Calcium-dependent protein kinase/NADPH oxidase activation circuit is required for rapid defense signal propagation

Abstract

In animals and plants, pathogen recognition triggers the local activation of intracellular signaling that is prerequisite for mounting systemic defenses in the whole organism. We identified that Arabidopsis thaliana isoform CPK5 of the plant calcium-dependent protein kinase family becomes rapidly biochemically activated in response to pathogen-associated molecular pattern (PAMP) stimulation. CPK5 signaling resulted in enhanced salicylic acid-mediated resistance to the bacterial pathogen Pst DC3000, differential plant defense gene expression, and synthesis of reactive oxygen species (ROS). Using selected reaction monitoring MS, we identified the plant NADPH oxidase, respiratory burst oxidase homolog D (RBOHD), as an in vivo phosphorylation target of CPK5. Remarkably, CPK5-dependent in vivo phosphorylation of RBOHD occurs on both PAMP-and ROS stimulation. Furthermore, rapid CPK5-dependent biochemical and transcriptional activation of defense reactions at distal sites is compromised in cpk5 and rbohd mutants. Our data not only identify CPK5 as a key regulator of innate immune responses in plants but also support a model of ROS-mediated cell-to-cell communication, where a self-propagating mutual activation circuit consisting of the protein kinase, CPK5, and the NADPH oxidase RBOHD facilitates rapid signal propagation as a prerequisite for defense response activation at distal sites within the plant.disease resistance | plant innate immunity | ROS signaling R eceptor-mediated recognition of pathogen-associated molecular patterns (PAMPs) triggers the activation of inducible defenses against microbial pathogens in both plants and animals. Some of the earliest PAMP-induced intracellular signaling responses are shared in these two kingdoms (1), including changes in ion fluxes, an increase in the intracellular calcium concentration, the activation of protein kinases, or the synthesis of reactive oxygen species (ROS). These rapid responses are a prerequisite for the subsequent transcriptional reprogramming and alterations in hormone status that ultimately lead to resistance (2, 3). In contrast, the role of calcium-regulated protein kinase signaling in local and systemic immune responses is less well characterized. In the animal system, stimulation of Toll-like receptors TLR2 or TLR4 is known to result in the recruitment and activation of distinct calcium-responsive kinases Ca 2+

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

“…A possible source could be NADPH oxidase-like and superoxide dismutase activity, as has been shown for elicitor and salt-induced accumulation of ROS (Leshem et al, 2006;Ashtamker et al, 2007;Suzuki et al, 2011). Similarly, biotic elicitors are known to initiate ROS, likely through activation of calcium and phosphorylation cascades (Sagi and Fluhr, 2001;Fluhr, 2009;Dubiella et al, 2013).…”

“…Superoxide can react with H 2 O 2 via a Haber-Weiss mechanism to form singlet oxygen (H 2 O 2 +O 2 2 → OH 2 + OH$ + 1 O 2 ; Kellogg and Fridovich, 1975;Khan and Kasha, 1994), although the efficiency of singlet oxygen production by these ROS in vivo is not known (MacManus-Spencer and McNeill, 2005). Nonetheless, the accumulation of superoxide and H 2 O 2 is readily detected in local or systemic wounded tissue (Sagi et al, 2004;Warwar et al, 2011) and in the immune responses (Dubiella et al, 2013). A possible source could be NADPH oxidase-like and superoxide dismutase activity, as has been shown for elicitor and salt-induced accumulation of ROS (Leshem et al, 2006;Ashtamker et al, 2007;Suzuki et al, 2011).…”

Aquaporins facilitate hydrogen peroxide entry into guard cells to mediate ABA- and pathogen-triggered stomatal closure

“…In molecular terms, flg22 activates ABA-independent signaling components, such as oxylipins and salicylic acid, together with specific protein kinases (19). These include BIK1 and CPK5, which were recently shown to phosphorylate RbohD (18,43), or CPK4, CPK6, and CPK11, which function as positive regulators of the PAMP-induced ROS burst (44). Along with these lines, our study suggests that ABA and flg22 induced distinct kinetics and intensities of H 2 O 2 accumulation in the guard cell cytoplasm.…”

Harnessing host ROS-generating machinery for the robust genome replication of a plant RNA virus

“…RBOH activity is tightly and coordinately regulated posttranslationally [e.g., activation by Ca 2+ , phosphatidic acid (PA), G proteins, or protein kinases] (9-12). It has recently been shown that receptor-like cytoplasmic kinases and calciumdependent protein kinases (CDPKs) regulate RBOHs activity via direct phosphorylation or through modulating regulators of RBOHs during recognition of MAMPs via corresponding surface-localized pattern recognition receptors (13)(14)(15)(16). RBOH-derived ROS induce stomatal closure to keep out invading bacteria, cross-linking the plant cell wall to block pathogen entry, and trigger local and systemic transcriptional reprogramming to activate plant immunity (1,4,9,10).…”