Biology subjects

Lai, E.-M.

Publications and source records attributed to Lai, E.-M..

14 recordsLinked to original sources

bioRxiv2026

Rhizobia transform legume nodules via native T-DNA transfer systems

Agrobacteria can deliver transfer DNA (T-DNA) into plant cells, causing tumorous growth. This process, agrobacteria-mediated transformation (AMT), underlies plant genetic engineering and has been regarded as an evolutionary innovation specific to these pathogens. By screening 63,759 alphaproteobacterial genomes for T-DNA border sequences, we identify mini-T-DNAs in diverse rhizobial genera. Each typically encodes a single cargo, a RolB/RolC family protein or a distant rhizobial homologue. We show that native rhizobia-mediated transformation (RMT) systems deliver these mini-T-DNAs into the root nodules of common bean, pea, and chickpea, where the cargo genes are expressed, with no signal in adjacent root tissue. RMT systems share VirG-dependent activation of the core virulence regulon but respond to distinct upstream signals. Our genome screen identifies many additional candidate T-DNA transfer systems, and phylogenetic analysis places their genes into two of the many type IV secretion system (T4SS) clades. RMT systems occur in both clades, together with other systems unrelated to T-DNA transfer, and typically have an ancestral-like T4SS configuration. In contrast, AMT systems occupy a single subclade, consistent with later acquisition of their T4SS module. AMT and RMT thus represent two specialized outcomes of an older, more widespread capability for bacteria-to-plant DNA transfer.

microbiology↗

bioRxiv2026

Abiotic cues modulate exopolysaccharides to influence type VI secretion system-mediated antagonism

The type VI secretion system (T6SS) enables Gram-negative bacteria to inject toxic effectors into neighboring cells, mediating contact-dependent antagonism and interbacterial competition. How the T6SS-mediated attack responds to environmental cues varies and remains unclear among different bacteria. Here, using Agrobacterium fabrum C58, a soil-borne phytopathogenic bacterium, we investigated the impact of osmolarity, moisture, surface stiffness, and glucose on T6SS-mediated antagonism. We show that these abiotic factors influenced the production of two polysaccharides, cyclic-{beta}-(1,2)-glucan (C{beta}G) and succinoglycan (SG), and modulated the T6SS-killing outcome. Mechanistically, high osmolarity inhibits C{beta}G production, thereby enhancing the expression and secretion of the T6SS. In contrast, SG biosynthesis, in response to moisture, surface stiffness, and glucose, does not impact T6SS expression and function but decreases T6SS-mediated killing efficacy. Electron microscopy revealed that SG creates a physical barrier between bacterial cells. Such physical distancing not only hinders the T6SS attack from Agrobacterium, but also confers protection against other competitors at both intra- and inter-species levels. Our results unravel the complexity of how specific environmental factors modulate the contact-dependent antagonism and highlight a balance between offensive and defensive behaviors. Significance StatementBacteria live in polymicrobial communities where they often need to fight off competitors to survive. One well-characterized weapon is the type VI secretion system (T6SS), a nanomachine mediating contact-dependent antagonism. In this study, we aim to study how the T6SS attack is influenced by environmental cues. We discovered that carbon sources, osmolarity, and surface stiffness modulate the T6SS efficacy through the secretion of sugar chains outside the cell. This sugar secretion increases the physical distance between cells, protecting against T6SS-mediated attack. However, such physical distancing also hinders the efficacy of the T6SS attack originating from Agrobacterium itself. Our results reveal a previously understudied offense-defense tradeoff between EPS production and T6SS-mediated attack. This finding underscores the intricate balance between bacterial offense and defense strategies.

microbiology↗

bioRxiv2026

A broad-host-range Rhizobium rhizogenes strain enables transient expression across diverse crops and establishes functional assays in faba bean

Agrobacterium-mediated transient expression has revolutionized plant research, enabling numerous landmark discoveries across diverse areas of plant biology. Yet this powerful approach remains largely confined to solanaceous species, leaving most economically important crop families without a comparable rapid assay platform. Here, we show that an engineered Rhizobium rhizogenes strain, AS109, mediates efficient transient expression across diverse dicot species spanning multiple taxonomic families, consistently outperforming commonly used laboratory agrobacterial strains. Leveraging the broad host range of AS109, we establish a suite of functional assays in faba bean (Vicia faba), including protein localisation, RNA interference-mediated gene silencing, cell-surface elicitor recognition screens, nucleotide-binding leucine-rich repeat receptor (NLR) activation, and infection cell biology at the host-pathogen interface. We further demonstrate that both singleton NLRs and sensor-helper NLR pairs from Solanaceae retain effector recognition and cell death activity when transferred into faba bean, establishing a rapid platform for evaluating cross-family transferability of disease-resistance genes. AS109 thus provides an accessible and versatile chassis for functional genomics in non-model crops, bridging the widening gap between hypothesis generation and experimental validation across diverse plant species.

plant biology↗

bioRxiv2026

An N-degron proteolytic pathway modulates recipient susceptibility to T6SS DNase effectors

The type VI secretion system (T6SS) is a contractile nanoweapon widely employed by Gram-negative bacteria to gain competitive advantages by injecting effector proteins into recipient cells. Although the biochemical activities of T6SS effectors have been well characterized, how recipient factors modulate effector toxicity remains poorly understood. Using Agrobacterium C58 as a model, previous work identified the Escherichia coli ClpAP protease as a recipient susceptibility (RS) factor that enhances T6SS-mediated interbacterial competition. Agrobacterium C58 deploys two DNase effectors, Tde1 and Tde2, as the major antibacterial weapon. Here, we demonstrate that the recipient ClpAP protease and its adaptor ClpS enhanced C58-mediated interbacterial competition in a Tde2-dependent manner in both intra- and interspecies competition. Ectopic expression of Tde2 in E. coli caused growth inhibition and DNA cleavage in the presence of a functional ClpAPS protease complex, but not in any of the clpP, clpA or clpS mutants. Notably, Tde2 accumulated in these mutants but not in wild-type cells, whereas a catalytic variant accumulated regardless of ClpAPS status, suggesting that Tde2 is not directly degraded by ClpAPS. Instead, Tde2 depends on ClpAPS for full toxicity, likely through degradation of inhibitory N-degron substrate(s). Affinity purification of His-tagged Tde2 in a clpP mutant background, followed by mass spectrometry, identified eight N-degron substrate candidates. Tde2-mediated interbacterial competition was significantly reduced by overexpression of three candidates. Among them, the Tde2 DNase domain directly associated with guanosine 5-monophosphate reductase GuaC, supporting a model in which Tde2 toxicity is blocked by binding of GuaC. Collectively, our findings reveal an unanticipated layer of recipient-mediated regulation in T6SS competition and highlight proteolytic control of inhibitory substrates as a determinant of bacterial susceptibility during interbacterial conflict.

microbiology↗

bioRxiv2025

Comparative Transcriptomics Reveals Context- and Strain-Specific Regulatory Programs of Agrobacterium During Plant Colonization

Agrobacterium is a genus of plant-associated bacteria capable of transferring DNA into host genomes to induce tumorigenesis. The process has been primarily studied in a few model strains, particularly C58, and developed into Agrobacterium-mediated transformation (AMT) for genetic manipulation. However, the diversity of wild-type strains and their context-specific regulatory responses remain poorly characterized. Here, we evaluated five wild-type strains and identified 1D1108 as superior in tumorigenesis on legumes and transient transformation in Nicotiana benthamiana. Under in vitro virulence induction with acetosyringone (AS), we identified 126 differentially expressed genes (DEGs) in 1D1108. Although the number of DEGs was comparable to those in C58 and the legume isolate 1D1609 under the same condition, only 22 DEGs, primarily within the vir regulon, were conserved, indicating extensive divergence among these Agrobacterium strains. Leaf infiltration of N. benthamiana revealed 1,134 DEGs specifically regulated in planta for 1D1108. These included genes involved in attachment, virulence regulation, type IV pilus, succinoglycan biosynthesis, and diverse nutrient transporters, providing new evidence on expression regulation during colonization. Comparative analyses of in planta transcriptomes with C58 and Pseudomonas syringae DC3000 revealed distinct secretion systems required for pathogenesis, namely type IV for Agrobacterium and type III for Pseudomonas, and only approximately 5-19% of DEGs were conserved. These limited transcriptomic overlaps underscore the importance of studying gene expression in strains and conditions directly relevant to the biological context, rather than relying on model systems. Together, this work reveals how environmental and host-associated cues shape transcriptional responses in plant-associated bacteria. IMPORTANCEAgrobacterium is a key tool for plant genetic engineering, yet much of our knowledge about its biology comes from a few strains studied under artificial conditions. This work combines tumor formation assays on legumes and transient transformation in Nicotiana benthamiana with transcriptomic analyses to explore how diverse Agrobacterium strains, particularly the high-performing wild-type strain 1D1108, respond to host and environmental cues. By comparing gene expression under in vitro and in planta conditions, we found that the bacterial response within plant tissue is more complex than previously appreciated. Cross-strain comparisons further revealed limited conservation in gene expression regulation, even under similar conditions. Comparative analysis with Pseudomonas syringae revealed the activation of distinct secretion systems for pathogenesis and differences in regulatory programs used during plant colonization. These findings underscore the importance of studying bacteria in biologically relevant contexts and highlight the limitations of relying solely on model strains to infer regulatory responses.

microbiology↗

bioRxiv2025

Floral Stage Optimization and Immune Evasion Enhance Agrobacterium-Mediated Genome Editing in Arabidopsis

O_LIAgrobacterium-mediated transformation via floral inoculation (AMT-FI) enables genetic engineering without tissue culture. It is widely used in the model plant Arabidopsis thaliana, yet its efficiency and broader applicability remain limited. C_LIO_LIHere, we used a dual-reporter system (RUBY and hygromycin resistance) to identify key floral stages and engineered Agrobacterium strains to evade plant immunity, leading to enhanced transient expression and genome editing. C_LIO_LIWe determined that flowers opened at 6 day-post-inoculation (DPI) are optimal for high transformation efficiency, with nearly 100% of siliques harboring transformants. However, Agrobacterium infection induced ovule abortion, particularly in wild-type (Col-0) plants, whereas efr mutants lacking the EF-Tu receptor (EFR)-mediated pattern-triggered immunity (PTI) showed reduced ovule abortion. Notably, efr mutants exhibited more RUBY-positive ovules and significantly enhanced genome editing efficiency. Two engineered stealth Agrobacterium strains (AS201 and AS202) expressing a chimeric EF- Tu for evading recognition by EFR enhanced both transient transformation and genome editing efficiency. Remarkably, genome-edited T1 plants could be recovered based on phenotype or direct sequencing without the need for antibiotic selection when targeting flowers opened at 6 DPI. C_LIO_LIBy integrating floral stage selection, immune evasion, and Agrobacterium engineering, this study provides a practical and versatile platform to advance plant genome engineering. C_LI

plant biology↗

bioRxiv2024

Rhizobium rhizogenes A4-derived strains mediate hyper-efficient transient gene expression in Nicotiana benthamiana and other solanaceous plants

Agroinfiltration, a method utilizing agrobacteria to transfer DNA into plant cells, is widely used for transient gene expression in plants. Besides the commonly used Agrobacterium strains, Rhizobium rhizogenes can also introduce foreign DNA into host plants for gene expression. While many R. rhizogenes strains have been known for inducing hairy root symptoms, their use for transient expression has not been fully explored. Here, we showed that R. rhizogenes A4 outperformed all other tested agrobacterial strains in agroinfiltration experiments on leaves of Nicotiana benthamiana and other solanaceous plants. By conducting an agroinfiltration screening in N. benthamiana leaves using various agrobacterial strains carrying the RUBY reporter gene cassette, we discovered that A4 mediates the strongest and fastest transient expression. Utilizing the genomic information, we developed a collection of disarmed and modified strains derived from A4. By performing vacuum infiltration assays, we demonstrated that these A4-derived strains efficiently transiently transform 6-week-old N. benthamiana leaves, showing less sensitivity to the age of plants compared to the laboratory strain GV3101. Furthermore, we performed agroinfiltration using AS109, an A4-derived disarmed strain, on the leaves of tomato, pepper, and eggplant. Remarkably, AS109 mediated transient gene expression on tested solanaceous plants more effectively than all the tested commonly used agrobacterial strains. This discovery paves the way for establishing R. rhizogenes A4-derived strains as a new option for enhancing transient expression in N. benthamiana and facilitating the functional study of plant genes in other solanaceous species.

plant biology↗

bioRxiv2023

Two classes of His-Me finger superfamily nuclease effectors exerting synergy for Agrobacterium T6SS-mediated interbacterial competition

Type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. 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. 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. Previous study reported that vgrGa-associated gene 2, named as v2a, encodes a His-Me finger nuclease toxin (also named as 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 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 in Escherichia 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 retains 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 activity in vivo and in vitro. Notably, V2c also mediated the DNA degradation and cell elongation of 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.

microbiology↗

bioRxiv2022

The antibacterial T6SS of Agrobacterium tumefacien promotes disease occurrence and influences crown gall microbiota composition

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 soil-borne phytopathogens causing crown gall disease on various plant species, deploys 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., 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 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 two Sphingomonas species and the family Burkhoderiaceae were enriched in the gallobiome induced by the mutants. Further in vitro competition and colonization assay 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 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 a Soil Inoculation method coupled with Blocker-mediated enrichment of Bacterial 16S rRNA gene Amplicon Sequencing, 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.

microbiology↗

bioRxiv2022

A glycine zipper motif governs translocation of type VI secretion toxic effectors across the cytoplasmic membrane of 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 harbours continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G39xxxG43 motif does 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 G39xxxG43 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.

microbiology↗

bioRxiv2021

Species boundaries in the Agrobacterium tumefaciens complex and multi-level modular evolution of their antibacterial type VI secretion system and tumor-inducing plasmids

BackgroundMany bacterial taxa are species complexes and uncertainties regarding the organization of their genetic diversity challenge research efforts. We utilized Agrobacterium tumefaciens, 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. ResultsWe utilized 35 genome assemblies to achieve a comprehensive and balanced sampling of A. tumefaciens. Our confident inference of gene content and core-genome phylogeny supported a quantitative guideline for delineating 12 species and allowed for robust 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. ConclusionsWe demonstrated 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. Our strategies for multi-level investigations at scales that range from whole-genomes to intragenic domains and phylogenetic depths of between- and within-species are applicable to other bacteria. Finally, modularity observed in the molecular evolution of genes and domains is useful for inferring functional constraints and informing experimental works.

genomics↗

bioRxiv2019

Redundancy and specificity of type VI secretion vgrG loci in antibacterial activity of Agrobacterium tumefaciens 1D1609 strain

Type VI secretion system (T6SS) is a contractile nanoweapon employed by many Proteobacteria to deliver effectors to kill or inhibit their competitors. One T6SS gene, vgrG, encodes a spike protein for effector translocation and is often present as multiple copies in bacterial genomes. Our phylogenomic analyses sampled 48 genomes across diverse Proteobacteria lineages and found [~]70% of them encode multiple VgrGs, yet only four genomes have nearly identical paralogs. Among these four, Agrobacterium tumefaciens 1D1609 has the highest vgrG redundancy. Compared to A. tumefaciens model strain C58 which harbors two vgrG genes, 1D1609 encodes four vgrG genes (i.e. vgrGa-d) with each adjacent to different putative effector genes. Thus, 1D1609 was selected to investigate the functional redundancy and specificity of multiple vgrG genes and their associated effectors. Secretion assay of single and multiple vgrG deletion mutants demonstrated that these four vgrGs are functionally redundant in mediating T6SS secretion. By analyzing various vgrG mutants, we found that all except for the divergent vgrGb could contribute to 1D1609s antibacterial activity. Further characterizations of putative effector-immunity gene pairs revealed that vgrGa-associated gene 2 (v2a) encodes an AHH family nuclease and serves as the major antibacterial toxin. Interestingly, C58s VgrG2 shares 99% amino acid sequence identity with 1D1609s VgrGa, VgrGc and VgrGd. This high sequence similarity allows 1D1609 to use an exogenous VgrG delivered from C58 to kill another competing bacterium. Taken together, Agrobacterium can use highly similar VgrGs, either produced endogenously or injected from its close relatives, for T6SS-mediated interbacterial competition. Authors SummarySelective pressure drives bacteria to develop adaptive strategies, which include competitive and cooperative behaviors. Type VI secretion system (T6SS) is one powerful antibacterial and anti-host nanoweapon employed by many Gram-negative bacteria for growth advantages or pathogenesis. A T6SS-harboring bacterium can encode one to multiple VgrG proteins for delivery of cognate effector(s) but the prevalence and biological significance of having sequence redundant vgrGs have not been comprehensively explored. In this study, we investigated the extensiveness of having multicopy vgrG genes for effector delivery among diverse Proteobacteria with T6SS. Moreover, a plant pathogenic bacterium Agrobacterium tumefaciens strain 1D1609 with highest vgrG redundancy was selected for detailed characterization of the roles of multiple VgrGs in T6SS secretion and antibacterial activity. We revealed that the majority of Proteobacterial genomes harbor multiple copies of vgrG and the expansion of vgrG gene clusters contributed to effector diversity and functional redundancy. Furthermore, the near identical VgrG proteins between 1D1609 and its sibling strain C58 can be exchanged for effector delivery in killing another competing bacterium. Such strategy in using exchangeable effector carriers injected from its isogenic sibling or close relatives during T6SS attacks may be a beneficial strategy for agrobacteria to compete in their ecological niche.

microbiology↗

bioRxiv2019

Identification of Escherichia coli ClpAP in regulating susceptibility to type VI secretion system-mediated attack by Agrobacterium tumefaciens

Type VI secretion system (T6SS) is an effector delivery system used by gram-negative bacteria to kill other bacteria or eukaryotic host to gain fitness. In Agrobacterium tumefaciens, T6SS has been shown to kill other bacteria such as Escherichia coli. Interestingly, the A. tumefaciens T6SS killing efficiency differs when using different E. coli strains as recipient cells. Thus, we hypothesize that a successful T6SS killing not only relies on attacker T6SS activity but also depends on recipient factors. A high-throughput interbacterial competition assay was employed to test the hypothesis by screening for mutants with reduced killing outcomes caused by A. tumefaciens strain C58. From the 3909 E. coli Keio mutants screened, 16 candidate mutants were filtered out. One strain, {Delta}clpP::Kan, showed ten times more resistant to T6SS-mediating killing but restored its susceptibility when complemented with clpP in trans. ClpP is a universal and highly conserved protease that exists in both prokaryotes and eukaryotic organelles. In E. coli, ClpP uses either ClpA or ClpX as an adaptor for substrate specificity. Therefore, the susceptibility of the {Delta}clpA::Kan and {Delta}clpX::Kan was also tested. The T6SS attack susceptibility of {Delta}clpA::Kan is at the same level as {Delta}clpP::Kan, while {Delta}clpX::Kan showed no difference as compared to that of wild-type E. coli BW25113. The data also suggest that ClpA-ClpP interaction, rather than its protease activity, is responsible for enhancing susceptibility to T6SS killing. This study highlights the importance of recipient factors in determining the outcome of T6SS killing.

microbiology↗

bioRxiv2019

Effector loading onto VgrG spike proteins is critical for the assembly of the type VI secretion system in Agrobacterium tumefaciens

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

microbiology↗