Rhs-toxin Abundance, Diversity, and Function in Four Genera of Plant Pathogenic Bacteria: Xanthomonas, Ralstonia, Pectobacterium, and Dickeya.

Rearrangement hotspot (Rhs) toxins are polymorphic bacterial toxins that inhibit competitor microbes, playing a critical role in interbacterial competition. Each toxin comprises a conserved N-terminal region for translocation and a hypervariable C-terminal domain harboring toxic activity, with a cognate immunity gene positioned downstream to prevent self-killing. The ubiquity and variability of Rhs toxins across bacterial genera suggest they contribute significantly to microbial fitness and niche exclusion. However, their distribution, diversity, and functional roles remain poorly understood in plant-pathogenic bacteria. Here, we used a profile Hidden Markov Model to systematically mine genomes from four agriculturally important plant-pathogenic genera: Xanthomonas, Ralstonia, Pectobacterium, and Dickeya, identifying 604, 294, 255, and 113 Rhs homologs, respectively, across 343 genomes. N-terminal sequence classification revealed multiple distinct families, including lineage-specific groups exclusive to Xanthomonas and Ralstonia. Notably, these were linked to the type II secretion system, diverging from the canonical association with type VI secretion. C-terminal domain analyses via sequence similarity networks revealed both conserved and lineage-specific toxic variants. Xanthomonas strains encoded the most diverse repertoire, including predicted DNases, RNases, proteases, and deaminases. However, the functions of 69.6% of C-terminal domains remain uncharacterized. Contrary to our initial hypothesis that soilborne bacterial pathogens would encode more abundant and diverse Rhs toxins due to intense microbial competition in soil, foliar pathogens exhibited greater Rhs diversity. This suggests that aboveground plant environments may impose stronger selective pressures for Rhs toxin diversification. These findings highlight the unexplored potential of Rhs toxins in shaping microbial ecology and underscore the need for functional characterization to elucidate their roles in bacteria-microbiome interactions. Author SummaryBacteria constantly interact and compete for resources with other microbial agents in plant-associated microbiomes. One way they can enhance their competitive fitness is by producing proteinaceous toxins that can harm or kill rival cells. One group of toxins used for intraspecies competition is the Rhs toxins, which have a unique structure and diversity of enzymatic actions encoded in their hypervariable protein tip. To better understand the diversity and abundance of Rhs toxins in plant pathogenic bacteria, a computational pipeline was developed and used to analyze publicly available genomes from four major bacterial plant pathogenic genera. Results confirm the ubiquity of Rhs toxins in bacterial genomes and show the lack of Rhs toxins in genomes from unique species. Furthermore, some Rhs toxin enzymatic functions were unique to a particular genus. The data presented here suggest that some Rhs toxins may be secreted through alternative pathways beyond the well-known Type VI secretion system. Together, this study on the abundance and diversity of Rhs toxins in plant pathogenic bacterial genera highlights the complexity and predicted functional diversity of Rhs toxins and provides fundamental knowledge to test hypotheses on the role Rhs toxins play in microbial ecology, community structure, and evolution in the context of plant disease.