New kids on the block-cysteine-rich receptor-like kinases in pattern-triggered immunity.

Plant-specific receptor-like protein kinases (RLKs) are essential for pathogen recognition during pattern-triggered immunity. Together with coreceptors and associated proteins, they act as bona fide immune receptors, perceiving a variety of microbe-associated molecular patterns or damage-associated molecular patterns. The cysteine-rich receptor-like kinases (CRKs) form one of the biggest subgroups of RLKs, but so far, their ligands have not been identified. It has been shown that CRKs play important roles in plant immunity and defense responses as well as in response to abiotic stimuli and in control of plant development. However, molecular information on how CRKs integrate with the known framework of signaling components controlling early defense responses remains enigmatic.

CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES: their evolution, structure, and roles in stress response and development.

Plant-specific receptor-like protein kinases (RLKs) are central components for sensing the extracellular microenvironment. CYSTEINE-RICH RLKs (CRKs) are members of one of the biggest RLK subgroups. Their physiological and molecular roles have only begun to be elucidated, but recent studies highlight the diverse types of proteins interacting with CRKs, as well as the localization of CRKs and their lateral organization within the plasma membrane. Originally the DOMAIN OF UNKNOWN FUNCTION 26 (DUF26)-containing extracellular region of the CRKs was proposed to act as a redox sensor, but the potential activating post-translational modification or ligands perceived remain elusive. Here, we summarize recent progress in the analysis of CRK evolution, molecular function, and role in plant development, abiotic stress responses, plant immunity, and symbiosis. The currently available information on CRKs and related proteins suggests that the CRKs are central regulators of plant signaling pathways. However, more research using classical methods and interdisciplinary approaches in various plant model species, as well as structural analyses, will not only enhance our understanding of the molecular function of CRKs, but also elucidate the contribution of other cellular components in CRK-mediated signaling pathways.

Poly(ADP-ribose)-binding protein RCD1 is a plant PARylation reader regulated by Photoregulatory Protein Kinases.

Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.

Stress-induced reactive oxygen species compartmentalization, perception and signalling.

Reactive oxygen species (ROS) are essential for life and are involved in the regulation of almost all biological processes. ROS production is critical for plant development, response to abiotic stresses and immune responses. Here, we focus on recent discoveries in ROS biology emphasizing abiotic and biotic stress responses. Recent advancements have resulted in the identification of one of the first sensors for extracellular ROS and highlighted waves of ROS production during stress signalling in Arabidopsis. Enzymes that produce ROS, including NADPH oxidases, exhibit precise regulation through diverse post-translational modifications. Discoveries highlight the importance of both amino- and carboxy-terminal regulation of NADPH oxidases through protein phosphorylation and cysteine oxidation. Here, we discuss advancements in ROS compartmentalization, systemic ROS waves, ROS sensing and post-translational modification of ROS-producing enzymes and identify areas where foundational gaps remain.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis.

Reactive oxygen species (ROS) are important messengers in eukaryotic organisms, and their production is tightly controlled. Active extracellular ROS production by NADPH oxidases in plants is triggered by receptor-like protein kinase-dependent signaling networks. Here, we show that CYSTEINE-RICH RLK2 (CRK2) kinase activity is required for plant growth and CRK2 exists in a preformed complex with the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) in Arabidopsis (Arabidopsis thaliana). Functional CRK2 is required for the full elicitor-induced ROS burst, and consequently the crk2 mutant is impaired in defense against the bacterial pathogen Pseudomonas syringae pv tomato DC3000. Our work demonstrates that CRK2 regulates plant innate immunity. We identified in vitro CRK2-dependent phosphorylation sites in the C-terminal region of RBOHD. Phosphorylation of S703 RBOHD is enhanced upon flg22 treatment, and substitution of S703 with Ala reduced ROS production in Arabidopsis. Phylogenetic analysis suggests that phospho-sites in the C-terminal region of RBOHD are conserved throughout the plant lineage and between animals and plants. We propose that regulation of NADPH oxidase activity by phosphorylation of the C-terminal region might be an ancient mechanism and that CRK2 is an important element in regulating microbe-associated molecular pattern-triggered ROS production.

Mechanistic insights into the evolution of DUF26-containing proteins in land plants.

Large protein families are a prominent feature of plant genomes and their size variation is a key element for adaptation. However, gene and genome duplications pose difficulties for functional characterization and translational research. Here we infer the evolutionary history of the DOMAIN OF UNKNOWN FUNCTION (DUF) 26-containing proteins. The DUF26 emerged in secreted proteins. Domain duplications and rearrangements led to the appearance of CYSTEINE-RICH RECEPTOR-LIKE PROTEIN KINASES (CRKs) and PLASMODESMATA-LOCALIZED PROTEINS (PDLPs). The DUF26 is land plant-specific but structural analyses of PDLP ectodomains revealed strong similarity to fungal lectins and thus may constitute a group of plant carbohydrate-binding proteins. CRKs expanded through tandem duplications and preferential retention of duplicates following whole genome duplications, whereas PDLPs evolved according to the dosage balance hypothesis. We propose that new gene families mainly expand through small-scale duplications, while fractionation and genetic drift after whole genome multiplications drive families towards dosage balance.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress.

Receptor-like kinases are important regulators of many different processes in plants. Despite their large number only a few have been functionally characterized. One of the largest subgroups of receptor-like kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among the CRKs has been suggested as major cause for functional redundancy. The genomic localization of CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extracellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not chloroplastic ROS production.

Transcriptional regulation of the CRK/DUF26 group of receptor-like protein kinases by ozone and plant hormones in Arabidopsis.

BACKGROUND: Plant Receptor-like/Pelle kinases (RLK) are a group of conserved signalling components that regulate developmental programs and responses to biotic and abiotic stresses. One of the largest RLK groups is formed by the Domain of Unknown Function 26 (DUF26) RLKs, also called Cysteine-rich Receptor-like Kinases (CRKs), which have been suggested to play important roles in the regulation of pathogen defence and programmed cell death. Despite the vast number of RLKs present in plants, however, only a few of them have been functionally characterized. RESULTS: We examined the transcriptional regulation of all Arabidopsis CRKs by ozone (O3), high light and pathogen/elicitor treatment - conditions known to induce the production of reactive oxygen species (ROS) in various subcellular compartments. Several CRKs were transcriptionally induced by exposure to O3 but not by light stress. O3 induces an extracellular oxidative burst, whilst light stress leads to ROS production in chloroplasts. Analysis of publicly available microarray data revealed that the transcriptional responses of the CRKs to O3 were very similar to responses to microbes or pathogen-associated molecular patterns (PAMPs). Several mutants altered in hormone biosynthesis or signalling showed changes in basal and O3-induced transcriptional responses. CONCLUSIONS: Combining expression analysis from multiple treatments with mutants altered in hormone biosynthesis or signalling suggest a model in which O3 and salicylic acid (SA) activate separate signaling pathways that exhibit negative crosstalk. Although O3 is classified as an abiotic stress to plants, transcriptional profiling of CRKs showed strong similarities between the O3 and biotic stress responses.

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.