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3 UBRARY
3 i» 7 Michigan State
University
This is to certify that the

dissertation entitled

Study of Pseudomonas syn'ngae effector protein HopM1 and
its host target AtMIN7 implicated in vesicle trafﬁcking in
Arabidopsis thaliana

presented by

Young Nam Lee

has been accepted towards fulﬁllment
of the requirements for the

 

 

PhD. degree in Plant BiologL
/2'

1‘
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Date

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5/08 K:lProj/Acc&Pres/ClRC/Dateoue.indd

. STUDY OF PSE UDOMONAS S YRINGAE EFFECTOR PROTEIN HOPMl AND
ITS HOST TARGET ATMIN7 IMPLICATED IN VESICLE TRAFFICKING IN
‘ ARABIDOPSIS T HALIANA

By

Young Nam Lee

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
Plant Biology

2009

ABSTRACT
STUDY OF PSEUDOMONAS S YRINGAE EFFECTOR PROTEIN HOPMl AND
ITS HOST TARGET ATMIN7 IMPLICATED IN VESICLE TRAFFICKING IN
ARABIDOPSIS T HALIANA
By
Young Nam Lee

In the pathogenic interaction between Arabidopsis thaliana and Pseudomonas
syringae pv. tomato DCBOOO (Pst DC3000), it has been suggested that one function of
type III secretion system (TTSS) effector proteins of Pst DC3000 is to interfere with the
defense-associated cellular trafﬁcking pathway(s) in Arabidopsis. This study focuses on
characterization of HopM1, a TTSS effector of Pst DC3000, and its host target AtMIN7,
a putative adenosine-diphosphate ribosylation factor-guanine nucleotide-exchanging
factor (ARF-GEF) implicated in intracellular vesicle trafﬁcking in Arabidopsis.

The localization of full—length and truncated HopM1 was studied using confocal
microscopy. In tobacco cells, full-length HopM1 fusion proteins were found in small,
punctate structures, which were co-localized with a trans-Golgi network (TGN) marker
VHA-al and an early endosome marker ARA6. The fusion protein of the N-terminus of
HopM1 (HopM1 1-300) was found in punctate structures co-localizing with VHA-al.
Approximately 5 hours after their expression, full-length HopM1 fusion proteins were
found in punctate structures, whereas truncated HopM1 fusion proteins (HopM1 1-300 and
HopM1301-712) were dispersed in the cells. F ull-length HopM1 fusions induced death of
tobacco leaf tissue by 48 hours after their induction. Some of the HopM1 fusion proteins

transformed into Arabidopsis (Col-O and atmin7 backgrounds) showed similar

localization patterns as in tobacco. It is suggested that HopM1 is localized in the

endosomes in the host cell and that HopM11-300 contains an organelle-targeting signal.

Whether other Arabidopsis ARF-GEFs besides AtMIN7 are involved in host
defense is not known. The roles of three ARF-GEFS (AtBIG2, AtBIG3 and AtBIG4) in
defense were studied by examination of bacterial multiplication in corresponding T-DNA
insertional mutants. None of the examined mutants showed a clear defect in defense
against Pst DC3000, the ACEL mutant (lacking HopM1), or the hrcC 'mutant (defective
in the TTSS). The HopM11-3oo was previously shown to interact with the C-terminus of
AtMIN7. The C-termini of four ARF-GEFS (AtBIGl, AtBIG3, GNLl and GNL2) were
examined by yeast two-hybrid assay to determine whether they also interacted with
HopM11-300, The C-terminus of GNL2 did not interact with HopM11-300. The interactions
between HopM11-300 and the C—termini of AtBIGl, AtBIG3 and GNLl were not
determined because of the failure to express these proteins in yeast.

To determine whether AtMIN 7 regulates the secretion of defense-related proteins,
three Arabidopsis proteins (PR1, a putative lipid-transfer protein encoded by At2g10940,
and FLA9, a putative arabinogalactan protein encoded‘by At1g03870) were fused with
GF P, and expressed in Arabidopsis (Col-0 and atmin7 background) followed by confocal
microscopic examination. PRl-GFP was localized in the intercellular space both in Col-O
and atmin7 backgrounds. At2g10940-GFP was localized in the intercellular space in C01-
0 background, but in atmz‘n 7 background, unidentiﬁed intracellular structures appeared.
FLA9-GFP fusion was not expressed in Arabidopsis. These results suggest that the
localization of At2g10940-GFP may be dependent on the AtMIN7-mediated vesicle

trafﬁcking pathway(s) whereas the localization of PRl-GFP is not.

Copyright by
YOUNG NAM LEE

2009

ACKNOWLEDGEMENTS

I show my sincere gratitude to my advisor Dr. Sheng Yang He, whose support led
me profound improvement of myself as a scientist. I thank my committee members, Dr.
Jonathan Walton, Dr. Rebecca Grumet and Dr. Ray Hammerschmidt, for their valuable
guidance and support during my Ph.D.

1 want to thank my previous and current colleagues of He laboratory for their kind
support and help. Especially the support from Dr. Kinya Nomura, Dr. Christy Mecey, and
Wei-Ning Huang was indispensable for my study. The help from Dr. Elena Bray Speth in
confocal microscopy was also valuable for me. I thank Dr. Roger Thilmony and Dr.
Maeli Melotto for their kind support. I also would like to mention the warm support
consistently shown to me from Lori Imboden, and the support from Francisco Uribe, Dr.
Weiqing Zeng, Dr. J ian Yao, John Withers, Matt Oney, and Xiufang Xin. The previous
and current undergraduate assistants provided great help to me.

I received warm and kind help from Dr. Federica Brandizzi for my confocal
microscopic experiments. I was also kindly helped from Dr. Melinda Frame.

The help I received from the Plant Research Laboratory was one of the greatest
supports in my life, and I am proud that I was in it.

I feel that I am lucky to share my Ph.D. time with precious friends, Janet Paper,
Jessica Koczan, Hoosun Chung, Jeongwoon Kim, Julie Bordowitz, Xinchun Zhang,
Eliana Gonzales-Vigil, Bill Underwood, Hiroshi Maeda, and Clarisa Bejar. I would like
to show my gratitude to my families in Korea and the Netherlands. Also, I deeply thank

to my husband Dr. Harrie vanErp, and our daughter Annemarie Soyeon.

TABLE OF CONTENTS

 

 

 

 

LIST OF TABLES ix
LIST OF FIGURES 1:
CHAPTER 1 -_ - -- - ................ - - -- 1
General aspects of plant-pathogen interactions ......................................................... 2
The Arabidopsis thaliana-Pst DC3 000 model ............................................................ 2
Pseudomonas syringae pv. tomato DC 3 000 as a bacterial phytopathogen ............ 3
Arabidopsis thaliana as a model plant in plant-pathogen interactions .................. 6
A four-part model for the interaction between Arabidopsis and Pst DC3000 ............ 6
I” step: the activation of PAMP-triggered immunity (PT I) ................................... 7
2nd step: the suppression of PT I by TTSS eﬂector proteins ................................... 9
3rd step: the recognition of TISS effectors by plants and activation of effector-
triggered immunity (ET I) ...................................................................................... 10
4th part: evasion or suppression of E TI by pathogens through TISS effectors 12
Systemic acquired resistance (SAR) ......................................................................... 13
Suppression of defense-associated cellular trafficking pathway in plants by TTSS
effectors .................................................................................................................... 15
[-10le and AtMIN 7: a TTSS eﬂector involved in the suppression of defense-
associated cellular traﬂicking pathway(s) and its target of Arabidopsis ............. 17
Summary of thesis research subjects ........................................................................ 18
REFERENCES ............................................................................................................. 20
CHAPTER 2 ..... - -- 32
ABSTRACT .................................................................................................................. 33
INTRODUCTION ........................................................................................................ 36
MATERIALS AND METHODS .................................................................................. 39
Construction of HopM1 fusions ............................................................................... 39
Introduction of Hole fusion ORFs into Agrobacterium ....................................... 42
Transient assays of the Hole fusion ORFs in tobacco plants ............................... 45
Transformation of HopM1 fusions into Arabidopsis plants ..................................... 45
Plant growth condition .............................................................................................. 46
Application of Dexamethasone (DEX) ..................................................................... 46
Confocal microscopy ................................................................................................ 47
Dual localization with cellular markers .................................................................... 47
Brefeldin A (BFA) treatment .................................................................................... 49
Protein extraction and western blot analyses ............................................................ 49
The color of the images in this dissertation ............................................................. 50

vi

RESULTS ..................................................................................................................... 51
Transiently expressed full-length HopM1 fusion proteins induce tissue death in

 

 

 

tobacco leaves ........................................................................................................... 51
Western blot analyses of HopM1 fusions in transient assay ..................................... 53
Transiently expressed fusions of ﬁill-length HopM1 are found in small, punctate
structures in tobacco leaves ...................................................................................... 53
Dual localization tests of full-length HopM1 fusions with a Golgi marker with or
without Brefeldin A (BFA) ....................................................................................... 58
Dual localization tests of YFP-Hole and endosome markers ............................... 61
Localization of truncated HopM1 fusion proteins .................................................... 63
Transgenic expression of HopM1 fusions in Arabidopsis .................................... 66
DISCUSSION ............................................................................................................... 75
REFERENCES ............................................................................................................. 82
ACKNOWLEDMENTS ............................................................................................... 33
CHAPTER 3 -- 86
ABSTRACT .................................................................................................................. 87
INTRODUCTION ........................................................................................................ 89
MATERIALS AND METHODS .................................................................................. 93
Identiﬁcation of homozygous T-DNA insertion mutants for Arabidopsis ARF-GEF
genes ......................................................................................................................... 93
Conﬁrmation of gene knockout of T-DNA insertion mutants by reverse transcription
and polymerase chain reaction (RT-PCR) ................................................................ 96
Construction of ARF-GEF gene clones for yeast two-hybrid assays ....................... 98
Yeast two-hybrid assay ........................................................................................... 104
Growth curve assay for determining multiplication of Pst DC3000 and its mutants in
Arabidopsis ............................................................................................................. 106
Crossing atmin7 knockout mutant (salk_012013) and knockout mutant of AtBIGZ
(salk_033446) ......................................................................................................... 107
RESULTS ................................................................................................................... 108
Identiﬁcation of homozygous T-DNA insertion mutants for Arabidopsis ARF-GEF
genes ....................................................................................................................... 108
Conﬁrmation of gene knockout in the homozygous T-DNA insertion mutants by
RT-PCR .................................................................................................................. 108
Determination of the multiplication of the ACEL mutant in the selected T-DNA
insertion mutants ..................................................................................................... 110
Determination of the multiplication of the ACEL mutant in the double knockout
mutant of AtMIN 7 and 8162 ................................................................................... 1 10
Yeast two- hybrid assay of selected Arabidopsis ARF-GEF genes with HopM1 .. 113
DISCUSSION ................................................................................................ 108
REFERENCES ........................................................................................................... 122
ACKNOWLEDMENTS ............................................................................................... 87
CHAPTER 4 -- - - - - - - 124
ABSTRACT ................................................................................................................ 125

vii

INTRODUCTION ...................................................................................................... 126

 

MATERIALS AND METHODS ................................................................................ 130
Construction of OF P fusions and their subcloning into plant expression vector.... 130
Transformation of recombinant plasmids into A grobacterium ............................... 133
Production of transgenic Arabidopsis lines expressing GF P ﬁisions ..................... 133
Confocal microscopy .............................................................................................. 133
Plasmolysis of leaf samples .................................................................................... 134
Protein extraction and western blot analyses .......................................................... 134

RESULTS ................................................................................................................... 136
Production of transgenic Arabidopsis expressing GFP fusion proteins ................. 136
The subcellular localization of PRl-GFP in Arabidopsis ....................................... 139
The subcellular localization of At2g10940-GF P in Arabidopsis ............................ 142

DISCUSSION ............................................................................................................. 145

REFERENCES ........................................................................................................... 150

CHAPTER 5 - - 153

REFERENCES ........................................................................................................... 160

APPENDICES- - 162

 

viii

LIST OF TABLES

Table 2-1. Summary of the fusion proteins created for the localization study of HopM1 40

Table 2-2. PCR primers for cloning the ORFs of sGF P and E YF P ................................. 40

Table 2-3. PCR primers for obtaining full-length or truncated hopMI ORFs for ﬁision
constructions ..................................................................................................................... 43

Table 2-4. Summary of transgenic Arabidopsis expressing HopM1 fusions ................... 73

Table 3-1. The Arabidopsis ARF-GEF genes, their selected T-DNA insertion mutants,
and the putative insertion site of T-DNA of each mutant ................................................. 94

Table 3-2. Primer sets designed for screening homozygous T-DNA insertion mutants for
ARF-GEF genes ................................................................................................................ 95

Table 3-3. Primers for RT-PCR for T-DNA insertion mutants of selected ARF—GEF
genes ................................................................................................................................. 97

Table 3-4. Primer sequences for obtaining clones of selected ARF-GEF genes for yeast
two-hybrid assay with the N-terminus of HopM1 ............................................................ 99

Table 4-1. Primers used for PCR to construct OF P fusions of selected Arabidopsis genes
........................................................................................................................................ 132

Table 4-2. Summary of transgenic Arabidopsis expressing P‘Rl-GFP fusion protein 137

Table 4-3. Summary of transgenic Arabidopsis expressing At2g1 0940-GF P fusion
protein ............................................................................................................................. I37

LIST OF FIGURES

Figure 2-1. Diagrams of HopM1 fusions used for localization studies ............................ 41

Figure 2-2. Appearance of Nbenthamiana leaves which were infiltrated Agrobacterium
carrying various plasmids ................................................................................................. 52

Figure 2-3. Immunoblot analyses of HopM1 fusions in transient assays in N.tabacum .. 54

Figure 2-4. HopM1-GFP and YFP-Hole transiently expressed in N.tabacum ............ 57

Figure 2-5. Dual localization results of full-length HopM1 fusion proteins and ST-RF P 59

Figure 2-6. Dual localization result of HopM1-GP P and ST-RF P with BFA treatment .. 62

Figure 2-7. Dual localization results of YFP-Hole and VHA-al-RFP ........................ 64
Figure 2-8. Dual localization results of YFP-Hole and ARA6-CFP ............................ 65
Figure 2-9. Localization of truncated HopM1 ﬁJSIOI‘I proteins ......................................... 68
Figure 2-10. Dual localization results of YFP-Hole-N and VHA-al-RFP .................. 70
Figure 2-11. HopM1 fusion proteins in Arabidopsis ........................................................ 74
Figure 3-1. A phylogenetic tree of Arabidopsis ARF-GEF genes .................................... 90

Figure 3-2. Predicted amino acid sequences of the ARF-GEFs for yeast two-hybrid
assays .............................................................................................................................. 100

Figure 3-3. Diagrams of four ARF—GEF genes analyzed by yeast two-hybrid assay ..... 105

Figure 3-4. Genomic PCR results of homozygous T-DNA insertional mutants for three
ARF-GEF genes (BIG2, BIG3 and 3104) ...................................................................... 109

Figure 3-5. RT-PCR results of selected T-DNA insertional mutants of AtBIGZ, AtBIG3
and AtBIG4 ..................................................................................................................... 1 l 1

Figure 3-6. Multiplication of Pst DC3000, the ACEL mutant and the hrcC' mutant in Col—
0, atmin7 and ARF-GEF T-DNA insertion lines ............................................................ 1 12

Figure 3-7. Genomic PCR results conﬁrming the double knockout of AtMIN 7 and
AtBIGZ ............................................................................................................................ 1 14

Figure 3-8. Multiplication of Pst DC3000, the ACEL mutant and the hrcC' mutant in Col-
0, atmin 7, atbig2, and the double knockout of AtMIN 7 and AtBIGZ ............................. 115

Figure 3-9. Yeast two-hybrid assay result of the C-termini of selected ARF-GEFs with
HopM1 1-300 ...................................................................................................................... I I 7

Figure 4-1. Diagrams of the PRl-GFP, At2g10940-GFP and FLA9—GFP fusions for the
subcellular localization study .......................................................................................... 131

Figure 4-2. Immunoblot analysis of PRl-GFP and At2g10940-GF P in Arabidopsis 138

Figure 4-3. Localization and expression of PR] -GF P in Arabidopsis (in Col-0
background) .................................................................................................................... 140

Figure 4-4. Localization of PRl-GF P in Arabidopsis (in atmin7 mutant background). 141

Figure 4-5. Localization of At2g10940-GFP in Arabidopsis (in Col-0 background) 143

Figure 4-6. The localization of At2g1 O940-GFP in Arabidopsis (in the atmin7
background) .................................................................................................................... 1 44

xi

 

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CHAPTER 1

Literature Review

General aspects of plant-pathogen interactions

During evolution, plants have been exposed to a variety of micro-organisms and
developed various types of interactions with them. Among these interactions, the
interactions between plants and pathogenic microorganisms have been extensively
studied in plant science, in order to advance our understanding of these processes and
how they have been evolved. Moreover, the study of plant-pathogen interactions is
important for agriculture, considering that the crop loss caused by plant disease is one of
the major limiting factors in food production (reviewed by Savary et al., 2006).

The aspects of the interactions between plants and pathogens are diverse
depending on types of plants and pathogens. However, an emerging theme of these
interactions is that plants and their pathogens are in a continuous cycle of attack, defense,
and counterattack. In this ongoing “battle”, the pathogens have continuously affected the
physiological state of the plants for obtaining nutrition for their survival and proliferation.
In many cases, these effects compromise or severely interfere with the normal physiology,
grth and proliferation of plants. Plants have evolved defense mechanisms in order to
cope with these pathogen attacks, as summarized in different reviews (Katagiri et al.,
2002; Chisholm et al., 2006; Speth et al., 2007; Boiler and He, 2009). This evolutionary
arms race enabled both pathogens and plants to develop multiple strategies for attack and
defense. As a result, this arms race has signiﬁcantly affected the evolution of plants and

pathogens at the genetic and molecular level (Ma et al., 2006; Stavrinides et al., 2008).

The Arabidopsis thaliana-Pst DC3000 model

In many cases the interactions between plants and pathogens are speciﬁc. Some of
these interactions have been studied as model systems, in which a speciﬁc plant species
and a speciﬁc pathogen are connected as an interaction pair. In this thesis, I focus on one
plant-pathogen interaction model, in which Arabidopsis thaliana is the host plant and
Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is the pathogen. This model
was introduced in 19903 with several other Arabidopsis thaliana-Pseudomonas syringae
interaction models, and has been widely used in plant-pathogen interaction studies since
then (Davis et al., 1991; Dong et al., 1991; Whalen et al., 1991; Katagiri et al., 2002).
Both Arabidopsis and Pst DC3000 have advantages for studying the molecular basis of
plant-pathogen interactions, because both of them have well-developed resources of
molecular genetic and genomic manipulations, including their full genome sequences (for
Arabidopsis genome, refer to Rhee et a1. (2003) and www.arabidopsis.org; for Pst

DC3000 genome, refer to Buell et al. (2003) and hgnﬂaseudomonas-syringae.org).

Pseudomonas syringae pv. tomato DC3000 as a bacterial phytopathogen

P.syringae is a gram-negative bacterium. It is included in the Pseudomonas genus,
which has approximately 50 pathogenic species with different host speciﬁcities (Deng et
al., 1998; Anzai et al., 2000). As a plant pathogen P.syringae has more than 50 pathovars
(Gardan et al., 1999) which differ largely in host range among different plants (Gardan et
al., 1999). Different races and strains are assigned to these pathovars, based on their
ability to infect different plant cultivars (Alfano and Collmer, 1997; http://pseudomonas-

syringaeorg). Pathovars of P. syringae infect various crops such as tomato, soybean, rice,

 

tobacco and wheat (Gardan et al., 1999).

Pst DC3000 is a strain included in the pathovar P. syringae pv. tomato, which is
the cause of speck disease in tomato (Cuppels, 1986). Speck disease on tomato is one of
the representative examples of disease caused by P..syringae, with characteristic
symptoms such as necrotic lesions on the tomato leaves and dark spots on the fruit
(Cuppels et al., 1990; http://pseudomonas-syringaeorg). Pst DC3000 is assigned to race
0, on the basis of its avirulence on tomato cultivars carrying Pto resistance (Ronald et al.,
1992). Pst DC3000 is also a pathogen of A. thaliana (Whalen et al., 1991; Katagiri et al.
2002).

Pst DC3000 is considered as a hemibiotrophic pathogen: it typically enters plant
leaves through openings such as stomata or through wounds and aggressively multiplies
(to the level of 108 cells/cm2 of leaf) in the intercellular space (also known as apoplast),
followed by formation of necrotic lesions (Whalen et al., 1991; Hirano and Upper, 2000;
Katagiri et al., 2002; Nomura et al., 2005; Melotto et al., 2008). Upon infection by Pst
DC3000, A. thaliana displays disease with visible symptoms, such as leaf yellowing
(chlorosis) and the water-soaking phenomenon, followed by eventual tissue necrosis
(Whalen et al., 1991; Katagiri et al., 2002).

The pathogenicity and virulence of Pst DC3000 depend on at least two
pathogenicity factors, the type III secretion system (TTSS) with its effector proteins and
the phytotoxin coronatine (Katagiri et al., 2002). Pst DC3000 injects 30-40 bacterial
proteins collectively called “TTSS effectors” into the plant cells through its TTSS
(Alfano and Collmer, 2004). The TTSS of Pst DC3000 is encoded by a cluster of
hypersensitive response and pathogenicity (hrp) genes; hrp' mutants of Pst DC3000 are

impaired in regulation or secretion of type III effectors, and lose the ability to multiply

and cause disease in compatible hosts (Yuan and He, 1996; Roine et al., 1997). The
TTSS (and its effector proteins) and hrp genes are not speciﬁc to Pseudomonas, but
found in other plant pathogens such as Xanthomonas, Ralstonia, and Erwinia spp.
(Alfano and Collmer, 1997; Alfano and Collmer, 2004) and in several animal pathogens
like Yersinia, Salmonella, and Shigella spp. (He et al., 2004).

The TTSS and its effectors are essential for the pathogenicity and virulence of Pst
DC3000 and other pathogens (Yuan and He, 1996; Deng et al., 1998; Collmer et al.,
2002). Accumulated research results indicate that TTSS effectors contribute to
pathogenicity and virulence by manipulating the physiological states of host plants
(Nobuta and Meyers, 2005; Cunnac et al., 2009). These manipulations will be further
discussed in later parts of this chapter.

On the other hand, coronatine is a phytotoxin made by several pathovars of
P.syringae including Pst DC3000 (Bender et al., 1999). It is composed of two moieties,
coronafacic acid and coronamic acid. The structure of coronatine shares a high similarity
to that of jasmonoyl isoleucine (JA-Ile), and it has been thought that coronatine is a
functional mimic of JA-Ile in plant-pathogen interactions (Katagiri et al., 2002; Melotto
et al., 2008). The role of coronatine in pathogenicity and virulence of Pst DC3000 has
been studied in Arabidopsis and tomato, and it is generally accepted that coronatine
contributes to symptom development, activation of jasmonate (JA)-related responses and
suppression of the expression of defense-related genes in plants (Mittal and Davis, 1995;
Zhao et al., 2003; Thilmony et al., 2006). The JA-mediated defense pathways and the
salicylic acid (SA)-mediated defense pathways are often antagonistic (Kunkel and Brooks,

2002; Heil and Bostock, 2002), and the suppression of SA-mediated defenses in plants by

coronatine was shown in multiple studies (Zhao et al., 2003; Uppalapati et al., 2007).
More recently a role of coronatine in the regulation of stomatal opening was shown

(Melotto et al., 2006; Melotto et al., 2008).

Arabidopsis thaliana as a model plant in plant-pathogen interactions

Arabidopsis thaliana has advantages in laboratory research due to its small plant
size, short generation time (around 8 weeks), high seed production, natural self-
pollination, small genome size and well-established genomic resource (Meyerowitz,
1987; Ausubel et al., 1995; Nobuta and Meyers, 2005). Arabidopsis has been used as a
model plant for studying the interactions with several pathogenic P.syringae strains
including Pst DC3000 and showed clear resistance or susceptibility to different strains of
P.syringae, depending on the genotypes of those strains (Davis et al., 1991; Dong et al.,
1991; Whalen et al., 1991; Ausubel et al., 1995). As mentioned above, Arabidopsis
exhibits susceptibility to Pst DC3000, showing symptom development including water-
soaking, chlorosis, and tissue necrosis, followed by death of whole plant (Katagiri et al.,

2002).

A four-part model for the interaction between Arabidopsis and Pst DC3000

The Arabidopsis-Pst DC3000 interaction can be explained by a recently suggested
“four-part model” (Bent and Mackey, 2007). This model shows the roles of TTSS
effector proteins of Pst DC3000 in inducing plant resistance and susceptibility (Chisholm

et al., 2006; Jones and Dangl, 2006; Bent and Mackey, 2007) and includes two major

concepts of two major plant defenses, PAMP-triggered immunity (PTI) and effector-
triggered immunity (ETI). This model also reﬂects the hypothetical evolutionary steps of

the interaction between Arabidopsis and Pst DC3000.

1‘" step: the activation of PAMP-triggered immunity (PT I)

In the ﬁrst part of four-part model, generic, conserved components of micro-
organismic pathogens called pathogen-associated molecular patterns (PAMPs), or more
recently called microbe-associated molecular patterns (MAMPs: Ausubel, 2005), are
perceived by plants. This recognition of PAMPs/MAMPs by plants activates a basal layer
of defense responses, which are believed to be sufﬁcient for preventing infections by a
wide range of microbes (Alfano and Collmer, 2004; Nomura et al., 2005; Bent and
Mackey, 2007). In recent reviews this layer of defense responses are referred to as
PAMP-triggered immunity (PTI) (Chisholm et al., 2006; Jones and Dangl, 2006; Bent
and Mackey, 2007). In earlier studies PTI was not an independent concept, but it was
incorporated in “nonhost” resistance, which allows a given plant species to confer broad-
spectrum resistance against most microorganisms (Heath, 2000; Niimberger and Brunner,
2002; Mysore and Ryu, 2004). In this context PAMPs/MAMPs were studied as “general
elicitors” activating defense responses in the species level (Boller, 1995; Nilmberger et
al., 2004). However, the PTI is now accepted as the ﬁrst line of plant defense against
different microorganisms.

Well-known PTI-associated defense responses include formation of papillae
(localized apposition of callose and other materials) in the plant cell wall, induction of

defense genes, accumulation of reactive oxygen species (ROS) and plant secondary

metabolites (Alfano and Collmer, 2004; Nomura et al., 2005). Modiﬁcation of the plant
cell wall is likely an important part of plant immunity (Hauck et al., 2003; DebRoy et al.,
2004; Keshavarzi et al., 2004).

Various PAMPs/MAMPS have been reported from different pathogens. They
include bacterial ﬂagellin, bacterial lipopolysaccharide (LPS), fungal cell wall
components like chitin, heptaglucosides of oomycetes, and fungal ergosterol (Nilmberger
et al., 2004; Bent and Mackey, .2007). Among these PAMPs/MAMPs, bacterial ﬂagellin
(especially the 22-amino-acid conserved epitope in the N-terrninus of ﬂagellin (ﬂg22))
has been well studied (Felix et al., 1999; Gomez-Gomez et al., 1999). The ﬂg22 peptide
is sufﬁcient to induce PTI (Gomez-Gomez et al., 1999). Recognition of ﬂg22 contributes
to enhanced resistance against non-pathogenic bacteria (Hann and Rathjen, 2007). F lg22
is recognized by and binds to Arabidopsis F L82, which is a transmembrane receptor
protein with extracellular leucine-rich repeats (LR) and intracellular protein kinase
(Gomez-Gomez et al., 1999; Gomez-Gomez and Boller, 2000; Zipfel et al., 2004;
Chinchilla et al., 2006). The recognition of flg22 by FLSZ enhances plant resistance
against Pst DC3000, presumably through activation of downstream signal transduction
pathways, including expression of defense-related genes (Asai et al., 2002; Navarro et al.,
2004; Zipfel et al., 2004; Livaja et al., 2008).

Besides ﬂagellin (or its ﬂg22), the elongation factor Tu (EF-Tu) of bacteria was
also reported as a PAMP/MAMP (Kunze et al., 2004). EF-Tu induces defense responses
in plants including Arabidopsis, such as ethylene biosynthesis and oxidative burst (Kunze
etal., 2004). Later, another Arabidopsis transmembrane LRR-receptor-like kinase, EFR,

was reported as the binding receptor of EF-Tu (Zipfel et al., 2006). EFR and FLS2

induce a common set of defense-related responses, including oxidative burst and MAP
kinase activation, aﬁer recognizing EF-Tu and ﬂ g22, respectively (Zipfel et al., 2006).
LPS, another example of bacterial PAMPs/MAMPS, extracted from Xanthomonas
campestris pv. vesicatoria induced papilla formation and delay of symptom development
in pepper (Keshavarzi et al., 2004).

Although not common, there are a few cases that PAMPs/MAMPs induce
localized cell death in nonhost plants. For example, the ﬂagellin monomer or ﬂg22 from
Pst DC3000 induces cell death in Nicotiana benthamiana, which is a nonhost plant to Pst
DC3000 (Taguchi et al., 2003; Ham and Rathjen, 2007). This cell death is dependent on
an orthologue ofArabidopsis FLSZ in Nbenthamiana, NbFls2 (Hann and Rathjen, 2007).
Also, certain LPS activates the generation of ROS and activations of defense genes

associated with local cell death (Desaki etal., 2006).

2nd step: the suppression of PT I by TTSS eﬂector proteins

In the 2Ind step of the “four-part model”, successful pathogens including Pst
DC3000 release pathogenic proteins, which target host plants and suppress PTI. TTSS
effectors of Pst DC3000 is among the most extensively studied examples of PTI
suppression by bacterial proteins.

As shown in previous reports, wild type bacterial pathogens with intact TTSS
(Kesharvarzi et al., 2004) or even speciﬁc TTSS effectors such as AvrPto (Hauck et al.,
2003; He et al., 2006; Hann and Rathjen, 2007) suppress PTI. AvrPto was later shown to
also suppress ﬂg22-induced MAP kinase signal transduction pathways and early gene

expressions for defense (He et al., 2006). Remarkably, it was recently shown that AvrPto

9

physically interacts with F L82 or EF R to block innate immunity of Arabidopsis (Xiang et
al., 2008). In addition to AvrPto, other TTSS effectors of Pst DC3000 like AvrPtoB (de
Torres et al., 2006; Ham and Rathjen, 2007), HopM1 (DebRoy et al., 2004), HopUl (Fu
et al., 2007), Aerpt2 and Aerme (Kim et al., 2005), or HopAOl (also known as
HothoDZ [Underwood et al., 2007; Guo et al., 2009]) suppress PTI.

The local cell death in nonhost plants is also a target of some TTSS effectors.
AvrPto and AvrPtoB expressed in Nicotiana benthamiana suppresses the localized
PAMP-induced cell death (Kang et al., 2004; Harm and Rathjen, 2007). There are cases
in which the induction of certain plant genes during PTI is suppressed by TTSS effectors
(de Torres et al., 2006; He et al., 2006; Underwood et al., 2007).

The suppression of PTI by the TTSS effectors often contributes to the enhanced
multiplication of non-pathogens or mutants of virulent pathogens, as shown in the cases
of AvrPto or AvrPtoB (Hauck et al., 2003; He et al., 2006; de Torres et al., 2006; Ham

and Rathjen, 2007).

3rd step: the recognition of IT SS effectors by plants and activation of reﬂector-
triggered immunity (E T I)

The third part of the four-part model is essentially the classic gene-for-gene
interaction model (Flor, 1971), in which TTSS effectors are recognized by plant
resistance (R) proteins, followed by the eliciting of defense responses called the
hypersensitive response (HR). It is a relatively recent trend that the gene-for-gene
interaction is incorporated in the bigger plant-pathogen interaction model emphasizing its

evolution as a counterattack to the suppression of plant defenses by TTSS effectors, and

10

it is called effector-triggered immunity (ETI) (Chisholm et al., 2006; Jones and Dangl,
2006; Bent and Mackey, 2007).

Gene-for-gene resistance was studied in the context of speciﬁc plant genotypes
(for instance, cultivars) and speciﬁc pathogen genotypes (strain or race). According to
this model, a plant-pathogen interaction can be compatible (leading to plant disease) or
incompatible (leading to plant resistance). The incompatible interaction occurs when the
plant expresses a speciﬁc plant resistance gene (R) and the (avirulent) pathogen expresses
a speciﬁc avirulence (avr) gene (Dangl and Jones, 2001). During the incompatible
interaction, downstream signal transduction pathways lead to defense responses, such as
the expression of pathogenesis-related (PR) genes (Uknes etal., 1992), the rapid and
localized cell death in the infected region (HR) (Heath, 2000), and the activation of
systemic acquired resistance (SAR).

Many avr and R gene pairs have been discovered in various plant-pathogen
interactions (Martin et al., 2003; Alfano and Collmer, 2004). In the four-part model,
bacterial Avr proteins (TTSS effectors) suppress basal defense, and the R proteins detect
this defense-suppressing virulence factors by detecting the effect of a TTSS effector on
the designated host protein. The host protein which is affected by the TTSS effector is
indispensable to the plant defense (Chisholm et al., 2006; Bent and Mackey, 2007).
Initially it was thought that Avr proteins and R proteins were directly interacting and that
this interaction activates downstream defense pathways. This is based on several
examples of direct interactions between Avr proteins and R proteins, such as the
interaction between Pi-ta of rice and AVR-Pita of rice blast fungus (Magnaporthe grisea)

(J ia et al., 2000) and that between R proteins of ﬂax and Aer567 variants of rust

11

(Melampsora lini) (Dodds et al., 2006). However, this was not the case for the majority
of Avr-R interactions, and an alternative explanation called “guard hypothesis” was
introduced (Dangl and Jones, 2001). In this hypothesis, Avr proteins interact with and
manipulate plant target proteins which are independent of, but often physically associated
with, R proteins. This indirect recognition of Avr protein by R protein through the third
plant target protein induces downstream defense pathways. The interaction between
AvrPto of P.syringae and tomato proteins Pto and Prf is explained by the guard
hypothesis: AvrPto physically interact with Pto (Scoﬁeld et al., 1996; Tang et al., 1996),
which may be a virulence target in tomato, and this interaction is recognized by Prf
followed by HR (Salmeron et al., 1996; van der Biezen and Jones, 1998; Dangl and Jones,
2001; Zipfel and Rathjen, 2008). Other TTSS effectors of P.syringae, Aerpml, AvrB or
Aerpt2 phosphorylates or eliminates RIN4, which is a regulator of basal defense of
Arabidopsis, and these modiﬁcations of RIN4 activate RPMI- or RPS2-mediated defense
responses (Mackey et al., 2002; Axtell and Staskawicz, 2003; Mackey et al., 2003).
Recently another model referred to as “decoy model” was introduced for
explaining the relationship between Avr protein (TTSS effector), its corresponding R
protein and the plant target protein of Avr protein. Compared to the guard hypothesis, in
the decoy model the plant protein targeted by Avr protein (TTSS effector) does not have
functions in virulence, and this target protein works as a decoy to detect pathogen

effector protein (Zipfel and Rathjen, 2008; van der Hoom and Kamoun, 2008).

4th part: evasion or suppression of E T 1 by pathogens through 7T SS effectors

12

In the ﬁnal part of the four-part model, ETI activated by the recognition of TTSS
effectors by plant R proteins are evaded or suppressed (Bent and Mackey, 2007). The
suppression of ETI is summarized in several reviews (Abramovitch and Martin, 2004;
Nomura et al., 2005; Grant et al., 2006).

The HR induced by gene-for-gene resistance can be suppressed by TTSS effectors.
Ritter and Dangl (1996) reported that Aerpt2 suppresses HR and disease resistance
activated by the Aerpml-RPMI interaction in Arabidopsis. This suppression of HR by
Aerpt2 is mediated by the action of Aerp2 as a cysteine protease, cleaving
Arabidopsis RIN4 which is required for HR activated by the Aerpml-RPMI interaction
(Axtell et al., 2003; Mackey et al., 2003). In addition, in the interaction between
P.syringae pv. phaseolicola and soybean, it was shown that the TTSS effectors of
P.syringae pv. phaseolicola can suppress HR induced by gene-for-gene resistance on the
cultivar level (Jackson et al., 1999; Tsiamis et al., 2000). On the other hand, Abramovitch
et a1. (2003) showed that HR induced by AvrPto-Pto was suppressed by AvrPtoB;
however, this result is based on the transient expression of AvrPto, Pto and AvrPtoB in

the nonhost plant N. benthamiana, not in the host plant tomato or Arabidopsis.

Systemic acquired resistance (SAR)

Systemic acquired resistance (SAR) is a long-lasting, broad-spectrum resistance
which is systemically induced in plant by pathogen infection (Durrant and Dong, 2004)
and not included in the four-step model. SAR is usually activated during the incompatible
interaction between a pathogen with an avr gene and a plant with an R gene (with HR).

However, non-HR-inducing bacteria also can activate SAR through their

13

PAMPs/MAMPS, showing the systemic resistance against bacterial infection,
accumulation of SA and defense-associated gene transcripts (such as ﬂg22 or LPS:
Mishina and Zeier, 2007). During SAR, local and systemic accumulation of salicylic acid
(SA) occurs, followed by expression of defense-related genes such as pathogenesis-
related (PR) genes and activation of signal transduction cascades (Durrant and Dong,
2004; Vlot et al., 2008).

Salicylic acid (SA) is an essential signal for SAR (Gaffney et al., 1993), and
exogenous application of SA (Ryals et al., 1995) or SA analogues such as
benzothiadiazole (BTH) or 2,6-dichloroisonicotinic acid (INA) can activate SAR as well
(Uknes et al., 1992; Lawton et al., 1996). However, SA itself is not a mobile signal for
activating SAR in systemic tissue (Vemooji et a1, 1994). Several metabolites such as
methyl salicylate (MeSA), jasmonic acid (JA) or azaleic acid were suggested as potential
mobile signals in SAR (Park et al., 2007; Truman et al., 2007; Jung et al., 2009).
Research results show that SA induces redox reactions to reduce the protein NPRl (non-
expressor of pathogenesis-related 1, also known as non-immunity 1 (N 1M1)), which is
activated and moves to the nucleus of the plant cell in order to activate transcription of
defense-related genes. This activation is regulated by several proteins in plants. Also,
there are NPRl-independent signaling pathways affecting SAR activation and regulation
(Durrant and Dong, 2004; Grant and Lamb, 2006; Loake and Grant, 2007). SA-mediated
defense signaling pathway is also affected by cross-talk with other signaling pathways,
including JA/ethylene, abscisic acid (ABA), and nitric oxide (NO)-mediated signaling

pathways (Loake and Grant, 2007).

14

Suppression of defense-associated cellular trafﬁcking pathway in plants by
TTSS effectors

Recent literature suggests that plant vesicle trafﬁcking machineries are associated
with PTI, ET] and SAR, and are a target of TTSS effector-mediated suppression. The
importance of vesicle trafﬁcking system in plant defense was initially suggested from
studies of cell wall-associated PTI, the formation of papillae. As brieﬂy mentioned above,
the papilla is a microscopic apposition which is formed in the intercellular space of the
plant (apoplast) (Smart et al., 1985); it consists of heterogeneous materials such as callose
(B-l,3-glucan), phenolics and proteins (reviewed by Schmelzer, 2002). Papilla formation
were reported in the interactions between fungal pathogens (such as Blumeria graminis or
Erysiphe graminis) and plants (Zeyen and Bushnell, 1979; Smart et al., 1985; Koga et al.,
1990; Mendgen et al., 1995; Collins et al., 2003; Gjetting et al., 2004), as well as those
between bacterial pathogens such as P. syringae or Xanthomonas campestris (Bestwick et
al., 1995; Brown et al., 1995) and plants. Papillae also can be induced by treatment of
PAMPs/MAMPS such as ﬂg22 (Gomez-Gomez et al., 1999; Zipfel et al., 2004; de Torres
et al., 2006).

It has been suggested that the cellular vesicle trafﬁcking pathway is important for
papilla deposition. Although callose is synthesized in the plasma membrane (Turner et al.,
1998; Jacobs et al., 2003; Nishimura et al., 2003), earlier research results suggested that
polarized vesicle trafﬁcking may be involved in the formation of papillae (Bestwick et al.,
1995; Mendgen etal., 1995). Later, more evidence showing that components of vesicle
trafﬁcking pathways of plants play roles in papilla formation was obtained. In particular,

mutation of the Arabidopsis PEN 1 gene which encodes a plasma membrane syntaxin

15

(SYP121), which is a member of soluble N—ethylmaleimide-sensitive factor adaptor
protein receptors (SNAREs) involved in vesicle docking to a target membrane (Bassham
et al., 2008), resulted in delayed papilla formation in response to a fungal pathogen
Blumeria graminis f.sp. hordei (th) (Collins et al., 2003). Also, both PEN] and its
closest homologue SYP122 accumulated at the papilla formation site upon th infection,
indicating that vesicle trafficking plays an important role in papilla formation (Collins et
al., 2003; Assaad et al., 2004).

Another syntaxin from Nicotiana benthamiana, NbSYPl32 (an orthologue of
SYP132 in Arabidopsis), is required for gene-for-gene resistance activated by the
AvrPto-Pto interaction and for extracellular accumulation of the PR1 protein. NbSYPl32
contributes to basal defense against the hrpA' mutant of Pst DC3000 and SA-associated
defense (Kalde et al., 2007). Also, an ER-chaperone BiP2 was required for SA-associated
defense responses and PR1 accumulation in Arabidopsis (Wang et al, 2005). In addition,
the importance of cellular trafﬁcking in plant defense is suggested in several studies of
gene expression proﬁling: microarray data of Wang et al. (2005) showed that protein
secretion pathway-associated genes were included in the primary targets of A rabidopsis
NPR], which is a key regulator of SAR.

Hauck et al. (2003) showed that around 40% of the Arabidopsis genes which are
suppressed by a TTSS-dependent manner encoded putative secretion-related proteins.
Similar results were obtained later in a whole-genome microarray study (Thilmony et al.,
2006). Supporting evidence for the hypothesis that TTSS effectors suppress defense-
associated cellular trafﬁcking pathway(s) comes from the following studies: one of the

putative host cellular targets of AvrPto is Arabidopsis RabEld, which is involved in

16

vesicle targeting (Speth et al., 2009). A TTSS effector of Xanthomonas campestris pv.
vesicatoria (XopJ) suppresses cell wall-associated PTI by interfering with plant protein
secretion (Bartetzko et al., 2009). Finally, research on the Pst DC3000 effector HopM1
provide more detailed information, as summarized below, about the action of a TTSS
effector on the defense-associated trafﬁcking mechanisms in host plants (Badel et al.,

2003; DebRoy et al., 2004; Badel et al., 2006; Nomura et al., 2006).

HopM1 and AtMIN 7: a T T SS eﬂector involved in the suppression of defense-
associated cellular traﬂicking pathway(s) and its target in Arabidopsis

HopM1 is one of the TTSS effectors whose contribution to the virulence of Pst
DC3000 was experimentally conﬁrmed (Badel et al., 2003; DebRoy et al., 2004). The
virulence-associated functions of HopM1 were mainly studied in cell wall-associated
PTI: transgenic expression of HopM1 in Arabidopsis plants caused suppression of papilla
formation, restored multiplication of the Pst DC3000 ACEL mutant, in which hopMI and
several adjacent TTSS effector genes are deleted (Alfano et al., 2000), and eventually
caused tissue death (Nomura et al., 2006). Nomura et al. (2006) found that HopM1
interacts and destroys several Arabidopsis proteins (AtMINs), including AtMIN 7.
AtMIN 7 is a member of the Arabidopsis adenosine-diphosphate ribosylation factor-
guanine nucleotide-exchanging factor (ARF-GEF) family, whose members have roles in
vesicle formation for intracellular trafﬁcking (Memon, 2004; Gillingham and Munro,
2007; Anders and Jiirgens, 2008; Bassham et al., 2008). T-DNA insertional mutants of
AtMIN 7 show enhanced multiplication of the ACEL mutant of Pst DC3000 (Nomura et

al., 2006). Also, callose deposition in papillae of atmin7 by the ACEL mutant is reduced

17

compared with that in wild-type Arabidopsis plants (Nomura et al., 2006). Therefore,
AtMIN7 functions in the defense-associated vesicle trafﬁcking pathway(s), and HopM1

interferes with the action of AtMIN7 by degrading it.

Summary of thesis research subjects

One of the major questions in the study of plant-pathogen interactions is how a
virulent pathogen suppresses the plant defense mechanisms. Various mechanisms of
suppression of plant defenses by pathogens have begun to be determined. In the
interaction between Pst DC3000 and Arabidopsis thaliana, it has been suggested that one
function of TTSS effector proteins of Pst DC3000 is to interfere with the defense-
associated cellular trafﬁcking pathway(s) in Arabidopsis. The strongest evidence for this
comes from the discovery of the action of HopM1 on AtMIN 7 in Arabidopsis (Nomura et
al., 2006). The activity of HopM1 on a regulator of vesicle trafﬁcking is intriguing and
raises a number of questions for further study. My thesis research focuses on answering
some of these questions.

In chapter 2, I describe the results of my study to determine the subcellular
localization of HopM1 in plant cells. In a membrane fractionation experiment, HopM1
was not present in the plasma membrane, but was detected in an unidentiﬁed
endomembrane compartment of Arabidopsis cells (Nomura et al., 2006). To more
precisely determine the localization of HopM1 in plant cells, I constructed ﬁisions of
HopM1 with ﬂuorescence proteins, and examined the localization of these fusion proteins

by confocal microscopy.

18

As mentioned above, there are seven other ARF GEF genes in Arabidopsis,
besides AtMIN7 (Cox et al., 2004; Anders and Jiirgens, 2008). In chapter 3, I describe
my study to determine whether any of these other ARF-GEF genes has a role in defense.
Nomura et al (2006) studied this possibility with a few ARF-GEF genes, but not all ARF-
GEF genes were studied. Therefore, I focused on the unstudied ARF-GEF genes using
two approaches: one was yeast two-hybrid assay between ARF-GEFs and HopM1 in
order to determine whether HopM1 interact with those ARF-GEFs in addition to AtMIN7.
The other was the investigation of multiplication of the ACEL mutant in T-DNA
insertional mutants of these ARF-GEF genes to determine if mutants of other ARF-GEF
genes, like atmin 7, also showed enhanced multiplication of the ACEL mutant.

In chapter 4, I describe a study of several defense-associated Arabidopsis
extracellular proteins. By tagging them with ﬂuorescence proteins and confocal
microscopic examination, I monitored the localization of those ﬂuorescence protein
fusions in wild type and atmin7 background to determine whether the secretion of these

selected Arabidopsis extracellular proteins is affected in the atmin7 background.

19

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31

CHAPTER 2

Subcellular Localization of HopM1 in Plant Cells

Christy Mecey contributed to Figure 2-4, Figure 2-5, Figure 2-7, Figure 2-8, Figure 2-9
and Figure 2-10.

32

ACKNOWLEDMENTS

I appreciate the great help of Dr. Christy Mecey who contributed to the ﬁgures of

this chapter by important confocal microscopic data.

I would like to thank Dr. Federica Brandizzi for her helpful suggestions and
discussion of the microscopy data presented in this chapter. She also kindly provided ST-
RF P, and helped me obtain endosome markers. 1 thank Dr. Takeshi Ueda (Tokyo
University, Tokyo, Japan) who kindly provided the information of ARA6-CFP, and Dr.
Jurgen Denecke (University of Leeds, Leeds, United Kingdom) kindly provided the
information for BP80-YFP, which was not used in this chapter. Dr. Karin Schmacher
(University of Heidelberg, Heidelberg, Germany) showed the kindness to provide the
VHA-al-RF P. I thank Dr. Jen Sheen (Harvard Medical School, Boston, MA) for
providing sGFP, and Dr. Jeff Dangl (University of North Carolina, Chapel Hill, NC) for
providing pBD vector. 1 also would like to thank Dr. Jianping Hu (Plant Research
Laboratory, MSU, MI), who kindly provided the ORFs of EYFP and ECFP, and PTSl-
dsRED2. Dr. Melinda Frame (Center for Advanced Microscopy, MSU, MI) helped me
perform confocal microscopy over several semesters. Xinchun Zhang in Hu laboratory
helped me perform confocal microscopy for dual localization of HopM1-GP P and PTSI-

dsRED2, and provided helpﬁil discussions.

1 would like to thank Krithika Shanmugasundaram who was in the High School
Honors Science/Mathematics/Enginnering Program of MSU (2007) and assisted the

subcloning of HopM1 ORF.

33

ABSTRACT

HopM1, a bacterial effector protein secredted through the type III secretion
system ("ITS S), is important for the virulence of Pseudomonas syringae pv. tomato (Pst)
DC3000. HopM1 interacts with and mediates degradation of several Arabidopsis proteins
(AtMINs), including AtMIN7. AtMIN 7 is a putative ARF-GEF protein, which has a role
in vesicle formation and budding during intracellular trafﬁcking. To further understand
the action of HopM1 in host cells, I studied the subcellular localization of HopM1 in
tobacco and Arabidopsis. F ull-length HopM1 and its truncated versions (HopM11-3oo and
HopM1 301-712) were fused with GFP or YF P, and they were transiently expressed in
tobacco followed by examination by confocal microscopy. After 8 hours of DEX-
induction, ﬁtll-length HopM1 fusion proteins caused tissue death in tobacco leaves, in
contrast to truncated HopM1 fusion proteins which did not induce tissue death.

Approximately between 2.5 hours and 4 hours after DEX-induction, full-length
HopM1 and HopM1 1-300, which were fused with GFP or YF P, were found in small,
punctate structures in tobacco cells. These small structures were co-localized with VHA-
al -RFP, a marker for trans—Golgi network (TGN), and ARA6-CFP, a marker for early
endosome. They were not co-localized with ST-RF P (a Golgi marker). After 4 to 6 hours,
ﬁJll-length HopM1 fusion proteins were still detected in punctate structures, but truncated
HopM1 fusion proteins were found dispersed in the tobacco cells. These data indicate
that HopM1 is localized in certain parts of the endosomal components, suggesting that its
virulence function affects vesicle trafﬁcking in host cells. Also, HopM1 [.300 is sufﬁcient

for the localization in the endosomal compartments.

34

HopM1 fusion constructs were transformed into Arabidopsis Col-0 (wild type),
and some were also transformed into the atmin7 plants. Eight hours after DEX-induction,
full-length HopM1 fusion constructs were found in small, punctate structures, whereas
the fusions of HopM1 1-300 were dispersed in Arabidopsis cells. This localization is
consistent with the localization of HopM1 fusion proteins in later time points of

microscopic observation in tobacco cells.

35

INTRODUCTION

Research on TTSS effector proteins of Pst DC3000 in Arabidopsis led to the
hypothesis that one of their virulence functions is to interfere with the secretion of
defense-associated proteins of host plants. This hypothesis was suggested based on a
cDNA microarray analysis of Arabidopsis (Hauck et al., 2003): Pst DC3000 suppressed
the expression of certain Arabidopsis genes, around 40% of which encoded putative
secreted proteins. This biased suppression of gene expression occurred in a TTSS-
dependent manner (Hauck et al., 2003).

Other reports also showed that intracellular trafﬁcking and protein secretion
pathways have roles in plant defense: several defense-associated proteins are components
of trafﬁcking pathways, such as PENI or NbSYPl32 (Collins et al., 2003; Assaad et al.
2004; Kalde et al., 2007). Also, protein secretion pathway-associated genes are among
the targets of Arabidopsis NPR], which is a key regulator of systemic acquired resistance
(SAR; Wang et al., 2005). Direct evidence for the suppression of trafﬁcking pathways
and protein secretion by TTSS effectors was provided by Nomura and colleagues
(Nomura et al., 2006), who showed that a Pst DC3000 effector, HopM1, mediates 26S-
proteasome-dependent degradation of AtMIN7, which is a member of the adenosine-
diphosphate ribosylation factor-guanine nucleotide-exchanging factor (ARF-GEF) family
of proteins necessary for the initiation of vesicle trafﬁcking (Memon, 2004; Gillingham
and Munro, 2007; Anders and J iirgens, 2008; Bassham et al., 2008).

The hopM] ORF is located in the conserved effector locus (CEL) cluster in the

genome of Pst DC3000 (Alfano et al., 2000; Buell et al., 2003). Other ORFs present in

36

the CEL encode additional TTSS effectors, including Aer, Hle, and HothoAl
(Alfano et al., 2000). HopM1 is translocated into plant cells with the assistance of its
chaperone Sth (Alfano et al., 2000; Badel et al., 2003). The hopM] ORF (also known
as ORF3: Alfano etal., 2000) consists of 2,139 bases, encoding a protein of 712 amino
acids (predicted molecular weight:75.23 kDa [Buell et al., 2003;
http://www.ncbi.nlm.nih.gov/, NC_004578.1]).

HopM1 is important for Pst DC3000 virulence. Reduced multiplication and
symptom development were observed when Arabidopsis was inoculated with the ACEL
mutant of Pst DC3000. This virulence defect of ACEL mutant was restored by a plasmid
carrying the hole gene and its chaperone gene sth whose product is required for the
translocation of HopM1 into plant cell (Badel et al., 2003; DebRoy et al., 2004).
Transgenic expression of HopM1 in Arabidopsis plants caused two phenotypes: 1) Low-
level expression restored multiplication of the ACEL mutant, and 2) High-level
expression induced host cell death (Nomura et al, 2006). Interestingly, when the N-
terrninal 300 amino acids of HopM1 (HopM1 1-300) were expressed in Arabidopsis, it did
not enhance the multiplication of the ACEL mutant, suggesting that HopM11-300 is not
functional. Moreover, transgenically expressed HopM1 1-300 suppressed the multiplication
of the ACEL mutant complemented with full-length hopMI and its chaperone sth
(Nomura et al., 2006). This dominant negative effect was not found with the C-terminus
of HopM1 (HopM1 301-712) (Nomura et al., 2006).

Immunoblot analyses by Nomura et al. (2006) showed that HopM1 is found in the
endomembrane ﬁactions of transgenic Arabidopsis cells. However, the endomembrane

system includes many different compartments, and the exact subcellular localization of

37

HopM1 remains elusive. In this study, I used the ﬂuorescence tagging approach
combined with confocal microscopy to investigate the subcellular localization of HopM1
in tobacco and Arabidopsis cells. By determining the cellular localization of HopM1 in
the plant cells, I expect to obtain a more complete understanding of the virulence action
of HopM1 inside the host cells. I also examined the localization of the N-tenninus of
HopM1 (HopM1 1-300), which has a dominant-negative effect, and that of the C-terminus
of HopM1 (Hothmmz) to explore a possible relationship between the cellular

localization and virulence function of HopM1.

38

MATERIALS AND METHODS

Construction of HopM1 fusions

F ull-length or truncated hopMI ORFs were selected for fusion construction.
Computer prediction based on the amino acid sequence of HopM1 did not give a clue of
subcellular targeting signals (http://www.cbs.dtu.dk/services/TargetP: Emanuelsson et al.,
2007; http://psort.ims.u-tokvo.ac.jp: Nakai and Kanehisa, 1991). Therefore, to avoid
possible interference with hidden subcellular targeting signals at the N- or C-terminus,
both N-terminal and C-terrninal GF P/YF P fusion were designed per each fusion construct
(Table 2-1, Figure 2-1).

Synthetic green ﬂuorescence protein (sGF P: Chiu et al., 1996) and enhanced
yellow ﬂuorescence protein (EYFP: Clontech, Mountain View, CA) were selected for
fusion construction. The ORFs of sGF P and EYF P were ampliﬁed by PCR using
PﬁrTurbo® DNA polymerase and relevant primers (Table 2-2). The PCR products were
subcloned into the pBluescript II SK(+) (Stratagene, La Jolla, CA) using T4 ligase (New
England Biolabs, Ipswich, MA), at its PstI/Spel sites and XhoI/EcoRI sites, respectively.
Mutation-free fusion ORFs were selected for the next cloning steps.

Full-length hopMI ORF was ampliﬁed by PCR with PfuTurbo® DNA polymerase
(Stratagene, La Jolla, CA) using the genomic DNA of Pst DC3000 and primers covering
the whole ORF (Table 2-3). The sequence of the PCR product was compared to the
sequence of hopM] ORF deposited in the NCBI (http://wwwncbinlm.nih. gov/,
NC_004578.1). Mutation-free hopM] ORF was cloned into a GATEWAY-compatible

vector pENTR/D-TOPO (Invitrogen, Carlsbad, CA) and used as a template for ﬁirther

39

Table 2-1. Summary of the fusion proteins created for the localization study of HopM1 .

 

 

Construct Description
HopM1-GFP 3’ end of the full-length hopM] ORF was fused with 5’ end of GFP ORF
YFP-HopM1 5’ end of the full-length hopM] ORF was fused with 3’ end of YFP ORF

HopM1-N-GF P 3’ end of the N-terminus of hopM] ORF was fused with 5’ end of GFP
ORF

YFP-HopM 1 -N 5’ end of the N-terminus of hopM] ORF was fused with 3’ end of YFP
ORF

HopM1-C-GFP 3’ end of the C-terminus of hopM] ORF was fused with 5’ end of GFP
ORF

YFP-Hole-C 5’ end of the C-terminus of hopM] ORF was fused with 3’ end of YFP ORF

 

Table 2-2. PCR primers for cloning the ORFs of sGFP and E YFP.

 

Primer name Primer sequence

 

Forward primer for 5’-GGGCTGCAGATGTGTGAGCAAGGGCGAGGAG-3’(Pstl)
sGFP ORF

Reverse primer for 5’- CGCACTAGTTTA"CTTGTACAGCTCGTCC-3’(Spel)
sGFP ORF

Forward primer for 5’- CTTCTCGAGATG*GTGAGCAAGGGCGAG-3’(Xhol)
EYFP ORF

Reverse primer 5’-CTAGAATTCCTT"GTACAGCTCGTCCATGCCGA-3’ (EcoRI)
for EYFP ORF

 

Bold and underlined letters indicate the restriction enzyme sites for subcloning and
fusions (EcoRI, PstI, SpeI and XhoI).

* Start codons are with bold and red letters.

*"‘ Stop codons are with bold and blue letters.

40

(A)
Xhol EcoRl Pstl Spel

E I _, . r E

 

(B)
Xhol EcoRl Spel

L YFP l” , j

 

(C)

 

 

 

 

 

 

Xhol EcoRl Pstl Spel
(D)
Xhol EcoRl Spel
I YFP It . great; 1
(E) XhOI EcoRl Pstl Spel
I C-terminus of hopM1 I m
(F) Xhol EcoRl Spel

 

YFP l C-terminus of hopM1 l

 

Figure 2-1. Diagrams of HopM1 fusions used for localization studies. hopM] indicates a
full-length ORF, which encodes 712 amino acids. The N-terminus of hopM] indicates a
truncated ORF encoding 1-300 amino acids. The C-terminus of hopM] indicates a
truncated ORF encoding 301-712 amino acids. Xhol, EcoRI, PstI, and Spel indicate the
restriction enzyme sites added for fusion construction. (A) A diagram of the HopM1-CF P
fusion. (B) A diagram of the YFP-HopM1 fusion. (C) A diagram of the HopM1-N-GFP
fusion. (D) A diagram of the YFP-Hole-N fusion. (E) A diagram of the HopM1-C-
GFP fusion. (F) A diagram of the YFP-Hole-C fusion.

41

PCRs to obtain full-length or truncated HopM1 ORFs. Primers for the PCRs are shown in
Table 2-3. Full-length and truncated hopMI ORFs were obtained by PCRs with
PfuTurbo® DNA polymerase (Stratagene, La Jolla, CA). The PCR products of full-length
or truncated HopM] ORFs were subcloned into the multiple cloning sites next to the 3’
end of the E YFP ORF in pBluescript II SK(+) (XhoI/EcoRI sites) in order to generate
YFP-hopM], YFP-hopMI-N and YFP-hopMI-C fusion ORFs. Alternatively, they were
subcloned into the multiple cloning sites next to the 5’ end of the sGFP ORF in
pBluescript II SK(+) (EcoRI/Spel sites) to construct hopMI-GFP, hopMI-N-GFP and
hopMI-C—GFP fusion ORFs. All of these ﬁision ORFs were sequenced and mutation-
free gene fusions were selected. The hapM] -C-GF P fusion was excluded from furtherh

experiments due to its mutations introduced during PCR.

Introduction of HopM1 fusion ORFs into Agrobacterium

The mutation-free fusion ORFs were subcloned into the Xhol/Spel sites of the
pBD vector (3 gift From Dr. Jeff Dangl laboratory, University of North Carolina, Chapel
Hill, NC). This is a binary vector containing a kanamycin-resistance gene for selecting
bacterial transformants, and a BASTA (glufosinate)-resistance gene for selection of
transgenic plants. The expression of the transgene in pBD vector depends on a promoter
inducible by the rat glucocorticoid hormone dexamethasone (DEX, Aoyama and Chua,
1997). The recombinant pBD plasmids were transformed into E.coli strains (DHSa, One
Shot® TOPIO competent cell (Invitrogen, Carlsbad, CA), or One Shot® MachTM-TlR
(Invitrogen, Carlsbad, CA)), and kanamycin-resistant colonies were obtained on the solid

Luria-Bertani (LB) medium with kanamycin (50 mg/ml). The kanamycin-resistant E. coli

42

Table 2-3. PCR primers for obtaining full-length or truncated hopM] ORFs for fusion

constructions.

 

Primer name

Primer sequence

 

Forward primer for the
hopMI ORF from Pst
DC3000

Reverse primer for the
hole ORF from Pst
DC3000

Forward primer for YFP-
HopM1

Reverse primer for YFP-
HopM1

Forward primer for
HopM1-GFP

Reverse primer for
HopM1-GFP

Forward primer for
HopM l -N-GFP

Reverse primer for
HopM1-N-GF P

Forward primer for YFP-
HopM1-N

Reverse primer for YFP-
HopM1-N

Forward primer for
HopM 1 -C-GFP

Reverse primer for
HopM 1 -C -G FP

Forward primer for YFP-
HopM1-C

5’-CACCATGATCAGTTCGCGGAT CGG-3’

5’- TTAACGCGGGTCAAGCAAGC-3 ’

5’ -—CGCGAATTCATG‘ATCAGTTCGCGGATCGG-3’(EcoRI)

5’-CCGACTAGTTTA“ACGCGG GTCAAGCAAGCC-3’(Spel)

5’-GCGCTCGAGATG*ATCAGTTCGCGGATCGGC-3 ’(Xhol)

5 ’-GGCGAATTCACGCGGGTCAAGCAAGCC-3 ’(EcoRl)

5’-GCGCTCGAGATG‘ATCAG TTCGCGGATCGGC-3’(Xhol)

5’-CTGGAATTCTGCACCTI'TCC AGCCAC-3’(EcoRl)

5’-CTTGAATI'CATG‘ATCAGTTCGCGGATCGGC-3 ’(EcoRI)

5’-GTCACTAGTTTA*'TGCACCTI‘TCCAGCCACCCJ’(Spel)

5’-CTACTCGAGATG‘GGGCCGATTGTCGCGG-3 ’(Xhol)

5’- GTAGAATTCACGCGGGTCAA GCAAGCC-3’(EcoRI)

5’- CTAGAATTCATG‘GGGCGATTGTCOCGG-3’ (EcoRI)

43

Table 2-3 (continued).

Reverse primer for YFP— 5’-CTAACTAGTTTA*‘ACGCGGGTCAAGCAAGCC-3’(Spel)
HopM 1 -C

 

Bold and underlined letters are corresponding to selected restriction enzyme sites (EcoRl,
Spel and Xhol) for cloning and gene fusions.

*Start codons are with bold and red letters.

“Stop codons are with bold and blue letters.

44

colonies containing the fusion ORFs were used for introducing recombinant plasmids
into Agrobacterium strain C58C1 by tri-parental mating using the E.coli helper strain

ka201 3.

Transient assays of the HopM1 fusion ORFs in tobacco plants

Transient assays were performed in the leaves of Nicotiana benthamiana or
Nicotiana tabacum plants. The protocol for Agrobacterium preparation for transient assay
was adapted from the experimental methods of Goodin et al. (2002). Each Agrobacterium
carrying desirable hopMI fusion plasmids was inoculated in LB liquid medium with
antibiotics (rifampicin, tetracycline and kanamycin) and incubated at 28-30°C for 8-9
hours with shaking at 250 rpm, until the OD600 of Agrobacterium reached 0.8-0.9. These
cultures were centrifuged at 3,000 rpm in a Beckman GS-6R tabletop centrifuge at room
temperature. The pellet of each culture was resuspended in the induction medium (10
mM MgC12, lOmM MES [pH 5.6], 150 11M acetosyringone [3’,5’-Dimethoxy-4’-
hydroxyacetophenone: Sigma-Aldrich, St. Louis, MO]) and the OD600 was adjusted to
0.1-0.2. These re-suspended cultures were collected in sterile test tubes, covered with
sterile caps, and left at room temperature for 2-3 hours prior to their injection into
tobacco leaves. Each culture was hand-inﬁltrated with 1 ml needless syringe into leaves

and inoculated plants were left for 36-48 hours prior to DEX treatment.

Transformation of HopM1 fusions into Arabidopsis plants
The HopM1 fusions were transformed into Arabidopsis plants (Col-0 or atmin7

knockout mutant) by ﬂoral dipping (Clough and Bent, 1998). T1 seeds were collected and

45

germinated in soil. Ten-day-old T1 plants were sprayed with 0.2 % BASTA solution
(glufosinate-ammonium, trade name Finale, AgroEvo Environemntal Health, Montvale,
NJ) containing 0.025% Silwet L-77. One leaf from each BASTA-survived T, plant was
detached and dipped in a DEX solution (30 pM) and observed with confocal microscopy,

in order to examine the expression of the ﬂuorescent fusion proteins.

Plant growth condition

Tobacco plants were raised in the laboratory, at the room temperature, using a
light of 300 microeinstein. Arabidopsis plants were grown in soil in grth chambers,
under a 12 hr dark/12 hr light cycle. The light was with 100 microeinstein, and the

temperature was 20°C.

Application of Dexamethasone (DEX)

DEX powder (Sigma-Aldrich, St. Louis, MO) was dissolved in 100% ethanol at
30 mM, and stored at -20°C. This solution was diluted in water to 15-30 11M just before
spraying, dabbing or dipping. For spraying, 0.01 % surfactant Tween-20 (Si gma-Aldrich,
St. Louis, MO) was added, as suggested by Aoyama and Chua (1997). DEX spray was
performed at least 36 hours after Agrobacterium inﬁltration. DEX solution was either
sprayed on the surface of tobacco leaves with Tween-20 or dabbed on the leaf surface
without Tween-20. The time allowed for DEX induction was variable, between 2 hours
and 48 hours, depending on experiments. Arabidopsis leaves were detached from the

plants and dipped in 3011M DEX solution, at room temperature for at least 8 hours.

46

Confocal microscopy

Leaf samples (5 mm x 5 mm) were cut from the Agrobacterium-inﬁltrated areas
of tobacco leaves and mounted in water. The confocal microscopy and imaging were
performed with a LSM510 META inverted confocal laser scanning microscope (hereafter
META: Carl Zeiss Microlmaging, Inc., Thomwood, NY) or FVlOOOD laser confocal
scanning microscope (hereafter FV1000D: Olympus America Inc., Center Valley, PA).
The 40x or 60x oil immersion objectives were used.

The OF P fusions were excited at 488 nm from the argon laser of META, and the
emission light was ﬁltered with a 505-530 nm band-pass ﬁlter for obtaining OF P
ﬂuorescence, and with a 615 nm long-pass ﬁlter for obtaining autoﬂuorescence from
chloroplasts. The YFP fusions were excited at 514 nm from the argon laser of META, or
at 515 nm from the multi-argon laser of FVlOOOD. The emission light of YFP was
ﬁltered with a 520-555 nm band-pass ﬁlter of META, or with a 535-565 nm band-pass
ﬁlter of F VlOOOD. For these experiments the autoﬂuorescence from chloroplasts was not
collected. The images obtained from the confocal microscopy with META were
examined and processed with Carl Zeiss AIM Version 3.2. Some images were adjusted

for brightness or contrast using Adobe Photoshop Element version 5.5 or 7.0.

Dual localization with cellular markers
The following subcellular markers fused with red ﬂuorescence protein (RF P,
monomer of dsRED: Campbell et al., 2002) or dsRED2 (Clontech, Mountain View, CA)

were selected for dual localization experiments with HopM1-GFP or YFP-HopM1 in

47

Agrobacterium-mediated transient expression assays: rat sialyl transferase fused to RFP
(ST-RF P [Saint-Jore et al., 2002; a gift from Dr. Federica Brandizzi, MSU, East Lansing,
MI]), dsRED2 ﬁised with the conserved C-terminal, peroxisome-targeting signal type 1
(PTSl) consisting of serine-lysine-leucine (PTSl-dsRED2 [Gould et al.,l989; Reumann,
2004; Fan et al., 2005]), a CF P-fusion of the endosomal compartment marker ARA6
(ARA6-CF P [Ueda et al., 2001]), and a RF P-fusion of the trans-Golgi network marker
VHA-al (VHA-al-GF P [Dettmer et al., 2006]). All of these markers are constitutively
expressed in plants from the CaMV 35S promoter. Procedures for dual localization
experiments were the same as those of transient assays for HopM1 ﬁrsions. The
Agrobacterium culture containing each marker gene was mixed with the Agrobacterium
culture of the desired HopM1 fusion gene in a 1:1 ratio.

Dual localization with HopM1-GFP and ST-RF P or PTSl-dsRED2 was
performed with META. The condition of confocal microscopy for HopM1-OF P was the
same as described in page 46. ST-RFP or PTSl-dsRED2 were excited at 543 nm Helium-
Neon laser, and emission light from the RF P or dsRED2 was ﬁltered with a 560 nm long-
pass ﬁlter or with the 560-615 nm band-pass ﬁlter. Dual localization of YFP-HopM1 and
ARA6-CFP was performed with META or FV 1000D. The condition of confocal
microscopy for YFP-HopM1 was the same as described in page 46. ARA6-CF P was
excited by 458 nm laser of META or F V1000D, and the emission light was ﬁltered by a
465-510 nm band-pass ﬁlter of META or by a 465-510 nm band-pass ﬁlter of FVlOOOD.
Dual localization was performed in a sequential mode to avoid false signal. The
examination and processing procedure for the images acquired from the confocal

microscopy for dual localization are identical to those described in page 46.

48

Brefeldin A (BFA) treatment

The fungal toxin Brefeldin A (BFA, y,4-Dihydroxy-2[6-hydroxy-1-heptenyl]-4-
cyclopentanecrotonic acid k-lactone) extracted from Penicillium brefeldianum was
purchased from Sigma-Aldrich (St. Louis, MO). A stock of 1.8 mM BFA (lOmg/ml) was
prepared in 100% methanol and stored at -20°C. For BFA treatment of tobacco leaf
samples, 360 11M BFA solution was prepared in water, and tobacco leaf samples which
had been hand-inﬁltrated with Agrobacterium cultures carrying HopM1-GP P and ST-
RFP plasmids were treated with DEX to induce the expression of HopM1 fusions, and

then immersed in the BFA solution for 30-35 minutes prior to confocal microscopy.

Protein extraction and western blot analyses

Total protein samples were obtained from tobacco leaves in transient assays or
from the leaves of transgenic Arabidopsis. Tobacco leaf samples were obtained from the
leaf areas inﬁltrated with Agrobacterium expressing HopM1 fusions (i.e., these leaf areas
were sprayed with 30 11M DEX solution with 0.01% Tween-20, and left at room
temperature for 8 hours to allow protein expression). Arabidopsis leaf samples were
obtained from the detached leaves which were dipped in DEX (30 uM) for 24 hours.
Twenty mg of each leaf sample (fresh weight) was ground in 200 pl SDS buffer [100 mM
Tris-HCI pH 6.8, 200 mM DTT, 4% SDS, 20% glycerol] for protein extraction. Extracts
were immediately heated at 90°C for 10 minutes and then frozen at -20°C.

Prior to loading of each protein sample on a protein gel, extracts were thawed on

ice, heated at 90°C for 3 minutes, and centrifuged at 10,000 X g for 1 minute. Ten to

49

twenty 11L of each sample was used for SDS-PAGE (equal volumes were used in a given
gel). Total proteins were separated on precast gradient gels (4-20%, ISC BioExpress) or
hand-made SDS-PAGE gels (7.5, 10, or 12% gels), then transferred onto Immobilon-P
membrane (Millipore, Billerica, MA) using a semi-dry transfer apparatus (SEMIPHOR,
Hoefer Scientiﬁc Instruments, San Francisco, CA). Immunoblot analyses were performed
using a HopM1-speciﬁc antibody (Nomura et al., 2006) or a GFP-speciﬁc antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For estimating the sizes of proteins,
PageRulerTM Prestained Protein Ladder Plus (#SM1811, Fermentas International Inc,

Ontario, Canada) was used.

The color of the images in this dissertation

The images in this dissertation (chapter 2, chapter 3, chapter 4 and appendices)

are presented in color.

50

RESULTS

Transiently expressed full-length HopM1 fusion proteins induce tissue death

in tobacco leaves

Agrobacterium strains containing pBD derivatives designed to express the
HopM1-GFP or YFP-HopM1 fUSIOfI were hand-inﬁltrated into the leaves of Nicotiana
benthamiana or Nicotiana tabacum. After 48 hours, the transgenes were sprayed with 30
11M DEX. The inﬁltrated areas of tobacco leaves started losing turgor approximately 8
hours after DEX induction. Two days after DEX spray, the inﬁltrated area died. This was
similar to a previous report of tissue death induced by another TTSS effector of Pst
DC3000, Aer, in tobacco (Badel etal., 2006). To conﬁrm that the tissue death was
caused by the HopM1 fusion proteins, the transient assay was repeated with following
controls: Agrobacterium C58C1 alone, C58Cl carrying pBD vector, C58C1 carrying the
sGFP ORF in pBD, and C58C1 with the hole ORF in pTA7002 (another DEX-
inducible vector which carries a hygromicyn resistance gene [DebRoy et al., 2004;
Nomura et al., 2006]). These strains were hand-inﬁltrated into the leaves of Nicotiana
benthamiana or Nicotiana tabacum, and sprayed with 30 M DEX. Two days after DEX
spray, only the areas injected with Agrobacterium carrying pTA7002-hopM], pBD-
Hole-GFP or pBD-YFP-Hole showed tissue death. C58C1, C58C1 carrying pBD
vector and C58Cl carrying pBD-sGF P did not show tissue death, and the appearance of
the inﬁltrated areas was indistinguishable from the uninﬁltrated areas (Figure 2-2).

The fusion constructs of truncated hopMI were also transiently expressed in

tobacco leaves. In contrast to HopM1-GFP or YFP-HopM1, none of the truncated

51

     

uninﬁltrated pBD pBD-sGFP pTA7002-6mis-

HopM1

(B)

           

  

 

If; a
pBD-HopM1- pBD-YFP- ' pBD-HopM1-N— pBD-YFP ' pBD-YF-P
GFP HopM1 GFP HopM1-N HopM1-C

Figure 2-2. Appearance of Nbenthamiana leaves which were inﬁltrated with
Agrobacterium carrying various plasmids. (A) Nbenthamiana leaves which were
inﬁltrated with nothing, Agrobacterium C58Cl, C58Cl transformed with pBD vector,
C58Cl with sGF P ORF in pBD vector, C58Cl with 6xHis-Hole in pTA7002 (DebRoy
et al., 2004; Nomura et al., 2006). (B) Nbenthamiana leaves which were inﬁltrated with
Agrobacterium carrying pBD derivatives designed to express HopM1 (full-length or
truncated) fused with GFP or YFP ORFs under the DEX-inducible promoter. Black lines
in the photos of pTA7002-6xHis-HopM l , pBD-HopM1 -GF P and pBD-YFP-Hole
mark the Agrobacterium-inﬁltrated area. Leaves were sprayed with 30 11M DEX solution
and left at the room temperature for 48 hours prior to photography.

52

HopM1 ﬁJSlOI‘IS induced tissue death (Figure 2-28). Therefore, only full-length HopM1

or HopM1 fusions were able to induce tissue death in tobacco plants.

Western blot analyses of Hole fusions in transient assay

The expression of the fusion proteins of full-length HopM1 or truncated Holein
transient assays was analyzed by immunoblot. Several controls were included:
N.tabacum leaf sample without bacterial inﬁltration, pEGAD-eGFP for constitutive
expression of eGF P and pTA7002-6XHis-HopM1 for DEX-inducible expression of
Hole. Immunoblotting was performed with an anti-HopM1 antibody or anti-GF P
antibody (Figure 2-3). The results showed approximately expected sizes of each HopM1
fusion (HopM1-OF P: 102 kDa, YFP-HopM1: 102 kDa, HopM1-N-GFP: 59 kDa, YFP-
Hole-C: 72 kDa), except that the expression of YFP-Hole-N was too low to detect.
The gel lanes of HopM1-N-GFP (Figure 2-3B, lane 6) and YFP-Hole-N (Figure 2-3B,

lane 7) had faint bands of the approximate size of GFP detected by OF P antibody.

Transiently expressed fusions of full-length HopM1 are found in small,
punctate structures in tobacco cells
To determine the localization of full-length HopM1 in plant cells, HopM1-GP P and YFP-
Hole were transiently expressed in tobacco plants. After 8 hours of DEX induction (on
average), some leaf tissue began to lose turgor, leading to tissue death eventually. Based
on this observation, DEX induction time for preparing confocal microscope samples did
not exceed 6 hours. Confocal microscopy was performed between 4 and 6 hours after

DEX spraying. Both epidermal cells and mesophyll cells of tobacco plants were

53

Figure 2-3. Immunoblot analyses of HopM1 fusions in transient assays in N. tabacum,
using (A) the anti-HopM1 antibody, and (B) the anti-GFP antibody. M: protein size
markers. Lane 1 to 8 represent N. tabacum leaf samples inﬁltrated with Agrobacterium
containing the following plasmids: lane 1, leaf sample without bacterial inﬁltration; lane
2, pEGAD; lane 3, pTA7002-6xHis-HopM1; lane 4, pBD—HopM1 -GFP; lane 5, pBD-
YFP-Hole; lane 6, pBD-Hole-N-GFP; lane 7, pBD-YFP-Hole-N; lane 8, pBD-
YFP-Hole-C. All leaf samples were collected after 8 hour of DEX induction with 30
M of DEX solution. None of the leaf samples were observed by confocal microscopy to
conﬁrm the expression of proteins prior to sample preparation.

54

 

(A)
95?."
130

95
72

 

 

55

examined, but the majority of images were from epidermal cells.

Neither HopM1-GP P (Figure 2-4A and B) nor YFP-HopM1 (Figure 2-4C) was
found in the plasma membrane or large organelles such as the chloroplast, nucleus,
endoplasmic reticulum (ER), or vacuole. Instead, both HopM1-GP P (Figure 2-4A and B)
and YFP-HopM1 (Figure 2-4C) were found in small, punctate structures in the cell.
These punctate structures were not concentrated in speciﬁc areas of a cell, but they were
not evenly dispersed in the cell, either. These structures actively moved in the cells. The
images of HopM1-0F P and YFP-HopM1 looked similar, therefore the N-terrninal or C-
terrninal fusion of ﬂuorescence protein tagging did not seem to affect the localization of
HopM1 fusion proteins.

To determine the localization of HopM1 at the earliest detectable stage, the
condition of DEX-induction was modiﬁed in some experiments: 30 11M DEX solution
was dabbed onto the surface of tobacco leaves without surfactant Tween-20, and the
confocal microscopic observation was limited within 5 hours after DEX induction. The
ﬂuorescence from full-length HopM1 fusions could be observed 3 hours after DEX
application (Christy Mecey and Sheng Yang He, unpublished). F ull-length HopM1 ﬁision
proteins were again found in small, punctate structures as well, although the density of
punctate structures was lower than that observed with longer DEX treatment (Figure 2-
4D: Christy Mecey and Sheng Yang He, unpublished). These punctate structures were
more even in size and shape (Figure 2-4D), compared to the punctate structures observed
5 hours after DEX treatment (Figure 2-4A, B and C). According to the observations made
at different time points of DEX treatment, I conclude that the ﬂuorescence intensity and

signal density of the punctate structures associated with full-length HopM1 fusion

56

 

Figure 2-4. HopM1—GFP and YFP-HopM1 transiently expressed in N.tabacum. (A)
Hole-GFP expressed in epidermal cells. (B) HopM1-GFP expressed in mesophyll cells.
(C) and (D) YFP-HopM1 expressed in epidermal cells. Panels A. B and C were from the
images of the leaf samples which were sprayed with DEX solution containing 0.01%
Tween-20. Panel D was from the images of the leaf sample which was dabbed with DEX
solution without Tween-20. HopM1—associated punctate structures are yellow in C and
green in other panels (indicated punctuate with red arrows in D). All images are from
single focal planes. Ch: chloroplast. S: stomata.

57

proteins increased over time. The movement of punctate structures was almost stopped

approximately 7 hours after DEX application.

Dual localization tests of full-length HopM1 fusions with a Golgi marker with
or without Brefeldin A (BFA)

The localization of full-length HopM1 fusions further was investigated by dual
localization tests with different subcellular marker proteins that are associated with small
organelles. First, I examined the possibility that HopM1-GFP may be localized to the
Golgi apparatus. The rat sialyl transferase (Wee et al., 1998; Saint-Jore et al., 2002) fused
to RFP (ST-RF P: the gift from Dr. Federica Brandizzi laboratory, Michigan State
University, East Lansing, MI) was selected as a marker for this purpose. ST-RF P was
transiently co-expressed with HopM1-GP P or YFP-HopM1 in tobacco leaves, and
confocal microscopy analyses were performed both at early time points (within 5 hours)
and later time points (after 5 hours) after the DEX-induction. In both conditions, there
was no signiﬁcant co-localization of full-length HopM1 ﬁJsions (Figure 2-5). At later
time point, punctate structures associated with HopM1 fusion protein (HopM1-GP P) and
ST-RFP showed distinct sizes, densities and distributions in the cells (Figure 2-5).

Fungal toxin Brefeldin A (BF A) was also tested to further investigate the
relationship between the localization of HopM1-GFP and Golgi, based on the known
effects of BF A on disruption of Golgi stacks and fusion of Golgi into ER (Brandizzi et al.,
2002; Ritzenthaler et al., 2002; Saint-Jore et al., 2002). The BF A solution was applied to
the leaf samples 5-6 hours after the leaves were co-inﬁltrated with Agrobacterium strains

carrying pBD-Hole-GFP and/or pVKH-ST-RFP. As expected, ST-RFP-associated

58

Figure 2-5. Dual localization results of full-length HopM1 fusion proteins and ST-RFP.
The ﬂuorescence from HopM1-GFP (A), ST-RFP (B) and their merged image (C) within
5 hours after DEX treatment; the ﬂuorescence from YFP-HopM1 (D), ST-RFP (E) and
their merged image (F) 5 hours after DEX treatment. Images A, B and C were from the
leaf sample which was dabbed with DEX solution without Tween-20. Image D, E and F
were from the leaf sample which was sprayed with DEX solution containing 0.01%
Tween-20. HopM1-associated punctate structures are green (A, C, D and F) and ST-RFP
are red (B, C, E and F). YFP-HopM1 in panel A and ST-RFP in panel B are indicated
with red arrows and white arrows, respectively. A, B and C are from Z-stack, and D, E
and F are from single focal planes of the epidermal cell layer of N.tabacum, respectively

59

I—-—I

l [I 1-1m

’l [1 tJI'I‘I

 

60

punctate structures disappeared and ﬂuorescence was changed into a mesh-like network
(Figure 2-6), reﬂecting the re-distribution of ST-RFP into the ER by BFA (Brandizzi et
al., 2002; Saint-Jore et al., 2002). Contrary to this change, HopM1-GFP did not disappear,
and there was no signiﬁcant morphological change of the punctate structures associated

with HopM1-GP P (Figure 2-6).

Dual localization tests of YFP-HopM1 and endosome markers

Besides Golgi, endosomal compartments were selected for determining the
localization of HopM1 fusions. Endosome is a collection of small, vesicular organelles
the main function of which is to transport and sort cargo materials between Golgi,
vacuole and plasma membrane (Jiirgens and Geldner, 2002; Bassham et al., 2008:
Robinson et al., 2008). The endosomes are shown as small, punctate structures (Ueda et
al., 2001: Ueda et al., 2004: daSilva et al., 2006; Dettmer et al., 2006). In eukaryotic cells
several different types of endosomes exist: they are different in structure, function and
biochemical composition (Bassham et al., 2008). There are three known subgroups of
endosomes (Robinson et al., 2008; Otegui and Spitzer, 2008): early endosome, late
endosome and recycling endosome. Early endosomes are produced from endocytosis of
the plasma membrane. In plants, trans-Golgi network (TGN) is considered to be early
endosomes (Dettmer et al., 2006: Lam et al., 2007). Late endosomes function in the
vesicle trafﬁcking from early endosomes to the vacuole, and include distinctive structures
such as multivesicular bodies (MVB) or prevacuolar compartment (PVC) (Otegui and
Spitzer, 2008: Robinson et al., 2008). Recycling endosomes are thought to function from

early and late endosomes to the plasma membrane (Otegui and Spitzer, 2008: Robinson

61

Ch

 

Figure 2-6. Dual localization result of HopM1-GFP and ST-RFP with BFA treatment.
HopM1-GFP (A), ST-RF P (B) and their merged image (C) are shown. The induction of
HopM1-GFP was by spraying of DEX solution with 0.01% of Tween-20. All images are
from single focal planes of the epidermal cell layer of N. tabacum, 5-6 hours after DEX
treatment. Ch: chloroplast.

62

et al., 2008). Currently known endosome markers area available both for different types
of endosomes and/or for different portions of an endosome subgroup (Otegui and Spitzer,
2008: Robert et al., 2008; Robinson et al., 2008).

VHA-al, a marker for TGN, was selected ﬁrst for dual localization with HopM1
fusion proteins. Tanaka et a1 (2009) recently showed that AtMIN 7, a member of
Arabidopsis ARF-GEF family and a cellular target of HopM1 (Nomura et al., 2006), is
co-localized with VHA-al. Moreover, like HopM1-GP P observed in this study, the
localization of VHA-al-GF P does not show signiﬁcant change upon BF A treatment,
although some aggregation was noted (Dettmer et al., 2006). YFP-HopM1 and VHA-al-
RFP (Dettmer et al., 2006) were transiently co-expressed in tobacco leaves, and the
expression of YFP-HopM1 was induced by DEX for 3 or 4 hours. The confocal
microscopy result showed that YFP-HopM1 and VHA-al -RFP were largely co-localized
(Figure 2-7: Christy Mecey and Sheng Yang He, unpublished). Next, Arabidopsis RabF l
(ARA6), another early endosome marker (Ueda et al., 2001; Kotzer et al., 2004; Haas et
al., 2007), was selected in co-localization study. The confocal microscopic observation
was performed at early time points (before 5 hours) and later time points (after 5 hours)
after the DEX-induction of YFP-HopM1. YFP-HopM1 and ARA6-CF P were co-
localized at earlier time points (Figure 2-8: Christy Mecey and Sheng Yang He,

unpublished), but they did not co-localize at later time point (Figure 2-8).

Localization of truncated HopM1 fusion proteins

Next, the localization of truncated HopM1 protiens fused to GFP or YF P was

studied: the truncated HopM1 proteins included HopM1-N-GFP, YFP-Hole-N, and

63

 

Figure 2-7. Dual localization results of YFP-HopM1 and VHA-al-RFP. The ﬂuorescence
from YFP-HopM1 (indicated with red arrows) (A), VHA-al -RFP (indicated with white
arrows) (B) and their merged image (co-localized punctate structures are marked with
blue arrows) (C) within 5 hours after dabbing DEX solution on the leaf surface. All of the
images are from Z-stack, from the epidermal cell layer of N.tabacum.

64

Figure 2-8. Dual localization results of YFP-HopM1 and ARA6-CF P. The ﬂuorescence
from YFP-HopM1 (A), ARA6-CF P (B) and their merged image (C) within 5 hours after
DEX treatment; the ﬂuorescence from YFP-HopM1 (D), ARA6-CF P (E) and their
merged image (F) 5 hours after DEX treatment. Images A, B and C were from the leaf
sample which was dabbed with DEX solution without Tween-20. Image D, E and F were
from the leaf sample which was sprayed with DEX solution containing 0.01% Tween-20.
YFP—Hole-associated punctate structures are yellow (A, C, D and F) and ARA6-CFP
are blue (B, C, E and F). YFP-HopM1 in panel A and ARA6-CFP in panel B are
indicated with green arrows and red arrows, respectively. Co-localized YFP-HopM1 and
ARA6-CFP are marked with blue arrows (C).All images are from single focal planes of
the epidermal cell layer of N. tabacum.

65

 

YFP-Hole-C (HopM1-C-GF P was excluded from localization study due to mutations
introduced during cloning). The confocal microscopic observations were performed at
both early time point (within 5 hours) and later time point (between 5 and 6 hours) after
DEX-induction. At early time point, HopM1-N-GF P and YFP-Hole-N were found in
small, punctate structures, which were similar to those observed with full-length HopM1
fusion proteins. YFP-Hole-C was shown in unidentiﬁed structures which were round
with uneven size (Figure 2-9). In later time point, however, all truncated HopM1 ﬁJsion
proteins were found dispersed in the cells, instead of punctate structures (Figure 2-9).
The localization of the N-terminus of HopM1 was further studied. The shape and
size of the punctate structures of YFP-Hole-N at the early time point were similar to
those of full-length HopM1 fusion proteins. This result suggested that YFP-Hole-N
might be localized in the same subcellular structures in which full-length HopM1 ﬁisions
are localized. To test this possibility, YFP-HopM 1 -N was co-expressed with VHA-al-
RFP into tobacco leaves, which were examined by confocal microscopy. As shown in
Figure 2-10, YFP-Hole-N and VHA-al -RFP co-localized, indicating that YFP-
Hole-N is localized in TGN. This result provides evidence that the N-terminal 300

amino acids of HopM1 contain all the information for localization to TGN.

Transgenic expression of HopM1 fusions in Arabidopsis

HopM1 fusions which were constructed and examined by confocal microscopy in
transient assays were transformed into Arabidopsis by ﬂoral dipping (Clough and Bent,
1998), to establish stable transgenic Arabidopsis plants. The T. plants of each HopM1

fusion were sprayed with BASTA solution. Surviving plants were obtained from T1 seeds

67

Figure 2-9. Localization of truncated HopM1 fusion proteins. The ﬂuorescence from (A)
HopM1-N—GFP at early time point (marked with red aqrrows); (B) YFP-Hole-C at
early time point (marked with blue arrows); (C) HopM1-N-GFP at later time point; (D)
YFP-Hole-N at later time point; (B) YFP-HopMI-C at later timer point. All images are
from single focal planes. Panels A and B are from the leaf samples which were dabbed
with DEX solution and examined within 5 hours. Panels C, D, and E are from the leaf
samples which were sprayed with DEX solution with 0.01% Tween-20 and observed 5
hours after DEX induction. S: Stomate. Chzchloroplast. G: guard cell.

68

(A) (B)

 

69

Figure 2-10. Dual localization results of YFP-Hole-N and VHA-al —RFP. The
ﬂuorescence from YFP-Hole-N (indicated with white arrows) (A), VHA-al-RFP
(indicated with green arrows) (B) and their merged image (co-localized punctate
structures are marked with blue arrows) (C) within 5 hours after dabbing DEX solution
on the leaf surface. All of the images are from Z-stack, from the epidermal cell layer of
N.tabacum.

70

 

71

for four constructs in Col-0 background (Table 2-4). One leaf of each plant was detached,
dipped in DEX solution, and examined by confocal microscopy after 24 hours (Table 2-4;
Figure 2-1 lA-D). F ull-length HopM1 fusions were found in punctate structures, and
truncated HopM1 fusion proteins were shown dispersed in the cell (Figure 2-11A-D).
These localization patterns were consistent with those in transient assay conducted at
later time point after DEX treatment (Figure 2-4 and 2-9). The HopM1 constructs were
transformed into the atmin7 mutant, and the T1 plants transformed with YFP-HopM1 and
YFP-Hole-N were obtained. YFP-HopM1 in the atmin7 background was examined by
confocal microscopy (Figure 2-11E); it was shown in small, punctate structures, which

were similar to those in Col-0 background.

72

Table 2-4. Summary of transgenic Arabidopsis expressing HopM1 fusions.

 

HopM1 fusion name Arabidopsis background Line number

 

HopM1-GFP Col-0 #I',l I, 12

YFP-HopM1 Col-O #2‘,3,6

HopM1-N-GFP Col-O #113

YFP-HopMI-N Col-0 #4",10

YFP-HopM1 atmin7 #3"

YFP-Hole-N atmin7 #100, 101,102, 103,104

 

"' indicates the transgenic lines which were examined by confocal microscopy (Figure 2-
11)

73

Figure 2-11. HopM1 fusion proteins in Arabidopsis (C ol-O and atmin7 background). The
ﬂuorescence of (A) HopM1-GFP in Col-0 (1ine#1), (B) YFP-HopM1 in Col-0 (line#2),
(C) HopM1-N-GFP in Col-0 (1ine#1), (D) YFP-Hole-N in Col-0 (line#4), and (E)
YFP-HopM1 in atmin7 (line#3). All images are from single focal planes of leaf samples
except A, which was from z-stack. Leaf samples were dipped in DEX solution for 24
hours prior to confocal microscopy.

74

 

75

DISCUSSION

To determine the subcellular localization of HopM1 in plant cells, full-length and
truncated versions of HopM1 were fused with selected ﬂuorescence protein tags (sGFP
and EYFP), and they were analyzed using transient expression assays in tobacco leaves
and stable expression in Arabidopsis plants. All HopM1 fusions were induced by DEX.
Under this condition, the expression of all HopM1 fusions can be regulated based on the
induction time and DEX concentration.

When full-length HopM1 ﬁlSlOI’lS (HopM1-GP P and YFP-HopM1) were
transiently expressed in tobacco, the leaf tissues began to lose turgor approximately 8
hours after DEX induction, and were dead 48 hours after DEX induction. Tissue death
was also induced by 6xHis-Hole expressed from pTA7002-6xHis-Hole. The tissue
death induced by HopM1 in Arabidopsis was previously reported (DebRoy et al., 2004;
Nomura et al., 2006), but the tissue death induction by HopM1 (and HopM1 fusions) in a
nonhost plant (tobacco) has not been reported before this study. The characteristics of the
tissue death in tobacco plants induced by 6xHis-Hole, HopM1-GFP and YFP-HopM1
are not clearly deﬁned in this study. Badel et al. (2006) showed that another TTSS
effector, Aer, induced tissue death in N.tabacum. Aer-induced tissue death was
interpreted as a kind of nonhost hypersensitive response (HR), but experiments for
determining the molecular characteristics of the tissue death were not performed (Badel
et al., 2006). Considering that Aer and HopM1 are both encoded in the CEL of Pst
DC3000 and that they have redundancy in function (DebRoy et al., 2004), it is possible

that the tissue death induced by Aer and HopM1 (or HopM1 fusion proteins) share the

76

similar mechanism in tobacco plants. The tissue death caused by DEX-induced HopM1-
GFP or YFP-HopM1 limited the DEX-induction time for confocal microscopy and
protein sampling for immunoblot analyses. In this thesis, the confocal microscopy was
performed within 6 hours after DEX spray, and protein sampling for western blot analysis
was done 8 hours after DEX induction.

Confocal microscopy in this study was performed at two different time points: an
early time point which was between 3 hours and 4 hours after DEX-induction, and a later
time point which was between 4 hours and 6 hours after DEX-induction. In the
experiments at later time point, the DEX solution was sprayed onto the leaf surface, and
the surfactant Tween 20 (0.01%) was added for enhanced uptake of DEX. Under this
condition, the GF P or YF P signal of full-length or truncated HopM1 fusion proteins was
intense, clearly distinguishable from the background. The density of the ﬂuorescence of
full-length HopM1 fusion was high as well. Because it is generally believed that bacteria
deliver only small amounts of effectors into host cells, it would be ideal to express
effectors at the lowest possible level that still allow microscopic observation. Therefore,
in some experiments, the observation was performed between 3 hours and 5 hours after
DEX application, and the DEX was dabbed onto the leaf surface without Tween-20.
Under this condition the intensity and density of the GF P/Y F P ﬂuorescence were reduced
compared to those obtained at later time point, but they were distinguished from the
background signal.

Full-length HopM1 fUSlOl'l proteins were found in small, punctate structures both
in early and later time points. However, in later time point the density and intensity of

GFP/YFP signal were higher than those in earlier time point. It is likely that, within the

77

time periods used in this study, the expression of full-length HopM1 fUSlOl‘l proteins is
proportional to the DEX exposure time. The difference in DEX concentration between
dabbing and spraying (with or without Tween-20, respectively) also could affect the
expression of HopM1 fusions.

The dual localization experiments show that YFP-HopM1 co-localizes with
VHA-al-RF P (a marker for TGN) and ARA6-CFP (a marker for early endosome). The
localization of HopM1 in TGN is similar to that of AtMIN7 (Tanaka et al., 2009),
consistent with the previous report that HopM1 interacts with AtMIN7 (Nomura et al.,
2006). This result also is consistent with the notion that HopM1 targets and interferes
with vesicle trafﬁcking to suppress host defenses. On the other hand, HopM1-GP P did
not co-localize with ST-RFP (a Golgi marker) and did not relocalize after BF A treatment
as ST-RF P did. These results indicate that HopM1 does not target Golgi apparatus for its
function. Finally, the dual localization assay of HopM1-GFP and PTSl-dsRED2 (a
marker for peroxisome: Gould et al., 1989; Reumann, 2004; Fan et al., 2005) showed that
HopM1 is not localized to the peroxisome (Appendix 2-1).

Besides full-length HopM1 fusions, the localization of truncated HopM1 fusions
(HopM1-N-GF P, YFP-Hole-N and YFP-Hole-C) was also studied. These truncated
HopM1 fusion proteins did not induce tissue death in tobacco plants, which is correlated
with a lack of virulence function of HopM1 1-300 and HopM1 301-712 in Arabidopsis. The
localization of truncated HopM1 fusion proteins provides interesting information. At
early time point of DEX treatment, the N-terminus (YFP-Hole-N) was localized in
small, punctate structures, whereas the C-terminus (YFP-Hole-C) was found in bigger,

irregular-sized structures. YFP-Hole-N co-localized with VHA-al-RFP and ARA-

78

CF P, indicating that the N-terminus of HopM1 is sufﬁcient for the localization to the
same endosomal compartments in which full-length HopM1 fusion protein is localized.
In a previous study to determine the intramolecular regions important for the virulence
function of HopM1, transgenic overexpression of the same N-terminus of HopM1 had a
dominant-negative effect on full-length HopM1 delivered from bacteria during infection,
suppressing the symptom development and multiplication of the ACEL mutant
complemented with a HopM1-expressing plasmid (Nomura et al., 2006). It was suggested
that the N-terminus of HopM1 might function as an independent domain, interfering with
the virulence function of the full-length HopM1 in plant cells (Nomura et al., 2006). The
same localization of the N-terminus of HopM1 and full-length HopM1 in the endosomal
compartments suggests that the dominant-negative effect may be caused in part by
competition for the same localization between the overexpressed, nonﬁmctional N-
terrninus of HopM1 and full-length, functional HopM1.

At later time point of DEX- treatment, the intensity and density of the
ﬂuorescence signal of full-length HopM1 fusion proteins increased, and the shape of the
punctate structures of hill-length HopM1 proteins became more irregular compared with
that at early time point of DEX treatment. Dual localization with a Golgi marker suggests
that full length HopM1 was not in the Golgi apparatus at later time points, but the precise
location was not precisely determined. It is possible that increased expression of full-
length HopM1 fusion proteins led to mislocalization to additional small organelles.
Alternatively, the high-density punctate structures at the later time point were caused by
the virulence action of full-length HopM1 in plant cells. In this case, by degrading

AtMIN7, HopM1 might have induced abnormal structures from endosomal

79

compartments. The punctate structures were found both in the transient expression assays
and in transgenic Arabidopsis plants, suggesting that the effect of HopM1 upon the
endomembrane structures is similar in nonhost and host plants. This putative distortion of
endomembrane structures could be related to the suppression of basal defense of
Arabidopsis by HopM1 (DebRoy et al., 2004; Nomura et al., 2006).

The different localization patterns between early and later time points were more
obvious for the fusion proteins of truncated HopM1: at later time point both the N-
tenninus and C-terminus of HopM1 were dispersed in the cell. The dispersed localization
of truncated HopM1 fusion proteins is similar to that of GFP alone (Appendix 2-2), but
this diffused localization is not likely caused by fortuitous degradation of fusion proteins,
which could generate a free GFP or YFP tag: western blot analysis did not indicate a
signiﬁcant amount of GFP or YFP. Thus, it seems that the localization of the N-terminus
of HopM1 in the endosomal compartments is transient, and that neither the N-terminus
nor the C-terminus of HopM1 is sufﬁcient for the long-terrn localization in the endosome.

The HopM1 fUSIOflS were transformed into Arabidopsis (Col-0 and atmin 7) for
establishing transgenic Arabidopsis plants. DEX-induction time was longer in
Arabidopsis (up to 24 hours) than in tobacco, because the expression of Hole fusions
was slower in Arabidopsis: for example, confocal microscopic examination of transgenic
plants expressing HopM1-GFP in Col-0 showed that, between 4-6 hours after DEX
induction, full-length HopM1 fusion was not detected. At least 8 hours were needed for
detecting ﬂuorescence above background, and at 24 hours after DEX induction the
ﬂuorescence was clearer. The localization patterns of various HopM1 fusions in

transgenic Arabidopsis were consistent with those in transient assays with tobacco at later

80

time point of DEX treatment: HopM1-OF P and YFP-HopM1 were associated with small,
punctate structures, whereas HopM1-N-GFP and YFP-Hole-N were dispersed in
Arabidopsis cells.

The transgenic Arabidopsis plants expressing full-length or truncated HopM1
fusion proteins need to be further characterized. First, it would be important to conﬁrm
tissue death of transgenic Arabidopsis plants expressing HopM1-GFP or YFP-HopM1
after DEX induction: this experiment is needed to determine whether full-length HopM1
fusion proteins act like untagged HopM1, which induces tissue death in Arabidopsis
(Nomura et al., 2006). Second, the localization of the HopM1 fusion proteins should be
determined at early time point and with a reduced DEX concentration to avoid potential

problems associated with overexpression of fusion proteins.

81

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85

CHAPTER 3

Studies of the roles of several Arabidopsis ARF-GEFs in the defense

against bacteria and their interaction with HopM1

Kinya Nomura contributed to Figure 3-9.

86

ACKNOWLEDMENTS

I appreciate the help of Dr. Kinya Nomura, who contributed to the Figure 3-9, and

gave me numemous and valuable advices during my Ph.D.

87

ABSTRACT

AtMIN 7, an adenosine-diphosphate ribosylation factor-guanine nucleotide-
exchange factors (ARF-GEF) gene of Arabidopsis, has a role in defense against the
ACEL mutant of Pst DC3000. AtMIN7 interacts with N-terminus of HopM1 (HopM1 1-
300) and it is degraded by full-length HopM1. In addition to AtMIN 7, there are seven more
ARF-GEF genes in Arabidopsis. To determine whether any of these genes have a role in
defense, the multiplication of the ACEL mutant was quantiﬁed in the T-DNA inserton
lines, and the yeast two-hybrid system was used for testing the interactions between
HopM1 and these ARF-GEFs.

I was able to obtain T-DNA insertion lines for three of the seven ARF-GEF genes,
BIGZ, BIG3 and 8104. The T-DNA insertion mutants of 3162 (salk_033446) and 3104
(salk_082249) were knockouts, and the BIG3 (salk_044617) insertion line was a knock-
down. Unlike atmin7 plants the multiplication of the ACEL mutant in these T-DNA
insertion mutants was not signiﬁcantly higher than wild type Arabidopsis (Col-O),
suggesting that none of the three genes have signiﬁcant role in the defense against the
ACEL mutant as AtMIN 7 did.

The C-terrninal portions of four ARF-GEFs (BIGl, BIG3, GNLI and GNL2)
were studied by yeast two-hybrid assay to determine whether they interacted with
HopM1 1.300_ The C-terminus of GNL2 did not interact with HopM1 (-300 The interactions
between HopM] (-300 and the C-termini of BIG], BIG3 and GNLl were not determined

because of the failure to express the proteins in yeast.

88

INTRODUCTION

Previous research results showed that twenty one Arabidopsis proteins interact
with the ﬁrst 300 amino acids of the TTSS effector HopM1 from the bacterial pathogen
Pseudomonas syringae pv. tomato (Pst) DC3000. These proteins were named AtMINs
(Arabidopsis thaliana HopM1 interactors). At least eight AtMINs were destabilized by
full-length HopM1 in plants and in yeast. Among them, AtMIN 7 was shown to be
important for Arabidopsis defense against the ACEL mutant of Pst DC3000 (Nomura et
al., 2006). In the ACEL mutant the conserved effector locus (CEL), encoding HopM] and
three other effectors, is deleted. As a result, the ACEL mutant does not cause symptoms
and its multiplication level is reduced in host plants (Alfano et al., 2000; Badel et al.,
2003; DebRoy et al., 2004). In the atmin7 plants, however, the multiplication of the
ACEL mutant is increased and the symptom development is partially restored (Nomura et
al., 2006). In addition, the atmin7 mutant the ACEL mutant is compromised in callose
deposition (a defense response) after the inoculation of the ACEL mutant compared with
wild-type Col-O plants (Nomura et al., 2006).

AtMIN 7 is one of the eight members of Arabidopsis gene family encoding
adenosine-diphosphate ribosylation factor-guanine nucleotide-exchange factors (ARF-
GEFs: Anders and Jiirgens, 2008; Figure 3-1). ARF-GEFs regulate adenosine-
diphosphate ribosylation factor (ARF) GTPase by promoting nucleotide exchange from
its inactive GDP-bound state to the active GTP-bound state. It has been shown that ARF-
GTPases function in vesicle budding necessary for intracellular vesicle trafﬁcking, and

ARF-GEF-dependent activation of ARF is critical for promoting the vesicle formation

89

At1901960
At3960860
4114938200
At4g35380
At3943300

Att 913980
{415939500

At5919610

 

rm

 

 

 

 

 

(BIG3)
(BIGZ)

(BIG1)

(BIG4)

(AtMIN7; BIG5)
(GNOM; EMB30; GBF3)

(GNL1; GBF1)

(GNL2; GBFZ)

Figure 3-1. A phylogenetic tree of Arabidopsis ARF-GEF genes with their names in

parenthesis (modiﬁed from Nomura et al., 2006).

90

and budding in trafficking pathways (Memon, 2004; Gillingham and Munro, 2007;
Casanova, 2007; Anders and J iirgens, 2008; Bassham et al., 2008). ARF-GEF proteins
share a conserved Sec7 domain that is sufﬁcient to catalyze GTP/GDP exchange on
ARFs (Chardin et al., 1996; Jackson and Casanova, 2000; Gillingham and Munro, 2007).
The Sec7 domains of some ARF-GEFs are sensitive to a fungal toxin Brefeldin A (BF A).
BFA binds to the ARF-GDP complex and interferes with the exchange of GTP for GDP
(Anders and Jiirgens, 2008).

The eight ARF-GEF genes including AtMIN 7 are grouped into two subfamilies:
Sec7/BIG-type and Gea/GNOM/GBF-type (Cox et al., 2004; Gillingham and Munro,
2007).AtM1N7 is in the Sec7/BIG-type subfamily. The Arabidopsis ARF-GEF genes are
closely clustered in a larger phylogenetic tree containing all ARF-GEF genes, and their
Sec7 domains show highly similarity (Cox et al., 2004). Currently, AtMIN 7 is the only
ARF-GEF gene which has been studied in plant-pathogen interactions. Two other ARF-
GEF genes have been characterized in growth and development ofArabidopsis: GNOM
(Steinmann et al., 1999; Geldner et al., 2003), and GNOM-LIKE 1 (GNLl: Richter et al.,
2007; Teh and Moore, 2007). GNOM is found in endosome/prevacuolar compartments
(PVCs) of the Arabidopsis cells, and it is involved in the polarized targeting of PIN ]
which is critical for polar auxin transport (Steinmann et al., 1999; Geldner et al., 2003).
GNLI is detected in the Golgi stack and has overlapping cellular functions with GNOM
(Richter et al., 2007; Teh and Moore, 2007).

Although the multiplication of the ACEL mutant is higher in atmin7 mutant plants
compared with that in wild-type Col-0 plants, it does not reach the level of Pst DC3000

grth in Arabidopsis, suggesting that AtMIN7 is not the only host target of HopM1

9l

(Nomura et al., 2006). Second, in preliminary experiments the product of another ARF-
GEF encoding gene (AtBIGZ [Cox et al., 2004]) had a weak yeast two-hybrid interaction
with HopM1 (Kinya Nomura and Sheng Yang He, unpublished). Third, BFA treatment of
Arabidopsis Col-O leaves restored the multiplication of the ACEL mutant to the level of
Pst DC3000, indicating that there are other host targets of HopM1 which are BFA-
sensitive, possibly including additional ARF-GEFs (Nomura et al., 2006). These
observations suggest that other ARF-GEFs may also have a role in the defense of
Arabidopsis and their products may be targeted by HopM1.

Nomura et al. (2006) tested the C-termini of three ARF-GEF genes (BIGZ, BIG4
and GNOM) in yeast two-hybrid system with HopM11-300; none of them showed an
interaction with HopM11_3oo. However, not all Arabidopsis ARF-GEF genes were tested.
Additionally, no study has been reported to directly determine the role of individual ARF-
GEF genes in Arabidopsis defense against the ACEL mutant.

In this chapter, Arabidopsis ARF-GEF genes that had not been previously studied
were analyzed with two approaches. First, to determine a possible role of other ARF-GEF
genes in Arabidopsis defense against the ACEL mutant, the grth of the ACEL mutant
in the T-DNA insertion mutants of ARF -GEF genes were compared with that of atmin7
plants. Second, yeast two-hybrid assays were performed to examine possible physical

interactions between these ARF-GEFs with HopM1 1-300.

92

MATERIALS AND METHODS

Identiﬁcation of homozygous T-DN A insertion mutants for Arabidopsis ARF-

GEF genes

The T-DNA insertion mutant of AtMIN 7 (salk_O l 2013) was previously
characterized by Nomura et al. (2006) and it was used in this chapter. The genomic
sequences and putative T-DNA insertions of the ARF-GEF mutants were examined in
The Arabidopsis Information Resource (TAIR: wwwarabidopsisorg), and the mutants
with putative T-DNAs located in exons were selected (Table 3-1). The seeds of the
selected T-DNA insertion mutants were purchased from Arabidopsis Biological Resource
Center (ABRC, The Ohio State University, Columbus, OH).

Plants were grown in soil for 4-5 weeks. Genomic DNA was extracted from leaf
tissue of each plant and prepared following the protocol provided with the SIGMA
REDExtract-N-AmpTM Plant PCR kit (Sigma-Aldrich, St. Louis, MO). The primers for
genomic PCR are summarized in Table 3-2. For each T-DNA insertion mutant, two pairs
of primers were used. One pair consists of two gene-speciﬁc primers that are before and
after the putative insertion site of T-DNA. The second primer set includes a gene-speciﬁc
primer and the primer designed from the left border of the T-DNA (5’-
GACCGCTTGCTGCAACTCTCTC-3’). The locations of the primers in each ARF-GEF
gene sequence are shown in the appendix (Appendix 3-1 to 3-7). Ten microliters of each
PCR product was run on a 1% agarose gel containing SYBR® Safe DNA gel stain
(1:10,000 dilution: Invitrogen, Carlsbad, CA). Gels were photographed with Quantity

One software (Bio-Rad Laboratories, Hercules, CA), and the images were processed with

93

Table 3-1. The Arabidopsis ARF-GEF genes, their selected T-DNA insertion mutants,
and the putative insertion site of T-DNA of each mutant.

 

 

Arabidopsis ARF-GEF gene T-DNA insertion mutants Insertion site
At4g3 8200 (BIGI) salk_066766 lst exon
At3g60860 (BIGZ) salk_033446 8th exon
At] g01960 (BIG3) salk_0446l7 7th exon
At4g35380 (BIG4) salk_082249 8th exon
At1g13980 (GNOM;EMB30;GBF 3) salk_103014 1stexon
At5g39500 (GNLI ;GBFI) salk_067415 lst exon
At5gl9610 (GNL2;GBF 2) salk_021757 3rd exon

 

94

Table 3-2. Primer sets designed for screening homozygous T-DNA insertion mutants for

 

 

ARF-GEF genes.

ARF-GEF T-DNA Primer sets

gene mutants

At] g01960 salk_0446l 7 Forward primer:

(BIG3) 5’-GGAGGAAGAAGGAAACTATCTACAAGATGC-3’
Reverse primer:
5’-GTTATAATTCGCCAACTCTI‘CCCGCTC-3’

At3g60860 salk_03 3446 Forward primer:

(BIGZ) 5’-GTCATTGTTATGCGTAGAAGTAATGATGTTGAGAT-
3’

Reverse primer:
5’-GCGTAAGGTATCAAACATAATCTGTAGTGC-3’

At4g3 8200 salk_066766 Forward primer:

(BIG!) 5'-CGTGAGAAGTCGAATATGCGTTAGATGTC -3'
Reverse primer:
5'-GAGCATGATCTGAGCCAGCACAGA IT] A -3'

At4g35380 salk_082249 Forward primer:

(3104) 5’- CTGTTGCAATAT’ITGTCATGGACTCGCTT-3’
Reverse primer:

5’- CTGGGAGAATGCTAGAGCTGAAGATTCCA-3 ’

At1g13980 salk_103014 Forward primer:

(GNOM'EMB30; 5’-GGAGGTCGATACATGTCTGGTGATGATC-3’

GBF 3) Reverse primer:
5’-CAGAGCCATACGGAGTCCCAAGTATG-3’

At5g39500 salk_067415 Forward primer:

(GNL];GBF1) 5 ’-GAATCATCCTTCGGGAAGTAACTCGTTCC-3 ’
Reverse primer:
5’-CTCATGCATTGTGTGGCGAGCTATAC -3’

At5gl9610 salk_021757 Forward primer:

(GNL2;GBF2) 5'-GCATTATCTAACGTCCACCG-3'

Reverse primer:
5'-GGAGACTAATGTGGTATGTGG-3'

 

95

Adobe Photoshop Element version 5.5 or 7.0.

Conﬁrmation of gene knockout of T-DNA insertion mutants by reverse
transcription and polymerase chain reaction (RT-PCR)

Homozygous T-DNA insertion mutants identiﬁed by PCR (salk_033446,
salk_0446l7, and salk_082249) were analyzed by RT-PCR to conﬁrm the loss of
transcript. Total RNA sample of each salk line was extracted from 100 mg of Arabidopsis
leaf tissue with the RNeasy® Plant Mini Kit (QIAGEN, Valencia, CA), followed by
genomic DNA removal using RQl RNase-free DNaseI (Promega, Madison, WI). RNA
was quantiﬁed with a NanoDrop ND-IOOO Spectrophotometer (NanoDrop, Wilmington,
DE).

RT-PCR was performed using the RNA LA PCR Kit (AMV), Ver. 1.1 (TaKaRa,
Japan). The reverse transcription reaction mixture was prepared following the modiﬁed
protocol of RNA LA PCR Kit (AMV), Ver. 1.1 (5mM MgClz, I X RNA PCR Buffer, 1
mM dNTP mixture, 1 unit/pl RNase Inhibitor, 0.25 units/u] AMV Reverse Transcriptase,
0.125 uM Oligo-dT Adaptor Primer, RNase-free water and 200-300 ng total RNA). The
reverse transcription reaction was performed in a total volume of 50 u], and incubated for
30 minutes at 45°C, followed by 5 minutes at 99°C and 5 minutes at 5°C. This cDNA
sample was used as template in the PCR reaction with gene-speciﬁc primer pairs (Table
3-3). The sequences of these primers were chosen after the putative T-DNA insertion
sites (Appendix 3-1 to 3-7). Each primer was checked by Basic Local Alignment Search
Tool (BLAST: http://blast.ncbi.nlm.nih.gov/blast.cg), to determine whether the primer

shares sequence identity with an unrelated area of the Arabidopsis genome. Only the

96

Table 3-3. Primers for RT-PCR for T-DNA insertion mutants of selected ARF-GEF
genes.

 

Primer Name Sequence

 

Forward primer for Actin8 (ACT 8, 5’- CTACATITGCTCCCTCTGTGC-3’
At1g49240)

Reverse primer for Actin8 (AC T 8, 5’- AGGAATGACCTGTGACGAGTG-3’
At] g49240)

Forward primer E for salk_044617 5’- GTTGACAATGTCAAGTCGGGATGGAAGAG -3’
(BIG3, Atl g01960)

Reverse primer E for salk_044617 5’ -CGTATGAGAAGTCAGGGGATGTAGCA -3’
(BIG3, At] g01960)

Forward primer H for salk_082249 5’-GCGTTGCATAGAAGTATTGTTCCACATTCTG-3’
(3104, At4g35380)

Reverse primer H for salk_082249 5’-GTAACCATGTCTTGGAGGACATCATGTAGG-3’
(BIG4, At4g35380)

Forward primer D3 for 5’-CGCCTGGCTCTACGAGACCT-3’
salk_033446 (BIGZ, At3g60860)

Reverse primer D3 for 5’-CTTGCATCTGTGTCATGGGTCCTAGC-3’
salk_03 3446 (8102, At3g60860)

 

97

primers which showed identity only with the selected ARF-GEF gene were used for RT-
PCR. The locations of the selected primers in each ARF-GEF gene sequence are shown
in the appendix. RT-PCR was performed using Arabidopsis ACT8 gene-speciﬁc primers
as a control (Table 3-3).

The second PCR reaction contained 2.5 mM MgC12, l X LA PCR Buffer II, 0.2

pM of each primer, sterilized distilled water and 10 pl of the previous reverse

transcription reaction sample in a ﬁnal volume of 50 pl. The reaction was performed as
follows: 94°C, 2 minutes (1 cycle), 94°C, 30 seconds, 53°C, 30 seconds, 72°C, 1 -2
minutes (25 cycles, 30 cycles, 40 cycles or 50 cycles) and 72°C, 10 minute (1 cycle). Ten
pl of the PCR reactions were loaded on a 1% agarose gel containing SYBR® Safe DNA
gel stain (1:10,000 dilution: Invitrogen, Carlsbad, CA). Gels were photographed with
Quantity One software (Bio-Rad Laboratories, Hercules, CA), and the images were

processed with Adobe Photoshop Element version 5.5 or 7.0.

Construction of ARF-GEF gene clones for yeast two-hybrid assays

Four ARF-GEF genes (BIGI, BIG3, GNL] and GNL2) were cloned for the yeast
two-hybrid assay. The 3’ ends downstream of the putative Sec7 domains were selected
for cloning, following the cloning procedure of the 3’ end of AtMIN 7 (Nomura et al.,
2006): the sequences of the ORFs of four selected ARF-GEF genes were aligned with
other Arabidopsis ARF-GEF genes by CLUSTALW (http://align.genome.jp/), and the
putative Sec7 domain and the C-terminus after the Sec7 domain was determined in each
ORF. The putative amino acid sequence of the C-terminus of each ARF-GEF is in Figure

3-2. The 3’ends were obtained by PCR with primers in Table 3-4. The diagrams of

98

Table 3-4. Primer sequences for obtaining clones of selected ARF-GEF genes for yeast
two-hybrid assay with the N-terminus of HopM].

 

 

ARF-GEF Template Primer

gene

BIG] cDNA Forward primer : EcoRI

(At4g38200) 5’-
CGTGAATTCATAGAGCATTTGCAATTATTGGGCGAGO-3’
Reverse primer: Clal
5’GTTATCGA'I'I l ATTCATCCATCATTGCACCCATACATG
TATGG-3’

BIG3 cDNA Forward primer: EcoRI

(Atl g01960) 5 ’-CGCGAA'I'I‘C‘ I 'I 'I GAGC ATCTTCATCTCTTGGGGGAAG-
3,
Reverse primer: EcoRI
5’-CTAGAATTCTTAGCAGCAAGAGCGGAGGAGAA-3’

GNLI Genomic Forward primer: XmaII

(At5g39500) DNA

GNL2 Genomic
(At5g19610) DNA

5’-TATCCCGGGAGCCTCAACAAACTCCACA I I'l"l'ACCA-
3"

Reverse primer: Xhol
5’-GAACTCGAGTCAGACCTCATTTCCCGGTACCG-3’

Forward primer: EcoRl
5’CTAGAA'I'I‘CAAACTTAGGAAGC'ITCAGCTTCTTCCACA
-3’

Reverse primer: Xhol
5’GAACTCGAGCTAAATCTCTTCATCGGGAAATAACTCA
TCCTTGAG-3’

 

The restriction sites inserted in the primers for subcloning are shown as bold and

underlined.

99

Figure 3-2. Predicted amino acid sequences of the ARF-GEES for yeast two-hybrid
assays. Green-colored letters indicate amino acids for the putative Sec7 domain, and
yellow-colored letters indicate amino acids for the C-tenninus of an ARF-GEF.
Asterisks(*) denote the stop codons.

100

BIG 1 (At4g38200)

MSSSQNLGGATRCGRVIGPSLDKIIKNAAWRKHTFLVSACKSVLDKLEALSDSPDPSSPLFGLTTSDADAV
LQPLLLSLDTGYAKVIEPALDCSFKLFSLSLLRGEVCSSSPDSLLYKLIHAICKVCGIGEESIELAVLRVL
LAAVRSPRILIRGDCLLHLVRTCYNVYLGGFNGTNQICAKSVLAQIMLIVFTRSEANSMDASLKTVNVNDL
LAITDKNVNEGNSVHICQGFINDVITAGEAAPPPDFALVQPPEEGASSTEDEGTGSKIREDGFLLFKNLCK
LSMKFSSQENTDDQILVRGKTLSLELLKVIIDNGGPIWLSDERQLTLPPQKICRFLNAIKQLLCLSLLKNS
ALSVMSIFQLQCAIFTTLLRKYRSGMKSEVGIFFPMLVLRVLENVLQPSFVQKMTVLSLLENICHDPNLII
DIFVNFDCDVESPNIFERIVNGLLKTALGPPPGSSTILSPVQDITFRHESVKCLVSIIKAMGTWMDQQLSV
GDSLLPKSLENEAPANNHSNSNEEDGTTIDHDFHPDLNPESSDAATLEQRRAv’=" " "” .

‘ ...., « _. .- -,,,-,,:";3 FDFKEMNFGEAIRFF ’1

RLPGEAQKIDRIMEKFAERFCKCNP ,J, ‘ nutg.,,n-w-- gq,.¢Y9,,9.~-

 

IQEKFRSKSGKSESAYHVVTDVAILRFMVEVSWGPMLAAFSVTLDQSDDRLAAVECLRGFRYAVHVTAVMG
MQTQRDAFVTSMAKFTNLHCAGDMKQKNVDAVKAIISIAIEDGNHLQDAWEHILTCLSRIEHLQLLGEGAP
SDASYFASTETEEKKALGFPNLKKKGALQNPVMMAVVRGGSYDSSTIGPNMPGLVKQDQINNFIANLNLLD
QIGSFQLNNVYAHSQRLKTEAIVAFVKALCKVSMSELQSPTDPRVFSLTKLVEIAHYNMNRIRLVWSRIWS
ILSDFFVSVGLSENLSVAIFVMDSLRQLSMKFLEREELANYNFQNEFLRPFVIVMQKSSSAEIRELIVRCI
SQMVLSRVSNVKSGWKSVFKVFTTAAADERKNIVLLAFETMEKIVREYFSYITETEATTFTDCVRCLITFT
NSTFTSDVSLNAIAFLRFCALKLADGGLVWNEKGRSSSPSTPVTDDHSPSTQNFMDADENISYWVPLLTGL
SKLTSDSRSAIRKSSLEVLFNILKDHGHIFSRTFWIGVFSSVIYPIFNSVWGENDLLSKDEHSSFPSTFSS
HPSEVSWDAETSAMAAQYLVDLFVSFFTVIRSQLSSVVSLLAGLIRSPAQGPTVAGVGALLRLADELGDRF
SENEWKEIFLAVNEAASLTLSSFMKTLRTMDDIPDEDTLSDQDFSNEDDIDEDSLQTMSYVVARTKSHITV
QLQVVQVVTDLYRIHQQSLLASHVTVILEILSSISSHAHQLNSDLILQKKVRRACSILELSEPPMLHFEND
TFQNYLDILQAIVTNNPGVSLELNVESQLMTVCMQILKMYLKCTLFQGDELEETRQPKNWILPMGAASKEE
AAARSPLVVAVLKALRELKRDSFKRYAPNFFPLLVELVRSEHSSSQVPQVLSTVFHTCMGAMMDE*

BIG3 (At1 901 960)

MASTEVDSRLGRVVIPALDKVIKNASWRKHSKLAHECKSVIERLRSPENSSPVADSESGSSIPGPLHDGGA
AEYSLAESEIILSPLINASSTGVLKIVDPAVDCIQKLIAHGYVRGEADPTGGPEALLLSKLIETICKCHEL
DDBGLELLVLKTLLTAVTSISLRIHGDSLLQIVRTCYGIYLGSRNVVNQATAKASLVQMSVIVFRRMEADS
STVPIQPIVVAELMEPMDKSESDPSTTQSVQGFITKIMQDIDGVFNSANAKGTFGGHDGAFETSLPGTANP
TDLLDSTDKDMLDAKYWEISMYKSALEGRKGELADGEVEKDDDSEVQIGNKLRRDAFLVFRALCKLSMKTP
PKEDPELMRGKIVALELLKILLENAGAVFRTSDRFLGAIKQYLCLSLLKNSASNLMIIFQLSCSILLSLVS
RFRAGLKAEIGVFFPMIVLRVLENVAQPDFQQKMIVLRFLDKLCVDSQILVDIFINYDCDVNSSNIFERMV
NGLLKTAQGVPPGTVTTLLPPQEAAMKLEAMKCLVAVLRSMGDWVNKQLRLPDPYSAKMLEIVDRNLEEGS
HPVENOKGDGGHGGFERSDSQSELSSGNSDALAIEQRRA ." g »
rm .,, ,,1. .. ,__ .,w_,. ,r FEFQGMEFDEAIRAFLRGFRLPGEAQKID'
EKFAERFC. 1~.- :3 1 1‘ ‘.“-r..- -.1. s. -- :..:» in .r - r:
:0 :. KMKDDGLGPQQKQPTNSSRLLGLDTILNIVVPRRGDDMNMETSDDLIRHMQERFKEKARKSE
SVYYAASDVIILRFMVEVCWAPMLAAFSVPLDQSDDAVITTLCLEGFHHAIHVTSVMSLKTHRDAFVTSLA
KFTSLHSPADIKQKNIEAIKAIVKLAEEEGNYLQDAWEHILTCVSRFEHLHLLGEGAPPDATFFAFPQTES
GNSPLAKPNSVPAIKERAPGKLQYAASAMIRGSYDGSGVAGKASNTVTSEQMNNLISNLNLLEQVGDMSRI
FTRSQRLNSEAIIDFVKALCKVSMDELRSPSDPRVFSLTKIVEIAHYNMNRIRLVWSSIWHVLSDFFVTIG
CSDNLSIAIFAMDSLRQLSMKFLEREELANYNFQNEFMKPFVVVMRKSGAVEIRELIIRCVSQMVLSRVDN
VKSGWKSMFMIFTTAAHDAHKNIVFLSFEMVEKIIRDYFPHITETETTTFTDCVNCLVAFTNCKFEKDISL
QAIAFLQYCARKLAEGYVGSSLRRNPPLSPQGGKIGKQDSGKFLESDEHLYSWFPLLAGLSELSFDPRAEI
RKVALKVLFDTLRNHGDHFSLALWERVFESVLFRIFDYVRQDVDPSEDDSTDQRGYNGEVDQESWLYETCS
LALQLVVDLFVNFYKTVNPLLKKVLMLFVSLIKRPHQSLAGAGIAALVRLMRDVGHQFSNEQWLEVVSCIK
EAADATSPDFSYVTSEDLMEDVSNEDETNDNSNDALRRRNRQLHAVVTDAKSKASIQIFVIQAVTDIYDMY
RMSLTANHMLMLFDAMHGIGSNAHKINADLLLRSKLQELGSSLESQEAPLLRLENESFQTCMTFLDNLISD
QPVGYNEAEIESHLISLCREVLEFYINISCSKEQSSRWAVPSGSGKKKELTARAPLVVAAIQTLGNMGESL
FKKNLPELFPLIATLISCEHGSGEVQVALSDMLQTSMGPVLLRSCC*

101

Figure 3-2 (continued).

GNL1 (At5939500)
MGYQNHPSGSNSFHGEFKRCHSKPSKGAVASMINSEIGAVLAVMRRNVRWGVRYIADDDQLEHSLIHSLKE
LRKQIFSWQSNWQYVDPRLYIQPFLDVILSDETGAPITGVALSSVYKILTLEVFTLETVNVGEAMHIIVDA
VKSCRFEVTDPASEEVVLMKILQVLLACVKSKASNGLSNQDICTIVNTCLRVVHQSSSKSELLQRIARHTM
HELIRCIFSQLPFISPLANECELHVDNKVGTVDWDPNSGEKRVENGNIASISDTLGTDKDDPSSEMVIPET
DLRNDEKKTEVSDDLNAAANGENAMMAPYGIPCMVEIFHFLCTLLNVGENGEVNSRSNPIAFDEDVPLFAL
GLINSAIELGGPSFREHPKLLTLIQDDLFCNLMQFGMSMSPLILSTVCSIVLNLYLNLRTELKVQLEAFFS
YVLLRIAQSKHGSSYQQQEVAMEALVDLCRQHTFIAEVFANFDCDITCSNVFEDVSNLLSKNAFPVNGPLS
AMHILALDGLISMVQGMAERVGFELPASDVPTHFFRYFFFWTVRCFNYGDPNFWVPFVRKV

-]
.l' "I"' U' U V- ' ‘ "' " " I"" I "'I' UV "

     
 

ifLATALRLFVGTFKLSGEAQKIHRVLEAFSERYYEQSPHI
H I :11: 1 AI " - 3-_ ‘ul I oTbFQLMIAbRWIbVIYKbKEISPYIQCDA

ASHLDRDMFYIVSOPIIAAISVVFEQAEQEDVLRRCIDGLLAIAKLSAYYHLNSVLDDLVVSLCKFTPFFA
PLSADEAVLVLGEDARARMATEAVFLIANKYGDYISAGWKNILECVLMGYQNHPSGSNSFHGEFKRCHSKP
SKGAVASMINSEIGAVLAVMRRNVRWGVRYIADDDQLEHSLIHSLKELRKQIFSWQSNWQYVDPRLYIQPF
LDVILSDETGAPITGVALSSVYKILTLEVFTLETVNVGEAMHIIVDAVKSCRFEVTDPASEEVVLMKILQV
LLACVKSKASNGLSNQDICTIVNTCLRVVHQSSSKSELLQRIARHTMHELIRCIFSQLPFISPLANECELH
VDNKVGTVDWDPNSGEKRVENGNIASISDTLGTDKDDPSSEMVIPETDLRNDEKKTEVSDDLNAAANGENA
MMAPYGIPCMVEIFHFLCTLLNVGENGBVNSRSNPIAFDEDVPLFALGLINSAIELGGPSFREHPKLLTLI
QDDLFCNLMQFGMSMSPLILSTVCSIVLNLYLNLRTELKVQLEAFFSYVLLRIAQSKHGSSYQQQEVAMEA
LVDLCRQHTFIAEVFANFDCDITCSNVFEDVSNLLSKNAFPVNGPLSAMHILALDGLISMVQGMAERVGEE
LPASDVPTHEERYEEFWTVRCENYGDPNFWVPFVRKVKHIKKKLMLGADRFNRDPNKGLQYLQGVHLLPEK
LDPKSVACFFRYTCGLDKNVMGDFLGNHDQFCIQVLHEFAKTFDFQNMNLATALRLFVGTFKLSGEAQKIH
RVLEAFSERYYEQSPHILIDKDAAFVLAYSIILLNTDQHNAQVKTRMTEEDFIRNNRTINGGADLPREYLS
EIYHSIRHSEIQMDEDKGTGFQLMTASRWISVIYKSKETSPYIQCDAASHLDRDMFYIVSGPTIAATSVVF
EQAEQEDVLRRCIDGLLAIAKLSAYYHLNSVLDDLVVSLCKFTPFFAPLSADEAVLVLGEDARARMATEAV
FLIANKYGDYISAGWKNILECVLSLNKLHILPDHIASDAADDPELSTSNLEQEKPSANPVPVVSQSQPSAM
PRKSSSFIGRFLLSFDSEETKPLPSEEELAAYKHARGIVKDCHIDSIFSDSKFLQAESLQQLVNSLIRASG
KDEASSVFCLELLIAVTLNNRDRILLIWPTVYEHILGIVQLTLTPCTLVEKAVFGVLKICQRLLPYKENLT
DELLKSLQLVLKLKAKVADAYCERIAQEVVRLVKANASHVRSRTGWRTIISLLSITARHPEASEAGFEALR
FIMSEGAHLLPSNYELCLDAASHFAESRVGEVDRSISAIDLMSNSVFCLARWSQEAKNSIGETDAMMKLSE
DIGKMWLKLVKNLKKVCLDQRDEVRNHAISMLQRAIAGADGIMLPQPLWFQCFDSAVFILLDDVLTFSIEN
SRKTLKKTVEETLVLATKLMSKAFLQSLQDISQQPSFCRLWVGVLNRLETYMSTEFRGKRSEKVNELIPEL
LKNTLLVMKATGVLLPGDDIGSDSFWQLTWLHVNKISPSLQSEVFPQEELDQFQRRNAKPEDPPVPGNEV*

GNL2 (At591 961 0)

MDRIAVRAKRKELGISCMLNTEVGAVLAVIRRPLSESYLSPQETDHCDSSVQQSLKSLRALIFNPQQDWRT
IDPSVYLSPFLEVIQSDEIPASATAVALSSILKILKIEIFDEKTPGAKDAMNSIVSGITSCRLEKTDLVSE
DAVMMRILQVLTGIMKHPSSELLEDQAVCTIVNTCFQVVQQSTGRGDLLQRNGRYTMHELIQIIFSRLPDF
EVRGDEGGEDSESDTDEIDMSGGYGIRCCIDIFHFLCSLLNVVEVVENLEGTNVHTADEDVQIFALVLINS
AIELSGDAIGQHPKLLRMVQDDLFHHLIHYGASSSPLVLSMICSCILNIYHFLRKFMRLQLEAFFSFVLLR
VTAFTGFLPLQEVALEGLINFCRQPAFIVEAYVNYDCDPMCRNIFEETGKVLCRHTFPTSGPLTSIQIQAF
EGLVILIHNIADNMDREEDEGNEEDDNNSNVIKPSPVEIHEYIPFWIDKPKEDFETWVDHIRVRKAQKRKL

AIAANHFNRDEKKGL r ‘ i'*i~--'--r-n- ., r,‘- -H "a in“.
~ . . ¥ ‘ SFRLPGESQKIBRMIEAFSERFYDQQS u 1 .s . . 1 mo..'%
use 0 :11:; 1; 1| -. e 1 1: 1 RSGPVEMNPNRWIELMNRTKTTQPFSLC

QFDRRIGRDMFATIAGPSIAAVSAFFEHSDDDEVLHECVDAMISIARVAQYGLEDILDELIASFCKFTTLL
NPYTTPEETLFAFSHDMKPRMATLAVFTLANTFGDSIRGGWRNIVDCLLKLRKLQLLPQSVIEFEINEENG
GSESDMNNVSSQDTKFNRRQGSSLMGRFSHFLALDNVEESVALGMSEFEQNLKVIKQCRIGQIFSKSSVLP
DVAVLNLGRSLIYAAAGKGQKFSTAIEEEETVKFCWDLIITIALSNVHRFNMFWPSYHEYLLNVANFPLFS
PIPFVEKGLPGLFRVCIKILASNLQDHLPEELIFRSLTIMWKIDKEIIETCYDTITEFVSKIIIDYSANLH
TNIGWKSVLQLLSLCGRHPETKEQAVDALIGLMSFNASHLSQSSYAYCIDCAFSFVALRNSSVEKNLKILD
LMADSVTMLVKWYKTASTDTANSYSPASNTSSSSSMEENNLRGVNFVHHLFLKLSEAFRKTTLARREEIRN
RAVTSLEKSFTMGHEDLGFTPSGCIYCIDHVIFPTIDDLHEKLLDYSRRENAEREMRSMEGTLKIAMKVLM

102

Figure 3—2 (continued).

NVFLVYLEQIVESAEFRTFWLGVLRRMDTCMKADLGEYGDNKLQEWPELLTTMIGTMKEKEILVQKEDDD
LWE.ITYIQIQWIAPALKDELE‘PDEEI *

103

ARF-GEF genes are in Figure 3-3. The genomic DNA or cDNA from Arabidopsis Col-0
ecotype was used as a template for PCR, depending on whether introns are present in the
region to be cloned (Table 34). Each PCR was performed with PﬁlTurbo® DNA
polymerase (Stratagene, La Jolla, CA). PCR products were subcloned into pB42AD-L
vector (pB42AD vector (Clontech, Mountain View, CA) with multicloning sites) and
sequenced. Mutation-free clones were transformed into yeast strain EGY48 p8oplacz
according to the manufacturer’s protocol (frozen-E2 Yeast Transformation lITM kit,

Zymo Research Corperation, Orange, CA).

Yeast two—hybrid assay

Twenty pl of each yeast culture expressing HopM11-3oo and the C-termini of ARF -
GEF genes or containing an empty vector (pBD42AD-L) were grown on the solid
SD+Glu-Ura-His-Trp medium (13.35g of minimal SD base, 0.35g of -Ura-His-Trp DO
supplement, 10g of Agar in 500 ml water: Clontech, Mountain View, CA) at 30 °C for
two days. A single colony of the each yeast was used to inoculate in 2 ml of SD+Glu-
Ura-His-Trp liquid medium (6.675g of minimal SD base, 0.175g of -Ura-His-Trp DO
supplement in 250 ml water: Clontech, Mountain View, CA) and cultured at 30 °C with
shaking for approximately 24 hours. One mililiter of this liquid culture was used to
inoculate into 6 ml of SD+Glu-Ura—His-Trp liquid medium, and re-cultured overnight (20
ul of each culture was spotted on the SD+Glu-Ura-His-Trp solid medium again, and
incubated at 30 °C for two days). One ml of each culture was collected and centrifuged at
the room temperature for 4 minutes at SOOxg. The pellet was washed with 10 ml of Tris-

EDTA buffer. The washed pellet was resuspended with 6 ml of SD+Gal-Ura-His-Trp

104

3’61 EcoRl

 

 

 

 

 

 

Clal
r11 |
8’63 EcoRl EcoRl
I
GNL1
Xmal Xhol
E l
GNL2

EcoRl Xhol

 

l I

Figure 3-3. Diagrams of four ARF-GEF genes analyzed by yeast two-hybrid assay. Green
box with S denotes the putative Sec7 domain of each gene. Yellow box indicates the C-
terrninus of each gene that was used for yeast two-hybrid assay. EcoRI, C Ial, Xmal and
Xhol indicate the restriction enzyme sites added for subcloning.

105

liquid medium (9.25g of minimal SD base Gal/Raf and 0.175g of -Ura-His-Trp
supplement in 250 ml water: Clontech, Mountain View, CA), and 20 ul of each
resuspended sample was spotted on the SD+Gal-Ura-His-Trp solid medium containing
X-gal and BU salts (9.25g or minimal SD base Gal/Raf, 0.175g of -Ura-His-Trp DO
supplement, 5g of agarose, 25 ml of 10 X BU salt solution (0.26 M of NazHPO4-7HZO

and 0.25 mM of NaHzPO4-HZO), and lml of 20mg/ml X-gal solution in 225 ml water:
Clontech, Mountain View, CA) incubated at 30 °C for 5 days.

Total proteins extracted from yeast were separated on sodium dodecyl sulfate
polyacrylamide gels (7.5%), then transferred onto lmmobilon-P membrane (Millipore,
Billerica, MA) using a semi-dry transfer apparatus (SEMIPHOR, Hoefer Scientiﬁc
Instruments, San Francisco, CA). Immunoblot analyses were performed with a LexA-
antibody for HopM1 or a HA-antibody for ARF-GEF proteins. For determining the
approximate sizes of proteins, PageRulerTM Prestained Protein Ladder Plus (Fermentas

International Inc, Ontario, Canada) was used.

Growth curve assay for determining multiplication of Pst DC3000 and its
mutants in Arabidopsis

Arabidopsis plants for bacterial growth curve assay were grown in pots in grth
chambers (a 12 h dark/12 h light cycle, 100 uE, 20°C), until they were approximately 5
weeks old. The bacteria selected for grth curve assay were cultured in low-sodium
Luria-Bertany (LB) medium (10g/l Tryptone, Sg/l Yeast Extract, 5g/1NaCl) plus
appropriate antibiotics, at 30°C with shaking at 250 rpm. Bacterial liquid culture was

incubated to the mid- to late-logarithmic phase. Bacteria were collected by centrifugation

106

(3,000xg, room temperature, 30 minutes) and resuspended in sterile, de-ionized water
with 0.03% Silwet L-77 (OSI Specialties, Friendship, WV). The bacterial inoculum was
1x108 colony forming units (CFUs)/ml, unless otherwise indicated. Plants were
inoculated by dipping, and bacteria in leaf samples were quantiﬁed according to the

protocol described in Katagiri et al. (2002).

Crossing atmin7 knockout mutant (salk_012013) and knockout mutant of
BIGZ (salk_033446)

For constructing double knockout mutant lines of AtMIN 7 and 3162, homozygous
T-DNA insertion mutant of AtMIN 7 (salk_012013: Nomura et al., 2006) and that of BIG2
(salk_033446) were grown on pots until they were ﬂowering. The pollens from the
mature stamens of salk_033446 were collected and applied to the stigmas of semi-mature
ﬂowerbuds ofsalk_012013. The artiﬁcially pollinated stigmas of salk_012013 were
sealed with plastic wrap, and the whole plants were put into the growth chamber (under a
12 h dark/12 h light cycle, 100 1.113, 20°C), until the artiﬁcially pollinated stigmas had
developed into siliques. F, seeds were collected from mature siliques and germinated on
the soil, and F1 plants were analyzed by genomic PCR (Table 3-2). F. plants which had
T-DNA insertion for both AtMIN 7 and 3102 were selected, and their F2 seeds were
collected. F2 plants were analyzed by genomic PCR, and homozygous knockouts for

AtMIN 7 and BIGZ were selected. F3 or F4 plants were used for further analyses.

'1 O7

RESULTS

Identiﬁcation of homozygous T-DNA insertion mutants for Arabidopsis ARF-

GEF genes

To determine a possible role of ARF -GEF genes other than AtMIN 7 in
Arabidopsis defense against the ACEL mutant, I attempted to identify T-DNA insertion
mutants of seven Arabidopsis ARF-GEF genes. Based on the genomic PCR results,
homozygous T-DNA insertion mutants of three genes (BIGZ, BIG3 and 3104) were
obtained (salk_033446, salk_0446l7, and salk_082249, respectively: Figure 3-4). These
homozygous mutants did not have visible phenotypes in grth and development.
Homozygous T-DNA insertion mutants of four ARF-GEF genes (AtBIGI, GNOM, GNL1
and GNL2) were not isolated and only heterozygote plants were observed by genomic
PCR (Wei-Ning Huang and Sheng Yang He, unpublished). Three heterozygous mutants
of the four ARF-GEF genes (salk_066766 of AtBIGI , salk_067415 of GNL1 and
salk_103014 of GNOM) had shrunken seeds in their siliques and their germination rate
were low (Wei-Ning Huang and Sheng Yang He, unpublished). The heterozygous T-

DNA insertion mutants were not included in further analyses.

Confirmation of gene knockout in the homozygous T-DNA insertion mutants
by RT-PCR

The homozygous salk lines chosen by genomic PCR were examined by RT-PCR
to determine whether they were true knockouts. RT-PCR was performed with the primers

which are speciﬁc for each ARF-GEF gene. After 25 cycles of PCR, transcript was not

108

Col-0 salk_033446

LR RT LR RT

 

 

 

Col-0 salk_082249

IR Rl iR E!

 

   

Col-O salk_04461 7

 

 

LR RT

LR RT

   

.'M ‘ ' n

3

Figure 3-4. Genomic PCR results of homozygous T-DNA insertional mutants for three
ARF-GEF genes (BIG2, BIG3 and BIG4). LR indicates the reactions performed with
gene-speciﬁc primer surrounding the T-DNA insertion site. RT indicates the reactions
performed with a gene-speciﬁc primer and the T-DNA left border primer.

109

detected for salk _033446, indicating that it was a knockout mutant of 3162. On the
contrary, transcript was detected in salk_0446l7 plant. However, the amount of transcript
was signiﬁcantly less than that of Col-0. This indicates that salk_044617 is not a
knockout of BIG3, but the transcript level was reduced (Figure 3-5A).

Neither Col-O nor big4 (salk_082249) samples had detectable transcript after 25
cycles (Figure 3-5A). However, aﬁer increasing the number of the PCR cycles to 30, 40,
and 50 cycles, transcript was detected in Col-0 but not in salk_082249 (Figure 3-SB).

This result indicates that salk_082249 is a 8104 knockout mutant.

Determination of the multiplication of the ACEL mutant in the selected T-
DNA insertion mutants

The multiplication of the ACEL mutant in the three homozygous T-DNA insertion
mutants was examined. For comparison, the multiplication of Pst DC3000 (wild type;
fully virulent) and the hrcC mutant (defective in the TTSS; nonpathogenic) were also
investigated. Besides selected salk lines, Col-O and atmin7 plants were included as
controls. Repeated assay results showed that none of the T-DNA insertion mutants
showed enhanced grth of ACEL as seen in atmin7 plants (Figure 3-6). This result
indicates that knockout of AtBIGZ and AtBIG4 and knockdown of AtBIG3 did not

increase the multiplication of the ACEL mutant.

Determination of the multiplication of the ACEL mutant in the double

knockout mutant of AtMIN 7 and BIGZ

The C-terminus of 3102 (the product of At3g60860) had shown a weak

llO

(A)

Act8 Primer D3 set

 

 

Col-0 sdt_(B34-48 Col-0 sdt_m3446

 

Act8 Primer E set

 

 

Col-0 salk_OMB‘l?

Col-0 salk_044617

    
 

 

Act8 Primer H set

 

 

Col-0 salk_082249 Col-0 sauna“
‘ ”1 . :_.:‘ I, 5, r a
1‘ .5 l .1 L__.il

(B)

 

Figure 3-5. RT-PCR results of selected T-DNA insertional mutants of AtBIGZ, AtBIG3
and AtBIG4. (A) RT-PCR results of salk-033446, salk_0446l7, and salk_082249 after 25
cycles. (B) RT-PCR result of salk_082249 after 30, 40 and 50 cycles. PCR products from
genomic DNA were detected (arrow) in addition to those from cDNA.

11]

0)

 

logCFU/Cm2
co 4: 01 on \1

 

 

PstDC3m0 ACEL HrcC-

 

7- Col-0

I atm'n7
u bigZ (salk_033446)
I: big3 (salk_044617)

‘ I big4 (salkg082249)

Figure 3-6. Multiplication of Pst DC3000, the ACEL mutant and the hrcC mutant in C01-
0, atmin7 and ARF-GEF T-DNA insertion lines. Results are displayed as means of 4

leaves with standard errors.

112

interaction with HopM1 1-300 in the yeast two-hybrid assay (Kinya Nomura and Sheng
Yang He, unpublished). Although the T-DNA insertion mutant of 8102 (salk_033446)
did not show an enhanced susceptibility to the ACEL mutant, it is still possible that the
role oftBlGZ in defense may be partially redundant to and masked by AtMIN7. If so, a
double knockout mutant of AtMIN 7 and AtBIG2 might support a higher level of
multiplication of the ACEL mutant than either atmin7 or atbig2 single mutant,. To test
this hypothesis, a double knockout mutant was constructed by crossing atmin7
(salk_012013) and atbigZ (salk_033446). The F 3 and F4 plants were tested by genomic
PCR and it was conﬁrmed that they have T-DNA insertions in both genes. Line #6-3 was
selected for further analysis (Figure 3-7).

The multiplications of Pst DC3000, ACEL mutant, and hrcC mutant in the F4
plants of line #6-3 were compared (Figure 3-8). The atmin 7/big2 plants showed higher
multiplication of ACEL mutant than in Col-0, but it was not signiﬁcantly higher than that
of atmin7 mutant. Therefore, the double knockout of AtMIN 7 and 3102 did not fully

complement the ACEL mutation.

Yeast two- hybrid assay of selected Arabidopsis ARF-GEF genes with
HopM1

Nomura et al. (2006) reported that the C-terminus of AtMIN7 interacted with
HopM1 1-300, whereas the corresponding C-termini of three other ARF-GEFs, BIGZ,
GNOM and BIG4, did not in the yeast two-hybrid assay. It is not known whether any of
the four remaining ARF-GEFs, BlGl, BIG3, GNL1 and GNL2, interacts with HopM1 1.

300. In this study, the sequences of the ORFs of these four genes were aligned with other

113

K5"
- Q0
AtMIN7 LR primers j

AtMIN7 LT primers

 

11"“: ‘5

.. .' " 3+1
rhrxrlur.b u-

      

AtBIG2 LR primers

AtBIGZ RT primers 1

 

Figure 3-7. Genomic PCR results conﬁrming the double knockout of AtMIN 7 and BIGZ.
LR primers are the forward and reverse primers for the genomic PCR products of
AtMIN 7 or 3102. Primer sets LT and RT denote the reactions performed with a gene-
speciﬁc primer and a T-DNA-speciﬁc primer. LR indicates the reactions performed with
gene-speciﬁc primers surrounding the insertion site. The double knockout plant was from
the F3 generation of line #6-3.

114

 

:-
l

bgCFU/Cm
Nuhmmwoo

 

PstDC3000 ACEL HrcC-

 

 

l Col-07 , 7

I atm'n7

El bigZ (salk_033446)
El atrrin7 x bigZ

Figure 3-8. Multiplication of Pst DC3000, the ACEL mutant and the hrcC mutant in C01-
0, atmin 7, big2, and the double knockout of AtMIN 7 and AtBIGZ. Results are displayed

as means of 4 leaves with standard errors.

115

Arabidopsis ARF-GEF genes by CLUSTALW (http://align.genome.ip/), and the
predicted C-terminus after the putative Sec7 domain of each gene was determined (Figure
3-2), following the previous experimental deSign (Nomura et al., 2006). The 3’-coding
regions of GNL1 and GNL2 were obtained by PCR from the genomic DNA of
Arabidopsis (Col-O ecotype), and those of BIG] and BIG3 were obtained by PCR from
the cDNA of Arabidopsis. The PCR products were subcloned into pB42AD-L vector and

sequenced, and the sequences were compared to those in TAIR (www.arabidopsisorg).

 

The sequences of PCR products for BIG], BIG3 and GNL1 were conﬁrmed to be
identical to those in the TAIR database, but the PCR product GNL2 had mismatches,
resulting in two amino acid changes (Appendix 3-8). However, these two changed amino
acids were found in two independent sets of PCR, and were therefore not likely to be
mutations introduced during PCR procedures.

The PCR products were cloned into pB42AD-L vector and the recombinant plasmids
were introduced into yeasts. Previously characterized ARF-GEF clones (BIG2, BIG4,
AtMIN 7 and GNOM: Nomura et al., 2006) were included in yeast two-hybrid assay with
HopM11-300. Among the four newly made ARF-GEF clones, BIGI, BIG3 and GNL1
were not expressed in yeast, and only GNL2 clone was expressed (Figure 3-9B). GNL2
did not show interaction with HopM1 1.300 (Figure 3-9A). In these experiments, BIGZ
clone which had previously been demonstrated to have a weak interaction with HopM1 1-
300 (Kinya Nomura and Sheng Yang He, unpublished) also did not interact with HopM1].

300 (Figure 3-9A).

116

(A)
BIG1 BIGZ BIG3 B|G4 AtMIN7(BIGS)

 

GNOM GNL1 GNL2 AD

(B)
3161 BIGZ 8163 3164 MINT GNOM GNL1 GNL2 AD

a-HA - H ‘n‘

HAzzARF-GEFs

a-LexA
LexA::HopM114m

Coomassie staining

Total yeast extract

 

Figure 3-9. Yeast two-hybrid assay result of the C-termini of selected ARF-GEFs with
HopM1 1.300. (A) Yeast two-hybrid assay between the C-termini of ARF-GEFs and
Hole 1-300, AD is the empty pB42AD-L vector. (B) Immunoblot analyses of ARF-GEF
clones expressed in yeast. ARF-GEFs were detected by an HA antibody, and HoleHoo

was detected by a LexA antibody.

117

DISCUSSION

In this chapter Arabidopsis ARF-GEF genes were studied to determine whether
they have roles in the defense against the ACEL mutant and whether their products
interact with HopM1.

To determine the roles of the ARF-GEF genes in Arabidopsis defense against the
ACEL mutant of Pst DC3000, homozygous T-DNA insertion mutants of seven ARF-GEF
genes were collected. Homozygous T-DNA insertion mutants of three ARF—GEF genes
(BIG2, BIG3 and BIG4) were conﬁrmed by PCR. RT-PCR results showed that the
homozygous T-DNA insertion mutants of 8102 (salk_033446) and BIG4 (salk_082249)
were knockout mutants and that the T-DNA insertion mutant of BIG3 (salk_044617) had
a reduction in transcript level. However, salk_0446l7 was included in further
experiments because it was the only available salk line for BIG3 (TAIR,
www.arabidopsis.org). The RT-PCR result of salk_082249 needed more PCR cycles than
those of the two other salk lines (25 cycles), indicating that the transcription level of the
BIG4 is low. It is consistent with the information available from the Bio-Array Resource
for Arabidopsis Functional Genomics. The expression of BIG4 is very low in vegetative
organs but high in anthers (the information of the expression of each ARF-GEF gene in

different tissue is in theAppendix 3-9: Winter et al., 2007; http://wwwbar.utoronto.ca

 

/efp/cgi-bin/epreb.cgi).
The multiplication of the ACEL mutant was quantiﬁed in the selected T-DNA
insertion mutants, and compared to those in Col-O and in atmin7 plants. In addition to the

ACEL mutant (a mutant with an intact TTSS, but a partially deleted CEL), Pst DC3000 (a

118

wild type pathogen with an intact TTSS) and the hrcC' mutant (a mutant without a
functional TTSS) were also tested. The multiplication of the ACEL mutant in the T-DNA
insertion mutants was similar to that seen in Col-O, and none of the mutants showed
increased multiplication of the ACELSmutant equivalent to the multiplication in atmin7
plants. These data show that the mutation of 3102 and BIG4 do not result in enhanced
multiplication of the ACEL mutant, and that these ARF-GEF genes do not have a role in
Arabidopsis defense against the ACEL mutant.

A prior yeast two-hybrid assay showed a weak interaction between BIG2 and
HopM1 1-300, suggesting that 3102 may contribute to Arabidopsis defense against the
ACEL mutant (Kinya Nomura and Sheng Yang He, unpublished). The double knockout
mutant of AtMIN 7 and BIG2 may therefore support increased multiplication level of the
ACEL mutant above that shown in atmin 7. However, the multiplication of the ACEL
mutant in the double knockout mutant was not signiﬁcantly different than that in atmin7
mutant. In addition, the yeast two-hybrid interaction between BIG2 and HopM11-3oo was
not reproduced. Based on these data, it can be interpreted that BIG2 does not signiﬁcantly
contribute to Arabidopsis defense against the ACEL mutant and it is not a host target of
HopM1.

BIG4 is not likely to contribute to Arabidopsis defense against the ACEL mutant
based on the ACEL mutant multiplication level in the T-DNA insertion mutant
(salk_082249). BIG4 also did not interact with HopM1 [.300 in the yeast two-hybrid assay
(Nomura et al., 2006), indicating that it is not a host target of HopM1. From its anther-
speciﬁc expression (Appendix 3-9 [Winter et al., 2007]), AtBIG4 may have roles in organ

development rather than in defense.

119

The role of BIG3 in Arabidopsis defense against the ACEL mutant remains
unclear. The T-DNA insertion mutant of BIG3 (salk_044617) was not a complete
knockout mutant. It did not support the multiplication of the ACEL mutant, and this may
be due to the presence of residual BIG3 transcript. The yeast two-hybrid assay between
BIG3 and HopM11-300 could not be performed, because the C-terminus of BIG3 was not
expressed in yeast for unknown reasons.

Homozygous T-DNA insertion mutants for BIG], GNOM, GNL1 and GNL2 were
not isolated although heterozygous mutant plants were obtained (Wei-Ning Huang and
Sheng Yang He, unpublished). These heterozygous mutants were not further analyzed.
The heterozygous mutants of BIG], GNOM and GNL1 had abnormal seed development
in the siliques (Wei-Ning Huang and Sheng Yang He, unpublished), suggesting that these
ARF-GEF genes may function in seed or embryo development. Indeed, GNOM functions
in embryo development, and its heterozygous mutant produces embryo-lethal seeds
(Mayer et al., 1993; Vielle-Calzada et al., 2000).

The C-termini of BIG], GNOM, GNL1 and GNL2 were tested by yeast two-
hybrid assay to determine whether they interact with HopM1 1-300. GNL2 was successfully
expressed in yeast, but it did not interact with HopM1 1-300 suggesting that GNL2 is not
likely to be targeted by HopM1. However, the C-termini of three of the ARF-GEFs,
BIGl, GNOM, GNL1, were not expressed in yeast. This could be due to the toxicity
generated by those C-termini to the yeast or structural defects in the truncated ARF-GEF
proteins. As a result, the interaction between AtBIGl, GNOM or GNL1 with HopM1 [.300
could not be tested in the yeast two-hybrid assay.

In this chapter it was conﬁrmed that AtBIGZ and AtBIG4 do not have a signiﬁcant

120

inﬂuence in Arabidopsis defense against the ACEL mutant. It was also demonstrated that
GNL2 does not interact with HopM1 1.300 The role of GNL2 in defense against the ACEL
mutant was not determined. However, thus far, we have been unable to demonstrate that
any ARF-GEFs other than AtMIN 7 function in the defense against the ACEL mutant or
that they are targeted by HopM1. However, it still cannot be concluded that AtMIN7 is
the only ARF GEF that is important for Arabidopsis defense and the only ARF-GEF

target of HopM 1.

121

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Jackson, CL. and Casanova, J.E. (2000) Turning on ARF: the Sec7 family of guanine-
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Mayer, U., Buttner, G., and Jiirgens, G. (1993) Apical-basal pattern formation in the
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123

CHAPTER 4

In vivo Subcellular Localization Imaging of Several Arabidopsis Proteins

That Are Putative Cargoes of Defense-Associated Cellular Trafficking

124

ABSTRACT

Confocal microscopy has been used for the visualization of plant-pathogen
interactions in living plant cells and enabled the understanding of the characteristics of
defense-associated proteins in the context of the cellular processes. In this chapter, three
known or predicted extracellular Arabidopsis proteins (PR1, a putative lipid transfer
protein (LTP) encoded by At2g10940 and F LA9, a putative arabinogalactan protein
(AGP) encoded by Atl g03870) were fused with GF P, and they were examined in
Arabidopsis by western blot analyses and confocal microscopy. The immunoblot analysis
results of PR1-GFP and At2g10940-GFP did not show any sign of degradation or
modiﬁcation of fusion proteins. The F LA9-GFP fusion was not expressed in Arabidopsis.
PR1-GF P was localized in the intercellular space both in Col-O and atmin7 backgrounds.
At2g10940-GFP was localized in the intercellular space in Col-0 background, but in
atmin7 background, unidentiﬁed round intracellular structures were observed. These
results suggest that the localization of At2gl 0940-GFP may be dependent on AtMIN7-

mediated vesicle trafﬁcking pathway(s), whereas the localization of PR1-GF P is not.

125

INTRODUCTION

Microscopic visualization of the components of pathogens or plants during
infection has been one of the most powerful experimental approaches in the study of
plant-pathogen interactions. In recent years, confocal microscopic analyses of cellular
proteins fused to ﬂuorescence proteins (such as GF P and its derivatives) have
revolutionized the ﬁeld of plant cell biology, in large part because confocal microscopy
enables monitoring the localization and dynamic changes of GFP fusion proteins in living
cells or organisms (Brandizzi et al., 2004).

The interactions between Arabidopsis and its pathogens have been also studied by
confocal microscopy. For instance, pathogens such as Pst DC3000 or cauliﬂower mosaic
virus (CaMV) were tagged with OF P and used for the microscopic detection of their
behaviors during infection (Badel et al., 2002; Love et al., 2007). Also, numerous
defense-associated proteins of Arabidopsis have been studied by confocal microscopy for
determining their localization in the cell.

Several known defense-associated proteins in Arabidopsis are components of the
cellular trafﬁcking system. PENI (SYP121), a protein having a role for the defense
against fungal pathogens, is a SNARE protein which acts in vesicle targeting and fusion
(Collins et al., 2003; Assaad et al., 2004; Mayer et al., 2009). SYP122, the closest
homologue of PEN l , is also involved in the regulation of defense-associated signaling
pathways (Assaad et al., 2004; Zhang et al., 2007). AtMIN7 plays a role in the defense
against the ACEL mutant of Pst DC3000, and it is a putative ARF-GEF which activates

ARF protein required for vesicle formation and budding (Nomura et al., 2006). Another

126

Arabidopsis protein, RabE 1 .d, which physically interacts with the type III secretion
system (TTSS) effector AvrPto, is a small GTPase functioning in vesicle targeting. The
constitutively active form of RabEl.d enhances the resistance against Pst DC3000 (Speth
et al., 2009). All of these proteins were studied by confocal microscopy and their
subcellular localizations were determined: PEN] and SYP122 are localized in the plasma
membrane (Collins et al., 2003; Assaad et al., 2004), AtMIN7 is localized in the trans-
Golgi network (Tanaka et al., 2009) and RabE] .d is localized in the Golgi and plasma
membrane (Speth et al., 2009). In addition to Arabidopsis, NbSYP132, a SNARE protein
in Nbenthamiana has role in defense (Kalde et al., 2007).

Accumulating evidence suggests that Pst DC3000 affects the components of
defense-associated cellular trafﬁcking pathways of Arabidopsis via its TTSS effectors.
For instance, AtMIN7 and RabE] .d are targeted by HopM1 and AvrPto, respectively.
AtMIN7 is degraded by HopM1, and the localization of RabEl.d is changed in transgenic
Arabidopsis expressing AvrPto (Nomura et al., 2006; Speth et al., 2009; Elena Bray
Speth and Sheng Yang He, unpublished). Furthermore, global gene expression analysis of
Arabidopsis upon infection of Pst DC3000 showed that there was a biased suppression of
genes encoding putatively secreted proteins in a TTSS-dependent manner (Hauck et al.,
2003: Thilmony et al., 2006). The TTSS-dependent effect on the host vesicle trafﬁc is
also observed in other bacterial pathogens. For example, a TTSS effector of
Xanthomonas campestris, XopJ, when expressed in tobacco, interfered with the secretion
of GFP which was introduced in tobacco (Bartetzko et al., 2009).

Despite these intriguing observations, it is still poorly understood how the

bacterial TTSS effector system alters the dynamic and spatial patterns of host vesicle

127

trafﬁc during infection. In the case of AtMIN7, currently neither the cargoes nor the
functional components of AtMIN7-dependent vesicle trafﬁcking pathway have been
elucidated. The only clue for understanding the characteristics of AtMIN7-mediated
vesicle trafﬁcking is that the atmin7 plant has reduced and altered callose deposition in
cell wall-associated PTI (Nomura et al., 2006).

In this chapter, the localization of three known or predicted extracellular
Arabidopsis proteins were studied by confocal microscopy: PR1 encoded by At2g14610
(Laird et al., 2004), a putative protease inhibitor/seed storage/lipid transfer protein (LTP)
family protein encoded by At2g10940 (Hauck et al., 2003), and a putative fasciclin-like
arabinogalactan 9 protein (F LA9) encoded by At1g03870 (Hauck et al., 2003; Johnson et
al., 2003). PR] is a representative gene which is induced in plant defense and has been
extensively used as a marker for the establishment of local and systemic plant resistance.
PR] is induced in both incompatible and compatible Arabidopsis-P.syringae interactions
(Uknes et al., 1992; Laird et al., 2004) and by SA or SA analogues such as 2,6-
dichloroisonicotinic acid (INA) or a benzothiadazole derivative (BTH) (Uknes et al.,
1992; Lawton et al., 1996). PR] is localized in the intercellular space of Arabidopsis
based on the analysis of apoplastic ﬂuid (Uknes et al., 1992), but its localization has not
been studied by confocal microscopy. The functions of PR] in plant resistance against
bacterial pathogens are not known despite its known contributions to the resistance
against fungi or oomycetes (Alexander et al., 1993; Niderrnan et al., 1995).

At2g10940 (a putative protease inhibitor/seed storage/lipid transfer protein (LTP)
family protein) and Atlg03870 (a putative fasciclin-like arabinogalactan 9 protein

(FLA9) were also selected in this study. The expression of both of them was suppressed

128

in a TTSS-dependent manner (Hauck et al., 2003; Roger Thilmony and Sheng Yang He,
unpublished), in contrast to the expression of PR], which was induced in a TTSS-
dependent manner. The products of these genes were suggested as putative cargoes of a
defense-associated secretion pathway or a defense-associated cell wall component in
Arabidopsis (Hauck et al., 2003): it was suggested that the TTSS-dependent suppression
the two genes is the reﬂection of the effect to the secretion of the gene products by TTSS
effectors.

These proteins were fused with OF P, and the GF P fusions were introduced into
Arabidopsis. Their expression and localization were studied by immunoblot analysis and

confocal microscopy, and their localization in Col-O and the atmin7 plants was compared.

129

MATERIALS AND METHODS

Construction of GFP fusions and their subcloning into plant expression

vector

The predicted amino acid sequences of PR], At2g10940 and FLA9 were

examined using the SignalP 3.0 software (http://www.cbs.dtu.dk/services/SignalP/)

 

(Bendtsen et al., 2004; Nielsen et al., 1997) to identify putative signal sequences at their
N-terrnini. Based on these analyses, fusions were designed to have GFP attached at the C-
termini of the proteins (Figure 4-1, Appendix 4-1 to 4-3).

The ORF of PR] was ampliﬁed by PCR using a PR] clone in pBluescript 11 KS
(+) (He laboratory culture collection #742) as the template. The ORFs of At2g10940 and
FLA9 were ampliﬁed by PCR using genomic DNA of Col-O as a template. TaKaRa LA
Taq (TaKaRa Biolnc, Japan) was used for PCR. The primers used for PCR are in table 4-
1. The PCR products were cloned into pCR®2. l -TOPO (Invitrogen, Carlsbad, CA), and
their sequences were determined. The ORFs were transferred to pBluescript 11 KS (+)
containing a sGFP ORF (Chiu et al., 1996), in which the sGFP ORF is followed by the
NOS terminator. In all cases, the 3’ end of the ORF was attached to the 5’ end of the
sGFP ORF. Mutation-free PR1-GFP, At2g10940-GFP and FLA 9-GFP fusions
(including the NOS terminator) were subcloned into the Xhol/Spel sites of the pBD
vector (a giﬂ From Dr. Jeff Dangl laboratory, University of North Carolina, Chapel Hill,
NC), a binary vector carrying a kanamycin resistance gene for selection in bacteria, a
resistance gene for herbicide BASTA (glufosinate), and a rat glucocorticoid hormone

dexamethasone (DEX [Aoyama and Chua, l997])-inducible promoter.

130

(A)

 

 

 

 

 

Xhol Ncol Spel
| PR1 l GFP 1
(B)
Xhol Ncol Spel
[ At2g1094o E GFP E]
(C)
Xhol Ncol Spel
[ FLA9 E GFP a

 

Figure 4-1. Diagrams of the PR1-GFP (A), At2g10940-GFP (B) and FLA9-GFP (C)
fusions. Xhol, Ncol, EcoRI, Hindlll, Sal] and Spel are the restriction enzyme sites.

131

Table 4-1. Primers used for PCR to construct GF P fusions of selected Arabidopsis genes.

 

Gene Primer

 

PR] Forward primer : Xhol
5 ’ -CGGCTCGAGGCTCTAGAAAAAATGAA l“l'l'-3 ’

Reverse primer: Ncol
5’-GTATCCATGGCGTATGGG TTCTCGTT-3 ’

At2g10940 Forward primer: Xhol
5’-CGCCTCGAG CACTCTACTCAACATG-3’

Reverse primer: Neal
5’- TACACCATGGCTATGGAACAAGTGTAGC-3’

F LA9 Forward primer: Xhol
5'-GCCCTCGAGACTAAGCAAACCAATAATGG -3'

Reverse primer: Ncol
5'-CGCACCATGGCAAAGAGAAATTTCAAACATAAG -
3!

 

The restriction sites which were inserted in the primers for subcloning are shown as bold
and underlined.

132

Transformation of recombinant plasmids into Agrobacterium

The pBD derivatives containing fusion ORFs were transformed into E. coli strain
DHSa, and kanamycin-resistant colonies were obtained on solid Luria-Bertani (LB)
medium supplemented with kanamycin (50 mg/ml). These recombinant plasmids were
then introduced into Agrobacterium strain C58Cl (resistant to rifampicin and tetracycline)

by tri-parental mating with the E.coli helper strain pRK2013.

Production of transgenic Arabidopsis lines expressing GFP fusions

PR1-GFP, At2g10940-GFP and FLA9-GFP gene fusions cloned in pBD vector
and transformed in Agrobacterium were transformed into Arabidopsis. pBD-PR1 -GF P
and pB‘D-At2glO940-GF P were introduced into both Col-0 and the atmin7 knockout
plant, and FLA9-GFP gene fusion was introduced into Col-0 only. The transformation
was performed following the protocol of ﬂoral dipping (Clough and Bent, 1998). Ten to
fourteen-day-old T; plants were sprayed with 0.2 % BASTA solution (glufosinate-
ammonium, trade name Finale, AgroEvo Environmental Health, Montvale, NJ) to select
putative T1 transformants. The leaf samples of BASTA-resistant T1 plants were dipped in
30 uM DEX solution and they were examined by confocal microscopy. T1 plants showing
GFP ﬂuorescence, an indication of GFP fusion expression, were chosen for further
analysis. Plants of T2 or later generations in each transgenic line were used for confocal

microscopy and western blot analyses.

Confocal microscopy

Leaf samples were prepared from stable transgenic Arabidopsis plants. Prior to

133

confocal microscopic observation the leaf samples were dipped into DEX solution (30pM,
24 hours), to induce the expression of GFP fusions. Leaf pieces were cut and mounted in
water, followed by imaging with LSMSIO META inverted confocal laser scanning
microscope (Zeiss). A 40x oil immersion objective was used.

The GFP fusions were excited at 488 nm from an argon laser, and the emission
light was ﬁltered with a 505-530 nm band-pass ﬁlter to acquire the GF P signal. The
autoﬂuorescence from chloroplasts of mesophyll cells was ﬁltered by a 615 nm long-pass
ﬁlter. All images were examined and processed with Carl Zeiss AIM Version 3.2. Some
images were adjusted using Adobe Photoshop Element version 5.5 or 7.0 in brightness

and contrasts.

Plasmolysis of leaf samples
Leaf samples were dipped in 1M Tris-HCl solution (pH 7.5) for up to 30 minutes
prior to confocal microscopic observation. The samples were mounted on slides in Tris-

HCl solution (pH 7.5) or water.

Protein extraction and western blot analyses

Total protein samples were extracted from transgenic Arabidopsis plants
expressing OF P fusions. A leaf from each transgenic plant was detached and dipped in
the DEX solution (30 uM). After 24 hours, 20 mg of each leaf sample (fresh weight) was
ground in 200 uL of the 2X SDS buffer [100 mM Tris-HCI pH 6.8, 200 mM DTT, 4%
SDS, 20% glycerol]. Extracts were immediately heated at 90°C for 10 minutes and then

frozen at -20°C.

134

Prior to the loading of each protein sample on protein gels, extracts were thawed
at room temperature, heated at 90°C for 3 minutes, and centrifuged at 10,000 X g for 1
minute. Ten to 20 uL of the supernatant of each sample was used for SDS-PAGE
electrophoresis (the volumes of different samples were the same in an immunoblot
analysis). Total proteins were separated on precast gradient gels (4-20%, ISC BioExpress,
Kaysville, UT) or hand-made SDS-PAGE gels (7.5, 10, or 12% gels), then transferred to
Immobilon-P membrane (Millipore, Billerica, MA) using a semi-dry transfer apparatus
(SEMIPHOR, Hoefer Scientiﬁc Instruments, San Francisco, CA). Immunoblot analyses
were performed with a GFP-speciﬁc antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA). For estimating the sizes of proteins, PageRulerTM Prestained Protein Ladder

Plus (#SM1811, F errnentas lntemational Inc, Ontario, Canada) was used.

135

RESULTS

Production of transgenic Arabidopsis expressing GFP fusion proteins

The ORF of sGFP (Chiu et al., 1996) was attached to the 3’ ends of the selected
Arabidopsis genes (Figure 4-1). These fusion ORFs were introduced into Arabidopsis,
and BASTA-resistant T1 plants were selected. More than 10 different T1 plants
transformed with pBD-PR] -GF P or pBD-At2g10940-GFP, respectively, survived after
BASTA spray. T1 plants showing the GFP-ﬂuorescence were collected. From these plants,
two independent PR1-GFP or At2g10940-GFP lines were selected for further
experiments (Table 4-2, Table 4-3 and Appendix 4-4 and 4-5). On the other hand,
ﬂuorescence was not detected in any of BA STA-resistant T] plants transformed with
pBD-FLA9-GFP. pBD-PRI-GFP and pBD-At2g10940-GFP were transformed into the
atmin7 mutant as well. Among the BASTA-resistant T. plants transformed with PR]-
GFP or At2g1 0940-GF P, two independent PR1-GFP or At2g10940-GF P lines showing
GFP ﬂuorescence were selected for further experiments (Table 4-2, Table 4-3 and
Appendix 4-4 and 4-5).

The transgenic Arabidopsis expressing PR1-GFP or At2g10940-GFP were
analyzed by western blot analysis. The western blot analysis results showed that the
protein bands of the expected sizes for both PR1-GFP (approximately 44 kDa) and
At2g10940-GFP (approximately 58 kDa) fusion proteins were detected in both Col-0 and
in the atmin7 mutant backgrounds using the GFP-speciﬁc antibody (Figure 4-2). Besides
the protein bands corresponding to PR1-GP P protein and At2g10940-GFP fusion proteins,

there were additional bands corresponding to the size of GFP. The intensity of the GFP-

136

Table 4-2. Summary of transgenic Arabidopsis expressing PR1-GFP fusion protein.

 

 

Genetic background of Arabidopsis Line number Generations used for
analyses
COl-O #l T2
#3 T6
atmin7 #1 T3
#5 T2

 

The lines described in this chapter are marked with red color.

Table 4-3. Summary of transgenic Arabidopsis expressing At2g10940-GFP fusion
protein.

 

 

Genetic background of Arabidopsis Line number Generations used for
analyses
Col-O #9 T6
#16 T3
atmin7 #41 T2
#71 T2

 

The lines described in this chapter are marked with red color.

137

(A) (B)

(“3985‘ M 1 2_ 3 M 1 2 3
72 (KDQasl
55 72

H 5"— 44 kDa 55 + 44 kD
36 ' a
a 36 -
28 . 28
17
17
(C) (D)

(KDa) M 1 2 3 M 1 2 3
25 fKDa)
13 95
95 2% " 4— 58 kDa
72 p . +58 kDa

55 36 "
36 Q

28 17

Figure 4-2. Immunoblot analysis of PR] -GFP and At2g10940-GFP in Arabidopsis using
a GFP antibody. (A) PR1-GFP in Col-0 background (line #1). M is marker, lane 1: Col-0,
lane 2: pEGAD transiently expressed in tobacco leaf, and lane 3: PR1-GFP in line #1. (B)
PR1-GF P in atmin7 background (line #1). M is marker, lane 1: the atmin7 plant, lane 2:
pEGAD transiently expressed in tobacco leaf, and lane 3:PR1 -GFP in Col-0 (line #1). (C)
At2g10940-GFP in Col-0 (line #9). M is marker, lane 1: Col-0 plant, lane 2: pEGAD
transiently expressed in tobacco leaf, and lane 3: At2g10940-GFP in Col-0 background
(line #9). (D) At2g10940-GFP in atmin7 background (line #41). M is marker, lane 1:
Col-0 plant, lane 2: pEGAD transiently expressed in tobacco leaf, and lane 3:1ine #41.

138

sized bands was variable in different leaf samples (Figure 4-2). The transgenic
Arabidopsis plants which were transformed with the FLA9-GF P plasmid did not shown

any GFP-speciﬁc bands in western blot analysis.

The subcellular localization of PR1-GFP in Arabidopsis

Confocal microscopic examination showed that PR1-GFP was located along the
cell edge in the Col-O genetic background (Figure 4-3A and B) in independent transgenic
lines. To determine whether the cell-edge ﬂuorescence represents localization of PR] -
GFP fusion at the plasma membrane or in the intercellular space between cells, I
performed plasmolysis to separate the plasma membrane from the cell edge. Under this
condition, PR1-GP P was clearly observed in the intercellular space (Figure 4-3C and D).
This intercellular localization of PR1-GF P is consistent with its localization based on the
intercellular wash ﬂuid analysis, as previously reported (Uknes et al., 1992). Besides the
intercellular space, PR1-GFP was also shown along the putative Arabidopsis cell wall.
Almost all epidermal cells aﬁer plasmolysis showed localization of PR1-GP P in the
intercellular space including the putative cell wall (Figure 4-3C). The examination of
mesophyll cells showed a similar localization pattern (Figure 4-3D), but the intensity of
ﬂuorescence from the intercellular space was often lower than that along the putative cell
wall.

The localization of PR1-GF P was also examined in transgenic lines with the
atmin7 background. PR1-GFP was found along the cell edge, like that in the Col-0
background (Figure 4-4A and B). After plasmolysis PR1 -GF P was found in the

intercellular space and the putative cell wall as shown in epidermal cells and mesophyll

I39

r—i
, 10mm

.003-

 

Figure 4-3. Localization and expression of PR] -GFP in Arabidopsis, line #1 (Col-0). (A)
and (C): Localization of PR1-GFP in epidermal cell layer before and after plasmolysis,
respectively. (B) and (D): Localization of PR1-GED in mesophyll cells before and after
plasmolysis. respectively. White arrows in panel A and B indicate PR1-GFP signal before
plasmolysis. Yellow arrows in panel C and D indicate putative cell wall. C indicates
cytoplasm which was shrunken after plasmolysis. Note that the shrunken cytoplasm of
mesophyll cells in panel D contains chloroplasts. Ch indicates chloroplast with red
autoﬂuorescence. 1 indicates the intercellular space. S indicates Stomate.

140

 

Figure 4-4. Localization of PR1-GFP in Arabidopsis, line#l (in atmin7 mutant
background). (A) and (C): Localization of PR1-GFP in epidermal cell layer before and
after plasmolysis, respectively. (B) and (D) are the localization of PR l-GFP in mesophyll
cells before and after plasmolysis. respectively. White arrows in panel A and B indicate
PR1-GFP before plasmolysis. Yellow arrows in panel C and D indicate putative cell wall.
C indicates cytoplasm which was shrunken after plasmolysis. Note that the shrunken
cytoplasm ofmesophyll cells in panel D contains chloroplasts. Ch indicates chloroplast
with red autoﬂuorescence. 1 indicates the intercellular space. S indicates Stomate.

141

cells of the Col-0 background (Figure 4-4C and D). Again, the intensity of ﬂuorescence
from the intercellular space was oﬁen lower than that along the putative cell wall. Thus,
the localization of PR1-GF P inCol-O and atmin7 knockout background did not appear to

be different.

The subcellular localization of At2g10940-GFP in Arabidopsis

The localization of At2g10940-GFP fusion protein in independent transgenic
Arabidopsis lines was examined by confocal microscopy. At2g10940-GF P was found
along the cell edge (Figure 4-5A and B), which was similar to the localization of PR1-
GFP. After plasmolysis At2g10940-GFP was detected in the intercellular space and along
the putative cell wall (Figure 4-5C and D). In mesophyll cells, the intensity of
ﬂuorescence of At2g10940 from the intercellular space was often lower than that along
the putative cell wall.

Next, the localization of At2gl O940-GFP in the atmin7 background was
examined in independent lines. Compared to that in Col-0 background, the At2g10940-
GFP was found in not only along the cell edge, but also in the unidentiﬁed, round
structures (Figure 4-6). This localization pattern was shown in two independent lines

(Appendix 4-4 and 4-5).

142

 

Figure 4-5. Localization of At2g10940-GFP in Arabidopsis. line #9 (in Col-0
background). (A) and (C): Localization of At2g10940-GFP in epidermal cell layer before
and after plasmolysis. respectively. (B) and (D): Localization of PR1-GFP in mesophyll
cells before and after plasmolysis. respectively. White arrows in panel A and B indicate
At2g10940-GFP before plasmolysis. Blue arrows in panel C and D indicate putative cell
wall. C indicates cytoplasm which was shrunken after plasmolysis. Note that the
shrunken cytoplasm ofmcsophyll cells in panel D contains chloroplasts. Ch indicates
chloroplast with red autolluorescence. 1 indicates the intercellular space. S indicates
Stomate.

143

 

Figure 4-6. The localization of At2g1 0940-GFP in the atmin7 background (line#4l ). The
image was from the epidermal cell layer.

144

DISCUSSION

In this chapter, as part of an effort to establish a GP P fusion-based, in planta
imaging system for long-term study of the dynamic effects of pathogen infection on
vesicle trafﬁcking and/or protein secretion in Arabidopsis, the subcellular locations of
GFP fusions of two Arabidopsis extracellular proteins were examined by confocal
microscopy and stable transgenic plants expressing these proteins were produced. These
proteins were selected based primarily on the hypothesis that they might be related to
extracellular defense or putative cargoes of defense-associated secretion pathways.

Numerous studies have shown that PR] is an extracellular protein associated with
various forms of plant disease resistance, although transgenic Arabidopsis plants stabling
expressing a PR1-GF P fusion has not yet been reported. Although direct evidence for an
association of At2g10940 and At] g03870 to plant defense is lacking, the products of
these genes, a putative protease inhibitor/seed storage/lipid transfer protein (LTP) family
protein and a putative fasciclin-like arabinogalactan 9 protein (FLA9), respectively, were
suggested as putative cargoes of a defense-associated secretion pathway or a defense-
associated cell wall component in Arabidopsis.

Both PR1-GFP and At2g10940-GFP were detected by western blot analysis in
transgenic plants. The detected bands were of expected sizes, without signiﬁcant
degradation. This result indicates that both GFP fusion proteins were successfully
expressed in the transgenic Arabidopsis, and the GF P ﬂuorescence detected by the
confocal microscopy reﬂected fusion proteins. There were additional bands

corresponding to the approximate size of OF P in several protein samples, but the

145

intensity of the GFP-sized bands was variable in different leaf samples. There was no
correlation between a speciﬁc transgenic line and the presence or intensity of the GFP-
sized band, suggesting that appearance of such bands is likely caused by sample
preparation. Consistent with this possibility, these bands were not detected in every
examined sample. I examined the images acquired by confocal microscopy to see
whether there is ﬂuorescence in the cytoplasm, in which GF P alone is known to reside
(Appendix 2-2), but there was no signiﬁcant ﬂuorescence detected in the cytoplasm.

The results from confocal microscopic analyses coupled with plasmolysis showed
that both PR1-GFP and the At2g10940-GFP were localized in the intercellular space of
Arabidopsis (Col-O). Such localization is consistent with their predicted localization
based on the presence of a putative N-terminal signal sequence, and, in the case of PR]-
GFP, to that based on the apoplastic ﬂuid analysis (Uknes et al., 1992). The plasmolysis
condition which was applied to the leaves expressing PR1 -GF P and At2g10940-GFP was
the same as that applied to the leaves expressing the PIPA2-GF P fusion, which is known
to be localized in the Arabidopsis plasma membrane (EGAD line Q8: Cutler et al, 2000).
It was previously shown that after plasmolysis PlP2A-GFP remains in the plasma
membrane (Speth et al., 2009), which looked clearly different from PR1-GF P or
At2g10940-GFP fusions found primarily in the intercellular space after plasmolysis.

I noticed that after plasmolysis, both PR1-GFP and At2g10940-GFP were found
not only in intercellular space but also in the putative cell wall. This result suggests that
PR] and the LTP encoded by At2g1940 may have interaction with the cell wall. Further
experiments may be performed with plasmolysis plus cell wall-speciﬁc marker or

detector, such as propidium iodide, which stains the plant cell wall (Chen et al., 2009). It

146

is possible that the putative cell wall localization is caused by transgenic overexpression
of GFP fusions. If so, future experiments should be performed to reduce the expression of
fusion proteins by reducing DEX concentration or induction time.

The localization of PR1-GF P and At2g10940-GF P was also determined in the
atmin7 knockout mutant. PR1-GFP was shown in the intercellular space and along the
putative cell wall, similar to its localization in Col-O. This result is supported by the
observation that PR] in the atmin7 plants was detected in the intercellular wash ﬂuid
sample of those transgenic plants by the immunoblot analysis (Kinya Nomura and Sheng
Yang He, unpublished). Together these results indicate that either PR] is not delivered to
the intercellular space and/or the cell wall via the AtMIN7-mediated vesicle trafficking
pathway, or PR] could be transported by multiple vesicle trafﬁcking pathways including
that mediated by AtMIN7.

In contrast, the At2g10940-GFP ﬁision protein expressed in the atmin7 mutant
showed interesting dual localization: multiple, round structures were found inside the
cells, besides the localization along the cell edge. It is unlikely that those round structures
were from the nonspeciﬁc aggregation or degradation of At2g10940-GF P, considering
that the western blot analysis result revealed a protein with the expected size. It is also
not likely that those round structures were from an aggregation of the putative GFP,
which was detected in western blot analysis, because the ﬂuorescence ﬁ'om the GF P
alone does not show such distinct localization pattern (Appendix 2-2). Also, there was no
signiﬁcant ﬂuorescence in the cytoplasm, in which GFP is known to reside (Appendix 2-
2). The possibility that the overexpression of At2g10940-GFP caused the appearance of

nonspeciﬁc structures can also be excluded, considering that the Arabidopsis plants

147

expressing PR1-OF P and At2g10940-GFP expressed in the Col-0 genetic background
and the plants expressing PR1-GFP in the atmin7 background did not show these
intracellular structures. In addition, the round intracellular structures were shown in two
independent transgenic lines in the atmin7 mutant background (Appendix 4-5).
Altogether, these results raise the possibility that the atmin7 mutation affected secretion
of the At2g10940-GFP, and, as a result, at least some At2g10940-GFP was retained in
the cytoplasm.

Several further experiments need to be performed to conﬁrm my localization
study of At2g10940-GFP. Speciﬁcally, plasmolysis of the transgenic atmin7 plant
expressing At2g10940-GFP is needed. Some At2g10940-GF P was detected along the cell
edge, but its exact localization was not determined. Without plasmolysis, it is difﬁcult to
distinguish the intercellular space from the plasma membrane or even the cytoplasm. If
the cell-edge localization of At2g] O940-GFP in atmin7 plants represents the intercellular
space, the result can be interpreted that the secretion of the At2g10940-GFP fusion
protein was blocked only partially in the atmin7 mutant and suggests existence of
multiple pathways for the secretion of At2g10940-GFP. On the other hand, if the cell-
edge localization of At2g10940-GFP represents the plasma membrane or the cytoplasm,
the result would suggest a more signiﬁcant block of At2g] 0940-GF P secretion in the
atmin7 background. The identity of the At2g10940-GFP-associated round structures is
unknown and remains to be elucidated. They could represent nonspeciﬁc aggregations of
At2g10940-GFP or can be some type of subcellular structures.

The fusion protein FLA9-GFP was not observed by confocal microscopy in any

of the BASTA-resistant plants. Western blot analysis also did not show any speciﬁc band

148

detectable by GFP antibody. These results suggest that the expression of FLA9-GFP in T1
plants was not successful. FLA9 is a member of fasciclin-like arabinogalactan protein
(FLA) family of Arabidopsis (Johnson et al., 2003). FLA proteins possess a C-terrninal
hydrophobic domain, which is cleaved and replaced by a glycosylphosphatidyl inositol
(GPI) anchor (Gaspar et al., 2001). Therefore, it is possible that the C-terrninal GFP in

F LA9-GF P was cleaved off, and as a result, F LA9-GFP fusion protein was not correctly

expressed.

149

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152

CHAPTER 5

Conclusions and Future Perspectives

153

In the pathogenic interactions between Arabidopsis and Pst DC3000, one
important question related to TTSS effector-associated virulence is how Pst DC3000
TTSS effector proteins affect cellular processes in the host plant. The recent study of
HopM1, a TTSS effector of Pst DC3000, and AtMIN7, its host target and a putative
ARF-GEF protein in Arabidopsis, supported the idea that one of the functions of TTSS
effectors is to contribute to the virulence of Pst DC3000 by suppressing the vesicle
trafﬁcking in plant cells. This led to the research direction that combined the study of
plant-pathogen interactions with a cell biological approach. In my dissertation the
localization of HopM1 in plant cells was studied by confocal microscopy in order to
understand how its action in host cells contributes to the virulence of Pst DC3000. I also
attempted to determine the speciﬁcity of the defense-associated role of AtMHV 7 compared
with that of other ARF-GEF gene family members. Finally, the localization of several
putative cargo proteins of defense-associated protein secretion pathways were compared
in Col-0 and the atmin7 backgrounds.

The results in the localization study of HopM1 show that hole is localized in
TGN and early endosome. The N-terminus of HopM1 (HopM11-3oo) is also localized in
the TGN and early endosome like the full-length HopM1, although it seems more
transient compared to that of full-length HopM1. With the previous study showing that
HopM1 interacts and destabilizes AtMIN 7 (Nomura et al., 2006), this is another
important clue for understanding the virulence-associated function of HopM1. A possible
further research direction could be the study of HopM1 fusion proteins in Arabidopsis.
After collecting transgenic Arabidopsis lines expressing all needed HopM1 fusion

proteins and conﬁrming the correct expression of the fusion proteins, 1 suggest the

154

following experiments. First of all, confocal microscopic examination of expression of
Hole fusions over time is essential. Microscopic observation at early and later time
points (within or after 5 hours post-DEX treatment) could be performed. It is also
necessary to determine whether tissue death of transgenic Arabidopsis plants is induced
by full-length HopM1 fusion proteins in a similar way as described for the full-length
HopM1 without ﬂuorescence protein tag (Debroy et al., 2004; Nomura et al., 2006).

Nomura et a1 (2006) showed that the multiplication of the ACEL mutant is
enhanced in transgenic Arabidopsis expressing HopM1. They also showed that the ﬁrst
300 amino acids of HopM1 expressed in Arabidopsis suppressed the multiplication of the
ACEL mutant complemented with hopM] and sth ORFs. These results could be
reconﬁrmed in the transgenic Arabidopsis expressing full-length or truncated HopM1
fusion proteins. Compared to previous research results with HopM1 without ﬂuorescence
tag, it is possible to detect the localization change of HopM] fusion proteins by confocal
microscopy with time courses. Related to this, it would be interesting to determine the
localization of HopM1 fusion proteins after the inﬁltration of the ACEL mutant
complemented with aer and its chaperone ORFs, considering that Aer and HopM1 are
both from the CEL of Pst DC3000 and are functionally redundant (Alfano et al., 2000;
DebRoy et al., 2004).

HopM1 suppresses the SA-dependent callose deposition in the Arabidopsis cell
wall (DebRoy et al., 2004), but the mechanism of this suppression was not determined in
previous studies. With ﬂuorescence protein-tagged HopM1, it is possible to detect the
localization change of HopM1 (full-length or truncated) associated with this suppression:

the GF P/YF P fusions of HopM1 can be compared before and after the application of SA

155

or its analogue, BTH. Also, considering that ﬂg22 induces callose deposition in
Arabidopsis (Gomez-Gomez et al., 1999; Underwood et al., 2007) and that
PAMP/MAMP-triggered signaling pathway in Arabidopsis has positive interplay with
SA—mediated signaling (Tsuda et al., 2008), it would be interesting to detect the
localization of HopM1 fusion proteins upon the treatment of ﬂg22. In addition, it would
be also interesting to detect the potential change of speciﬁc marker proteins of endosomal
compartments, including AtMIN7, after the inﬁltration of bacterial pathogens or in
transgenic Arabidopsis expressing HopM1 or its fusion proteins.

The experimental design for determining the localization of HOle in plant cells
is not a natural situation. It depends on the in planta overexpression of a speciﬁc TTSS
effector (or its fusions) and the amount of the effector protein is unnaturally high
compared to that translocated by the TTSS. The ideal approach is to introduce HopM1
fusions in Pst DC3000 and let it be delivered through the TTSS, but this approach was
not successful in previous studies. In this study, instead, the timing of microscopic
observation was limited, to avoid the overexpression of HopM1 fusion proteins as much
as possible.

The results of the study of Arabidopsis ARF-GEF genes in this dissertation are
not sufﬁcient to obtain a clear determination of the role and importance of each ARF-
GEF gene in defense. None of the studied ARF-GEF genes, however, showed the
characteristics indicating that they are involved in Arabidopsis defense against the ACEL
mutant. The possibility that AtBIG2 has additive role in the defense against the ACEL
mutant was not supported, based on the results from the multiplication of the ACEL

mutant in the T-DNA insertional mutant and the yeast two-hybrid assay with HopM1.

156

From the multiplication result in the knockout mutant and from the yeast two-hybrid
assay data, respectively, we can conclude that AtBIG4 and GNL2 are not important for
defense against the ACEL mutant. The studies of T-DNA insertional mutants of several
ARF-GEF genes suggest their possible roles in development and grth of Arabidopsis
(Steinmann et al., 1999; Geldner et al., 2003; Teh and Moore, 2008; Richter et al., 2008;
Tanaka et al., 2009), but this was not studied in this dissertation. Although the defense-
associated functions of Arabidopsis ARF-GEF genes and their physical interactions with
HopM1 1-300 were not completely determined in this chapter, it is still possible that these
ARF-GEF genes are involve in the defense against other pathogens for Arabidopsis
which are not Pst DC3000, such as fungal pathogens. It would be interesting to study the
roles of these genes in defense against the non-bacterial pathogens.

The location of several Arabidopsis proteins which are putative cargoes of
defense-associated protein secretion was studied by confocal microscopy. In this study
the localization was determined in Col-0 and the atmin7 backgrounds. PR1-GFP was
localized in the intercellular space, and this localization was not changed in the atmin7
background. On the other hand, At2g10940-GF P showed an interesting intracellular
localization in the atmin7 background, suggesting that its location is affected by the
AtMIN7-mediated trafﬁcking. It would be interesting to study the role of At2g10940 in
Arabidopsis defense. Unfortunately, we do not have sufﬁcient information for
understanding the function of At2g] 0940 besides its expression pattern (induced by the
hrpS mutant and suppressed by Pst DC3000 [Hauck et al., 2003; Roger Thilmony and
Sheng Yang He, unpublished]). At2g10940 is annotated as a lipid transfer protein (LTP),

but the nucleotide sequence of At2g10940 does not share high similarity with other LTP

157

genes (The Arabidopsis Information Resource, htg)://www.arabidopsisorg). Considering
the altered papilla formation in atmin7 mutant after the inﬁltration of the ACEL mutant
(Nomura et al., 2006) and the old annotation of At2g10940 as a putative cell wall
component (Hauck et al., 2003), At2g10940 might encode a putative component of
papillae. This idea can be tested by the inﬁltration of mutant bacteria which induce cell
wall-associated PTI into transgenic plants expressing Atg210940-GFP (the promoter can
be substituted by its native promoter). The study of T-DNA insertional mutant of
At2g10940 is challenging, because mutants containing T-DNA insertion in the ORF of
At2g10940 are not available (The Arabidopsis lnforrnation Resource,
http://www.arabidopsis.org). In the case of FIA9, this study does not provide data for its
localization in Arabidopsis cells, possibly due to its structural characteristics. There is an
example of GP P fusion design for an arabinogalactan protein (AGP), in which the GFP
was inserted after the putative N-terminal signal sequence (Sun et al., 2004), but this
approach requires an exact prediction of N-terrninal signal sequence. Like At2g10940,
F LA9 is induced by htpS' mutant and suppressed by Pst DC3000 (Hauck et al., 2003), but
other characteristics of FLA9 are unknown, and there is no available mutant containing
T-DNA in its ORF (The Arabidopsis Information Resource, http://www.arabidopsis.org).
The experimental system used in this study, (i.e., DEX-induced production of
GP P fusions and confocal microscopy) has advantages in that GFP fusion construction
enables the visualization of the localization of selected Arabidopsis proteins in living
cells under different conditions. For instance, the factors affecting Arabidopsis defense
such as the inﬁltration of different bacteria or application of chemical agents such as

BTH or ﬂg22 can be used for the localization study of GFP/YF P fusion proteins: some of

158

these suggested conditions were preliminarily applied to the transgenic Arabidopsis
expressing PR1-GFP (Appendix 4-6 to 4-8). On the other hand, using a DEX-inducible
promoter had advantages and limitations. It was useful to control the expression and
timing of transgenes, which is advantageous compared to using a constitutive promoter.
At the same time, the DEX-inducible promoter drives high expression of transgenes
resulting in excessive amount of fusion proteins. This situation can make the localizations
of fusion proteins incorrect by the “over-spill” of the overexpressed fusions into unrelated
subcellular compartments (Crofts et al., 1999; Brandizzi et al., 2004). Also, compared to
constitutive expression or native promoter-dependent expression, DEX-induction of
transgenes depends on the uptake of the DEX into the plant tissue through stomata. The
uptake of DEX in each experiment was not the same, so the expression level of GP P or

YFP fusion proteins was not completely predictable.

159

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161

APPENDICES

(A)

F—II

j I] 1-1 m '2 1:1 L1 ITI

   

Appendix 2—l. Dual localization result of Hole-GFP and PTSl-dsREDZ. (A) HopM1-
GFP expressed in mesophyll cells of N. benthamiana (marked with red arrows). The
autoﬂuorescence from chloroplasts is also shown (Ch). (B) PTSl-dsREDZ expressed in
mesophyll cells ofN.benthamiana (marked with blue arrows). (C) Merged image. Note
that PTS 1 -dsRED2 is excited by both 488 nm and 543 nm lasers (yellow punctuate
structures pointed by yellow arrows).

162

(A

 

Appendix 2-2. Transiently expressed of EGF P in pEGAD vector in Nbenthamiana. (A)
Fluorescence ofthe EGFP detected in (A) epidermal cells and (B) mesophyll cells. The
leaf samples were examined 48 hours after Agrobacterium inﬁltration. All images were
from single focal planes.

163

Appendix 3-1. The genomic sequence of AtBIG] (At4g38200). Start codon (ATG) and
stop codon (TGA) are in red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The partial 5’-UTR is indicated by black and small
letters. The putative T-DNA insertion of salk_066766 is highlighted with green color.
The forward and reverse primers for genomic PCR to screen homozygous salk_066766
are bold, underlined and blue-colored.

164

    

 

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Appendix 3-l (continued).

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167

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Appendix 3-3 (continued).

 

 

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172

Appendix 3-4. The genomic sequence of AtBIG4 (At4g35380). Start codon (ATG) and
stop codon (TGA) are in red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The putative T-DN‘A insertion of salk_082249 is
highlighted with green color. The forward and reverse primers for genomic PCR to
screen homozygous salk_082249 are bold, underlined and black-colored. The forward
and reverse primers for RT-PCR are in bold, underlined and blue letters.

173

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174

Appendix 3-4. The genomic sequence of AtBIG4 (At4g35380). Start codon (ATG) and
stop codon (TGA) are in red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The putative T-DNA insertion of salk_082249 is
highlighted with green color. The forward and reverse primers for genomic PCR to
screen homozygous salk_082249 are bold, underlined and black-colored. The forward
and reverse primers for RT-PCR are in bold, underlined and blue letters.

173

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174

Appendix 3—4 (continued).

 

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175

Appendix 3-5. The genomic sequence of GNOM (At1g13980). Start codon (ATG) and
stop codon (TGA) are in red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The putative T-DNA insertion of salk_103014 is
highlighted with green color. The forward and reverSe primers for genomic PCR to
screen homozygous salk_103014 are bold, underlined and black-colored.

176

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Appendix 3-6. The genomic sequence of GNL1 (At5g39500). Start codon (ATG) and
stop codon (TGA) are red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The putative T-DNA insertion of salk_067415 is
highlighted with green color. The forward and reverse primers for genomic PCR to
screen homozygous salk_067415 are bold, underlined and black-colored.

179

 

 
 
 

 
 

 
 

 

 
 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

 

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A “I _ _ V. .

Appendix 3-6 (continued).

‘TGA

18]

Appendix 3-7. The genomic sequence of GNL2 (At5g1 9610). Start codon (ATG) and
stop codon (TGA) are in red and bold letters. Exons are in yellow color and capitals, and
introns are in violet and small letters. The putative T-DNA insertion of salk_021757 is
highlighted with green color. The forward and reverse primers for genomic PCR to
screen homozygous salk_021757 are bold, underlined and black-colored.

182

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"”""“T"G“GJTAA£AJGAAAFAAJAn.cAG'AAuua. GTTURACACSEA3:HT32
~~1~,V’1w» ‘ ".“CZA v-mrv vA ~1~wv-r—nM~—A, 1‘? v. , 7. pg‘ a». ﬁ'ﬁf‘
J- "hALJX;A.H_ A.\.ALL,\_«_AAH/-.\_7’JZ‘ .."AA "At.;“‘-1..G.A*‘.-'qlu
”A”, . V. . ,7. A A. A., r 1. Va ﬂ~:(.r:y-~ lY-n “-1!“ qAA—Anm 7 ~-\ ”V‘L,
C.RnCA’L". A x A .A _ x. A AA uA JG 9 H. \HHAA 4..- CHM)“: '. A GU .1...
m w, r-w-w-m- m- « w-AA». A A "A.A., ,-,A ‘,An- v.r~:Cr~~; 1"
Axl1l) AAA-» '11 A .A 'K—R‘AJVnHl PM]! .\.\.::i .."A LA”
“F.,‘V. ‘Vnynﬂw. A. A‘.‘ ,—.:-A A.“ m, m. A .A «NHTTW , , ~ ::vyuvwr‘r~‘ «AA-AA , ~ . A.,. -n
not, GALGA. 1v . MT A A .“A‘UAAMAA A. . mm. ‘—-.l A ".'aA’E AU mun-1.- 3.1“- .. AHA ,I-AI A '"SAAA .AV

“'Y‘
'1.
y Nirvrn‘ mehr-wwwxn-q-a-y-v. m -p- r: ~ a... m n ﬂ-‘n
Aha; AtzvrhAAJ-ﬁr'AAAA/A: AAJA., k \ V1,,7ur..-ﬁu AN ‘11 '1' ("Curl—1L: JhJILIV‘AHH“ “it: TTF'J‘:I‘JI“.T.1A:1

GAUEC'I‘GTAAATD GA 1'5 Atl ART T51"; PASAQTTT‘; AAA "“IT‘TJCFAA'éf‘ t,.A%cP"" ’ TCCU Tgtac gt tcatt
aactataatctttttactacaaatgtttattttgtcc tcttttaagacagcaat aataagttatg tattaa

   

   

   

aacag‘.3A“AL‘-"“ A A. I." L513? JAT GAL. 1 “CG TTTGLA l..“.x“\"V+“A 3’"'.".“~‘~ «A l“.5.uIATC"I““A“T “CPA-A3" A A3" . 31: » “FAFA'TCT
A" “TESTAS. . ":fiz'A EBT'TTG " Z“: ’“VAJ‘A "53.3.9317 “PK“AA TESL? 3.-'AC“-CT~'E-2'l“"A‘_“.“\. 3 “ATV":A ‘“ ““ 1' 1' 1'1“":
GAG-a" THAC'.“ “" “53"“? .TI7."T“C-AG EAL T’JASEAIJ“ (" .TGAA'A AC“ l'f _7‘E. .TCAAA" 12G
ACA’“SASCGGGGCATAl“ "3133 ":T‘CA""T~"T’ “ECG“?"L'IG’," if} T A-AA- TGTT
aTTL“HA:AG'."‘L‘L5’A ’T“’":/'~A :rAAC _ 1.1.1A'A_“3.'—A~A;3.“"‘~.A (“1 A ("1113.71 3."’."'".“".“".“3’“"""’."

   

 

AA}. . - m?" A' 1.21. 9.17363 . _EA“A(£TT.FA.~.,~V'_ A; " "31‘33'11‘"GIL”F‘M- ’“‘“ "1:111; 313'“ " 1‘” "T2, u "AA 313
T CAAGGI—‘j. SAT TTZAT’I‘T’IK '“"""f~JAT‘~TAT’TA'IGGALEC’I‘E :1“ 1' " “AA"
TSTA 311 GIATACAGAM.ATKWWTI“S””¢“‘“CGCArgtaagtcctcrgttttttataacattaaaacct

aaaactctgtttat tct:caactttgtxaacaacaa‘cgtgttgatttattag'ST‘ L‘CA’A'L'GI-A 1;.3'171‘1‘A’TIAJA17’TC

 

   

V,‘ rrn “PCT“ r< rm pr vH- f‘
'r' 'T‘ AGGlh“. hi: ..I

 

 

A ,. mp." m t- mm ~ an "..A". .mwhp... "A. u a, EMTWA r. ”A“ A AA in” ~ A A A n, A fan; .n
1’" :11" ‘ TL'“ I H "AHA A‘ v, A\'.'\ JnuA’IA't A TAD‘..3'.‘-’G1U.TT'_TIVGUlll“:"A.AAA“AA ﬁnal AAA»
"“71”,”. A A A ‘, ,A An». ~,—,.~V ”..A A, . .Aivv Adv" Ar". AV ~~~n ﬁnrv'nvn m A. A A” “A. w '5'
ALVA _1rAA;n;Lv;CA:-‘AJ:A:- r. .’l“_ .AsrAC,,;A".VVr'AH_ GA'_'-,_ A’lkrtzrtuA: -.--r“-:: 1 .A.-GAA:.A".:'.,VAA".;\:A"~A-A :AJ
I'rm‘r-w -"m'~mr"' ""|T“"‘y"A‘-T"“."'7*' ~ﬁA‘.vv r-rv' rr ‘v"’Y '1': “Fr
A. L‘ .cA-‘A’I’ A‘.:Ar‘A‘\1h3ArT..TrA A A --;:'_~*A:.~. :r'.--. :n‘u-“x-‘AA ANA A _, AAAA’" G Hdrph-ﬁt.I-\ - {op AC- ' 'A

 

_V‘nrr—N‘ mr‘ “A. am“, ”A. - 5.‘
t r-‘A. v.1»). ., r . x} A aH r . ‘1: A . -.Vr‘A\,I~\_vH‘.\,/'\

~ 1 (1

 

J
nnmumm ‘Vnn—T‘nr—n—nnm « ‘q—Aw—‘A ~ h" vyn-m
A . AVA A _A

. A .H. A AA rAI-Ar‘: A ”GI-Arm‘s VV

       

r. .V. r“ a A”, - _, ”,1 V‘ P“, ”a”. Annnnmnm-«n
k1‘1H.._111 '1T7AI‘A’“1::.A‘\H 1M." A - 'AJU :M A "AA":— . :‘AAEAJA‘IA 2.13:1 A 3A1 TAIR I‘M-ﬁt T:A/r"A.-‘A A ‘1. .‘ . r‘Ar‘mJ V 'V L‘. _ H». - J - A :1
“VA,” VA - , I,'\I\'\Fr\- n — .«n—‘p-AA an; «an. ~Irv‘n ,Am‘H .,~.\,-.,,Vv.~,~v "7., ,n5
hur‘A yr'A- AAA 1 A MIA 351’: ..\, A “:37.“ :r‘. A 3'1:A"'. A #1:". ax, A A“ Art!“ «A ."A L‘. A "A" 1 1:93.". _ ‘o’l but. Artur. FL A“. l": AA'".
Av :25}? .“mﬁi’ ~G/“AAA‘ 31"" ; "AGE.“ G.A.jﬁ- .41.. AF? TEAS; G712“ "GUT ’“ I."«J\“<.T(1‘A’ITTCAA A "13'":- GAT 3.A<’,“:P‘~A.“‘.l-'L1A.“~IGG 3;

 

W“ AZTA -C::“AGTA.TTSEAA :TATAACTACTT r“:G’l".“"’"VA 31AA(“l’“"”““"‘““*-AI’“’“HJA’I _:CC“‘.’.“"“AL".’“TTCA'TTTTTCA
GA'I” "A" A"’“GGGTT-GL§A ‘. Hz-SACA.“"A“'3F~ 'TTA" GA" JAT" . ’.“-’“'f- ““ SGA EATCGTGAA"GAA -“ AA fiA'I'“'l'CA GT

JTI'lf'l‘"l“A “NAAGC’” '“A 13")“; AACATTTJASTT" .3: ‘3’.“1"FAT jkA""T‘TC§F-.TA".C“ IG'“FA“.‘ A“3“A’_“AVJVA1"A‘"TTC’I“

     

 

 

 

 

   

     

Tit-TAX“; "ATT'I"“-’§G’I"I“P. v1“".“.’“.GG.“A'5." ’3": “ T"AAAA .TGLEEL-iA“ 1:1..Tl’iA‘T“"“Ea.~.-GC“’“'“T _ T’" TGAGPGIAT‘ CIT
TGA'ATCAAAAA A? TCA A G “ ’“ "AA“ '“AGI TA‘CT’ZT { IA-CA'I“ I TTCTSTTAG ”3’3”.“ 1' 'l ETA-“HATC
"’7’“ AA “AC “SEAT".FAALTATPFA 137:9“. V. A A.A\A\1.AAacAAAALL-A CA ’“CA‘AGFLA :A'Tt ACT"'A“.“A'“’S 2’31: A.A' fA“"“I—'.C
G’E‘". .TTE—JAI “Ft _1I§’¥A‘“AF«_“ "A .“I’§A“A’”“':"“ “'“ArAA "115.5-“A1‘A _“;“.'.“C“TC'I SA'3""""TT’LA’:’Z”"L"A’T "SCFLACAAA’"3
Cr“ "A" ' "3"“T’“. 'A“A‘“A'T“C“V"AC,1L\CIF\"“‘A A 2'2“"7‘31 "V 33;"- WASP-A SGA:TG“.ST“ I'Ff-A’.‘ 3. “SATA “AA
Al’fAA-a" ’_:'P-.'“'l /“A(““‘-AC“ F: .TAC."’““““ . " ’I‘ASTT ."AAT‘S" A C“; A . ““A" ".'l‘uw'“'AAk J “~.T.~“A’l"“' l'TT-G “.A..A_.“C;“A’l’“
T'". _‘f‘zAE-G'I i“.'“A‘“-“C“A~u “ “ TAG. “.’“A’ECIA""A“TTL“ "‘1':"l““A:“:CTT~"r";\ULAL\ “.'TCI‘AGA' "“42“ TGAA’" A GE‘AG'" ’3CTTCA'I'GA'LA"
G'TG “‘" _t’EANT"“C“’I“A A "2AA,""71,“" A Ali-“ASIA.“ GA ART-.1 l a"? t C?".“:“A’.“i":{“.£7(.“ . TERAGACA’I ACT" “ “."C
G"T"‘C“’“. ’l“ ;"'T JA “J-WT _“F-"I’JZCG TGTTAAAE". Cc“ .’TT “VAC“.A ".’l‘r \ ACG’TSF'JAGA 3A’f""’1“’.“ (““3 g "TAG

   

A, "V., a V. ..,.V [V "AHA."- A, ”M., A-nm “WM .rmh V w‘ I A mM,-.‘A" -. A. r v.

T"A'GP anm AC G GAAAuuf A ('CGUA'I A .;r A ”WS.TT cs.A ' r"; Vn
AA,“ “("1” A .

6:111th ,J'J; _: 1A

AT T’““'.3T A‘TSAGA

     

 

"WT/“\l—‘rTr-ev-r' -—A- y A “Z";Lr;~ ’“vn‘W‘PnTT': 11m x 321:. “‘1'”?er

“.Z"l"’“LL.: ‘h:- " A-‘-Jr,rx .‘A; Huu‘"‘.::n A A ’" I“, .Al" A
"AMV. ,wmn . A..”- V” r‘rvf‘«~-\
.A’: Hle'iA

,_

 

 

   

"rv ."V(V1 —- v wnxr'
.,\1_::‘\.AV.‘.\~:A"Lr* H- AH. -AH‘XA'JAAL Pt'JLGAWlHA -V.

 

,. . Tmm A A A ,An- mrwnmu V~ —A.~/—,~'—,~V /-<~!~Uvr“ﬁv n w*"1f.""\F‘PT/‘r~r-Ar—FP
AHALAA- A A‘ At). V :r‘.» J.- lr‘AA-AA-J-Jx: .-..~._71A; A . A - _H..A’ ..A , c A A J 7-_,. “\ :HL RAGAIACS
A , MAT—2w. »,.‘-—‘...—~,., . A "A., m~7.,~..,. A‘qm,, A-,,.,HA..-,..T .‘ A VV‘~.~—.
.L‘AA’AUA'LI‘AA AM 1. A A A \Jlj. “.h.‘ '[\‘.I‘ .131 1A l". urn". .MJ-Adrtx. A A Arh‘f‘hv . ,Ar- .I‘AJ'U'L'MJU‘A- .1: .:1.1:11“‘A- _A J~1 .
1‘ A «a. v A Anna ,V~-v‘,<A.-A~. AMT ~11" . AA-ny- <- , Ann ’7‘ -.~ an —Am<‘n-‘~H nnvm v-

kHLLA'XANA T::1:“\2‘ ru '24.; a-“ _LJK', A‘srtA A); A .1- "71“JA H1321. »TLKJ‘~JA ,‘_1::A\ . .‘.”.;‘.A‘_ Ar‘.""‘_1‘

 

AAGCTGC (“AGAGAIAAGGVAT‘ A3931: TT" AGT.A"AGCTF\’I‘C 1A A..}‘GAA AG “ELAGAGAACGGTTF- GTT"T 'I‘GTTGGGAATT
TGA"CATAA-AC CA A 'I'GCATTATCTAACGTCCACCGGTTCAAACA'I G A

GAAA'“ cc
TC'I'G'TTGAGAAGAUAC T’EPASAAAWITGGATCT'; .ATGGCFA “AA" TGT’"G("’“PAAAA'"GG"FAGAP

 

   

 

 

     

   

AA:”3C1TCTA1 umt':“s"wAA:A:r-A.nArccsacAa: . 3393T7$A“‘ ‘

LAAACAAF“TWA:ASG“_T'HA'““"HYT?A CATJTT” ';A AAAﬂmCT"‘qAH

ACI"TTGC CSCCGAGAHJAGAIAA1\A ﬁSA”ZAGTC:CZTCTC.A?AGP“AEGT“W‘lPTATGGFT?F

”GA,GAT“TT""»TIFACAC T7"”GG:TGTA75TAT:GCA:“HECGATE c37111wvcznvnnrccr'
CT'GCAT AGArCFT T'ch:.c:nr:" AAA :ziAAA - ASS :
cc:r G.AGATAGGTATwrw VTGCTFAT ”TGCT?“ACTT37AACAAJTL3.1UAAAJTI?

 

Appendix 3-7 (continued).

~-_‘*IT I‘ 1V;.‘> “-TIT v'~'~f--":"1"--"w';r~

_v ..A. A ~ & I v V v A .l 4 2‘ >1 .1
.

TAGacttttattattgtactatgtaattcatgat

ttaatgctcaaattatatgatcaacttcaagaagtcatgagattttggatatatatagcttctactaattc
catagagcaatcatcaattttatggcaaataatccatattaaatatatatacccttataagtctcattcaa
ttaccacataccacattagtctccatttttttagacaacacattaactccatagaagagtgatttgaagca
a

184

Appendix 3-8. Comparison of the nucleotide sequences and amino acid sequences of the
C—termini of GNL2. (A) The comparison of the nucleotide sequences of the C-termini of
GNL2 in TAIR (GNL1-T) and that of the clone used for yeast two-hybrid assay (GNL1-
C). The different nucleotides are highlighted in yellow color. (B) The comparison of the
amino acid sequences of the C-termini of GNL2 in TAIR (GNL1-T) and that of the clone
used for yeast two-hybrid assay (GNL1-C). The different amino acids are highlighted in
blue color.

185

(A)
GNL1-T
GNL1-C

GNL1—T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1—C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1—T
GNL1-C

GNL1—T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1—T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

AAACTTAGGAAGCTTCAGCTTCTTCCACAGTCTGTTATTGAGTTTGAGATAAATGAAGAA
AAACTTAGGAAGCTTCAGCTTCTTCCACAGTCTGTTATTGAGTTTGAGATAAATGAAGAA

AATGGCGGATCAGAATCCGACATGAACAATGTTTCGAGCCAGGACACAAAATTTAACCGA
AATGGCGGATCAGAATCCGACATGAACAATGTTTCGAGCCAGGACACAAAATTTAACCGG

CGACAAGGTTCTAGTCTAATGGGTCGGTTTTCACACTTCTTGGCATTAGACAACGTGGAA
CGACAAGGTTCTAGTCTAALbbblbbblllTLACALliLlibbLATTAGACAGCGTGGAA

 

GAATCTGTAGCTCTTGGAATGAGTGAGTTTGAACAAAACCTTAAAGTCATAAAACAATGC
GAATCTGTGGCTCTTGGAATGAGTGAGTTTGAACAAAACCTTAAAGTCATAAAACAATGC

 

AGGATCGGTCAAATATTCAGCAAAAGCTCAbfbllbLLbbAlbllbLbblblleATCTT
AGGATCGGCCAAATATTCAGCAAAAbLTLbbTblTbbLbbAlbllbbbbiblLbAATCTT

 

GGTCGGTCTTTAATCTATGCAGCTGCTGGGAAAGGACAGAAGTTTAGTACAGCTATCGAA
GGTCGGTCTTTAATCTATGCAGCTGCTGGGAAAGGACAGAAGTTTAGTACAGCTATAGAA

GAAGAAGAGACGGTTAAGTTTTGTTGGGATTTGATCATAACCATTGCATTATCTAACGTC
GAAGAAGAGACGGTCAAGTTTTGTTGGGATTTGATCATAACCATTGCATTATCTAACGTC

CACCGGTTCAACATGTTTTGGCCAAGTTACCATGAATATCTACTCAACGTTGCAAACTTT
CACCGGTTCAACATGTTCTGGCCAAGTTACCATGAATATCTACTCAACGTTGCAAACTTT

 

 

LLbLiLllllLACCCAllLLblllblLbAAAAAbbbLlLLLbbbTTTATTTAGGGTATGC
LLbLiLllliLACCCAllLLbllelLbAAAAAbbbLTLLLbUULilﬂTTTAGGGTATGC

 

 

ATCAAGATTCTTGCTTCTAACCTTCAAGACCATCTACCAGAGGAGTTGATTTTTAGGTCC
ATCAAGATTCTTGCTTCTAACCTTCAAGACCATCTACCAGAGGAGTTGATTTTTAGGTCC

TTGACAATAATGTGGAAGATAGACAAAGAAATCATTGAAACATGTTATGACACAATAACA
TTGACAATAATGTGGAAGATAGACAAAGAAATCATTGAAACATGTTATGACACAATAACA

GAGTTTGTCAGCAAGATCATTATAGACTACTCTGCGAATCTGCATACCAATATAGGGTGG
GAGTTTGTCAGCAAGATCATTATAGACTACTCTGCGAATCTGCATACCAATATAGGGTGG

AAAAGTGTTTTACAACTACTTTCTTTATGTGGAAGACATCCAGAGACCAAAGAGCAAGCG
AAAAGTGTTTTACAACTACTTTCTTTATGTGGAAGACATCCAGAGACCAAAGAGCAAGCG

GTGGATGCTCTCATCGGTTTAATGTCATTTAACGCATCGCATCTATCGCAGTCAAGCTAC
GTGGATGCTCTCATCGGTTTAATGTCATTTAACGCATCGCATCTATCGCAGTCAAGCTAC

GCTTATTGCATCGATTGCGCCTTCAGTTTTGTTGCTTTAAGAAATAGCTCTGTTGAGAAG
GCTTATTGCATCGAlibLbLLliLAbllrlelbblTlAAGAAATAGCTCTGTTGAGAAG

 

AACTTGAAAATATTGGATCTCATGGCAGATTCAGTGACAATGTTGGTAAAATGGTACAAA
AACTTGAAAATATTGGATCTCATGGCAGATTCAGTGACAATGTTGGTAAAATGGTACAAA

ACTGCCTCTACTGATACCGCGAATAGCTATAGCCCGGCAAGCAACACAAGCAGTTCATCA
ACTGCCTCTACTGATACCGCGAATAGCTATAGCCCGGCAAGCAACACAAGCAGTTCATCA

 

TCCATGGAAGAAAACAACTTGAGAbbbelAALillblleTCAlLllilLLlLAAACTC
TCCATGGAAGAAAACAACTTGAGAGGGGTTAALlElbIILATCAlLililLLTLAAACTC

 

186

Appendix 3-8 (continued).

GNL1-T
GNL1-C

GNL1—T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

(B)
GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1~T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

TCCGAGGCTTTTCGAAAAACGACCCTTGCACGCCGAGAAGAGATAAGGAACAGAGCAGTG
TCCGAGGCTTTTCGAAAAACGACCCTTGCACGCCGAGAAGAGATTAGGAACAGAGCAGTG

ACGTCTCTAGAGAAAAGTTTCACCATGGGTCATGAAGATCTTGGATTCACACCTTCTGGT
AGGTCTCTAGAGAAAAGTTTCACCATGGGTCATGAAGATCTTGGATTCACACCTTCTGGT

TGTATATACTGCATAGACCATGTCATATTCCCAACAATCGATGACTTGCATGAGAAGCTT
TGTATATACTGCATAGACCATGTCATATTCCCAACAATTGATGACTTGCATGAGAAGCTT

CTCGACTATTCAAGGCGCGAAAACGCGGAAAGAGAGATGAGAAGCATGGAGGGGACGTTG
CTAGACTATTCAAGGCGCGAAAACGCGGAAAGAGAGATGAGAAGCATGGAGGGGACGTTA

 

AAGATAGCTATGAAAQIbLlLATGAALbL111L1LbblllACTTGGAACAAATTGTAGAA
AAGATAGCTATGAAAGTGCTCATGAACGTTTTCTTGGTTTACTTGGAACAAATTGTAGAA

AGTGCTGAGTTTAGAACTTTTTGGTTAGGAGTGTTGAGGAGAATGGATACGTGTATGAAG
AGTGCTGAGTTTAGAACTTTTTGGTTAGGAGTGTTGAGGAGAATGGATACGTGTATGAAG

GCGGATTTGGGAGAGTATGGAGATAACAAACTTCAAGAGGTTGTCCCTGAACTTTTGACC
GCGGATTTGGGAGAGTATGGAGATAACAAACTTCAAGAGGTTGTGCCTGAACTTTTGACC

ACCATGATTGGTACCATGAAGGAGAAAGAGATTTTGGTGCAGAAGGAAGACGATGACCTT
ACCATGATTGGTACCATGAAGGAGAAAGAGATTTTGGTGCAGAAGGAAGACGATGACCTT

TGGGAGATTACGTATATTCAGATTCAATGGATTGCTCCAGCGCTCAAGGATGAGTTATTT
TGGGAGATTACGTATATTCAGATTCAGTGGATTGCTCCAGCGCTCAAGGATGAGTTATTT

CCCGATGAAGAGATTTAG
CCCGATGAAGAGATTTAG

KLRKLQLLPQSVIEFBINEENGGSESDMNNVSSQDTKFNRRQGSSLMGRFSHFLAL E
KLRKLQLLPQSVIEFBINEENGGSESDMNNVSSQDTKFNRRQGSSLMGRFSHFLALD E

ESVALGMSEFEQNLKVIKQCRIGQIFSKSSVLPDVAVLNLGRSLIYAAAGKGQKFSTAIE
ESVALGMSEFEQNLKVIKQCRIGQIFSKSSVLPDVAVLNLGRSLIYAAAGKGQKFSTAIE

EEETVKFCWDLIITIALSNVHRFNMFWPSYHEYLLNVANFPLFSPIPFVEKGLPGLFRVC
EEETVKFCWDLIITIALSNVHRFNMFWPSYHEYLLNVANFPLFSPIPFVEKGLPGLFRVC

IKILASNLQDHLPEELIFRSLTIMWKIDKEIIETCYDTITEFVSKIIIDYSANLHTNIGW
IKILASNLQDHLPEELIFRSLTIMWKIDKEIIETCYDTITEFVSKIIIDYSANLHTNIGW

KSVLQLLSLCGRHPETKEQAVDALIGLMSFNASHLSQSSYAYCIDCAFSFVALRNSSVEK
KSVLQLLSLCGRHPETKEQAVDALIGLMSFNASHLSQSSYAYCIDCAFSFVALRNSSVEK

NLKILDLMADSVTMLVKWYKTASTDTANSYSPASNTSSSSSMEENNLRGVNFVHHLFLKL
NLKILDLMADSVTMLVKWYKTASTDTANSYSPASNTSSSSSMEENNLRGVNFVHHLFLKL

 

SEAFRKTTLARREEIRNRAV SLEKSFTMGHEDLbbrkbbLlYLlDHVlkPTlDDLHEKL
SEAFRKTTLARREEIRNRAV SLEKSFTMGHEDLGFTPSGCIYCIDHVIFPTIDDLHEKL

l87

Appendix 3-8 (continued).

GNL1-T
GNL1-C

GNL1-T
GNL1-C

GNL1-T
GNL1-C

LDYSRRENAEREMRSMEGTLKIAMKVLMNVFLVYLEQIVESAEFRTFWLGVLRRMDTCMK
LDYSRRENAEREMRSMEGTLKIAMKVLMNVFLVYLEQIVESAEFRTFWLGVLRRMDTCMK

ADLGEYGDNKLQEVVPELLTTMIGTMKEKEILVQKEDDDLWEITYIQIQWIAPALKDELF
ADLGEYGDNKLQEVVPELLTTMIGTMKEKEILVQKEDDDLWEITYIQIQWIAPALKDELF

PDEEIX
PDEEIX

188

Appendix 3-9. The expression patterns of ARF -GEF genes from the Bio-Array Resource
for Arabidopsis Functional Genomics (Winter et al., 2007; http://www.bar.utoronto.ca

/efE/cgi-bin/efg Web.c2i).

 

 

 

ARF-GEF gene Tissues with high expression of ARF-GEF gene
At4g38200 (BIG!) All tissue

At3g60860 (8102) All tissue

Atl g01960 (BIG3) All tissue

At4g35380 (BIG4) Anther

At3g43300 (BIGS, AtMIN 7) All tissue

Atlgl 3980 All tissue

(GNOM;EMB30;GBF 3)

At5g39500 (GNL1 ;GBF1) All tissue

Athl 9610 (GNL2;GBF 2) Anther

 

189

 

 

W1 (mg, --:g.-,'.'.;--.-: 1; 411 \-“""'."'.«".'.L..‘_..?r';"- _.. ":1: 5:91. 1. 1" ,. '.‘... 1'13.“ .1 .‘v' '1'. 1.~.'-.J-‘"-."- 3.1111115 :‘L'Sv 3111'!» a. HTS

.t ﬂTCAAGATAGCCCACAAGATTATCTAAGGGTTCACAACCAGGCACGAGGAGCGGTAGGCGTAGGTC
CCATGCAGTGGGACGAGAGGGTTGCAGCCTATGCTCGGAGCTACGCAGAACAACTAAGAGGCAACTGCAGA
CTCATACACTCTGGTGGGCCTTACGGGGAAAACTTAGCCTGGGGTAGCGGTGACTTGTCTGGCGTCTCCGC
CGTGAACATGTGGGTTAGCGAGAAGGCTAACTACAACTACGCTGCGAACACGTGCAATGGAGTTTGTGGTC
ACTACACTCAAGTTGTTTGGAGAAAGTCAGTGAGACTCGGATGTGCCAAAGTGAGGTGTAACAATGGTGGA
ACCATAATCAGTTGCAACTATGATCCTCGTGGGAATTATGTGAACGAGAAGCCATACGCCATGGTGAGCAA
GGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGT
TCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACC
GGCAAGCTGCCCG TGCCCTGGCCCACCCTCGTGACCACC TTCACCTACGGCGTGCAGTGCTTCAGCCGCTA
CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG GAGCGCACCA
TCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAAC
CGCATCGAGCTC AAGG GCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTA
CAACAGCCACAACGTCTATATCATGG CCGACAAGCAGAAC JAACGGCATCAAGGTGAACT TCAAG ATCCGCC
ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCC
GTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCCA
TCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

Appendix 4-1. The sequence of PR1-GFP fusion ORF. The PR1 ORF is in black letters
and the GFP ORF is in green letters. The start codons and stop codon are in red and bold
letters. The three nucleotides connecting PR1 ORF without stop codon and GF P ORF are
underlined. The putative N-terminal signal sequence of PR1 is highlighted with blue
color.

190

 

J. um"; 21,-. W:_.-l.'" 7""..17 ..-.. “a. "T.. ..-..r" I. 3". _‘TKT'.’ ' 1"." €711.31“ .1'.‘ “57.7.37 7:15."... 7.712" 7517.27.77. T. _':.".--.)-..'-..1-.‘F". V7.2 .-‘;:-.- 1.73.7”. ' -' «'17..- .19.“) F‘

“_._u-__ﬁuGCGGCLCTTGCAACCCACGGAAGGGCGGAAAGCACTCCCCTAAAGCCCCTAAGCTACCAG
TTCCTCCGGTGACCGTCCCTAAGCTACCAGTTCCTCCGGTGACCGTCCCTAAGCTACCAGTCCCTCCGGTG
ACCGTCCCTAAGCTACCCGTTCCTCCTGTGACCATCCCTAAGCTACCCGTTCCACCAGTGACTGTACCTAA
GCTACCCGTTCCTCCTGTGACCGTCCCCAAGCTACCCGTTCCTCCAGTGACCGTCCCCAAGCTACCCGTTC
CTCCAGTGACAGTCCCTAAGCTACCCGTTCCCCCGGTAACTGTACCTAAGCTACCCGTTCCTCCAGTGACC
GTCCCTAAGCTACCCCTTCCTCCGATTTCAGGGCTACCCATACCTCCAGTGGTAGGTCCCAATCTGCCATT
GCCACCTTTGCCAATTGTAGGTCCTATTCTTCCACCGGGAACAACCCCACCAGCCACAGGAGGGAAGGACT
GTCCTCCACCGCCAGGGAGCGTAAAGCCACCATCAGGGGGCGGGAAGGCGACATGTCCAATAGACACGCTG
AAGTTAGGTGCTTGCGTCGACTTGTTGGGAGGTTTAGTAAAGATAGGGCTTGGGGATCCAGCAGTTAACAA
ATGTTGTCCGTTACTTAAAGGCCTCGTTGAAATCGAAGCCGCGGCTTGTCTCTGCACTACCCTCAAGCTCA
AAGCTCTTGACCTCAATCTTTATGTCCCTGTTGCTCTTCAGCTTCTCCTTACCTGTGGCAAAAATCCACCT
CCGGGCTACACTTGTTCCATAGCCAGCCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCA
TCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCC
ACCTACG CCAACCTGACCCTGAACTTCATCTGCACCACCGCCAAGC GCCCGTGCCCTGGCCCACCCTCGT
GACCACCTTCACC ACCGCGTGCAGTGCTTCAGCCGCTACCCCCACCACATCAAGCAG ACGACTTCTTCA
ACTCCCCCATGCCCGAACGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACCACG CAACTACAAGACC
CGCGCCGAGGTGAAG GAGGGCGACACCCTGGTGAAC CATCGAGCTGAAGGGCATCGACTTC GG
GGACGG CAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACA
AGCAG AAGAACGGC ATCAAGGTGAACTTCA.AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCC
GACCACTACCAGCA GAACACCCCCATCGGCGACGGCCCCG TGCTGCTGCCCGACAACCACTACCTGAGCAC
CCAG TCCGC CTGAGC“AAGAC CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCG
CCGGGATCACTC TCGGCATGGA.CC GAGCTGTACAAGTAA

 

 

Appendix 4-2. The sequence of At2g] 0940-GFP fusion ORF. The At2g10940 ORF is in
black letters and the GFP ORF is in green letters. The start codons and stop codon are in
red and bold letters. The three nucleotides connecting At2g10940 ORF without stop
codon and GFP ORF are underlined. The putative N-terminal signal sequence of

At2g10940 is highlighted with blue color.

191

 

.—. . . .r _ ..- ..n- _,. .. .. -. T . ._ ,C. - .. r ... .._ .—.. . . _. . - . .. . .. - . .., .. . . .....-
-.7‘. - -_\ _- 7 ..~ ,- . -‘ Ian-Ln" -- -" ".4. ‘ 1". " ¢-. . :13." Te V— -'.c ---o".-‘.rT¢-.2- .1. -- - . .1'l’r‘r". 7- r . -" v - - "- 3‘ 1."3\ -" .-
. -.

£____3GCCGGCAGCCCCCGCTCCAGAGCCTGCTGGTCCCATCAACCTCACTGCGATCCTCGAAAAAGGTG
GTCAATTCACTACTTTCATCCATCTTCTAAACATCACTCAAGTCGGTAGTCAAGTGAACATTCAAGTCAAT
AGTTCATCCGAAGGTATGACGGTGTTCGCACCAACAGACAATGCTTTTCAAAACCTTAAACCCGGAACCCT
AAACCAGTTAAGCCCTGACGATCAAGTTAAACTCATTCTCTACCACGTTAGCCCCAAATATTACAGTATGG
ATGATCTCCTCTCCGTGAGTAACCCGGTTAGGACTCAAGCTTCTGGCCGAGACAACGGTGTTTACGGGCTT
AACTTCACCGGCCAAACAAACCAAATCAATGTCTCTACTGGTTATGTGGAGACACGTATTAGCAATTCGTT
GAGGCAACAACGTCCTCTCGCAGTTTATGTTGTCGACATGGTTTTGTTGCCCGGTGAGATGTTCGGAGAGC
ACAAGCTTTCGCCGATTGCTCCTGCCCCTAAATCTAAATCCGGTGGGGTTACCGATGACTCCGGCTCCACT
AAGAAGGCAGCGTCACCGTCGGATAAGTCAGGCTCCGGTGAGAAGAAAGTCGGACTAGGGTTTGGTCTTGG
ACTTATTGTCTTATGTTTGAAATTTCTCTTTCCATGGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGG
TGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAG
GGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC
CACCCTCGTGACCACCTTCACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACG
ACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAAC
TACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGA
CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCA
TGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTG
CAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTA
CCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCG
TGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA

hi":

Appendix 4-3. The sequence of F LA9-GFP fusion ORF. The FLA] ORF is in black
letters and the GFP ORF is in green letters. The start codons and stop codon are in red
and bold letters. The six nucleotides connecting F LA9 ORF without stop codon and GFP
ORF are underlined. The putative N-terminal signal sequence of F LA9 is highlighted
with blue color.

192

 

(C)

   

Appendix 4-4. PRl-GFP in Arabidopsis. (A) PR1-GFP in Col-0 (line#l). (B) PR1-GFP
in Col-0 (line #3). (C) PR1-GFP in the atmin,7 plant (line#l). (D) PR1-GFP in the atmin7
plant (line#S). All images are from single focal planes. Ch: chloroplast. S: stomata.

193

20pm

 

Appendix 4—5. At2g|0940-GFP in Arabidopsis. (A) At2gl0940-GFP in Col-0 (line#9).
(B) At2g10940-GFP in Col-0 (line #16). (C) At2g10940—GFP in the atmin7 plant
(line#4l). (D) At2g10940-GFP in the atmin7 plant (line#7l). All images are from single
focal planes except D. which was from Z-stack. Ch: chloroplast. S: stomata.

(A)
water-inﬁltrated ACEL-inﬁltrated hrcC' inﬁltrated

20pm

     

(B)
water—inﬁltrated ACEL-inﬁltrated hroC' inﬁltrated

t—I
20pm

20pm

   

Appendix 4-6. Comparison of the localization of PR1-GFP in Arabidopsis aﬁer
infiltrating hrc(' 0r ACEL mutant, in Col-0 (A) or in atmin7 background (B).

195

Sprayed with water
24 hours

7mm

72 hours
mm

     

Sprayed with BTH
24 hours 48 hours 72 hours

10.1.71

 

Appendix 4-7. Comparison ofthe localization of PR1-GFP in Col-0 background, after
spraying water or 300 uM BTH.

196

Sprayed with water
24 hours 48 hours 72 hours

    

 

mm

10pm

   

Sprayed with BTH
24 hours 48 hours 72 hours

Appendix 4-8. Comparison of the localization of PR 1 -GF P in atmin7 background. after
spraying water or 300 uM BTH.

    

10pm

 

197

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