Calcium-dependent and independent phospho-control of plant immunity by integrating kinase signalling with transcriptional regulation.

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Plant immunity is a continuum of varying resistance intensities, spreading from local infection sites to distal tissues, which is augmented by effector-triggered immunity (ETI, i.e. the ability to recognize pathogen effectors) and autoimmunity (typically resulting from loss of an effector target). The Arabidopsis thaliana calcium-dependent protein kinase (CDPK), CPK5, is a highly calcium sensitive signalling hub in plant immunity. It controls immune responses at the plasma membrane (PM) via direct phosphorylation of the NADPH-oxidase RBOHD for ROS production and phosphorylation of EXO70B1 to modulate exocyst function in the transport of key PM immune components. In a collaborative effort, the Tang and Romeis groups could show that CPK5, but not other CDPK isoforms, is required for exo70B1-activated and TN2-dependent autoimmunity. Additionally, CPK5 signalling triggers transcription-based resistance leading to systemic immunity (via SARD1 and CAMTA3 transcription factors). Comparative in vivo phosphoproteomics analysis of cpk5 mutant lines identified defence-related transcriptional regulator CAMTA3 as well as MAPK-signalling cascade components as potential CPK5 substrate proteins. Joint experiments verify that CPK5 phosphorylates CAMTA3 directly, and both proteins co-localize in the plant nucleus. Furthermore, enhanced disease resistance to powdery mildew, reported for CPK5-OE lines and for exo70B1 mutants, is compromised in the presence of the dominant camta3-3D (A855V) allele. These data are consistent with the reported CAMTA3 function as a negative transcriptional regulator of plant immunity; as validated by the Lee group recently, CAMTA3 nuclear localization and protein stability is controlled by phosphorylation through MAPKs. In this joint proposal, we will synergize our efforts to investigate:(i) EXO70B-mediated transport of immune components at the PM in dependency of CPK5. (ii) Dual control of nuclear negative immune regulator CAMTA3 by dynamic phosphorylation through CPK5 and MAPKs. (iii) Mechanistic basis for CPK5 enzyme location and shuffling between the PM and the nucleus. (iv) Cross-interference between CDPK and MAPK signalling in local signal initiation to long-term systemic defence manifestationGiven the detailed biochemical, molecular, and genetic characterization of the immune components investigated, and the broad technical expertise spanning from genetics to in vivo phosphoproteomics, this proposal provides a unique opportunity to uncover underlying mechanisms of long discussed regulatory principles prevalent to (but not only) immune signalling – that is the interplay of pathways (CDPK/MAPK cascades), the dynamic modulation of multiple post-translational modifications converging on a common key regulatory substrate (e.g. CAMTA3), and the role of subcellular re-distribution of signalling components - facilitating an unpreceded resolution on the molecular level.