BRAULT, A. | bioRxiv Science

bioRxiv2026

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

microbiology↗

bioRxiv2026

Seed Microbiota Diversity and Culture Collection of Four Major Crops Covering Different Genotypes and Production Modes

Seed microbiota play a crucial role in plant health and development, yet remain understudied compared to other plant-associated microbial communities. This study aimed to characterize seed microbiota diversity across four major crops (common bean, rapeseed, tomato, and wheat) and establish a comprehensive strain collection of seed-borne microorganisms (bacteria and fungi). We employed a combination of culture-dependent and culture-independent approaches to analyze 68 seed samples representing diverse genotypes and production modes. Our results revealed highly variable seed microbiota, with bacterial colonization ranging from 10 to 100 million bacterial CFUs per gram of seeds, and microbial richness varying from 4 to 351 bacterial and 16 to 138 fungal amplicon sequence variants (ASVs) per sample. Both plant genotype and production mode significantly influenced microbiota composition, with each seed sample produced harboring a distinct microbial assemblage. Interestingly, seeds produced in confined environments exhibited lower bacterial colonization but higher microbial richness compared to field-produced seeds. We observed divergent ecological drivers shaping bacterial and fungal communities. Bacterial assemblages were more host-specific and variable, while fungal communities showed greater stability and a substantial core microbiome shared across plant species. Our culturomics approach yielded a collection of 2,510 bacterial and 837 fungal isolates, representing 10-21% of the seed microbiota diversity detected by metabarcoding and the majority of the prevalent and abundant taxa. Notably, 44-60% of cultured bacterial isolates were not detected by metabarcoding, highlighting the complementary nature of these approaches to detect rare or under amplified taxa in PCR. This study provides insights into the complexity and variability of seed microbiota across different crops and production conditions. Our findings emphasize the importance of combining culturomics and sequencing methods for comprehensive characterization of seed microbiota to uncover the potential of seed-borne microorganisms as bioinoculants for sustainable agriculture.

ecology↗