Regulation of Microbe-Associated Molecular Pattern-Induced Hypersensitive Cell Death,Phytoalexin Production,and Defense Gene Expression by Calcineurin B-Like Protein-Interacting Protein Kinases,OsCIPK14/15, in Rice Cultured Cells

Although cytosolic free Ca²⁺ mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca²⁺ and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca²⁺ sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn²⁺-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.Calcium ions regulate diverse cellular processes in plants as a ubiquitous internal second messenger, conveying signals received at the cell surface to the inside of the cell through spatial and temporal concentration changes that are decoded by an array of Ca²⁺ sensors (Reddy, 2001; Sanders et al., 2002; Yang and Poovaiah, 2003). Several families of Ca²⁺ sensors have been identified in higher plants. The best known are calmodulins (CaMs) and CaM-related proteins, which typically contain four EF-hand domains for Ca²⁺ binding (Zielinski, 1998). Unlike mammals, which possess single molecular species of CaM, plants have at least three distinct molecular species of CaM playing diverse physiological functions and whose expression is differently regulated (Yamakawa et al., 2001; Luan et al., 2002; Karita et al., 2004; Takabatake et al., 2007). The second major class is exemplified by the Ca²⁺-dependent protein kinases, which contain CaM-like Ca²⁺-binding domains and a kinase domain in a single protein (Harmon et al., 2000). In addition, a new family of Ca²⁺ sensors was identified as calcineurin B-like (CBL) proteins, which consists of proteins similar to both the regulatory β-subunit of calcineurin and the neuronal Ca²⁺ sensor in animals (Liu and Zhu, 1998; Kudla et al., 1999).Unlike CaMs, which interact with a large variety of target proteins, CBLs specifically target a family of protein kinases referred to as CBL-interacting protein kinases (CIPKs) or SnRK3s (for sucrose nonfermenting 1-related protein kinases type 3), which are most similar to the SNF family protein kinases in yeast (Luan et al., 2002). A database search of the Arabidopsis (Arabidopsis thaliana) genome sequence revealed 10 CBL and 25 CIPK homologues (Luan et al., 2002). Expression patterns of these Ca²⁺ sensors and protein kinases suggest their diverse functions in different signaling processes, including light, hormone, sugar, and stress responses (Batistic and Kudla, 2004). AtCBL4/Salt Overly Sensitive3 (SOS3) and AtCIPK24/SOS2 have been shown to play a key role in Ca²⁺-mediated salt stress adaptation (Zhu, 2002). The CBL-CIPK system has been shown to be involved in signaling pathways of abscisic acid (Kim et al., 2003a), sugar (Gong et al., 2002a), gibberellins (Hwang et al., 2005), salicylic acid (Mahajan et al., 2006), and K⁺ channel regulation (Li et al., 2006; Lee et al., 2007; for review, see Luan, 2009; Batistic and Kudla, 2009). However, physiological functions of most of the family members still remain largely unknown.Plants respond to pathogen attack by activating a variety of defense responses, including the generation of reactive oxygen species (ROS), synthesis of phytoalexins, expression of pathogenesis-related (PR) genes, cell cycle arrest, and mitochondrial dysfunction followed by a form of hypersensitive cell death known as the hypersensitive response (Nürnberger and Scheel, 2001; Greenberg and Yao, 2004; Kadota et al., 2004b). Transient membrane potential changes and Ca²⁺ influx are involved at the initial stage of defense responses (Kuchitsu et al., 1993; Pugin et al., 1997; Blume et al., 2000; Kadota et al., 2004a). Many kinds of defense responses are prevented when Ca²⁺ influx is compromised by Ca²⁺ chelators (Nürnberger and Scheel, 2001; Lecourieux et al., 2002). Since complex spatiotemporal patterns of cytosolic free Ca²⁺ concentration have been suggested to play pivotal roles in defense signaling (Nürnberger and Scheel, 2001; Sanders et al., 2002), multiple Ca²⁺ sensor proteins and their effectors should function in the defense signaling pathways. Although possible involvement of some CaM isoforms (Heo et al., 1999; Yamakawa et al., 2001), Ca²⁺-dependent protein kinases (Romeis et al., 2000, 2001; Ludwig et al., 2005; Kobayashi et al., 2007; Yoshioka et al., 2009), as well as Ca²⁺ regulation of EF-hand-containing enzymes such as ROS-generating NADPH oxidase (Ogasawara et al., 2008) have been suggested, other Ca²⁺-regulated signaling components still remain to be identified. No CBLs or CIPKs have so far been implicated as signaling components in defense signaling.N-Acetylchitooligosaccharides, chitin fragments, are microbe-associated molecular patterns (MAMPs) that are recognized by plasma membrane receptors (Kaku et al., 2006; Miya et al., 2007) and induce a variety of defense responses, such as membrane depolarization (Kuchitsu et al., 1993; Kikuyama et al., 1997), ion fluxes (Kuchitsu et al., 1997), ROS production (Kuchitsu et al., 1995), phytoalexin biosynthesis (Yamada et al., 1993), and induction of PR genes (Nishizawa et al., 1999), without hypersensitive cell death in rice (Oryza sativa) cells. In contrast, a fungal proteinaceous elicitor, xylanase from Trichoderma viride (TvX)/ethylene-inducing xylanase (EIX), which is recognized by two putative plasma membrane receptors, LeEix1 and LeEix2 (Ron and Avni, 2004), triggers hypersensitive cell death along with different kinetics of ROS production and activation of a mitogen-activated protein kinase, OsMPK6, previously named as OsMPK2 or OsMAPK6, in rice cells (Kurusu et al., 2005). These two fungal MAMPs thus provide excellent model systems to study innate immunity in rice cells.This study identified two CIPKs involved in various MAMP-induced layers of defense responses, including PR gene expression, phytoalexin biosynthesis, mitochondrial dysfunction, and cell death, in rice. Molecular characterization of these CIPKs, including interaction with the putative Ca²⁺ sensors as well as their physiological functions, is discussed.