Calcium-dependent Protein Kinases, CDPKs

Heterodimeric complexes incorporating the lipase-li ke proteins EDS1 wi th PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in A. thaliana, bolstering of signaling and resistance mediated by cell-s u r face pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. •We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in N. benthamiana. •We do not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad can abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation is sufficient for N. benthamianaTNL (Roq1) immunity.•Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-f orming RNLs remains unknown.

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Whereas the role of calcium ions (Ca2+) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined.Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca2+ and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca2+- and pH-dynamics revealed interconnections previously not described.In tobacco PTs, we demonstrated Ca2+-dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca2+. In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca2+ elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca2+-increases and cytosolic acidification by c. 2 min, suggesting a Ca2+/pH-independent early pathogen signaling step. Imaging Ca2+ and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca2+- and pH-responses.We propose close interrelation in Ca2+- and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.

Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self‐maintaining phytohormone salicylic acid (SA)‐defense loop.In spatial and temporal resolution, we show that calcium‐dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR‐inducing metabolite N‐hydroxy‐L‐pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1)Plants of increased CPK5, but not CPK6, signaling display an ‘enhanced SAR’ phenotype towards a secondary bacterial infection. In the sard1‐1 background, CPK5‐mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost.The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca2+], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity.

The plasma membrane (PM)‐localized receptor kinase FLAGELLIN SENSING 2 (FLS2) recognizes bacterial flagellin or its immunogenic epitope flg22, and initiates microbe‐associated molecular pattern‐triggered immunity, which inhibits infection by bacterial pathogens. The localization, abundance and activity of FLS2 are under dynamic control.Here, we demonstrate that Arabidopsis thaliana EXO70B1, a subunit of the exocyst complex, plays a critical role in FLS2 signaling that is independent of the truncated Toll/interleukin‐1 receptor‐nucleotide binding sequence protein TIR‐NBS2 (TN2). In the exo70B1‐3 mutant, the abundance of FLS2 protein at the PM is diminished, consistent with the impaired flg22 response of this mutant. EXO70B1‐GFP plants showed increased FLS2 accumulation at the PM and therefore enhanced FLS2 signaling.The EXO70B1‐mediated trafficking of FLS2 to the PM is partially independent of the PENETRATION 1 (PEN1)‐containing secretory pathway. In addition, EXO70B1 interacts with EXO70B2, a close homolog of EXO70B1, and both proteins associate with FLS2 and contribute to the accumulation of FLS2 at the PM.Taken together, our data suggest that the exocyst complex subunits EXO70B1 and EXO70B2 regulate the trafficking of FLS2 to the PM, which represents a new layer of regulation of FLS2 function in plant immunity.

Ca2+ is a ubiquitous second messenger for cellular signalling in various stresses and developmental processes. Here, we summarize current developments in the roles of Ca2+ during plant immunity responses. We discuss the early perception events preceding and necessary for triggering cellular Ca2+ fluxes, the potential Ca2+‐permeable channels, the decoding of Ca2+ signals predominantly via Ca2+‐dependent phosphorylation events and transcriptional reprogramming. To highlight the complexity of the cellular signal network, we briefly touch on the interplay between Ca2+‐dependent signalling and selected major signalling mechanisms – with special emphasis on reactive oxygen species at local and systemic levels.