Abstract
Ca(2+) 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(2+)-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(2+) rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca(2+)/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca(2+) 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(2+) elevation, excluding the possibility of CaM acting upstream from Ca(2+). The CaM antagonist and Ca(2+) chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca(2+)/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 Ca(2+) 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.
Highlights
During their life cycle, plants must respond to the threat of invasion from numerous types of pathogenic microorganisms
hypersensitive response (HR) development in Arabidopsis was evaluated by visual observation of necrosis in ethanol-bleached leaves and ion leakage associated with programmed cell death (PCD)
Plants exposed to avirulent pathogen (Pst avrRpt2+) developed visually observable HR at a time point post inoculation when plants inoculated with virulent pathogen (Pst avrRpt2-) displayed no observable necrotic symptoms
Summary
Plants must respond to the threat of invasion from numerous types of pathogenic microorganisms. Perception of a pathogen or conserved components of microbial cells (pathogen-associated molecular pattern (PAMP) molecules) induces Ca2+ influx across the plasma membrane leading to cytosolic Ca2+ elevation as an early step in this signaling pathway (Ali et al, 2007; Ma and Berkowitz, 2007; Lecourieux et al, 2006; Hu et al, 2004; Lecourieux et al, 2002). AtCNGC11/12 was shown to conduct Ca2+ and application of Ca2+-channel blockers abolished the AtCNGC11/12 induced PCD/HR (Urquhart et al, 2007) This evidence supports Ca2+ acting as an upstream signaling molecule in the cytosol that is necessary for HR
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