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Type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion.Agrobacterium tumefaciensspecies complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels.A. tumefaciensstrain 1D1609 genome encodes four effector genes, in which all four are specialized effector harboring a conserved N-terminal PAAR-like DUF4150 domain but distinct C-terminal effector domains. Previous study reported that V2a is a His-Me finger nuclease toxin contributing to DNase-mediated antibacterial activity. However, it remains unknown about the functions and roles of other three effectors. In this study, we identified V2c is another His-Me finger nuclease encoded by 1D1609 genome but with distinct SHH motif differed from AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation inEscherichia coli. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues phenotypes of the growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retain the cell elongation phenotype while V2a induced cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activityin vivoandin vitro. Notably, V2c also mediated the DNA degradation and cell elongation of target cell in the context of interbacterial competition. Importantly, V2a and V2c function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen,Dickeya dadantii. |
