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

The type VI secretion system (T6SS) is deployed by many proteobacteria to secrete effector proteins into bacterial competitors for competition or eukaryotic cells for pathogenesis. Agrobacterium tumefacien, a soil-borne phytopathogen causing crown gall disease on various plant species, deploys its T6SS to attack closely- and distantly-related bacterial species in vitro and in planta. Current evidence suggests that the A. tumefaciens T6SS is not essential for pathogenesis under direct inoculation but it remains unknown whether T6SS influences natural disease incidence and microbial community of crown galls. To address these two key questions, we established a soil inoculation method on wounded tomato seedlings that mimics natural infections and performed 16S rRNA gene amplicon sequencing. By comparing the A. tumefaciens C58 wild-type strain with two T6SS mutants, we demonstrate that the T6SS influences disease occurrence and microbiota composition within crown galls. 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 gall microbiota (i.e., gallobiome). The influence of T6SS was evident in summer, in which two bacterial families (Sphingomonadaceae and Burkhoderiaceae) were enriched in the gallobiome induced by the T6SS mutants. Further in-vitro competition and colonization assay demonstrated the T6SS-mediated antagonism to a tomato isolate Sphingomonas sp. R1. In conclusion, this study informs that the A. tumefaciens T6SS may promote tumorigenesis in natural infection process and provide competitive advantages in gall-associated microbiota.