Gomez Cabrera, A.; Ameen, Y. O.; DAmico-Willman, K.; Joglekar, P.; Huguet-Tapia, J.; Carbone, I.; Huerta Vazquez, A. I.

Rearrangement hot spot (Rhs) toxins are specialized proteins bacteria use to inhibit rival microbes. Each toxin has two parts: a conserved N-terminal region used for protein translocation and delivery; and a hypervariable C-terminal tip that carries the toxic domain. Directly downstream of the Rhs toxin is a cognate immunity protein that protects the producing cell from autoinhibition. Rhs toxin ubiquity and diversity across bacterial genera suggest they play an essential role in bacterial fitness and niche exclusion. However, little is known about Rhs toxin abundance, diversity, and function within and among bacterial plant pathogens with different life histories. Here, we used a profile Hidden Markov Model to systematically mine publicly available genomes from four agriculturally significant plant-pathogenic bacterial genera: Xanthomonas, Ralstonia, Pectobacterium, and Dickeya. This approach identified 604, 294, 255, and 113 Rhs toxin homologs, respectively, from 165, 87, 60, and 31 genomes. N-terminal sequence analysis classified these homologs into distinct families, including two lineage-specific groups--one exclusive to Xanthomonas and another to Ralstonia--both unexpectedly linked to the type II secretion system instead of the canonical type VI secretion system. Sequence similarity network analysis of the C-terminal tip domains revealed conserved and lineage-specific toxin variants highlighting substantial diversification of Rhs C-terminal tips across taxonomic levels. Xanthomonads harbored the most extensive and diverse repertoire of predicted toxic domains, including DNases, RNases, proteases, and deaminases. Notably, the expected function of 69.64% of the C-terminal tip sequences across all genera remains unknown. Contrary to our hypothesis, our findings suggest that foliar pathogens encode more diverse Rhs toxin repertoires than their soilborne counterparts. However, only comprehensive functional analyses can provide a framework for understanding how these pathogens use Rhs toxins to establish themselves within agricultural ecosystems.