-down assay by affinity

chromatography which utilizes the anti-BON1/CPN1 antibody we generated will allow us

to isolate BON1/CPN1-interacting proteins/protein complexes which may be present

particularly during incompatible or compatible plant-pathogen interactions. Following

affinity chromatography, protein sequencing by mass spectrometry can determine the

identity of the interacting proteins (Chen et al., 2006; Soo et al., 2007). The advantage of

using the anti-BON1/CPN1 antibody that specifically recognizes BON1/CPN1 in a pull-

down assay is the ability to avoid any artificial effect that could arise due to the

overexpression of epitope-tagged BON1/CPN1 in transgenic plants. Certainly, more

characterization of the interacting mechanism between BON1/CPN1 and its associating

protein/protein complex is needed. The establishment of functional relationships between

BON1/CPN1 and downstream target proteins will help to decipher the corresponding

Ca2+ signaling cascades mediated by BON1/CPN1.

81

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Appendix A

Changes in BON1/CPN1 protein level in response to abiotic stimuli

A.1 Background information

Besides biotic stimuli, BON1/CPN1 gene expression is also regulated by abiotic

stimuli including low humidity and low temperature (Hua et al., 2001; Jambunathan and

McNellis, 2003). Accumulation of BON1/CPN1 transcript is increased in wild-type Col-

0 plants grown under low humidity (35-45% RH) or low temperature (21°C±0.5°C)

compared to that in high humidity, high temperature conditions (75-85% RH,

24.5°C±0.5°C) (Jambunathan and McNellis, 2003). A possible explanation for the

temperature- and humidity-regulated BON1/CPN1 expression came from the search of

putative cis-acting element motifs in the BON1/CPN1 promoter regions (Chapter 5).

There are two low temperature-responsive elements (LTR) (Brown et al., 2001) and two

MYB binding sites involved in drought-inducibility (MBS) (Yamaguchi-Shinozaki and

Shinozaki, 1993) in the 1.9kb BON1/CPN1 promoter region. However, experimental analysis

is needed to determine whether these putative regulatory elements are responsible for

BON1/CPN1 expression regulation in response to low temperature and low humidity.

The motif search in the BON1/CPN1 promoter region also identified five putative

abscisic-acid-responsive elements (ABRE) (Shen et al., 1993; Yamaguchi-Shinozaki and

Shinozaki, 1993) which may be responsive to Ca2+ stimulus (Kaplan et al., 2006). This

was further supported by the promoter activity analysis, which demonstrated the induced

91

GUS expression by calcium ionophore A23187 treatment in BON1/CPN1 promoter-GUS

transgenic plants (Chapter 5). In this study, we examined the BON1/CPN1 protein level

in response to calcium ionophore A23187 infiltration in wild-type Col-0 plants. The

BON1/CPN1 protein level in response to low temperature and low humidity is also

examined. Our results show that the accumulation of BON1/CPN1 protein was increased

by low humidity/low temperature (LH/LT) growth conditions and A23187 treatment.

A.2 Materials and methods

A.2.1 Plant materials and growth conditions

For the permissive HH/HT conditions, wild-type Col-0 plants and cpn1-1 mutant

plants were grown at 25°C and 90% RH under an 8 h photoperiod with 100 µmol m-2 s

-1

light intensity. For the non-permissive LH/LT conditions, wild-type Col-0 plants and

cpn1-1 mutant were grown at 21°C and 35% RH under a 10 h photoperiod with 75 µmol

m-2 s

-1 light intensity. 5-week-old plants were used for all experiments.

A.2.2 Chemical, cold treatments and western analysis

50 µM calcium ionophore A23187 (Sigma) in 0.25% DMSO and 100 µM

A23187 in 0.5% DMSO were used for leaf infiltration. 0.5% DMSO was used as solvent

control. 10 mM EGTA (Sigma) was used alone or in combination with 50µM A23187.

Cold treatment was administered by placing potted plants at 4°C for duration indicated in

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the Results. Plant protein extraction and western blot analyses were performed as

described in Chapter 4.

A.3 Results

A.3.1 BON1/CPN1 protein level in response to low temperature and low humidity

The BON1/CPN1 protein level was monitored in wild-type Col-0 and cpn1-1

mutant plants grown under LH/LT or HH/HT conditions by western analysis. As shown

in Figure A1, the BON1/CPN1 protein level was slightly higher in LH/LT grown Col-0

plants compared to that in HH/HH-grown plants. However, the difference on the

BON1/CPN1 level caused by LH/LT was not great, since it can only be observed when a

large amount the total plant protein was loaded on the SDS-PAGE gels (Figure A1 and

data not shown). BON1/CPN1 was not detected in the cpn1-1 mutant plants grown under

both LH/LT and HH/HT conditions as expected.

Figure A1: BON1/CPN1 protein level in LH/LT or HH/HT-grown Col-0 plants. BON1/CPN1

level in Col-0 wild-type and cpn1-1 mutant plants was monitored by western analysis. Arrow

indicates the position of the BON1/CPN1 band. 100 µg of total soluble protein were in each

lane.

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We speculated that the 3°C temperature difference between LT and HT conditions

may not be enough to cause major effect on BON1/CPN1 accumulation. Therefore, we

subjected wild-type Col-0 plants to continuous, non-freezing 4°C temperature for 6h or

24h (Figure A2). Similar BON1/CPN1 protein levels were observed in untreated and

cold-treated Col-0 plants at the indicated time points.

A.3.2 BON1/CPN1 protein level in response to calcium ionophore treatment

The effect of calcium ionophore A23187 on BON1/CPN1 accumulation in wild-

type Col-0 plants grown under LH/LT or HH/HT conditions was examined by western

analysis (Figure A3). Total protein extract was isolated from leaves that showed no

collapse in response to A23187 treatment. In LH/LT-grown Col-0 plants, 50 µM A23187

caused increased BON1/CPN1 accumulation at 48 hpi, while 100µM A23187 caused

increased BON1/CPN1 accumulation at 24 hpi and 48 hpi compared to that in untreated

Col-0 plants. At 24hpi, 100µM A23187 caused higher accumulation of BON1/CPN1

Figure A2: BON1/CPN1 protein level in Col-0 plants after 4°C cold treatment. Col-0 wild-type

plants were grown at 21°C for 5 weeks, and moved to a 4°C chamber for indicated periods. Un,

untreated Col-0 plants. Arrow indicates the position of the BON1/CPN1 band. 75 µg of total

soluble protein were in each lane.

94

protein than 50µM A23187, indicating a dosage effect of calcium ionophore on

BON1/CPN1 protein level. Furthermore, 50µM A23187 also caused increased

BON1/CPN1 accumulation in HH/HT-grown Col-0 plants at 24 hpi (Figure A3 B).

Next, we tested whether chelation of extracellular Ca2+ could attenuate the effect

of A23187 on BON1/CPN1 accumulation. When Ca2+ chelator EGTA was co-infiltrated

with A23187 into leaves of HH/HT-grown Col-0 plants, the increased BON1/CPN1

accumulation caused by A23187 was attenuated (Figure A3 B, compare lanes 50A to

10G+50A). This result showed that calcium ionophore-induced BON1/CPN1

accumulation was caused by the influx of extracellular Ca2+.

A

B

Figure A3: BON1/CPN1 protein level in wild-type Col-0 plants after A23187 treatment. A;

Western blots showing the effect of calcium ionophore A23187 on BON1/CPN1 accumulation

in LH/LT-grown wild-type Col-0 plants. Un, untreated Col-0 plants. Treatments: 0.5% DMSO

(D), 50 µM A23187 in 0.25% DMSO (50A), or 100µM A23187 in 0.5% DMSO (100A). Time,

hours after chemical infiltration. B, Suppression of calcium ionophore-induced BON1/CPN1

accumulation by EGTA. HH/HT-grown wild-type Col-0 plants were treated with 0.25% DMSO

(D), 50 µM A23187 in 0.25% DMSO (50A), 10 mM EGTA (10G), and 10 mM EGTA mixed

with 50 µM A23187 (10G+50A). Samples were collected at 24hpi. Arrow indicates the position

of the BON1/CPN1 band. 75 µg of total soluble protein were in each lane.

95

A.4 Dicussion

Low temperature and low humidity conditions (21°C, 35% RH) caused a slight

increase in BON1/CPN1 accumulation compared to high humidity, high temperature

conditions (21°C, 90% RH). This is in agreement with a previous study which showed

increased BON1/CPN1 transcript accumulation under LH or LT conditions (Jambunathan

and McNellis, 2003).

We suspected the degree of temperature change may contribute to the effect of

low temperature treatment on BON1/CPN1 level. However, when Col-0 plants grown at

25°C were subjected to cold treatment at 4°C for 6 h or 24 h, BON1/CPN1 protein level

remained unchanged compared to that in untreated control plants. It is notable that the

BON1/CPN1 RNA level had a twofold increase when Col-0 plants were shifted from

28°C to 22°C or 28°C to 16°C for 12h (Hua et al., 2001). Thus, the 24h duration of the

cold treatment in our experiment should be long enough to cause changes in BON1/CPN1

transcript or protein level if there are any. It is possible that changes in the BON1/CPN1

transcript level are not reflected on the BON1/CPN1 protein level. Also, unchanged

protein level after 4°C cold treatment may result from significantly reduced metabolic

activities including transcription and translation at such a low temperature.

Our results showed that calcium ionophore A23187 was able to induce

BON1/CPN1 accumulation in wild-type Col-0 plants grown under either LH/LT or

HH/HT conditions, and that EGTA could attenuate the A23187 effect. This result is in

accordance with the A23187-induced GUS expression in BON1/CPN1 promoter-GUS

activity analysis (Chapter 5). However, high concentrations of A23187 sometimes

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triggered necrotic cell death in LH/LT grown wild-type plants at 24 hpi (data not shown).

Therefore, the secondary effect of cell death caused by high concentration A23187 may

partially account for the increased accumulation of BON1/CPN1. Replacing A23187

with CaCl2 (Kim et al., 2003) or other calcium ionophores for calcium stimulus may help

to elucidate the actual effect of Ca2+ on BON1/CPN1 regulation.

97

Appendix B

Subcellular redistribution of YFP::BON1/CPN1 protein in response to calcium

ionophore and cold stimuli

B.1 Background information

The Arabidopsis BON1/CPN1 has been shown to localize to the plasma

membrane (PM) when expressed in Arabidopsis protoplasts or transgenic plants (Hua et

al., 2001). To further investigate the subcellular localization of BON1/CPN1 protein in

response to various stimuli, transgenic plants expressing a yellow fluorescence protein

(YFP) fused to the N-terminus of BON1/CPN1 were generated and the localization of the

YFP-tagged BON1/CPN1 protein (YFP::BON1/CPN1) was examined by confocal

microscopy (Liu, 2007). Previous study has shown that the YFP::BON1/CPN1 protein

has a focal localization in Arabidopsis epidermal cells after pathogen challenge. Here,

the subcellular localization of YFP::BON1/CPN1 protein was monitored after A23187

and brief cold treatments. Our results showed that A23187 and cold treatments

modulated the subcellular localization of the BON1/CPN1 protein in a manner similar to

that triggered by bacteria pathogen inoculation.

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B.2 Materials and methods

B.2.1 Plant materials and growth conditions

T2 transgenic Arabidopsis lines expressing YFP::BON1/CPN1 protein were used

in this study (Liu, 2007). In short, the Gateway vector was used to create the plasmid

with YFP fused to the N-terminal of the BON1/CPN1 protein (Hartley et al., 2000; Curtis

and Grossniklaus, 2003). The T2 plants were grown for 4 weeks at 21°C and 35% RH

under a 10h photoperiod with 70 µmol m-2 s

-1 light intensity. 4-week-old plants were

used in the experiments.

B.2.2 Chemical and cold treatments and confocal microscopy

300µM of calcium ionophore A23187 in 1.5% DMSO (Sigma) was used for leaf

infiltration. 1.5% DMSO (Sigma) was used as solvent control. The Pseudomonas

syringae pv tomato DC3000 strain was suspended in sterilized water at a concentration of

1×108 cfu/mL. Sterilized water was used as infiltration control. Leaves were observed

under a confocal microscope after the treatments. For cold stimuli, detached leaves were

subjected to 0ºC with ice water for 1 min and then observed under the confocal

microscope immediately.

For YFP::BON1/CPN1 subcellular localization, the fluorescence of YFP was

observed using a LSM510 META confocal laser scanning microscope (Carl Zeiss

MicroImaging, Inc., Thornwood, NY, USA). The YFP was excited by the 488-nm Argon

laser line and the emitted light was collected for imaging.

99

B.3 Results

To visualize the BON1/CPN1 localization in the plant cells, a yellow fluorescence

protein (YFP)-tagged BON1/CPN1 construct was made and stably expressed in

Arabidopsis plants (Liu, 2007). The YFP::BON1/CPN1 protein was mostly localized to

the cell periphery of epidermal cells in untreated transgenic plants (Figure B1 A). We

investigated the subcellular localization of YFP::BON1/CPN1 in response to various

stimuli including A23187 and cold. There was no distinguishable change in

YFP::BON1/CPN1 localization after water or 1.5% DMSO treatment (Figure B1 B and

C, respectively). Interestingly, YFP::BON1/CPN1 showed a localized accumulation

pattern along the cell periphery after 300µM A23187 treatment (Figure B1 D and E).

The localized accumulation pattern was observed 3 to 4 hpi and persisted until 24 hpi

(data not shown). Close-up views showed that YFP::BON1/CPN1 accumulated

intracellularly along the cell periphery, with substantial focal accumulation at the cell

junctions and lobes of the epidermal cells (Figure B1 F to I).

100

101

A brief cold treatment by ice water for 1 min also affected YFP::BON1/CPN1

subcellular localization. YFP::BON1/CPN1 had a cell peripheral localization in

epidermal cells before the detached leaf was treated (Figure B1 J). A localized

accumulation pattern of YFP::BON1/CPN1 was observed minutes after the detached leaf

was treated with ice water for 1 min (Figure B1 K and L). The YFP::BON1/CPN1

showed a focal accumulation at the cell junctions and lobes of the epidermal cells, similar

Figure B1: Subcellular localization of YFP::BON1/CPN1 in response to bacterial pathogen,

calcium ionophore, and cold stimuli in transgenic plant leaves. For bacteria and calcium

ionophore stimuli, leaves were syringe-infiltrated with water, DMSO, A23187, or P.s.t.DC3000.

Then the YFP::BON1/CPN1 localization in the epidermal cells were observed 6h post

infiltration. For cold stimuli, detached leaves were treated with 0ºC ice water for 1 min and then

observed immediately. A; untreated Arabidopsis epidermal cells expressing YFP::BON1/CPN1.

B and C; 6h post infiltration with water and 1.5% DMSO, respectively. D and E; the focal

accumulation of YFP::BON1/CPN1 in the epidermal cells 6h after 300µM A23187 infiltration.

F and H; close-up views of selected areas in E showed the intracellular accumulation of

YFP::BON1/CPN1 at the cell junctions and lobes when compared with the bright-field images

(G and I, respectively). J; the cell periphery-localized YFP::BON1/CPN1 in the epidermal cells

before the detached leaf was treated. K and L; focal accumulation of YFP::BON1/CPN1 was

observed minutes after the detached leaf was treated with cold. M and N; a close-up image from

the selected area in L showing cold-induced, intracellular YFP::BON1/CPN1 accumulation at

the lobes and cell junction of the epidermal cells. O; 1h after cold treatment, YFP::BON1/CPN1

showed a cell-periphery localization similar to that in untreated epidermal cells as shown in J.

P; the localized YFP::BON1/CPN1 accumulation patterns 6hr after treatment with 1×108 cfu/mL

of P.s.t. DC3000. Q; close-up views of the selected area in P showed the intracellular

accumulation of YFP::BON1/CPN1 at the cell junctions and lobes in the epidermal cells when

compared with the bright-field image R. Scale bar, 10µm.

102

to the patterns triggered by A23187 stimulus (Figure B1 M and N). The effect of cold on

YFP::BON1/CPN1 localization was transient, since the focal accumulation was no longer

detected in the same leaf 1 h after the cold stimulus (Figure B1 O). Consistent with the

results from Liu (2007), YFP::BON1/CPN1 protein exhibited a localized accumulation

pattern along the cell periphery after treated with 1x108 cfu mL

-1 of P.s.t. DC3000

(Figure B1 P to R). The localized accumulation of YFP::BON1/CPN1 was mainly at the

cell junctions and lobes of the epidermal cells (Figure B1 Q), which was first observed

from 3 to 4 hpi and persisted until 24 hpi (data not shown).

B.4 Discussion

Fluorescent proteins (FPs) have been widely used as fusion protein tags to

determine the subcellular localization and behavioral properties of another protein of

interest (Mathur, 2007; Berg and Beachy, 2008). The soluble, free (untargeted) FPs in

plant cells are usually localized in the cytoplasm (Haseloff et al., 1997; Davis and

Vierstra, 1998). However, to verify our results of this study, transgenic Arabidopsis

plants expressing only the YFP protein using the same vectors as other fluorescence

protein construct (Liu, 2007) should be generated. They will be controls for different

treatments along with the YFP::BON1/CPN1 expressing plants.

YFP::BON1/CPN1 exhibited remarkably similar, localized accumulation pattern

in response to A23187, bacterial pathogen, and cold stimuli. This result indicated that

BON1/CPN1 subcellular distribution may be modulated by Ca2+ influx triggered by both

abiotic and biotic stimuli. Interestingly, both A23187 and bacterial pathogen triggered a

103

similar temporal pattern for YFP::BON1/CPN1 accumulation, which was observed at 3 to

4hpi and persisted to 24 hpi. In contrast, a 1 minute-cold treatment triggered a similar

YFP::BON1/CPN1 spatial accumulation but in a much faster and transient manner. The

difference in the YFP::BON1/CPN1 temporal accumulation patterns by pathogen and

cold may be correlated to the unique Ca2+ perturbations triggered by different stimuli

(White and Broadley, 2003). While pathogens generate a relatively slow (hours) and

sustained cytosolic Ca2+ elevation, cold shock generates a immediate, brief spike on

cytosolic Ca2+ level (seconds).

In response to cold and pathogen stimuli, YFP::BON1/CPN1 protein showed a

distinct, focal accumulation at the cell junctions and lobes along the cell periphery of the

epidermal cells. It was unclear whether the accumulation was due to the redistribution of

YFP::BON1/CPN1 from previous cell peripheral locations or the transportation of de

novo-synthesized YFP::BON1/CPN1 to the focal accumulation sites. Therefore, future

experiments using inhibitors for protein synthesis and protein degradation will help to

investigate the origin of these accumulated YFP::BON1/CPN1 proteins.

The other challenge is to determine whether the distinct YFP::BON1/CPN1

accumulation pattern is functionally relevant. There are two possible explanations for the

formation of YFP::BON1/CPN1 accumulation patterns based on the biochemical

properties of copines and other C2-domain containing proteins. First, lipid selectivity of

the C2 domain could contribute to the localized YFP::BON1/CPN1 distribution along the

plasma membrane at the cell periphery. Human copines exhibit Ca2+-dependent lipid

binding with preference for negatively-charged phospholipids like phosphatidylserine

over neutral phospholipids like phosphatidylcholine (Creutz et al., 1998; Tomsig and

104

Creutz, 2002). Besides the structural role in membranes, PS has been shown to

participate in many important biological processes including protein kinase C activation

and apoptosis (Vance and Steenbergen, 2005). Although the physiological importance of

lipid composition is not well understood, studies have shown a change of phospholipid

composition in response to low temperature (Somerville, 1995). Along the same line, it

is possible that other stimuli could also affect the lipid composition by altering the

abundance and distribution of certain phospholipids in membranes. Therefore, the focal

accumulation pattern may result from preferable YFP::BON1/CPN1 binding to certain

part of the plasma membrane where phosphatidylserine is enriched in response to cold or

pathogen stimuli.

The other possible explanation is that YFP::BON1/CPN1 may bind to target

proteins located at certain domains in the membranes. Studies have demonstrated that

Nedd4, a C2-domain containing ubiquitin protein ligase, is localized specifically to the

apical region of epithelial cells where its interacting partner annexin XIII is located in the

presence of Ca2+ (Plant et al., 2000). The authors also showed that Nedd4 associated

with the lipid raft microdomain in a Ca2+ dependent manner. Lipid rafts are specific

membrane domains formed by stable association of certain lipids such as sphingolipids

and cholesterol. In animals and yeast cells, extensive studies have revealed the important

roles of lipid rafts in protein sorting, signal transduction, or pathogen infection (Simons

and Ikonen, 1997). For instance, these rafts may function as sorting platforms for the

attachment of acylated, glycosylphosphatidylinositol-anchored, or palmitoylated

signaling molecules. In higher plants, many recent studies have presented compelling

evidence for the existance of lipid rafts (Mongrand et al., 2004; Borner et al., 2005;

105

Grennan, 2007). In barley epidermal cells, powdery mildew challenge triggers the

formation of a plasma membrane microdomains enriched in plant sterols at pathogen

entry sites (Bhat et al., 2005). Furthermore, the barley mildew resistance locus O (MLO)

protein, which is required for host cell invasion by the powdery mildew fungus, shows a

focal plasma membrane accumulation beneath the fungal penetration sites (Bhat et al.,

2005). Other plant proteins involved in penetration resistance also redistribute to the

fungal entry sites, including a calmodulin that is known to positively regulate the MLO

activity (Kim et al., 2002). Together, the data suggest a scenario where both positive and

negative regulatory components of defense co-localize at the plasma membrane

microdomains that develop at pathogen entry sites. To determine if BON1/CPN1

functions in a similar way to the MLO machinery, it is necessary to identify whether

sterol-rich microdomains occur at the cell junction and lobes where the

YFP::BON1/CPN1 accumulates in response to A23187, cold, and pathogen stimuli.

Another important task will be to investigate the functional relationship of BON1/CPN1

and its interaction partners. It would be interesting to see if any of the BON1/CPN1

interacting proteins are modified with specific lipid groups or located in the lipid

microdomains so they could actually function together with BON1/CPN1 at the

membranes. In conclusion, the link between lipid rafts and the BON1/CPN1 localization

may provide more insights about the possible mode of action of Arabidopsis copines.

VITA

Tzuu-fen Lee

EDUCATION

PhD, Pennsylvania State University (2001-present, expected August 2008) Integrative Biosciences program, Ecological and Molecular Plant Physiology option

Thesis title: “Characterization of the pathogen-regulated Arabidopsis BONZAI1/COPINE1

protein and its role in calcium signaling”

Advisor: Dr. Timothy W. McNellis

MS, National Taiwan University, Taipei, Taiwan (1995-1997) Horticultural Science, Biotechnology group

Thesis title: “Characterization of banana ripening-related protein kinases”

Advisor: Dr. Pung-Ling Huang

BS, National Taiwan University, Taipei, Taiwan (1991-1995) Horticultural Science

RESEARCH EXPERIENCES

Doctoral Research, Integrative Biosciences program in Pennsylvania State University (2001-

present)

Research assistant, Department of Internal Medicine and Endocrinology in National Taiwan

University Hospital, Taipei, Taiwan (2001)

Research assistant, Department of Medical Genetics and Pediatrics in National Taiwan University

Hospital, Taipei, Taiwan, (1999-2000)

Research assistant, Institute of Biological Chemistry in Academia Sinica, Taipei, Taiwan (1997-

1999)

Master Research, Institute of Horticultural Science National Taiwan University, Taipei, Taiwan

(1995-1997)

PUBLICATIONS

T.-f. Lee and T.W. McNellis. Evidence that BONZAI1/COPINE1 is a calcium- and pathogen-

responsive protein. 2008 (Submitted, Plant Molecular Biology)

T.-f. Lee and T.W. McNellis. Eliminationof keratin artifact bands from western blots by using

low concentration of reducing agents. 2008 (Submitted, Analytical Biochemistry)

Leu, J.H, Yan, S.J, Lee, T.-f., Chou, C.M., Chen, S.T., Hwang, P.P., Chou, C.K. and C.J. Huang.

2000. Complete genomic organization and promoter analysis of the round-spotted pufferfish

JAK1, JAK2, JAK3, and TYK2 genes. DNA Cell Biol 19(7):431-46

Documents

CHARACTERIZATION OF THE PATHOGEN-REGULATED …