Biochemical characterisation of PARP and pseudo-PARP function in plant responses to biotic and abiotic stress

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Plants constantly respond to abiotic and biotic stressors in their environment to ensure survival. Poly-ADP-ribose-polymerases (PARPs) function as signal integrators of multiple stress signals including genotoxic and pathogen stress. PARPs catalyse the covalent post-translational attachment of ADP-ribose moieties onto themselves and onto target proteins. However, target proteins of plant PARPs remain unknown hampering our understanding of how PARPs influence plant stress responses. PARPs primarily function as sensors and signal amplifiers of DNA damage but additional functions in transcriptional regulation in response to stress are emerging. Plant infection by pathogens triggers DNA damage. parp mutants are insensitive to the DNA damaging agent bleomycin and we found that bleomycin primes transcriptional activation of salicylic acid-dependent defence genes in Arabidopsis. We will test the hypothesis that PARPs function as sensors and signal amplifiers of pathogen-induced DNA damage in plant immunity. To gain a better understanding of PARP function we will identify PARP target proteins and determine which amino acids of PARPs and target proteins are subject to ADP-ribosylation under stress.In addition to PARPs, plant genomes encode an additional protein family with a predicted PARP domain. This PARP-related protein family with Arabidopsis RADICAL INDUCED CELL DEATH 1 (RCD1) as founding member influences plant responses to salt and osmotic stress. Whether RCD1-type proteins are enzymatically active is debated. We have solved the crystal structure of the RCD1 PARP domain and provide molecular and structural evidence that RCD1-type proteins have lost ADP-ribosylation activity and can therefore be classified as pseudo-PARPs. Using the RCD1 pseudo-PARP domain structure as foundation we will define molecular features that distinguish active PARPs from pseudo-PARPs. We will test if the postulated active site of RCD1 is required for RCD1 function in abiotic stress responses. We found that RCD1 and its paralogue SIMILAR TO RCD1 1 (SRO1) form homo- and heteromers, likely mediated by their N-terminal WWE domains, and that SRO1 stabilises RCD1 in plant cells. We will define the structural basis for RCD1 and SRO1 homo- and heteromer formation and test the biological relevance of these interactions in Arabidopsis. In addition, we identified MUT9-like kinases (MLKs) as the first in planta interactors of RCD1. MLKs phosphorylate histone H3 thereby mediating transcriptional stress responses to salt and osmotic stress. We will test whether RCD1 together with MLKs influences histone H3 modifications of RCD1-dependent abiotic stress response genes.