Innate immunity signaling:: Cytosolic Ca2+ elevation is linked to downstream nitric oxide generation through the action of calmodulin or a calmodulin-like protein

Ca2+ 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 Ca2+-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 Ca2+ rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca2+/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca2+ 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 Ca2+ elevation, excluding the possibility of CaM acting upstream from Ca2+. The CaM antagonist and Ca2+ chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca2+/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. Pathogen-associated molecular pattern- mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca2+ 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.