Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum
Academy of Jiangsu Agricultural Sciences
, Nanjing 210014,
The State Key Laboratory of Crop Genetics & Germplasm enhancement, Nanjing Agricultural University
, Nanjing 210095,
College of Life Sciences, Nanjing Agricultural University
, Nanjing 210095,
Sspg1d, one of endopolygalacturonases, is an important fungal effector secreted by the necrotrophic fungus Sclerotinia sclerotiorum during early infection. Using sspg1d as bait, a small C2 domain protein (designated as IPG-1) was identified by yeast two-hybrid screening of a canola cDNA library. Deletion analysis confirmed that the C-terminus of IPG-1 is responsible for its interaction with sspg1d in the yeast two-hybrid assay. The sspg1d/IPG-1 interaction was further confirmed in plant cells by a biomolecular fluorescence complementation (BiFC) assay. A transient expression assay showed that the IPG-1-GFP fusion protein was targeted to the plasma membrane and nucleus in onion epidermal cells. Following treatment with a Ca2+ ionophore, it was distributed throughout the cytosol. Real-time PCR assay demonstrated that IPG-1 was highly induced by Sclerotinia sclerotiorum in canola leaves and stems. Southern blot analysis indicated the presence of about five homologues of IPG-1 in the canola genome. Two additional members of the IPG-1gene family were isolated by RT-PCR. Their sequence similarity with IPG-1 is as high as 95%. However, they did not interact with sspg1d in the yeast two-hybrid assay. Possible roles of IPG-1 and its association with sspg1d in the defence signalling pathway were discussed.
The necrotrophic fungal pathogen Sclerotinia sclerotiorum
exhibits little host specifity and has a range of more than
400 plant species among 75 families, primarily dicotyledons,
including many economically important crops such as the
grain legumes (soybean, pea, and bean) and oilseeds (canola
and sunflower) (Boland and Hall, 1994). Stem rot caused by
the fungus is an important disease of canola in China. There
is a lot of evidence to show that complete resistance to this
pathogen has not been identified in canola germplasm,
although partial resistance or tolerance to the pathogen in
different breeding lines has been reported (Liu et al., 1991;
Chen et al., 1993).
Sclerotinia sclerotiorum secretes several types of effector
proteins such as polygalacturonases (PGs) during its
development and plant infection. These PGs can be classified into
endopolygalacturonases (endo-PGs) and exo-polygalacturonases
(exo-PGs) (Li et al., 2004). Each of them has different
expression levels under pathogenic conditions; one of the
endoPGs, named sspg1d, was highly expressed during early
infection and thus may play an important role in pathogen
development and pathogenicity (Li et al., 2004; Hegedus and
Rimer, 2005). Zuppini et al. (2005) reported that an endo-PG
from Sclerotinia sclerotiorum can induce calcium-mediated
signalling and programmed cell death in soybean cells.
* These authors contributed equally to this work.
y To whom correspondence should be addressed. E-mail: ,
Abbreviations: BiFC, biomolecular fluorescence complementation; PG, polygalacturonase; endo-PG, endo-polygalacturonase; GFP, green fluorescent protein; YFP,
yellow fluorescent protein; CaMV, cauliflower mosaic virus.
2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/bync/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The immediate response of plant cells to pathogen attack
is the increase in cytosolic Ca2+ concentration, which can be
decoded by various Ca2+-binding proteins (Ca2+ sensors)
(Reddy, 2001). Once activated by Ca2+ ions, these Ca2+
sensors interact with downstream effectors that modulate
the numerous biochemical and cellular functions involved in
defence responses (Lecourieux et al., 2006). Many of these
Ca2+-binding proteins contain C2 domains (Ca2+-regulatory
domains) (Kretsinger, 1980). In mammals, C2 domains
have been found in more than 100 proteins, most of which
are involved in lipid metabolism, signal transduction or
membrane trafficking (Rizo and Su dhof, 1998). In response
to Ca2+ being imported, the C2 domains of phospholipases,
synaptotagmin I, and protein kinase C were shown to bind
Ca2+ and migrate from the cytosol to the plasma
membrane, thereby transducing the foreign signal into the cells
(Clark et al., 1991; Davletov and Sudhof, 1993; Edwards
and Newton, 1997; Lomasney et al., 1999; Pepio and Sossin,
2001; Ananthanarayanan et al., 2002; Teruel and Meyer,
2002). In plants, copine and phospholipase D, two proteins
that contain C2 domains, have been shown to be involved
in defence responses (Young et al., 1996; Laxalt et al., 2001;
Laxalt and Munnik, 2002; Jambunathan and McNellis,
2003). In rice, the small protein OsERG1 contains a single
C2 domain and is induced by treatment with a fungal
elicitor resulting in protein migration from the cytosol to
the plasma membrane in a Ca2+-dependent manner (Kim
et al., 2003). In mung beans (Vigna radiata L.), the C2
domain of V3-PLC3 plays an important role in the
translocation of the protein to the membrane in response to
abiotic stress (Kim et al., 2004). However, only a few C2
domain-containing proteins have been identified or
investigated in plants.
Using sspg1d as bait, a C2 domain protein, designated
IPG-1, was identified by screening a canola cDNA library
with the yeast two-hybrid assay (Wang et al., 2008). In the
current report, the sspg1d/IPG-1 interaction was confirmed
in living plant cells by bimolecular fluorescent
complementation (BiFC) assay (Walter et al., 2004). The C-terminus of
IPG-1 was demonstrated to be responsible for its
interaction with sspg1d. In addition, the dynamic subcellular
localization of IPG-1 protein was analysed in response to
Ca2+. Finally, the expression of IPG-1 in different tissues of
canola in response to Sclerotinia sclerotiorum inoculation
was characterized and the copy number of IPG-1 in the
canola genome was investigated. The possible roles of PG
and IPG-1, and their interaction in defence signalling, are
Materials and methods
Construction of IPG-1 deletion mutants and the
Two IPG-1 mutants, IPG-1/del-C (contains residues 187 aa)
and IPG-1/del-N (contains residues 89168 aa) were
produced. The coding sequences of each of the two mutants
were fused with the GAL4 activating domain in the
pGADT7 vector and individually transformed into yeast
AH109 cells. The full-length coding sequence of sspg1d was
fused with the GAL4 DNA binding domain in the pGBKT7
vector to generate the pGBKT7sspg1d construct and it
was transformed into yeast Y187 cells. Yeast mating
between AH109 and Y187 was used to test the IPG-1
domain that interacts with sspg1d.
The vectors (PUC-pSPYNE, PUC-pSPYCE) used in the
BiFC assay were kind gifts of Jorg Kudla. For BiFC
analysis, the full-length coding sequence of sspg1d was
fused with the N-terminal fragment of YFP in
PUCpSPYNE vector to form the YFPNsspg1d construct. The
full-length coding sequence of IPG-1 was cloned into
PUCpSPYCE as a fusion with the C-terminal fragment of YFP
to form the YFPCIPG-1 construct. The plasmids YFPN
sspg1d and YFPCIPG-1 were cotransformed into onion
epidermal cells by bombardment. YFP fluorescence was
analysed 16 h later using a Leica TCS SP2 laser confocal
Dynamic subcellular localization of IPG-1 in plant cells in
response to Ca2+
The full-length coding region of IPG-1was cloned into
pJIT166GFP vector as a fusion with the N-terminus of
GFP to form the IPG-1GFP construct. The IPG-1GFP
construct was transformed into onion epidermal cells by
bombardment, and after GFP was detected, the cells were
treated with the calcium ionophore (5 mM Ca2+ and 10 lM
ionomycin) and incubated in the dark at 22 C for 58 h
and then the GFP fluorescence was monitored using a Leica
TCS SP2 laser confocal scanning microscope.
Real-time quantitative RT-PCR
The eukaryotic translation elongation factor 1-a (EF-1a)
was used as the internal control in real-time quantitative
RT-PCR. The sequences of the forward and the reverse
primers were 5#-AGACCACCAAGTACTACTGCAC-3#
and 5#-CCACCAATCTTGTACACATCC-3#, respectively.
The primers used to amplify IPG-1 gene were 5#-GAG CCT
CGC CAT CAG AGA TA-3# and 5#-GTC CTC ATG
GAC TTG CAC ACT-3#. PCR was performed using
Multicolor Real-Time PCR Detection System, iQ 5
Full-length IPG-1 and its C-terminal region interact with
sspg1d in yeast cells
IPG-1 was a small C2 domain protein containing 168 amino
acid residues (Wang et al., 2008). BLAST analysis in
Genbank predicted that the 187 aa is the C2 domain in
which there are five conserved aspartic acid residues (D)
(Fig. 1), the potential Ca2+-binding sites. The 88168 aa is
the C-terminal region that may be involved in protein
protein interaction. To verify this hypothesis, two deletion
mutants were constructed: IPG-1-del-N, which contained
the C-terminal domain (89168 aa) and IPG-1-del-C, which
contained the N-terminal domain (C2 domain, 187 aa).
The coding sequence of the two mutants and the full-length
IPG-1 were individually fused with an activating domain
(AD) in the pGADT7 vector and transformed into yeast
strain AH109 separately. The pGBKT7sspg1d construct
was transformed into yeast strain Y187. The domain of
IPG-1 necessary for its interaction with sspg1d was
determined by yeast mating between Y187 and AH109 yeast
Yeast cells with pGBKT7sspg1d and IPG-1-del-N
plasmids, and cells with pGBKT7sspg1d and pGADT7
IPG-1 plasmids were able to grow on
SD/-Ade/-His/-Leu/Trp and turned blue with the X-aGal overlay assay, but
yeast cells with pGBKT7sspg1d and IPG-1-del-C plasmids
were not (Fig. 2). The experiments were replicated three
times and each obtained the same results. It means that the
full-length IPG-1 and the C-terminal region of IPG-1 can
Fig. 1. The structure of the IPG-1 protein. The domain consisting
of residues 187 is the C2 domain, in which the asterisks
represent the five conserved aspartatic residues (the positions are
22, 73, 75, 80, and 81, respectively). The C-terminus is shown in
black box consisting of residues 88168.
Fig. 2. sspg1d/IPG-1 interaction and the domain of IPG-1
necessary for its interaction with sspg1d analysed by yeast
twohybrid assay. 1, sspg1d/IPG-1 interaction; 2, positive control
(pGBKT7-53/ pGADT7-RecT interaction); 3, negtive control
(pGBKT7-Lam/pGADT7-RecT interaction); 4, 5,
sspg1d/IPG-1delN interaction; 6, sspg1d/IPG-1-delC interaction. Yeast cells
transformed with combinations of various AD and BD constructs
were subjected to b-galactosidase overlay activity assay. The blue
colour of the yeast cells and yeast growth on
SD/-Ade/-His/-Leu/Trp media indicate the activation of reporter genes and therefore
a positive proteinprotein interaction.
interact with sspg1d, while the C2 domain was not
necessary for the interaction.
Verification of sspg1dIPG-1 interaction in living plant
BiFC was used to test weather sspg1d and IPG-1 can
associate in plant cells. sspg1d and IPG-1 was fused with the
N-terminal 154 amino acid or C-terminal 84 amino acids of
yellow fluorescent protein (YFP), respectively, driven by
the CaMV 35S promoter. The two constructs were
cobombarded into onion epidermal cells. YFP fluorescence
was monitored using laser confocal scanning microscopy.
As shown in Fig. 3, YFP fluorescence could be detected
in onion epidermal cells co-transformed with YFPNsspg1d
and YFPCIPG-1. No YFP fluorescence was detected in the
negative controls (i.e. transformed with YFPCIPG-1/
YFPN, YFPNIPG-1/YFPC) (data not shown). These
results confirm that sspg1d interacts with IPG-1 in living
Dynamic subcellular localization of IPG-1 in response to
It is known that C2 domain proteins play a role in
Ca2+dependent spatio-temporal targeting in different regulatory
signal transduction chains (Evans et al., 2004).
Furthermore, it has been shown that the small rice C2 domain
protein OsERG1 is translocated to the plasma membrane of
plant cells in a Ca2+-dependent manner (Kim et al., 2003).
To determine whether the newly identified C2 domain
protein IPG-1 also exhibits calcium-dependent subcellular
localization, the IPG-1GFP construct driven by the CaMV
35S promoter was introduced into onion epidermal cells by
bombardment for transient expression. As shown in Fig. 4,
IPG-1 protein is mainly targeted to the plasma membrane
and nucleus. After treatment with ionomycin, a calcium
ionophore, for 5 h, the IPG-1GFP signals was observed
distributing throughout the cytosol (Fig. 5). It means that
the Ca2+ ionophore treatment induced translocation of the
green fluorescence signal emitted from the IPG-1-GFP from
the plasma membrane and the nucleus to the cytosol.
IPG-1 is highly induced by Sclerotinia sclerotiorum
Semi-quantitative PCR revealed that IPG-1 was highly
induced by Sclerotinia sclerotiorum in canola leaf and stem
(Wang et al., 2008). Here, real-time PCR was used to
analyse the expression of IPG-1 in leaf, stem, and flower
organs of canola following inoculation with Sclerotinia
sclerotiorum. As shown in Fig. 6, the expression of IPG-1
in flowers is about three times higher than that in leaves and
stems before inoculating with Sclerotinia sclerotiorum,
whereas the expression level of IPG-1 in leaves and stems
was about 23 times higher than in flowers following
inoculation with Sclerotinia sclerotiorum. The experiment
again provided evidence that IPG-1 is significantly induced
in leaves and stems by Sclerotinia sclerotiorum.
Fig. 3. Bimolecular fluorescence complementation assays in onion epidermal cells. The reconstitute YFP signals show that IPG-1 and
sspg1d can associate in plant cells.
Determine the copy numbers of IPG-1 in the canola
The copy numbers of IPG-1 in the canola genome were
analysed by Southern blot of canola genomic DNA probed
with IPG-1 cDNA (Fig. 7). The results showed that IPG-1
has about 25 copies in the canola genome, which means
that the canola genome may have a C2 domain gene family.
To test whether other members in the gene family interact
with sspg1d, an additional two genes in the gene family
were isolated by RT-PCR and designated as BnC2d1 and
BnC2d2. BLAST analysis indicated that the two genes also
contain the C2 domain. Sequence comparison of the two
genes with IPG-1 revealed that their identity is as high as
95% in amino acids (Fig. 8). The coding sequences of
BnC2d1 and BnC2d2 were individually fused into the GAL4
activating domain of the pGADT7 vector. Yeast mating
was used to test the interaction between sspg1d and the two
genes. To our surprise, the two genes did not interact with
sspg1d, although the experiment was replicated several
times. This means that the two IPG-1 homologues may
have evolved functions different from IPG-1. This may
explain why only one gene that interacts with sspg1d in the
canola cDNA library was isolated by yeast two-hybrid
PGs produced by fungi belong to cell wall-degrading
enzymes and some of them from several necrotrophic fungal
pathogens have been implicated as potential virulence
factors unrelated to their enzyme activity (Shieh et al.,
1997; Have et al., 1998; Poinssot et al. 2003; Kikot et al.,
2008). They can cause Ca2+ elevation in the cell cytosol and
subsequent cell death (Zuppini et al., 2005). In order to
identify host factors involved in PG signalling, sspg1d, one
of the important PGs secreted by Sclerotinia sclerotiorum
during its early development and the plant infection
process, was used as bait to screen PG-interacting proteins in
the canola cDNA expression library by using a yeast
twohybrid technique. A C2 domain protein was identified that
interacts with sspg1d, and which was further confirmed in
plant cells by BiFC. The C2 domain was first described in
protein kinase C (PKC), representing a large family
Fig. 4. Localization of IPG-1-GFP fusion in onion epidermal cell.
Plant cell wall and membrane were separated by treatment with
Fig. 5. Subcellular localization of IPG-1-GFP fusion transiently
expressed in onion epidermal cells before (A) and after (B)
treatment with Ca2+ ionophore.
comprising the most studied of all protein kinases in
animals. Animal PKC isoforms include 34 conserved
domains, C1C4, representing catalytic and regulatory
Fig. 7. Southern blot of canola genomic DNA using 32P labelled
IPG-1 cDNA as probe. Genomic DNA was digested with BamHI
(1), EcoRI (2), EcoRV (3), HindIII (4), and XbaI (5). M, molecular
Fig. 8. Amino acid sequence alignment of IPG-1 with its
homologues BnC2d1 and BnC2d2.
modules. Domains C1, C3, and C4 are present in all PKC
isoforms, whereas the C2 domain is unique to the
Ca2+dependent isoforms PKCa, PKCb, and PKCc, thus
identifying the C2 domain as a potential Ca2+-regulatory motif
(Hug and Sarre, 1993). C2 domains interact with
phospholipids in a Ca2+-dependent manner and thereby modulate
a diverse range of cellular actions. They mediate the
Ca2+dependent translocation of soluble proteins to membranes,
the Ca2+- and phospholipid-dependent activation of
enzymes, Ca2+- and phospholipid-dependent interaction
between proteins, or promote Ca2+-triggered self-association
(Kopka et al., 1998). The C2 domain proteins found in
human and animals generally consist of 13 C2 domains
(Nalefski and Falke, 1996). Only a few C2 domain proteins
are discovered in plants, and many of these proteins contain
only single C2 domain, known as small C2 domain protein.
The functions of already-described plant small C2 domain
proteins are not yet very clear. In pumpkin, a small C2
domain protein has been reported to increase the size of
mesophyll plasmodesmata to enable transport of cellular
materials, including RNA molecules, from cell to cell
(Xoconostle-Cazares et al., 1999). In Arabidopsis, the
C2domain protein BAPl negatively regulates defence responses
(Yang et al., 2006). In rice, two small C2-domain rice
proteins named OsERG1a and OsERG1b, are significantly
induced by a fungal elicitor (Kim et al., 2003), implicating
a functional role in defence signalling systems in plant cells.
The HvC2d, a C2-domain protein identified in barley
(Hordeum vulgare L.) was induced by exposure to different
heavy metals and its mRNA was accumulated during leaf
senescence (Ouelhadj et al., 2006). In this study, the novel
C2 domain protein identified, IPG-1, was first shown to
interact with sspg1d, a fungal effector of Sclerotinia
sclerotiorum. It is closely related in sequence to a
C2domain protein in Arabidopsis (NP198590) with unknown
functions (Wang et al., 2008). The expression studies (Fig. 6)
showed that the mRNA of this gene was highly induced by
Sclerotinia sclerotiorum inoculation. The subcellular
localization of IPG-1GFP fusion protein is dynamic in response
to Ca2+ elevation. These results implicate a role of IPG-1 in
Ca2+-dependent defence signalling.
Localization of proteins to distinct subcellular
compartments, including membranes, is a critical event in multiple
cellular pathways. The C2 domain has been identified in
many cellular proteins involved in signal transduction or
membrane trafficking. A majority of C2 domains bind the
membrane in a Ca2+-dependent manner and thereby play an
important role in Ca2+-dependent membrane targeting
(Nalefski and Falke, 1996; Stahlen and Cho, 2001).
Analyses of mammalian C2 proteins, for example,
phospholipases, synaptotagmin I and protein kinase C, also
showed that these proteins migrate after binding of Ca2+
from the cytosol to the plasma membrane and thus are able
to transduce foreign signals into the cell (Pepio and Sossin,
2001; Ananthanarayanan et al., 2002; Teruel and Meyer,
2002). By using GFP constructs, Kim et al. (2003) showed
that the small C2 domain protein OsERG1 is translocated
to the plasma membrane of plant cells by treatment with
a Ca2+ ionophore and also by a fungal elicitor.
Immunocytochemical analyses with the other known small C2 domain
protein from pumpkin (CmPP16-1) also suggested an
association with the plasma membrane (Xoconostle-Cazares
et al., 1999). Interestingly, there are several reports showing
that such calcium-binding proteins are not only localized in
the cytoplasm but also in the nucleus. Among these proteins
with nuclear localization are calcium-dependent protein
kinases (Dammann et al., 2003; Chehab et al., 2004), a novel
calmodulin-binding protein (Perruc et al., 2004), and a novel
C2 domain protein HvC2d1 found in barley (Ouelhadj
et al., 2006). In addition, the calcium-dependent protein
kinase McCPK1 from the ice plant was shown to undergo
a reversible change in subcellular localization from the
plasma membrane to the nucleus, endoplasmic reticulum,
and actin filaments of the cytoskeleton in response to
environmental stimuli (Chehab et al., 2004). In the present
study, another novel protein was identified with a
calciumbinding C2 domain-like motif that was shown to be located
in a calcium-dependent manner in the cytosol and also in
the nucleus. Our data indicate that IPG-1 plays a role in
cytosolic and nuclear localized calcium signalling processes
in response to external stressors.
It has been reported that the Arabidopsis C2 domain
proteins, BAP1 and BAP2, are general inhibitors of
programmed cell death (PCD) (Yang et al., 2007).
Overexpression of BAP1 or BAP2, with their partner BON1,
inhibits PCD induced by pathogens. Thus, the BAP genes
function as general negative regulators of PCD induced by
biotic stimuli. The BAP1 and BON1 molecules might
become targets of pathogen effector proteins because of
their ancestral role in cell death control during the evolution
of plant innate immune system (Jones and Dangl, 2006).
Considering that an endo-PG of Sclerotinia sclerotiorum
could induce PCD in plant cells (Zuppini et al., 2005), and
here, IPG-1 is shown to be the target of an endo-PG of
Sclerotinia sclerotiorum, implicating IPG-1 in the PCD
process in plant cells. It has been suggested that effective
defence against biotrophic pathogens is largely due to PCD
in the host, whereas necrotrophic pathogens benefit from
host cell death; they can utilize dead tissue and are not
limited by cell death (Govrin and Lexine, 2000; Glazebrook,
2005). Plants expressing animal PCD inhibitor genes, such
as the human Bcl-2 and Bcl-xl and the nematode CED-9,
confer resistance to several necrotrophic fungal pathogens
including Sclerotinia sclerotiorum (Dickman et al., 2001).
Considering that the plant material used here is susceptible
to the fungus, it is postulated that the PG/IPG-1 interaction
may interfere with the binding of IPG-1 with Ca2+ and the
subsequent Ca2+-dependent signal transduction might be
involved in PCD processes in the host. Overexpression of
IPG-1 might inhibit PCD and produce resistance to
At present, there are two models explaining pathogen
perception by plants: the Guard Model and the recently
proposed Decoy Model (van der Hoorn and Kamoun,
2008). The Guard Model proposes that pathogen effectors
may have a common target; the plant R-protein indirectly
perceives the presence of the pathogen effector by
monitoring the state of the effector target that associates with the
R-protein. In the absence of the R-protein, the effector
target will enhance pathogen fitness in plants. The Decoy
Model implies that some host targets of fungal effectors
have evolved to act as a decoy to trap the pathogen into
a recognition event. These decoys only function in the
perception of pathogen effectors without contributing to
pathogen fitness in the absence of its cognate R-protein in
plant. What types of effector target the IPG-1 belongs to is
an interesting question to be explored. Whether IPG-1
associates with plant R-proteins remains to be investigated.
Nowadays, numerous laboratories are determining the
enzymatic functions of pathogen effectors as well as their
host targets (Chisholm et al., 2006), which will benefit
elucidation of the molecular basis of plant resistance or
susceptibility. These works are of great importance for both
basic and applied research. This study lays the foundation
for further investigation of the functions of PG, IPG-1, and
the roles of PGIPG-1 interaction in plant defence
responses, which may also involve other factors such as
calcium, phospholipids, and other proteins.
This work was partially supported by the National Natural
Science Foundation of China (30571153). The experiments
were performed at the Institute of Cotton Genome
Research, the State Key Laboratory of Crop Genetics and
Germplasm Enhancement, Nanjing Agricultural University.
We thank Professor Tianzhen Zhang for providing
instruments and some reagents for the experiments.
Ananthanarayanan B, Das S, Rhee SG, Murray D, Cho W. 2002.
Membrane targeting of C2 domains of phospholipase C-d isoforms.
Journal of Biological Chemistry 277, 35683575.
Boland GJ, Hall R. 1994. Index of plant hosts of Sclerotinia
sclerotiorum. Canadian Journal of Plant Pathology 16, 94108.
Chehab EW, Patharkar OR, Hegeman AD, Taybi T, Cushman JC.
2004. Autophosphorylation and subcellular localisation dynamics of
a salt- and water deficit-induced calcium-dependent protein kinase
from ice plant. Plant Physiology 135, 14301446.
Chen YQ, Zhang JF, Wu YM, Hou QS, Zhou YJ, Han H. 1993.
Studies on virus and Sclerotinia sclerotiorum resistance of rapeseed
germplasm in Brassica genus. Chinese Journal of Oil Crop Science
15, 47 (in Chinese with English abstract).
Chisholm ST, Coaker G, Day B, Staskawicz BJ. 2006. Host
microbe interactions: shaping the evolution of the plant immune
response. Cell 124, 803814.
Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, Lin AY,
Milona N, Knopf JL. 1991. A novel arachidonic acid-selective
cytosolic PLA2 contains a Ca2+-dependent translocation domain with
homology to PKC and GAP. Cell 65, 10431051.
Dammann D, Ichida A, Hong B, Romanowsky S, Hrabak EM,
Harmon AC, Pickard BG, Harper JF. 2003. Subcellular targeting of
nine calcium dependent protein kinase isoforms from Arabidopsis.
Plant Physiology 132, 18401848.
Davletov BA, Su dhof TC. 1993. A single C2 domain from
synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.
Journal of Biological Chemistry 268, 2638626390.
Dicman MB, Park YK, Oltersdorf T, Li W, Clemente T, French R.
2001. Abrogation of disease development in plants expressing animal
antiapoptotic genes. Proceedings of the National Academy of
Sciences, USA 98, 69576962.
Edwards AS, Newton AC. 1997. Regulation of protein kinase Cb II
by its C2 domain. Biochemistry 36, 1561515623.
Evans JH, Gerber SH, Murray D, Leslie CC. 2004. The calcium
binding loops of the cytosolic phospholipase A2, C2 domain specify
targeting to Golgi and ER in live cells. Molecular Biology of the Cell 15,
Glazebrook J. 2005. Contrasting mechanisms of defense against
biotrophic and necrotrophic pathogens. Annual Review of Phytopathology
Govrin EM, Lexine A. 2000. The hypersensitive response facilitates
plant infection by the necrothophic pathogen Botrytis cinerea. Current
Biology 10, 751757.
Have AT, Mulder W, Visser J, Jan AL, Kan V. 1998. The
endopolygalacturonase gene Bcpg1 is required for full virulence of
Botrytis cinerea. Molecular PlantMicrobe Interactions 11,
Hegedus DD, Rimer SR. 2005. Sclerotinia sclerotiorum: when to
be or not to be a pathogen? FEMS Microbiology Letters 251,
Hug H, Sarre TF. 1993. Protein kinase C isoenzymes: divergence in
signal transduction? Biochemical Journal 291, 329343.
Jambunathan N, McNellis TW. 2003. Regulation of Arabidopsis
COPINE 1 gene expression in response to pathogens and abiotic
stimuli. Plant Physiology 132, 13701381.
Jones JD, Dangl JL. 2006. The plant immune system. Nature 444,
Kikot GE, Hours RA, Alconada TM. 2008. Contribution of cell wall
degrading enzymes to pathogenesis of Fusarium graminearum:
a review. Journal of Basic Microbiology 48, 111.
Kim CY, Koo YD, Jin JB, et al. 2003. Rice C2 domain proteins are
induced and translocated to the plasma membrane in response to
a fungal elicitor. Biochemistry 42, 1162511631.
Kim YJ, Kim JE, Lee JH, Lee MH, Jung HW, Bahk YY,
Hwang BK, Hwang I, Kim WT. 2004. The Vr-PLC3 gene encodes
a putative plasma membrane-localized phosphoinositide-speciWc
phospholipase C whose expression is induced by abiotic
stress in mung bean (Vigna radiata L.). FEBS Letters 556,
Kopka J, Pical C, Hetherington AM, Mu ller-Ro ber B. 1998. Ca2+/
phospholipids-binding (C2) domain in multiple plant proteins: novel
components of the calcium-sensing apparatus. Plant Molecular
Biology 36, 627637.
Kretsinger RH. 1980. Structure and evolution of calcium-modulated
proteins. Critical Reviews in Biochemistry 8, 119174.
Laxalt AM, Riet BT, Verdonk JC, Parigi L, Tameling WI,
Vossen J, Haring M, Musgrave A, Munnik T. 2001.
Characterization of five tomato phospholipase D cDNAs: rapid and specific
expression of LePLDb1 on elicitation with xylanase. The Plant Journal
Laxalt AM, Munnik T. 2002. Phospholipid signalling in plant defense.
Current Opinion in Plant Biology 5, 332338.
Lecourieux D, Ranjeva R, Pugin A. 2006. Calcium in plant
defencesignalling pathways. New Phytologist 171, 249269.
Li RG, Rimmer R, Buchwalolt L, Sharpe AG, Seguin-Swartz G,
Hegedus DD. 2004. Interaction of Sclerotinia sclerotiorum with
Brassica napus: cloning and characterization of endo- and
exopolygalacturonase expressed during saprophytic and parasitic mode.
Fungal Genetics and Biology 41, 754765.
Liu CQ, Du DZ, Huang YJ, Wang CH. 1991. Study on tolerance to
Sclerotinia sclerotiorum and the hereditary properties in B. napus.
Agricultural Sciences in China 24, 4349 (in Chinese with English
Lomasney JW, Cheng HF, Roffler SR, King K. 1999. Activation
of phospholipase Cd1 through C2 domain by a
Ca2+-enzymephosphatidylserine ternary complex. Journal of Biological Chemistry
Nalefski EA, Falke JJ. 1996. The C2 domain calcium-binding motif:
structure and functional diversity. Protein Science 5, 23752390.
Ouelhadj A, Kusch KP, Humbeck K. 2006. Heavy metal stress and
leaf senescence induce the barley gene HvC2d 1 encoding a
calciumdependent novel C2 domain-like protein. New Phytologist 170,
Perruc E, Charpenteau M, Ramirez BC, Jauneau A, Galaud JP,
Ranjeva R, Ranty B. 2004. A novel calmodulin-binding protein
functions as a negative regulator of osmotic stress tolerance in
Arabidopsis thaliana seedlings. The Plant Journal 38, 410420.
Pepio AM, Sossin WS. 2001. Membrane translocation of novel
protein kinase Cs is regulated by phosphorylation of the C2 domain.
Journal of Biological Chemistry 276, 38463855.
Poinssot B, Vandelle E, Bente jac M, Adrian M, Levis C,
Brygoo Y, Garin J, Sicilia F, Coutos-The venot P, Pugin A. 2003.
The endopolygalacturonase 1 from Botrytis cinerea activates
grapevine defense reactions unrelated to its enzymatic activity. Molecular
PlantMicrobe Interaction 16, 553564.
Reddy ASN. 2001. Calcium: silver bullet in signaling. Plant Science