ADP ribosylation of proteins during pathogens sensing and plant immunity: assays using swapped-domain receptors in plants.

ADP ribosylation is a post-translational modification of proteins shared by animals, plants and bacteria, and catalysed by mono- or poly-ADP- ribosyl transferases (ART). In plants, little is known about the targets of the various ART enzymes. The DAWDLE (DDL), a Forkhead-associated (FHA) domain protein, has been identified as a target of PARP2, with multiple PARylation sites, which was enhanced upon treatment of bacterial flagellin. Forkhead-associated (FHA) domains can bind and recognise ADP ribose units (Readers). DDL positively regulates plant defense to both adapted and non-adapted pathogens. DDL PARylation is required for its function in plant immunity. In addition to the endogenous ADP-ribosylation, plant pathogens contain effectors with mono ART activity (HopU1, HopF2, AvrRpmi1), while HopM1 interacts and induce degradation of an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) involved in vesicle trafficking. In parp triple mutants, Parylation was found increased, pointing to PARP-like domain proteins, Radical Cell Death 1 (RCD-1) and Similar to RCD One (SRO). Two LRR Receptors, FLAGELLIN SENSING 2 (FLS2) and EF-Tu RECEPTOR (EFR) are activated by flagellin peptide and by EF-Tu protein. Otherwise, CLADOSPORIUM FULVUM-9(Cf-9) R protein senses Cf-9. EFR/Cf-9 chimeric receptor is able to trigger in tobacco (Nicotiana tabacum) an HR in response to elf18C conferring resistance to different strains of P. syringae. We study FLS2/Cf-9 and EFR/Cf-9 fused with GFP, testing for immune response in tobacco plants expressing the avirulence gene Avr-9 and infiltrated with Agrobacterium carrying the native Cf-9 gene. SOBIR1 forms a constitutive complex with FLS2/Cf-9 and EFR/Cf-9, and BAK1/SERK3 plays a crucial role in plant immune response (PCD). Responsive plants show leaves with a Hypersensitive Response (HR). We are studying the requirement of signaling partners in the activation of immune response, in the presence of PARP inhibitors. In parallel, we will establish plants transformed with GFP-Macrodomain to detect subcellular localization of mono- and poly- ADP-ribosylated protein targets.