Type VI secretion system (T6SS) is a macromolecular machine used by many Gram-negative bacteria to inject effectors/toxins into eukaryotic hosts or prokaryotic competitors for survival and fitness. To date, our knowledge of the molecular determinants and mechanisms underlying the transport of these effectors remains limited. Here, we report that two T6SS encoded valine-glycine repeat protein G (VgrG) paralogs in Agrobacterium tumefaciens C58 specifically control the secretion and interbacterial competition activity of the type VI DNase toxins Tde1 and Tde2. Deletion and domain-swapping analysis identified that the C-terminal extension of VgrG1 specifically confers Tde1 secretion and Tde1-dependent interbacterial competition activity in planta, and the C-terminal variable region of VgrG2 governs this specificity for Tde2. Functional studies of VgrG1 and VgrG2 variants with stepwise deletion of the C terminus revealed that the C-terminal 31 aa (C31) of VgrG1 and 8 aa (C8) of VgrG2 are the molecular determinants specifically required for delivery of each cognate Tde toxin. Further in-depth studies on Tde toxin delivery mechanisms revealed that VgrG1 interacts with the adaptor/chaperone-effector complex (Tap-1-Tde1) in the absence of proline-alanine-alanine-arginine (PAAR) and the VgrG1-PAAR complex forms independent of Tap-1 and Tde1. Importantly, we identified the regions involved in these interactions. Although the entire C31 segment is required for binding with the Tap-1-Tde1 complex, only the first 15 aa of this region are necessary for PAAR binding. These results suggest that the VgrG1 C terminus interacts sequentially or simultaneously with the Tap-1-Tde1 complex and PAAR to govern Tde1 translocation across bacterial membranes and delivery into target cells for antibacterial activity.type VI secretion system | VgrG | DNase effector | interbacterial competition | Agrobacterium tumefaciens

The type VI secretion system (T6SS) is used by many bacteria to engage in social behavior and can affect the health of its host plant or animal. Because activities associated with T6SSs are often costly, T6SSs must be tightly regulated. However, our knowledge regarding how T6SS assembly and contraction are regulated remains limited. Using the plant pathogen Agrobacterium tumefaciens, we show that effectors are not just passengers but also impact on T6SS assembly. The A. tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major weapons for interbacterial competition. Here, we demonstrate that loading of Tde effectors onto their cognate carriers, the VgrG spikes, is required for active T6SS secretion. The assembly of the TssBC contractile sheath occurs only in the presence of Tde effectors. The requirement of effector loading for efficient T6SS secretion was also validated in other A. tumefaciens strains. We propose that such a mechanism is used by bacteria as a strategy for efficacious T6SS firing and to ensure that effectors are loaded onto the T6SS prior to completing its assembly.

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28The type VI secretion system (T6SS) is used by many bacteria to engage in social behaviors 29 with others and can directly or indirectly affect the health of plants and animals. Because 30activities associated with T6SS are often costly, the assembly and activation of the T6SS must 31 be highly regulated. However, our knowledge regarding how T6SS assembly and contraction 32 are regulated remains limited. Here we show that the loading of effectors onto their cognate 33 carriers is critical for the assembly of a functional T6SS in Agrobacterium tumefaciens. A. 34tumefaciens strain C58 encodes one T6SS and two Tde DNase toxin effectors used as major 35weapons for interbacterial competition. We found that loading of Tde effectors onto their 36 cognate carrier, the VgrG spike, is required for active T6SS secretion. Our data also suggest 37 the assembly of the TssBC contractile sheath occurs only after Tde effectors are loaded onto 38the VgrG spike. The requirement of effector loading for efficient T6SS secretion was also 39 validated in other A. tumefaciens strains. Such a mechanism may be used by bacteria as a 40 strategy for efficacious T6SS firing. Given the prevalence of T6SS-encoding loci in host-41 associated bacteria, these findings inform on mechanisms that influence the composition of 42 microbial communities and the services provided to hosts. 43 44