Innate Immunity Signaling: Cytosolic Ca Elevation Is Linked to Downstream Nitric Oxide Generation through the Action of Calmodulin or a Calmodulin-Like Protein

Ca rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca/ calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca elevation, excluding the possibility of CaM acting upstream from Ca. The CaM antagonist and Ca chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogenassociated molecular pattern-mediated NO generation in cells of cml24-4mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.