Bacterial pathogens manipulate NAD+ in the host to promote disease

The small molecule nicotinamide adenine dinucleotide (NAD) is a crucial component in both prokaryotic and eukaryotic immune systems. Recently, the discovery that Toll/interleukin-1 receptor (TIR) proteins function as NAD hydrolases (NADase) has linked NAD-derived small molecules with plant immune signalling. Here we investigated how the bacterial pathogen Pseudomonas syringae may manipulate host NAD metabolism as a virulence strategy. We searched the P. syringae pangenome for type III effectors (T3Es) with potential NADase activity. Thirteen T3Es were identified, including five new candidates, that possess potential NAD-hydrolysing domains. Most P. syringae strains encode at least one NAD-manipulating T3E. One novel T3E, named HopBY, showed structural similarity to adenosine diphosphate ribose (ADPR) cyclases. This domain has not been found in pathogen proteins before to our knowledge. HopBY has enzymatic activity and efficiently hydrolyses NAD in vitro and in planta to produce cyclic ADP-ribose (cADPR). cADPR is also produced by TIR immune receptors of plants and other bacteria. Intriguingly, HopBY promoted bacterial virulence, indicating that cADPR is unlikely to directly initiate plant immune signalling. Evolutionary analysis revealed hopBY is likely to be mobile in P. syringae and homologues were identified in the genomes of Enterobacteriaceae plant pathogens such as Pectobacterium and Brenneria. This suggests past horizontal gene transfer of this effector between bacterial genera. Interestingly, additional homologues were predicted to be Type VI effectors in diverse bacteria. These proteins possessed the NADase enzymatic domain and therefore may also target NAD in alternative biotic interactions. The ADPRC domain of HopBY may have been shuffled by recombination in different bacteria to facilitate secretion through distinct secretion systems. NAD is therefore a major target in diverse interspecies antagonistic interactions