Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum

Journal of Experimental Botany, Jul 2009

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.

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Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum

Xinyu Wang 0 1 2 Qian Li 2 Xiaowei Niu 1 Haiyan Chen 2 Langlai Xu 2 Cunkou Qi 0 0 Academy of Jiangsu Agricultural Sciences , Nanjing 210014, China 1 The State Key Laboratory of Crop Genetics & Germplasm enhancement, Nanjing Agricultural University , Nanjing 210095, China 2 College of Life Sciences, Nanjing Agricultural University , Nanjing 210095, China 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 discussed. Materials and methods Construction of IPG-1 deletion mutants and the twohybrid assay 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 scanning microscope. 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 (Bio-Rad). 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 cells. 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 cells 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 plant cells. Dynamic subcellular localization of IPG-1 in response to Ca2+ 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 genome 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 screening. 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 20% sugar. 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 weight marker. 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 Sclerotinia sclerotiorum. 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. Acknowledgements 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, 371383. Glazebrook J. 2005. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology 43, 205227. 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, 10091016. Hegedus DD, Rimer SR. 2005. Sclerotinia sclerotiorum: when to be or not to be a pathogen? FEMS Microbiology Letters 251, 177184. 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, 323329. 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, 127136. 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 26, 237247. 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 abstracts). 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 274, 2199522001. 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, 261273. 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 160, 381404.


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Xinyu Wang, Qian Li, Xiaowei Niu, Haiyan Chen, Langlai Xu, Cunkou Qi. Characterization of a canola C2 domain gene that interacts with PG, an effector of the necrotrophic fungus Sclerotinia sclerotiorum, Journal of Experimental Botany, 2009, 2613-2620, DOI: 10.1093/jxb/erp104