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Summary: The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL‐INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)‐induced defense genes and alters plant growth responses to light. HaRxL106‐mediated suppression of immunity is abolished in RCD1 loss‐of‐function mutants. We report that RCD1‐type proteins are phosphorylated, and we identified Mut9‐like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1‐interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA‐induced defense marker gene expression compared with wild‐type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity. [ABSTRACT FROM AUTHOR]

We have assembled approximately 20 000 Arabidopsis thaliana promoter regions, compatible with functional studies that require cloning and with microarray applications. The promoter fragments can be captured as modular entry clones (MultiSite Gateway format) via site-specific recombinational cloning, and transferred into vectors of choice to investigate transcriptional networks. The fragments can also be amplified by PCR and printed on glass arrays. In combination with immunoprecipitation of protein–DNA complexes (ChIP–chip), these arrays enable characterization of binding sites for chromatin-associated proteins or the extent of chromatin modifications at genome scale. The Arabidopsis histone acetyltransferase GCN5 associated with 40% of the tested promoters. At most sites, binding did not depend on the integrity of the GCN5 bromodomain. However, the presence of the bromodomain was necessary for binding to 11% of the promoter regions, and correlated with acetylation of lysine 14 of histone H3 in these promoters. Combined analysis of ChIP–chip and transcriptomic data indicated that binding of GCN5 does not strictly correlate with gene activation. GCN5 has previously been shown to be required for light-regulated gene expression and growth, and we found that GCN5 targets were enriched in early light-responsive genes. Thus, in addition to its transcriptional activation function, GCN5 may play an important role in priming activation of inducible genes under non-induced conditions. [ABSTRACT FROM AUTHOR]

Membrane-associated, integral membrane and secreted proteins are of key importance in many cellular processes. For most of the 28 952 predicted proteins in Arabidopsis, the actual subcellular localization has not been demonstrated experimentally. So far, their potential membrane-association has been deduced from algorithms that predict transmembrane domains and signal peptides. However, the comprehensiveness and accuracy of these algorithms is still limited. The majority of membrane-associated and secreted proteins is synthesized on membrane-bound polysomes. Therefore, the isolation and characterization of mRNA associated with membrane-bound polysomes offers an experimental tool for the genome-wide identification of these proteins. Here we describe an efficient method to isolate mRNA from membrane-bound polysomes and report on the validation of the method to enrich for transcripts encoding membrane-associated and secreted proteins. The sensitivity and reproducibility of the isolation method was investigated by DNA microarray analysis. Pearson correlations between transcript levels obtained from three replicate isolations showed that the method is highly reproducible. A significant enrichment for mRNAs encoding proteins containing predicted transmembrane domains and signal peptides was observed in the membrane-bound polysomal fraction. In this fraction, 301 transcripts were classified by gene ontologies as ‘cellular component unknown’, and potentially encode previously unrecognized secreted or membrane-associated proteins. [ABSTRACT FROM AUTHOR]

Summary The calcium‐dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28‐associated proteins in Arabidopsis thaliana. We used affinity‐based proteomics and identified several potential CPK28 binding partners, including the C7 Raf‐like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune‐triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans‐phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf‐like kinases share some features with mitogen‐activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans‐phosphorylate any of the 10 Arabidopsis mitogen‐activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf‐like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs. [ABSTRACT FROM AUTHOR]

Plant genomes encode hundreds of secreted peptides; however, relatively few have been characterised. We report here an uncharacterised, stress-induced family of plant signalling peptides, which we call CTNIPs. Based on the role of the common co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) in CTNIP-induced responses, we identified in Arabidopsis thaliana the orphan receptor kinase HAESA-LIKE 3 (HSL3) as the CTNIP receptor via a proteomics approach. CTNIP-binding, ligand-triggered complex formation with BAK1, and induced downstream responses all involve HSL3. Notably, the HSL3-CTNIP signalling module is evolutionarily conserved amongst most extant angiosperms. The identification of this novel signalling module will further shed light on the diverse functions played by plant signalling peptides and will provide insights into receptor-ligand co-evolution. [ABSTRACT FROM AUTHOR]

Plant calcium (Ca2+)-dependent protein kinases (CPKs) represent the primary Ca2+-dependent protein kinase activities in plant systems. CPKs are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a calmodulin-like domain (CLD) via an autoinhibitory junction (J). Although regulation of CPKs by Ca2+ has been extensively studied, the contribution of autophosphorylation in controlling CPK activity is less well understood. Furthermore, whether calmodulin (CaM) contributes to CPK regulation, as is the case for Ca2+/CaM-dependent protein kinases outside the plant lineage, remains an open question. We therefore screened a subset of plant CPKs for CaM binding and found that CPK28 is a high affinity Ca2+/CaMbinding protein. Using synthetic peptides and native gel electrophoresis, we coarsely mapped the CaM-binding domain to a site within the CPK28 J domain that overlaps with the known site of intramolecular interaction between the J domain and the CLD. Peptide kinase activity of fully dephosphorylated CPK28 was Ca2+-responsive and was inhibited by Ca2+/CaM. Using in situ autophosphorylated protein, we expand on the known set of CPK28 autophosphorylation sites, and we demonstrate that, unexpectedly, autophosphorylated CPK28 had enhanced kinase activity at physiological concentrations of Ca2+ compared with the dephosphorylated protein, suggesting that autophosphorylation functions to prime CPK28 for Ca2+ activation and might also allow CPK28 to remain active when Ca2+ levels are low. Furthermore, CPK28 autophosphorylation substantially reducedsensitivity of the kinase to Ca2+/CaM inhibition. Overall, our analyses uncover new complexities in the control of CPK28 and provide mechanistic support for Ca2+ signaling specificity through Ca2+ sensor priming. [ABSTRACT FROM AUTHOR]