Biology subjects

Dharmasena, T.

Publications and source records attributed to Dharmasena, T..

3 recordsLinked to original sources

bioRxiv2025

The Ralstonia solanacearum E3 ligase effector RipV1 targets subfamily IXb receptor-like cytoplasmic kinases that negatively regulate immunity in Nicotiana benthamiana.

Plants detect microbe-associated molecular patterns from pathogens via plasma membrane- localized receptors which activate multiple signaling cascades that lead to pattern-triggered immunity (PTI). Receptor-like cytoplasmic kinases (RLCKs) are essential hubs of plant immune signaling, associating with receptors and intracellular proteins through phosphorylation events. As a consequence, RLCKs have emerged as common targets of pathogen effectors. To improve our knowledge on Solanaceae responses to the bacterial wilt pathogen Ralstonia solanacearum, we conducted a yeast two-hybrid screen between tomato RLCKs and effectors conserved in R. solanacearum Korean isolates. Several members of RLCK subfamily IXb, which contain a ubiquitin-ligase plant U-box domain in addition to the kinase domain, interacted with RipV1, an effector containing a novel E3 ligase domain (NEL). In vitro assays revealed that RLCK-IXb-1 displayed ubiquitin-ligase activity but no detectable kinase activity. RipV1 could trans- ubiquitinate RLCK-IXb-1 in vitro and promote its stability in planta. Using virus-induced gene silencing of RLCK-IXb homologs in Nicotiana benthamiana, we could further show that several RLCK-IXb proteins act as negative regulators of early PTI signaling. RipV1 was previously reported to contribute to R. solanacearum virulence in potato and to elicit cell death in an E3 ligase activity-dependent manner in N. benthamiana. Here we show that RipV1-induced cell death occurred in plants impaired for effector recognition but could be suppressed by over-expression of RLCK-IXb-1, suggesting that this response is related to the virulence function of RipV1. Altogether, our work identifies possible substrates of an NEL effector and underlines the complex roles of RLCKs in plant immune signaling.

plant biology↗

bioRxiv2024

Catalytically inactive subgroup VIII receptor-like cytoplasmic kinases regulate the immune-triggered oxidative burst in Arabidopsis thaliana

Protein kinases are key components of multiple cell signaling pathways. Several protein kinases of the receptor-like cytoplasmic kinase (RLCK) family have demonstrated roles in immune and developmental signaling across various plant species, making them a family of interest in the study of phosphorylation-based signal relay. Here, we present our investigation of a subfamily of RLCKs in Arabidopsis thaliana. Specifically, we focus on subgroup VIII RLCKs: MAZ and its paralog CARK6, as well as CARK7 and its paralog CARK9. We found that both MAZ and CARK7 associate with the calcium-dependent protein kinase CPK28 in planta, and furthermore that CPK28 phosphorylates both MAZ and CARK7 on multiple residues in areas that are known to be critical for protein kinase activation. Genetic analysis suggests redundant roles for MAZ and CARK6 as negative regulators of the immune-triggered oxidative burst. We find evidence that supports homo- and hetero-dimerization between CARK7 and MAZ, which may be a general feature of this protein family. Multiple biochemical experiments suggest that neither MAZ nor CARK7 demonstrate catalytic protein kinase activity in vitro. Interestingly, we find that a mutant variant of MAZ incapable of protein kinase activity is able to complement maz-1 mutants, suggesting noncatalytic roles of MAZ in planta. Overall, our study identifies subgroup VIII RLCKs as new players in Arabidopsis immune signaling and highlights the importance of noncatalytic functions of protein kinases.

plant biology↗

bioRxiv2023

Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in Arabidopsis thaliana

The calcium-dependent protein kinase CPK28 is a regulator of immune homeostasis in multiple plant species. Here, we used a proteomics approach to uncover CPK28-associated proteins. We found that CPK28 associates with subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39, and trans-phosphorylates RAF26 and RAF39. Metazoan Raf kinases function in mitogen-activated protein kinase (MAPK) cascades as MAPK kinase kinases (MKKKs). Although Raf-like kinases share some features with MKKKs, we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis MKKs. We show that MRK1, RAF26, and RAF39 localize to the cytosol and endomembranes, and we define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal immunity, and possess substrate specificities distinct from canonical MKKKs.

plant biology↗