A conserved Rhs module associated with the type VI secretion system facilitates effector innovation and immunity acquisition for ecological adaptation of Stenotrophomonas species

The type VI secretion system (T6SS) is a widespread antibacterial weapon whose evolutionary flexibility promotes bacterial survival in competitive environments. Here, we characterized the vgrG6 cluster of Stenotrophomonas rhizophila CFBP13503, which encodes a putative PAAR-Rhs-fused effector (Rhs-Ct, Tse6) along with a poly-immunity cluster. Comparative genomics across Stenotrophomonas genus revealed strong conservation of the vgrG6 core genes, contrasting with a striking variability in the downstream poly-immunity region, consistent with rapid diversification and niche-specific adaptation. In silico structural analysis of Rhs-Ct domains from 158 Stenotrophomonas strains identified 14 distinct effector families with diverse putative enzymatic activities, including nucleases and deaminases. The Rhs-Ct from strain CFBP13503, Tse6, harbors a domain of unknown function that is rare across the bacterial diversity. Its closest orthologs were found in Gram-positive Actinomycetes and halophilic Gram-negative Planctomycetia. Strikingly, saline conditions significantly enhanced both S. rhizophila growth and T6SS activity. Functional assays demonstrated that Tse6 is specifically active against the phytopathogen Clavibacter michiganensis and the plant beneficial strains Curtobacterium herbarum and Plantibacter flavus, abolishing their resistance to S. rhizophila T6SS attacks. Furthermore, the immunity protein Tsi6 was predicted to interact with Tse6 orthologs from phylogenetically distant taxa, indicating a broad protective capacity. Taken together, our results establish the vgrG6 cluster as a flexible adaptive module that links effector innovation and immunity diversification to ecological specialization. This work highlights the previously unrecognized role for the S. rhizophila T6SS in mediating bacterial competition in saline niches dominated by certain Gram-positive species. IMPORTANCES. rhizophila CFBP13503 carries a large set of T6SS effectors, some of which are specifically targeting bacterial species. It is important to determine which species are targeted and how effectors adapt to changing environments. Stenotrophomonas species possess a conserved vgrG6 cluster composed of a Rhs-fused effector (Rhs-Ct) and a poly-immunity region. Comparative analysis showed that the Rhs-Ct effector varies from stain together with the poly-immunity content. This diversity underlies the adaptive potential of the vgrG6 cluster in Stenotrophomonas species, notably through targeting of Gram-positive bacteria in a very distinct ecological niche. This study reveals a new effector repertoire to target Gram-positive phytopathogens that can be investigated for biocontrol studies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/738213v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ada5b9org.highwire.dtl.DTLVardef@15b5211org.highwire.dtl.DTLVardef@68ef4dorg.highwire.dtl.DTLVardef@136bdfb_HPS_FORMAT_FIGEXP M_FIG C_FIG