A glycine zipper motif is required for the translocation of a T6SS toxic effector into target cells.

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here, we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbors continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39 xxxG43 motif do not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G39 xxxG43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper-mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

Modular evolution of secretion systems and virulence plasmids in a bacterial species complex.

BACKGROUND: Many named species as defined in current bacterial taxonomy correspond to species complexes. Uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized the Agrobacterium tumefaciens species complex (a.k.a. Agrobacterium biovar 1), a taxon known for its phytopathogenicity and applications in transformation, as a study system and devised strategies for investigating genome diversity and evolution of species complexes. RESULTS: We utilized 35 genome assemblies, including 14 newly generated ones, to achieve a phylogenetically balanced sampling of A. tumefaciens. Our genomic analysis suggested that the 10 genomospecies described previously are distinct biological species and supported a quantitative guideline for species delineation. Furthermore, our inference of gene content and core-genome phylogeny allowed for investigations of genes critical in fitness and ecology. For the type VI secretion system (T6SS) involved in interbacterial competition and thought to be conserved, we detected multiple losses and one horizontal gene transfer. For the tumor-inducing plasmids (pTi) and pTi-encoded type IV secretion system (T4SS) that are essential for agrobacterial phytopathogenicity, we uncovered novel diversity and hypothesized their involvement in shaping this species complex. Intriguingly, for both T6SS and T4SS, genes encoding structural components are highly conserved, whereas extensive diversity exists for genes encoding effectors and other proteins. CONCLUSIONS: We demonstrate that the combination of a phylogeny-guided sampling scheme and an emphasis on high-quality assemblies provides a cost-effective approach for robust analysis in evolutionary genomics. We show that the T6SS VgrG proteins involved in specific effector binding and delivery can be classified into distinct types based on domain organization. The co-occurrence patterns of VgrG-associated domains and the neighboring genes that encode different chaperones/effectors can be used to infer possible interacting partners. Similarly, the associations between plant host preference and the pTi type among these strains can be used to infer phenotype-genotype correspondence. Our strategies for multi-level investigations at scales that range from whole genomes to intragenic domains and phylogenetic depths from between- to within-species are applicable to other bacteria. Furthermore, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

Effector loading onto the VgrG carrier activates type VI secretion system assembly.

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.

Agrobacterium tumefaciens Deploys a Versatile Antibacterial Strategy To Increase Its Competitiveness.

The type VI secretion system (T6SS) is a widespread antibacterial weapon capable of secreting multiple effectors for inhibition of competitor cells. Most of the effectors in the system share the same purpose of target intoxication, but the rationale for maintaining various types of effectors in a species is not well studied. In this study, we showed that a peptidoglycan amidase effector in Agrobacterium tumefaciens, Tae, cleaves d-Ala-meso-diaminopimelic acid (mDAP) and d-Glu bonds in peptidoglycan and is able to suppress the growth of Escherichia coli recipient cells. The growth suppression was effective only under the condition in which E. coli cells are actively growing. In contrast, the Tde DNase effectors in the strain possessed a dominant killing effect under carbon starvation. Microscopic analysis showed that Tde triggers cell elongation and DNA degradation, while Tae causes cell enlargement without DNA damage in E. coli recipient cells. In a rich medium, A. tumefaciens harboring only functional Tae was able to maintain competitiveness among E. coli and its own sibling cells. Growth suppression and the competitive advantage of A. tumefaciens were abrogated when recipient cells produced the Tae-specific immunity protein Tai. Given that Tae is highly conserved among A. tumefaciens strains, the combination of Tae and Tde effectors could allow A. tumefaciens to better compete with various competitors by increasing its survival during changing environmental conditions.IMPORTANCE The T6SS encodes multiple effectors with diverse functions, but little is known about the biological significance of harboring such a repertoire of effectors. We reported that the T6SS antibacterial activity of the plant pathogen Agrobacterium tumefaciens can be enhanced under carbon starvation or when recipient cell wall peptidoglycan is disturbed. This led to a newly discovered role for the T6SS peptidoglycan amidase Tae effector in providing a growth advantage dependent on the growth status of the target cell. This is in contrast to the Tde DNase effectors that are dominant during carbon starvation. Our study suggests that combining Tae and other effectors could allow A. tumefaciens to increase its competitiveness among changing environmental conditions.

Cyclic di-GMP inactivates T6SS and T4SS activity in Agrobacterium tumefaciens.

The Type VI secretion system (T6SS) is a bacterial nanomachine that delivers effector proteins into prokaryotic and eukaryotic preys. This secretion system has emerged as a key player in regulating the microbial diversity in a population. In the plant pathogen Agrobacterium tumefaciens, the signalling cascades regulating the activity of this secretion system are poorly understood. Here, we outline how the universal eubacterial second messenger cyclic di-GMP impacts the production of T6SS toxins and T6SS structural components. We demonstrate that this has a significant impact on the ability of the phytopathogen to compete with other bacterial species in vitro and in planta. Our results suggest that, as opposed to other bacteria, c-di-GMP turns down the T6SS in A. tumefaciens thus impacting its ability to compete with other bacterial species within the rhizosphere. We also demonstrate that elevated levels of c-di-GMP within the cell decrease the activity of the Type IV secretion system (T4SS) and subsequently the capacity of A. tumefaciens to transform plant cells. We propose that such peculiar control reflects on c-di-GMP being a key second messenger that silences energy-costing systems during early colonization phase and biofilm formation, while low c-di-GMP levels unleash T6SS and T4SS to advance plant colonization.

The RNase YbeY Is Vital for Ribosome Maturation, Stress Resistance, and Virulence of the Natural Genetic Engineer Agrobacterium tumefaciens.

Riboregulation involving regulatory RNAs, RNA chaperones, and ribonucleases is fundamental for the rapid adaptation of gene expression to changing environmental conditions. The gene coding for the RNase YbeY belongs to the minimal prokaryotic genome set and has a profound impact on physiology in a wide range of bacteria. Here, we show that the Agrobacterium tumefaciensybeY gene is not essential. Deletion of the gene in the plant pathogen reduced growth, motility, and stress tolerance. Most interestingly, YbeY is crucial for A. tumefaciens-mediated T-DNA transfer and tumor formation. Comparative proteomics by using isobaric tags for relative and absolute quantitation (iTRAQ) revealed dysregulation of 59 proteins, many of which have previously been found to be dependent on the RNA chaperone Hfq. YbeY and Hfq have opposing effects on production of these proteins. Accumulation of a 16S rRNA precursor in the ybeY mutant suggests that A. tumefaciens YbeY is involved in rRNA processing. RNA coimmunoprecipitation-sequencing (RIP-Seq) showed binding of YbeY to the region immediately upstream of the 16S rRNA. Purified YbeY is an oligomer with RNase activity. It does not physically interact with Hfq and thus plays a partially overlapping but distinct role in the riboregulatory network of the plant pathogen.IMPORTANCE Although ybeY gene belongs to the universal bacterial core genome, its biological function is incompletely understood. Here, we show that YbeY is critical for fitness and host-microbe interaction in the plant pathogen Agrobacterium tumefaciens Consistent with the reported endoribonuclease activity of YbeY, A. tumefaciens YbeY acts as a RNase involved in maturation of 16S rRNA. This report adds a worldwide plant pathogen and natural genetic engineer of plants to the growing list of bacteria that require the conserved YbeY protein for host-microbe interaction.

TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha.

The bacterial type VI secretion system (T6SS) delivers effectors into eukaryotic host cells or toxins into bacterial competitor for survival and fitness. The T6SS is positively regulated by the threonine phosphorylation pathway (TPP) and negatively by the T6SS-accessory protein TagF. Here, we studied the mechanisms underlying TagF-mediated T6SS repression in two distinct bacterial pathogens, Agrobacterium tumefaciens and Pseudomonas aeruginosa. We found that in A. tumefaciens, T6SS toxin secretion and T6SS-dependent antibacterial activity are suppressed by a two-domain chimeric protein consisting of TagF and PppA, a putative phosphatase. Remarkably, this TagF domain is sufficient to post-translationally repress the T6SS, and this inhibition is independent of TPP. This repression requires interaction with a cytoplasmic protein, Fha, critical for activating T6SS assembly. In P. aeruginosa, PppA and TagF are two distinct proteins that repress T6SS in TPP-dependent and -independent pathways, respectively. P. aeruginosa TagF interacts with Fha1, suggesting that formation of this complex represents a conserved TagF-mediated regulatory mechanism. Using TagF variants with substitutions of conserved amino acid residues at predicted protein-protein interaction interfaces, we uncovered evidence that the TagF-Fha interaction is critical for TagF-mediated T6SS repression in both bacteria. TagF inhibits T6SS without affecting T6SS protein abundance in A. tumefaciens, but TagF overexpression reduces the protein levels of all analyzed T6SS components in P. aeruginosa Our results indicate that TagF interacts with Fha, which in turn could impact different stages of T6SS assembly in different bacteria, possibly reflecting an evolutionary divergence in T6SS control.

Plant-Pathogenic Agrobacterium tumefaciens Strains Have Diverse Type VI Effector-Immunity Pairs and Vary in In-Planta Competitiveness.

The type VI secretion system (T6SS) is used by gram-negative bacteria to translocate effectors that can antagonize other bacterial cells. Models predict the variation in collections of effector and cognate immunity genes determine competitiveness and can affect the dynamics of populations and communities of bacteria. However, the outcomes of competition cannot be entirely explained by compatibility of effector-immunity (EI) pairs. Here, we characterized the diversity of T6SS loci of plant-pathogenic Agrobacterium tumefaciens and showed that factors other than EI pairs can impact interbacterial competition. All examined strains encode T6SS active in secretion and antagonism against Escherichia coli. The spectra of EI pairs as well as compositions of gene neighborhoods are diverse. Almost 30 in-planta competitions were tested between different genotypes of A. tumefaciens. Fifteen competitions between members of different species-level groups resulted in T6SS-dependent suppression in in-planta growth of prey genotypes. In contrast, ten competitions between members within species-level groups resulted in no significant effect on the growth of prey genotypes. One strain was an exceptional case and, despite encoding a functional T6SS and toxic effector protein, could not compromise the growth of the four tested prey genotypes. The data suggest T6SS-associated EI pairs can influence the competitiveness of strains of A. tumefaciens, but genetic features have a significant role on the efficacy of interbacterial antagonism.

The Agrobacterium Type VI Secretion System: A Contractile Nanomachine for Interbacterial Competition.

The bacterial type VI secretion system (T6SS) is a contractile nanomachine dedicated to delivering molecules out of bacterial cells. T6SS-encoding loci are in the genome sequences of many Gram-negative bacteria, and T6SS has been implicated in a plethora of roles. In the majority of cases, the T6SSs deliver effector proteins in a contact-dependent manner to antagonize other bacteria. Current models suggest that the effectors are deployed to influence social interactions in microbial communities. In this chapter, we describe the structure, function, and regulation of the T6SS and its effectors. We provide focus on the T6SS of Agrobacterium tumefaciens, the causative agent of crown gall disease, and relate the role of the T6SS to the ecology of A. tumefaciens.

VgrG C terminus confers the type VI effector transport specificity and is required for binding with PAAR and adaptor-effector complex.

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.

Functional Exploration of the Bacterial Type VI Secretion System in Mutualism: Azorhizobium caulinodans ORS571-Sesbania rostrata as a Research Model.

The bacterial type VI secretion system (T6SS) has been considered the armed force of bacteria because it can deliver toxin effectors to prokaryotic or eukaryotic cells for survival and fitness. Although many legume symbiotic rhizobacteria encode T6SS in their genome, the biological function of T6SS in these bacteria is still unclear. To elucidate this issue, we used Azorhizobium caulinodans ORS571 and its symbiotic host Sesbania rostrata as our research model. By using T6SS gene deletion mutants, we found that T6SS provides A. caulinodans with better symbiotic competitiveness when coinfected with a T6SS-lacking strain, as demonstrated by two independent T6SS-deficient mutants. Meanwhile, the symbiotic effectiveness was not affected by T6SS because the nodule phenotype, nodule size, and nodule nitrogen-fixation ability did not differ between the T6SS mutants and the wild type when infected alone. Our data also suggest that under several lab culture conditions tested, A. caulinodans showed no T6SS-dependent interbacterial competition activity. Therefore, instead of being an antihost or antibacterial weapon of the bacterium, the T6SS in A. caulinodans ORS571 seems to participate specifically in symbiosis by increasing its symbiotic competitiveness.

Fha interaction with phosphothreonine of TssL activates type VI secretion in Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a widespread protein secretion system found in many Gram-negative bacteria. T6SSs are highly regulated by various regulatory systems at multiple levels, including post-translational regulation via threonine (Thr) phosphorylation. The Ser/Thr protein kinase PpkA is responsible for this Thr phosphorylation regulation, and the forkhead-associated (FHA) domain-containing Fha-family protein is the sole T6SS phosphorylation substrate identified to date. Here we discovered that TssL, the T6SS inner-membrane core component, is phosphorylated and the phosphorylated TssL (p-TssL) activates type VI subassembly and secretion in a plant pathogenic bacterium, Agrobacterium tumefaciens. Combining genetic and biochemical approaches, we demonstrate that TssL is phosphorylated at Thr 14 in a PpkA-dependent manner. Further analysis revealed that the PpkA kinase activity is responsible for the Thr 14 phosphorylation, which is critical for the secretion of the T6SS hallmark protein Hcp and the putative toxin effector Atu4347. TssL phosphorylation is not required for the formation of the TssM-TssL inner-membrane complex but is critical for TssM conformational change and binding to Hcp and Atu4347. Importantly, Fha specifically interacts with phosphothreonine of TssL via its pThr-binding motif in vivo and in vitro and this interaction is crucial for TssL interaction with Hcp and Atu4347 and activation of type VI secretion. In contrast, pThr-binding ability of Fha is dispensable for TssM structural transition. In conclusion, we discover a novel Thr phosphorylation event, in which PpkA phosphorylates TssL to activate type VI secretion via its direct binding to Fha in A. tumefaciens. A model depicting an ordered TssL phosphorylation-induced T6SS assembly pathway is proposed.

Overexpression of the HspL Promotes Agrobacterium tumefaciens Virulence in Arabidopsis Under Heat Shock Conditions.

Agrobacterium tumefaciens transfers a specific DNA fragment from the resident tumor-inducing (Ti) plasmid and effector virulence (Vir) proteins to plant cells during infection. A. tumefaciens VirB1-11 and VirD4 proteins assemble as the type IV secretion system (T4SS), which mediates transfer of the T-DNA and effector Vir protein into plant cells, thus resulting in crown gall disease in plants. Previous studies revealed that an α-crystallin-type, small heat-shock protein (HspL) is a more effective VirB8 chaperone than three other small heat-shock proteins (HspC, HspAT1, and HspAT2). Additionally, HspL contributes to efficient T4SS-mediated DNA transfer and tumorigenesis under room-temperature growth. In this study, we aimed to characterize the impact of HspL on Agrobacterium-mediated transformation efficiency under heat-shock treatment. During heat shock, transient transformation efficiency and VirB8 protein accumulation were lower in the hspL deletion mutant than in the wild type. Overexpression of HspL in A. tumefaciens enhanced the transient transformation efficiency in root explants of both susceptible and recalcitrant Arabidopsis ecotypes. In addition, the reduced transient transformation efficiency during heat stress was recovered by overexpression of HspL in A. tumefaciens. HspL may help maintain VirB8 homeostasis and elevate Agrobacterium-mediated transformation efficiency under both heat-shock and nonheat-shock growth.

Acid-induced type VI secretion system is regulated by ExoR-ChvG/ChvI signaling cascade in Agrobacterium tumefaciens.

The type VI secretion system (T6SS) is a widespread, versatile protein secretion system in pathogenic Proteobacteria. Several T6SSs are tightly regulated by various regulatory systems at multiple levels. However, the signals and/or regulatory mechanisms of many T6SSs remain unexplored. Here, we report on an acid-induced regulatory mechanism activating T6SS in Agrobacterium tumefaciens, a plant pathogenic bacterium causing crown gall disease in a wide range of plants. We monitored the secretion of the T6SS hallmark protein hemolysin-coregulated protein (Hcp) from A. tumefaciens and found that acidity is a T6SS-inducible signal. Expression analysis of the T6SS gene cluster comprising the imp and hcp operons revealed that imp expression and Hcp secretion are barely detected in A. tumefaciens grown in neutral minimal medium but are highly induced with acidic medium. Loss- and gain-of-function analysis revealed that the A. tumefaciens T6SS is positively regulated by a chvG/chvI two-component system and negatively regulated by exoR. Further epistasis analysis revealed that exoR functions upstream of the chvG sensor kinase in regulating T6SS. ChvG protein levels are greatly increased in the exoR deletion mutant and the periplasmic form of overexpressed ExoR is rapidly degraded under acidic conditions. Importantly, ExoR represses ChvG by direct physical interaction, but disruption of the physical interaction allows ChvG to activate T6SS. The phospho-mimic but not wild-type ChvI response regulator can bind to the T6SS promoter region in vitro and activate T6SS with growth in neutral minimal medium. We present the first evidence of T6SS activation by an ExoR-ChvG/ChvI cascade and propose that acidity triggers ExoR degradation, thereby derepressing ChvG/ChvI to activate T6SS in A. tumefaciens.

Complete genome sequence of Sphingomonas sp. strain R1, a bacterium isolated from tomato (Solanum lycopersicum).

Sphingomonas sp. strain R1 was isolated from the stem of a tomato plant and exhibited antagonism with Agrobacterium. The complete genome sequence of this bacterium consists of one 3,874,532 bp circular chromosome and two plasmids.

Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors.

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species 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. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa-associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc-associated gene 2 (v2c) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces 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 activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii.

IcmF family protein TssM exhibits ATPase activity and energizes type VI secretion.

The type VI secretion system (T6SS) with diversified functions is widely distributed in pathogenic Proteobacteria. The IcmF (intracellular multiplication protein F) family protein TssM is a conserved T6SS inner membrane protein. Despite the conservation of its Walker A nucleotide-binding motif, the NTPase activity of TssM and its role in T6SS remain obscure. In this study, we characterized TssM in the plant pathogen Agrobacterium tumefaciens and provided the first biochemical evidence for TssM exhibiting ATPase activity to power the secretion of the T6SS hallmark protein, hemolysin-coregulated protein (Hcp). Amino acid substitutions in the Walker A motif of TssM caused reduced ATP binding and hydrolysis activity. Importantly, we discovered the Walker B motif of TssM and demonstrated that it is critical for ATP hydrolysis activity. Protein-protein interaction studies and protease susceptibility assays indicated that TssM undergoes an ATP binding-induced conformational change and that subsequent ATP hydrolysis is crucial for recruiting Hcp to interact with the periplasmic domain of the TssM-interacting protein TssL (an IcmH/DotU family protein) into a ternary complex and mediating Hcp secretion. Our findings strongly argue that TssM functions as a T6SS energizer to recruit Hcp into the TssM-TssL inner membrane complex prior to Hcp secretion across the outer membrane.

The Tzs protein and exogenous cytokinin affect virulence gene expression and bacterial growth of Agrobacterium tumefaciens.

The soil phytopathogen Agrobacterium tumefaciens causes crown gall disease in a wide range of plant species. The neoplastic growth at the infection sites is caused by transferring, integrating, and expressing transfer DNA (T-DNA) from A. tumefaciens into plant cells. A trans-zeatin synthesizing (tzs) gene is located in the nopaline-type tumor-inducing plasmid and causes trans-zeatin production in A. tumefaciens. Similar to known virulence (Vir) proteins that are induced by the vir gene inducer acetosyringone (AS) at acidic pH 5.5, Tzs protein is highly induced by AS under this growth condition but also constitutively expressed and moderately upregulated by AS at neutral pH 7.0. We found that the promoter activities and protein levels of several AS-induced vir genes increased in the tzs deletion mutant, a mutant with decreased tumorigenesis and transient transformation efficiencies, in Arabidopsis roots. During AS induction and infection of Arabidopsis roots, the tzs deletion mutant conferred impaired growth, which could be rescued by genetic complementation and supplementing exogenous cytokinin. Exogenous cytokinin also repressed vir promoter activities and Vir protein accumulation in both the wild-type and tzs mutant bacteria with AS induction. Thus, the tzs gene or its product, cytokinin, may be involved in regulating AS-induced vir gene expression and, therefore, affect bacterial growth and virulence during A. tumefaciens infection.

Soil Inoculation and Blocker-Mediated Sequencing Show Effects of the Antibacterial T6SS on Agrobacterial Tumorigenesis and Gallobiome.

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. Agrobacteria, a group of soilborne phytopathogens causing crown gall disease on various plant species, deploy the T6SS to attack closely and distantly related bacterial species in vitro and in 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., the 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 the Agrobacterium wild-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 induced 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 the T6SS was evident in summer, during which two Sphingomonadaceae species and the family Burkholderiaceae were enriched in the gallobiome induced by the mutants. Further in vitro competition and colonization assays demonstrated the T6SS-mediated antagonism to a Sphingomonas sp. R1 strain isolated from tomato rhizosphere in this study. In conclusion, this work demonstrates that the Agrobacterium T6SS promotes tumorigenesis in infection processes and provides competitive advantages in gall-associated microbiota. IMPORTANCE The 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 the 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 a soil inoculation method coupled with blocker-mediated enrichment of bacterial 16S rRNA gene amplicon sequencing, named 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.