Activation of Plant Immune Responses by a Gain-of-Function Mutation in an Atypical Receptor-Like Kinase

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.Receptor-like kinases (RLKs) are a large group of kinases with a variable extracellular domain and a cytoplasmic kinase domain linked by a single transmembrane motif. RLKs have been shown to play diverse roles in regulating plant innate immunity as well as growth and development (Morillo and Tax, 2006). The extracellular domains of RLKs are believed to bind directly to ligands to perceive extracellular signals, whereas the cytoplasmic kinase domains transduce these signals into the cell. There are over 600 RLKs (Shiu and Bleecker, 2001) in Arabidopsis (Arabidopsis thaliana). The biological functions of most RLKs are unknown.Several RLKs have been identified to be receptors of microbe-associated molecular patterns (MAMPs). FLS2 and EFR are two well-characterized RLKs with extracellular Leu-rich repeats (LRRs) that recognize bacterial flagellin and translation elongation factor EF-Tu, respectively (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006). BAK1 is also an RLK with extracellular LRRs. BAK1 seems to function as an adaptor protein for multiple RLKs including BRI1, FLS2, and BIR1 (Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Gao et al., 2009). Interestingly, knocking out BIR1 activates cell death and defense responses mediated by another RLK, SOBIR1 (Gao et al., 2009). Recently, the rice (Oryza sativa) RLK Xa21 was also suggested to be a MAMP receptor. Xa21 recognizes a peptide derived from the secreted effector protein AvrXa21, which is conserved among different Xanthomonas species (Lee et al., 2009). Unlike FLS2, EFR, and Xa21, the putative receptor for chitin is an RLK with three extracellular LysM domains instead of LRRs that are required for the perception of chitin as well as resistance against bacterial pathogens (Miya et al., 2007; Wan et al., 2008; Gimenez-Ibanez et al., 2009).Perception of MAMPs by receptors leads to the rapid activation of mitogen-activated protein (MAP) kinases including MAP KINASE3 (MPK3), MPK4, and MPK6 (Boller and Felix, 2009). MAP KINASE SUBSTRATE1 (MKS1) was identified as an MPK4-interacting protein that positively regulates defense responses (Andreasson et al., 2005). Silencing of MKS1 compromises basal resistance to Pseudomonas syringae pv tomato DC3000, whereas overexpression of MKS1 leads to enhanced pathogen resistance. Activation of MAMP receptors also induces a number of responses such as oxidative burst, callose deposition, and increased salicylic acid synthesis (Boller and Felix, 2009). Defense responses induced by different MAMPs seem similar, suggesting that they may share common signaling components. Identification of the signaling components downstream of RLK receptors remains a major task in understanding MAMP-triggered immunity.Here we report the identification and characterization of suppressor of npr1-1, constitutive4-1D (snc4-1D), a gain-of-function mutant of an atypical RLK that is autoactivated by a mutation in its kinase domain. The snc4-1D mutant plants constitutively express defense maker genes PR1, PR2, and PDF1.2, and display enhanced resistance to Hyaloperonospora arabidopsidis (H. a.) Noco2. Epistasis analysis showed that snc4-1D-mediated defense responses are dependent on multiple factors including NON-RACE-SPECIFIC DISEASE RESISTANCE1 (NDR1), MKS1, and OPR3.     

PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei

Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.In plants, organelles move within the cell and become appropriately positioned to accomplish their functions and adapt to the environment (for review, see Wada and Suetsugu, 2004). Light-induced chloroplast movement (chloroplast photorelocation movement) is one of the best characterized organelle movements in plants (Suetsugu and Wada, 2012). Under weak light conditions, chloroplasts move toward light to capture light efficiently (the accumulation response; Zurzycki, 1955). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response; Kasahara et al., 2002; Sztatelman et al., 2010; Davis and Hangarter, 2012; Cazzaniga et al., 2013). In most green plant species, these responses are induced primarily by the blue light receptor phototropin (phot) in response to a range of wavelengths from UVA to blue light (approximately 320–500 nm; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). Phot-mediated chloroplast movement has been shown in land plants, such as Arabidopsis (Arabidopsis thaliana; Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001), the fern Adiantum capillus-veneris (Kagawa et al., 2004), the moss Physcomitrella patens (Kasahara et al., 2004), and the liverwort Marchantia polymorpha (Komatsu et al., 2014). Two phots in Arabidopsis, phot1 and phot2, redundantly mediate the accumulation response (Sakai et al., 2001), whereas phot2 primarily regulates the avoidance response (Jarillo et al., 2001; Kagawa et al., 2001; Luesse et al., 2010). M. polymorpha has only one phot that mediates both the accumulation and avoidance responses (Komatsu et al., 2014), although two or more phots mediate chloroplast photorelocation movement in A. capillus-veneris (Kagawa et al., 2004) and P. patens (Kasahara et al., 2004). Thus, duplication and functional diversification of PHOT genes have occurred during land plant evolution, and plants have gained a sophisticated light sensing system for chloroplast photorelocation movement.In general, movements of plant organelles, including chloroplasts, are dependent on actin filaments (for review, see Wada and Suetsugu, 2004). Most organelles common in eukaryotes, such as mitochondria, peroxisomes, and Golgi bodies, use the myosin motor for their movements, but there is no clear evidence that chloroplast movement is myosin dependent (for review, see Suetsugu et al., 2010a). Land plants have innovated a novel actin-based motility system that is specialized for chloroplast movement as well as a photoreceptor system (for review, see Suetsugu et al., 2010a; Wada and Suetsugu, 2013; Kong and Wada, 2014). Chloroplast-actin (cp-actin) filaments, which were first found in Arabidopsis, are short actin filaments specifically localized around the chloroplast periphery at the interface between the chloroplast and the plasma membrane (Kadota et al., 2009). Strong blue light induces the rapid disappearance of cp-actin filaments and then, their subsequent reappearance preferentially at the front region of the moving chloroplasts. This asymmetric distribution of cp-actin filaments is essential for directional chloroplast movement (Kadota et al., 2009; Kong et al., 2013a). The greater the difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts becomes, the faster the chloroplasts move, in which the magnitude of the difference is determined by fluence rate (Kagawa and Wada, 2004; Kadota et al., 2009; Kong et al., 2013a). Strong blue light-induced disappearance of cp-actin filaments is regulated in a phot2-dependent manner before the intensive polymerization of cp-actin filaments at the front region occurs (Kadota et al., 2009; Ichikawa et al., 2011; Kong et al., 2013a). This phot2-dependent response contributes to the greater difference in the amount of cp-actin filaments between the front and rear regions of chloroplasts. Similar behavior of cp-actin filaments has also been observed in A. capillus-veneris (Tsuboi and Wada, 2012) and P. patens (Yamashita et al., 2011).Like chloroplasts, nuclei also show light-mediated movement and positioning (nuclear photorelocation movement) in land plants (for review, see Higa et al., 2014b). In gametophytic cells of A. capillus-veneris, weak light induced the accumulation responses of both chloroplasts and nuclei, whereas strong light induced avoidance responses (Kagawa and Wada, 1993, 1995; Tsuboi et al., 2007). However, in mesophyll cells of Arabidopsis, strong blue light induced both chloroplast and nuclear avoidance responses, but weak blue light induced only the chloroplast accumulation response (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In Arabidopsis pavement cells, small numbers of tiny plastids were found and showed autofluorescence under the confocal laser-scanning microscopy (Iwabuchi et al., 2010; Higa et al., 2014a). Hereafter, the plastid in the pavement cells is called the pavement cell plastid. Strong blue light-induced avoidance responses of pavement cell plastids and nuclei were induced in a phot2-dependent manner, but the accumulation response was not detected for either organelle (Iwabuchi et al., 2007, 2010; Higa et al., 2014a). In both Arabidopsis and A. capillus-veneris, phots mediate nuclear photorelocation movement, and phot2 mediates the nuclear avoidance response (Iwabuchi et al., 2007, 2010; Tsuboi et al., 2007). The nuclear avoidance response is dependent on actin filaments in both mesophyll and pavement cells of Arabidopsis (Iwabuchi et al., 2010). Recently, it was shown that the nuclear avoidance response relies on cp-actin-dependent movement of pavement cell plastids, where nuclei are associated with pavement cell plastids of Arabidopsis (Higa et al., 2014a). In mesophyll cells, nuclear avoidance response is likely dependent on cp-actin filament-mediated chloroplast movement, because the mutants deficient in chloroplast movement were also defective in nuclear avoidance response (Higa et al., 2014a). Thus, phots mediate both chloroplast (and pavement cell plastid) and nuclear photorelocation movement by regulating cp-actin filaments.Molecular genetic analyses of Arabidopsis mutants deficient in chloroplast photorelocation movement have identified many molecular factors involved in signal transduction and/or motility systems as well as those involved in the photoreceptor system for chloroplast photorelocation movement (and thus, nuclear photorelocation movement; for review, see Suetsugu and Wada, 2012; Wada and Suetsugu, 2013; Kong and Wada, 2014). CHLOROPLAST UNUSUAL POSITIONING1 (CHUP1; Oikawa et al., 2003) and KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT (KAC; Suetsugu et al., 2010b) are key factors for generating and/or maintaining cp-actin filaments. Both proteins are highly conserved in land plants and essential for the movement and attachment of chloroplasts to the plasma membrane in Arabidopsis (Oikawa et al., 2003, 2008; Suetsugu et al., 2010b), A. capillus-veneris (Suetsugu et al., 2012), and P. patens (Suetsugu et al., 2012; Usami et al., 2012). CHUP1 is localized on the chloroplast outer membrane and binds to globular and filamentous actins and profilin in vitro (Oikawa et al., 2003, 2008; Schmidt von Braun and Schleiff, 2008). Although KAC is a kinesin-like protein, it lacks microtubule-dependent motor activity but has filamentous actin binding activity (Suetsugu et al., 2010b). An actin-bundling protein THRUMIN1 (THRUM1) is required for efficient chloroplast photorelocation movement (Whippo et al., 2011) and interacts with cp-actin filaments (Kong et al., 2013a). chup1 and kac mutant plants were shown to lack detectable cp-actin filaments (Kadota et al., 2009; Suetsugu et al., 2010b; Ichikawa et al., 2011; Kong et al., 2013a). Similarly, cp-actin filaments were rarely detected in thrum1 mutant plants (Kong et al., 2013a), indicating that THRUM1 also plays an important role in maintaining cp-actin filaments.Other proteins J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE1 (JAC1; Suetsugu et al., 2005), WEAK CHLOROPLAST MOVEMENT UNDER BLUE LIGHT1 (WEB1; Kodama et al., 2010), and PLASTID MOVEMENT IMPAIRED2 (PMI2; Luesse et al., 2006; Kodama et al., 2010) are involved in the light regulation of cp-actin filaments and chloroplast photorelocation movement. JAC1 is an auxilin-like J-domain protein that mediates the chloroplast accumulation response through its J-domain function (Suetsugu et al., 2005; Takano et al., 2010). WEB1 and PMI2 are coiled-coil proteins that interact with each other (Kodama et al., 2010). Although web1 and pmi2 were partially defective in the avoidance response, the jac1 mutation completely suppressed the phenotype of web1 and pmi2, suggesting that the WEB1/PMI2 complex suppresses JAC1 function (i.e. the accumulation response) under strong light conditions (Kodama et al., 2010). Both web1 and pmi2 showed impaired disappearance of cp-actin filaments in response to strong blue light (Kodama et al., 2010). However, the exact molecular functions of these proteins are unknown.In this study, we characterized mutant plants deficient in the PMI1 gene and two homologous genes PLASTID MOVEMENT IMPAIRED1-RELATED1 (PMIR1) and PMIR2. PMI1 was identified through molecular genetic analyses of pmi1 mutants that showed severe defects in chloroplast accumulation and avoidance responses (DeBlasio et al., 2005). PMI1 is a plant-specific C2-domain protein (DeBlasio et al., 2005; Zhang and Aravind, 2010), but its roles and those of PMIRs in cp-actin-mediated chloroplast and nuclear photorelocation movements remained unclear. Thus, we analyzed chloroplast and nuclear photorelocation movements in the single, double, and triple mutants of pmi1, pmir1, and pmir2.     

Sterol-Dependent Induction of Plant Defense Responses by a Microbe-Associated Molecular Pattern from Trichoderma viride

Plant-microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. An ethylene-inducing xylanase (Eix) of Trichoderma viride is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). We demonstrate that tomato cyclopropyl isomerase (SlCPI), an enzyme involved in sterol biosynthesis, interacts with the LeEix2 receptor. Moreover, we examined the role of SlCPI in signaling during the LeEix/Eix defense response. We found that SlCPI is an important factor in the regulation of the induction of defense responses such as the hypersensitive response, ethylene biosynthesis, and the induction of pathogenesis-related protein expression in the case of LeEix/Eix. Our results also suggest that changes in the sterol composition reduce LeEix internalization, thereby attenuating the induction of plant defense responses.Plant innate immunity is activated upon the recognition of pathogen- and microbe-associated molecular patterns by surface-localized immune receptors or the stimulation of cytoplasmic immune receptors by pathogen effector proteins (Jones and Dangl, 2006; Thomma et al., 2011). Leucine-rich repeat (LRR) receptor kinases and leucine-rich repeat receptor proteins (LRR-RLPs) respond to conserved microbe-associated molecular patterns by producing a defense response upon detection (Altenbach and Robatzek, 2007; Bittel and Robatzek, 2007; Robatzek et al., 2007; Geldner and Robatzek, 2008). One such LRR-RLP is the ethylene-inducing xylanase (Eix) receptor LeEix2. The fungal protein Eix (Dean et al., 1989) is a well-known protein elicitor of defense response reactions in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum; Bailey et al., 1990; Avni et al., 1994). Eix induces ethylene biosynthesis, extensive electrolyte leakage, pathogenesis-related (PR) gene expression, reactive oxygen species (ROS), and the hypersensitive response (HR; Bailey et al., 1990; Ron et al., 2000). Eix was shown to specifically bind to the plasma membrane of responsive cultivars of both tomato and tobacco (Hanania and Avni, 1997). The response to Eix in tobacco and tomato cultivars is controlled by an LRR-RLP encoded by a single locus, termed LeEix (Ron and Avni, 2004). Previously, we showed that Eix triggers internalization of the LeEix2 receptor and its localization to endosomes (Bar and Avni, 2009).Endocytic processes and vesicular transport in general require the participation of membrane components that form transport vesicles with a capability to store and process a number of molecules known to participate in cell signaling (Anderson, 1993; Patel et al., 2008; Hansen and Nichols, 2010). Sterols are lipophilic membrane components that have many important functions in an array of eukaryotes. Changes in membrane-bound sterol levels and composition can have effects on the activity of membrane proteins and on signal transduction processes. The interaction between sterols and phospholipids forms microdomains termed lipid rafts (Simons and Ikonen, 1997). In response to cellular stimuli, lipid rafts can change the protein microenvironment, leading to the initiation of signaling cascades (Simons and Toomre, 2000; Mongrand et al., 2010; Simon-Plas et al., 2011). Sterols also provide precursors for the biosynthesis of steroid hormones such as mammalian estrogens and glucocorticoids and plant brassinosteroids (Bishop and Yokota, 2001; Benveniste, 2004; Suzuki and Muranaka, 2007). One of the enzymes involved in sterol biosynthesis is cyclopropyl isomerase (CPI; Lovato et al., 2000; Benveniste, 2004).A variety of endocytic pathways have been described in mammalian and fungal cells that differ mainly in the size, shape, and composition of endocytic vesicles and in the participation of different proteins (Conner and Schmid, 2003; Soldati and Schliwa, 2006). Cholesterol, the main mammalian sterol, has an important role in most internalization steps through both caveolae and clathrin-coated pits (Murata et al., 1995; Subtil et al., 1999). Cholesterol depletion alters endocytic structures and reduces the polar delivery of target proteins (Keller and Simons, 1998; Pichler and Riezman, 2004). Plant sterols are reported to be internalized into endosomes and distributed throughout the endocytic pathway in an actin-dependent manner (Grebe et al., 2003). The sterol endocytic pathway has been shown to interrupt the internalization, trafficking, and polar recycling of PIN2, an auxin efflux facilitator and polarity marker, in developing root epidermal cells of Arabidopsis (Arabidopsis thaliana; Grebe et al., 2003; Men et al., 2008). Sterols were shown to function in the trafficking of an ATP-binding cassette (ABCB19) from the trans-Golgi to the plasma membrane (Yang et al., 2013).Here, we report the isolation of SlCPI, which interacts with the LeEix2 receptor. Modulating the expression or function of SlCPI affects the induction of plant defense responses mediated by Eix.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.