Development of a soil inoculation method coupled with blocker-mediated 16S rRNA gene amplicon sequencing reveals the effect of antibacterial T6SS on agrobacteria tumorigenesis and gallobiome composition

Abstract

ABSTRACTThe type VI secretion system (T6SS) is deployed by many proteobacteria to secrete effector proteins into bacterial competitors for competition or eukaryotic cells for pathogenesis. Agrobacteria, a group of soil-borne phytopathogens causing crown gall disease on various plant species, deploys the T6SS to attack closely- and distantly-related bacterial speciesin vitroandin planta. Current evidence suggests that the T6SS is not essential for pathogenesis under direct inoculation but it remains unknown whether the T6SS influences natural disease incidence or the microbial community within crown galls (i.e., gallobiome). To address these two key questions, we established a soil inoculation method on wounded tomato seedlings that mimics natural infections and developed a bacterial 16S rRNA gene amplicon enrichment sequencing platform. By comparing theAgrobacteriumwild-type strain C58 with two T6SS mutants, we demonstrate that the T6SS influences both disease occurrence and gallobiome composition. Based on multiple inoculation trials across seasons, all three strains could induce tumors but the mutants had significantly lower disease incidences. The season of inoculation played a more important role than the T6SS in shaping the gallobiome. The influence of T6SS was evident in summer, in which twoSphingomonasspecies and the family Burkhoderiaceae were enriched in the gallobiome induced by the mutants. Furtherin vitrocompetition and colonization assay demonstrated the T6SS-mediated antagonism to aSphingomonassp. R1 strain isolated from tomato rhizosphere in this study. In conclusion, this work demonstrates that theAgrobacteriumT6SS promotes tumorigenesis in infection process and provides competitive advantages in gall-associated microbiota.IMPORTANCEThe T6SS is widespread among Proteobacteria and used for interbacterial competition by agrobacteria, which are soil inhabitants and opportunistic bacterial pathogens causing crown gall disease in a wide range of plants. Current evidence indicates that the T6SS is not required for gall formation when agrobacteria are inoculated directly on plant wounding sites. However, in natural settings, agrobacteria may need to compete with other bacteria in bulk soil to gain access to plant wounds and influence microbial community inside crown galls. The role of the T6SS in these critical aspects of disease ecology have remained largely unknown. In this study, we successfully developed aSoilInoculation method coupled withBlocker-mediated enrichment ofBacterial 16S rRNA gene AmpliconSequencing, named as SI-BBacSeq, to address these two important questions. We provided evidence that the T6SS promotes disease occurrence and influences crown gall microbiota composition by interbacterial competition.