Proteome-wide identification of protein poly(ADP-ribosyl)ation targets reveals an important role of FHA domain protein DAWDLE in Arabidopsis immunity

Protein poly(ADP-ribosyl)ation (PARylation) primarily catalyzed by poly(ADP-ribose) polymerases (PARPs) plays a crucial role in controlling various cellular responses. However, PARylation targets and their functions remain largely elusive. In this study, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation to globally identify PARylation targets in plants. In line with nuclear localization of Arabidopsis PARPs, 56% of PARylation targets are predicted to localize in nucleus. Consistent with the essential role of protein PARylation in plant innate immunity, forkhead-associated (FHA) domain protein DAWDLE (DDL), one of the PARylation targets, positively regulates plant defense to both adapted and non-adapted pathogens. Arabidopsis PARP2 interacts and PARylates DDL, which was enhanced upon treatment of microbe-associated molecular pattern. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of DDL by PARP2. Genetic complementation assays indicate that DDL PARylation is required for its function in plant immunity. In contrast, DDL PARylation appears to be dispensable for its previously reported function in plant development likely mediated by the regulation of microRNA biogenesis. Our study uncovers many previously unknown PARylation targets and points to the distinct functions of DDL in plant immunity and development mediated by protein PARylation and small RNA biogenesis respectively. Two mutants (parg1-1 and parp1parp2) and one wild type control with and without treatment with 100nM flg22 (total 6 samples)