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. Here, we deployed an Arabidopsis protein microarray coupled with in vitro PARylation assays to globally identify PARylation targets in plants. Consistent with the essential role of PARylation in plant immunity, the forkhead‐associated (FHA) domain protein DAWDLE (DDL), one of PARP2 targets, positively regulates plant defense to both adapted and non‐adapted pathogens. Arabidopsis PARP2 interacts with and PARylates DDL, which was enhanced upon treatment of bacterial flagellin. Mass spectrometry and mutagenesis analysis identified multiple PARylation sites of style="fixed-case">DDL by style="fixed-case">PARP2. Genetic complementation assays indicate that style="fixed-case">DDL PARylation is required for its function in plant immunity. In contrast, style="fixed-case">DDL PARylation appears to be dispensable for its previously reported function in plant development partially mediated by the regulation of micro style="fixed-case">RNA biogenesis. Our study uncovers many previously unknown style="fixed-case">PARylation targets and points to the distinct functions of style="fixed-case">DDL in plant immunity and development mediated by protein style="fixed-case">PARylation and small style="fixed-case">RNA biogenesis, respectively.
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