Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae

et al. (2009) Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae. PLoS Pathog 5(2): e1000301. doi:10.1371/journal.ppat.1000301 Arabidopsis CaM Binding Protein CBP60g Contributes to MAMP-Induced SA Accumulation and Is Involved in Disease Resistance against Pseudomonas syringae Lin Wang 0 Kenichi Tsuda 0 Masanao Sato 0 Jerry D. Cohen 0 Fumiaki Katagiri 0 Jane Glazebrook 0 Frederick M. Ausubel, Massachusetts General Hospital, United States of America 0 1 Department of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota , St. Paul , Minnesota, United States of America, 2 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Meguro-ku, Tokyo, Japan, 3 Department of Horticultural Science, Microbial and Plant Genomics Institute, University of Minnesota , St. Paul, Minnesota , United States of America Salicylic acid (SA)-induced defense responses are important factors during effector triggered immunity and microbeassociated molecular pattern (MAMP)-induced immunity in plants. This article presents evidence that a member of the Arabidopsis CBP60 gene family, CBP60g, contributes to MAMP-triggered SA accumulation. CBP60g is inducible by both pathogen and MAMP treatments. Pseudomonas syringae growth is enhanced in cbp60g mutants. Expression profiles of a cbp60g mutant after MAMP treatment are similar to those of sid2 and pad4, suggesting a defect in SA signaling. Accordingly, cbp60g mutants accumulate less SA when treated with the MAMP flg22 or a P. syringae hrcC strain that activates MAMP signaling. MAMP-induced production of reactive oxygen species and callose deposition are unaffected in cbp60g mutants. CBP60g is a calmodulin-binding protein with a calmodulin-binding domain located near the N-terminus. Calmodulin binding is dependent on Ca2+. Mutations in CBP60g that abolish calmodulin binding prevent complementation of the SA production and bacterial growth defects of cbp60g mutants, indicating that calmodulin binding is essential for the function of CBP60g in defense signaling. These studies show that CBP60g constitutes a Ca2+ link between MAMP recognition and SA accumulation that is important for resistance to P. syringae. - Funding: This work was supported by grant IOB-0419648 from the National Science Foundation Arabidopsis 2010 program to JG and FK. JDC was supported by grants from the U.S. National Science Foundation (MCB-0725149) and the U.S. Department of Agriculture National Research Initiative (2005-35318-16197). MS was supported by a Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. Competing Interests: The authors have declared that no competing interests exist. Plant innate immunity is multi-layered and tightly regulated by a complex signaling network [1]. Defense against biotrophic or hemibiotrophic bacterial pathogens can be thought of as consisting of two branches: the broad and nonspecific defenses triggered by the perception of microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), and the robust and relatively more specific resistance mediated by resistance (R) genes [2,3]. MAMPs are proteins and other molecules characteristic of microbes. MAMP-triggered defense is initiated by perception of MAMPs by pattern-recognition receptors (PRRs). Well-characterized examples in Arabidopsis include recognition of flagellin by the receptor kinase FLS2 [4], of Ef-Tu by the receptor kinase EFR [5], and of chitin by the LysM receptor kinase CERK1. Direct binding has been demonstrated for FLS2 and EFR, but not for CERK1 [6,7]. FLS2 and EFR require a second kinase, BAK1, to initiate defense signaling [810]. Signaling activation results in an oxidative burst produced by the NADPH oxidase encoded by AtrbohD, which is in turn required for deposition of callose at the cell wall [11]. Other responses include closure of stomata, activation of a MAP kinase cascade, and a suite of gene expression changes [1214]. MAMP responses are effective in limiting pathogen growth, as pre-treatment with flg22, a peptide derived from flagellin, dramatically reduces growth of Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in an FLS2-dependent manner [15], efr plants are more susceptible to Agrobacterium tumefaciens [5], and cerk1 mutants are more susceptible to Alternaria brassicicola [6,7]. Bacterial pathogens produce numerous virulence effector proteins that are secreted into the host cytoplasm, where many of them disrupt plant defense responses [2,3,16]. Plants can counter this if they have one or more appropriate Resistance (R) genes. R proteins detect effectors by directly binding effector proteins or by sensing the cellular disturbance caused by effector activity [17]. R protein activation results in induction of additional layers of defenses, including production of reactive oxygen species (ROS) and activation of the hypersensitive response (HR), a programmed cell death response thought to limit pathogen access to water and nutrients [18]. R gene recognition of an effector also results in activation of the salicylic acid (SA)-dependent defense signaling pathway, which plays an important role in resistance [19]. Several components of the SA signaling circuitry have been identified through genetic analysis in Arabidopsis. ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) and PHYTOALEXIN DEFICIENT 4 (PAD4) are physically-interacting proteins that are required for SA synthesis in response to some, but not all, pathogens [2023]. PAD4 and EDS1 are also required for pathogen-induced expression of many SA-independent genes [24]. SALICYLIC ACID Plants respond to attack by microbial pathogens through activation of a battery of defense responses. This activation is controlled by a complex signaling network. Disease resistance depends on rapid activation of plant defense responses. Improved understanding of the signaling network may lead to development of crops with improved disease resistance. Here, we used the model plant Arabidopsis thaliana to study activation of defense responses after infection by a bacterial pathogen, Pseudomonas syringae. We found that a gene not previously known to function in defense signaling, CBP60g, is needed for resistance. By studying plants with mutations in this gene, we found that CBP60g contributes to the increases in levels of the important signaling molecule, salicylic acid, that occur after pathogen recognition. We also found that the CBP60g protein binds calmodulin, a protein that mediates calcium regulation of protein function. Calmodulin binding was necessary for the function of CBP60g in disease resistance. We conclude that CBP60g is a protein that mediates calmodulin-dependent activation of salicylic acid signaling in response to pathogen recognition. INDUCTION DEFICIENT 2 (SID2), which encodes isochorismate synthase, and ENHANCED DI (...truncated)