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+

(J.A.F.).Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca 2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca 2+ -dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca 2+ -dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth.

SUMMARYReactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col-0 and rbohh-1 rbohj-2 pollen tubes. Growth rate oscillations of rbohh-1 rbohj-2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh-1 rbohj-2 pollen tubes exhibit high-frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell-wall exocytosis precedes pollen tube collapse. Time-lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca 2+ reporter Yellow Cameleon 3.6, we demonstrate that high-amplitude growth rate oscillations in rbohh-1 rbohj-2 pollen tubes are correlated with growth-dependent Ca 2+ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K + homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca ) concentration ([Ca ] ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca ] orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca and anion imaging. We triggered Ca -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca ] increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca -permeable channels eliminated the [Ca ] increase, resulting in the abrogation of anion channel activity via Ca -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca ] gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.