Functional Characterization and Conservation of RLCK-VII/PBL Family Members and Substrates in Land Plants

Abstract

Members of the receptor-like cytoplasmic kinase subfamily VII/AVRPPHB-SUSCEPTIBLE 1 (PBS1)-LIKE (RLCK-VII/PBL) family are central immune signaling protein kinases that function downstream of plasma membrane (PM)-localized pattern-recognition receptors (PRRs). Upon activation by PRRs, PBLs directly interact and phosphorylate diverse substrates to regulate a wide range of cellular immune responses, such as rapid production of apoplastic reactive oxygen species (ROS), ion fluxes, callose deposition, phosphorylation of mitogen-activated protein kinases (MAPKs), as well as transcriptional reprogramming. While the functions of PBLs and their substrates have been comprehensively characterized in the angiosperm model species Arabidopsis thaliana, little is known about the conservation of PBL-mediated immune signaling in land plants. Therefore, my PhD work focused on the functional characterization and conservation of PBL family members and their substrates in land plants, using the liverwort Marchantia polymorpha as the main model species. First, by using a combination of genetic and biochemical approaches, I identified the single isoforms of Marchantia PBL and RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) families that are genetically required for chitin-induced ROS production and provided the molecular mechanism underlying MpPBLa-mediated activation of MpRBOH1 in ROS production, showing the deep origin of this key regulatory step for plant NADPH activation in immunity. In corroboration with the findings of conserved MpPBLa-phosphorylation sites on MpRBOH1 N-termini, comparative biochemical analysis revealed that MpPBLa and the major Arabidopsis immune PBL isoform, BOTRYTIS-INDUCED KINASE 1 (BIK1) share identical core substrate sequence specificity. As such, I selected two families of BIK1 substrates that are conserved in Marchantia and genetically probed the functions of corresponding orthologous isoforms in early immune signaling. Lastly, given the broad implication of the CYCLIC NUCLEOTIDE-GATED CHANNEL (CNGC) family members in immune signaling and the lack of systematic examination of this calcium-permeable channel family in land plants, I present a thorough phylogenetic analysis of CNGC family members in charophyte algae and land plants. Furthermore, I identified a Marchantia CNGC-IVb isoform, MpCNGC-IVb.1, that might function as a constitutively active channel, causing cell death when heterologously expressed in Arabidopsis thaliana and Nicotiana benthamiana, which provides an instrumental model for future investigation on CNGC structure-function relationships.