Little is known about molecular steps linking perception of pathogen invasion by cell surface sentry proteins acting as pattern recognition receptors (PRRs) to downstream cytosolic Ca 2+ elevation, a critical step in plant immune signaling cascades. Some PRRs recognize molecules (such as flagellin) associated with microbial pathogens (pathogen-associated molecular patterns, PAMPs), whereas others bind endogenous plant compounds (damage-associated molecular patterns, DAMPs) such as peptides released from cells upon attack. This work focuses on the Arabidopsis DAMPs plant elicitor peptides (Peps) and their receptors, PEPR1 and PEPR2. Pep application causes in vivo cGMP generation and downstream signaling that is lost when the predicted PEPR receptor guanylyl cyclase (GC) active site is mutated. Pep-induced Ca 2+ elevation is attributable to cGMP activation of a Ca 2+ channel. Some differences were identified between Pep/PEPR signaling and the Ca 2+ -dependent immune signaling initiated by the flagellin peptide flg22 and its cognate receptor Flagellin-sensing 2 (FLS2). FLS2 signaling may have a greater requirement for intracellular Ca 2+ stores and inositol phosphate signaling, whereas Pep/PEPR signaling requires extracellular Ca 2+ . Maximal FLS2 signaling requires a functional Pep/PEPR system. This dependence was evidenced as a requirement for functional PEPR receptors for maximal flg22-dependent Ca 2+ elevation, H 2 O 2 generation, defense gene [WRKY33 and Plant Defensin 1.2 (PDF1.2)] expression, and flg22/FLS2-dependent impairment of pathogen growth. In a corresponding fashion, FLS2 loss of function impaired Pep signaling. In addition, a role for PAMP and DAMP perception in bolstering effector-triggered immunity (ETI) is reported; loss of function of either FLS2 or PEPR receptors impaired the hypersensitive response (HR) to an avirulent pathogen.calcium | cyclic nucleotide | leucine-rich repeat | transduction

Ca 2+ influx is an early signal initiating cytosolic immune responses to pathogen perception in plant cells; molecular components linking pathogen recognition to Ca 2+ influx are not delineated. Work presented here provides insights into this biological system of non-self recognition and response activation. We have recently identified a cyclic nucleotide-activated ion channel as facilitating the Ca 2+ flux that initiates immune signaling in the plant cell cytosol. Work in this report shows that elevation of cAMP is a key player in this signaling cascade. We show that cytosolic Ca 2+ elevation, nitric oxide (NO) and reactive oxygen species generation, as well as immune signaling, lead to a hypersensitive response upon application of pathogens and/or conserved molecules that are components of microbes and are all dependent on cAMP generation. Exogenous cAMP leads to Ca 2+ channel-dependent cytosolic Ca 2+ elevation, NO generation, and defense response gene expression in the absence of the non-self pathogen signal. Inoculation of leaves with a bacterial pathogen leads to cAMP elevation coordinated with Ca 2+ rise. cAMP acts as a secondary messenger in plants; however, no specific protein has been heretofore identified as activated by cAMP in a manner associated with a signaling cascade in plants, as we report here. Our linkage of cAMP elevation in pathogen-inoculated plant leaves to Ca 2+ channels and immune signaling downstream from cytosolic Ca 2+ elevation provides a model for how non-self detection can be transduced to initiate the cascade of events in the cell cytosol that orchestrate pathogen defense responses.

Endogenous plant elicitor peptides (Peps) can act to facilitate immune signaling and pathogen defense responses. Binding of these peptides to the Arabidopsis (Arabidopsis thaliana) plasma membrane-localized Pep receptors (PEPRs) leads to cytosolic Ca2+ elevation, an early event in a signaling cascade that activates immune responses. This immune response includes the amplification of signaling evoked by direct perception of pathogen-associated molecular patterns by plant cells under assault. Work included in this report further characterizes the Pep immune response and identifies new molecular steps in the signal transduction cascade. The PEPR coreceptor BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 contributes to generation of the Pep-activated Ca2+ signal and leads to increased defense gene expression and resistance to a virulent bacterial pathogen. Ca2+-dependent protein kinases (CPKs) decode the Ca2+ signal, also facilitating defense gene expression and enhanced resistance to the pathogen. Nitric oxide and reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent reactive oxygen species generation (due to the function of Respiratory Burst Oxidase Homolog proteins D and F) are also involved downstream from the Ca2+ signal in the Pep immune defense signal transduction cascade, as is the case with BRASSINOSTEROID-INSENSITIVE1 Associated Kinase1 and CPK5, CPK6, and CPK11. These steps of the pathogen defense response are required for maximal Pep immune activation that limits growth of a virulent bacterial pathogen in the plant. We find a synergism between function of the PEPR and Flagellin Sensing2 receptors in terms of both nitric oxide and reactive oxygen species generation. Presented results are also consistent with the involvement of the secondary messenger cyclic GMP and a cyclic GMP-activated Ca2+-conducting channel in the Pep immune signaling pathway.

Ca 2+ and nitric oxide (NO) are essential components involved in plant senescence signaling cascades. In other signaling pathways, NO generation can be dependent on cytosolic Ca 2+ . The Arabidopsis (Arabidopsis thaliana) mutant dnd1 lacks a plasma membrane-localized cation channel (CNGC2). We recently demonstrated that this channel affects plant response to pathogens through a signaling cascade involving Ca 2+ modulation of NO generation; the pathogen response phenotype of dnd1 can be complemented by application of a NO donor. At present, the interrelationship between Ca 2+ and NO generation in plant cells during leaf senescence remains unclear. Here, we use dnd1 plants to present genetic evidence consistent with the hypothesis that Ca 2+ uptake and NO production play pivotal roles in plant leaf senescence. Leaf Ca 2+ accumulation is reduced in dnd1 leaves compared to the wild type. Early senescence-associated phenotypes (such as loss of chlorophyll, expression level of senescence-associated genes, H 2 O 2 generation, lipid peroxidation, tissue necrosis, and increased salicylic acid levels) were more prominent in dnd1 leaves compared to the wild type. Application of a Ca 2+ channel blocker hastened senescence of detached wild-type leaves maintained in the dark, increasing the rate of chlorophyll loss, expression of a senescence-associated gene, and lipid peroxidation. Pharmacological manipulation of Ca 2+ signaling provides evidence consistent with genetic studies of the relationship between Ca 2+ signaling and senescence with the dnd1 mutant. Basal levels of NO in dnd1 leaf tissue were lower than that in leaves of wild-type plants. Application of a NO donor effectively rescues many dnd1 senescence-related phenotypes. Our work demonstrates that the CNGC2 channel is involved in Ca 2+ uptake during plant development beyond its role in pathogen defense response signaling. Work presented here suggests that this function of CNGC2 may impact downstream basal NO production in addition to its role (also linked to NO signaling) in pathogen defense responses and that this NO generation acts as a negative regulator during plant leaf senescence signaling.