A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity

Abstract

The type VI secretion system (T6SS) of many gram‐negative bacteria injects toxic effectors into adjacent cells to manipulate host cells during pathogenesis or to kill competing bacteria. However, the identification and function of the T6SS effectors remains only partly known. Pantoea ananatis, a gram‐negative bacterium, is commonly found in various plants and natural environments, including water and soil. In the current study, genomic analysis of P. ananatis DZ‐12 causing brown stalk rot on maize demonstrated that it carries three T6SS gene clusters, namely, T6SS‐1, T6SS‐2, and T6SS‐3. Interestingly, only T6SS‐1 secretion systems are involved in pathogenicity and bacterial competition. The study also investigated the T6SS‐1 system in detail and identified an unknown T6SS‐1‐secreted effector TseG by using the upstream T6SS effector chaperone TecG containing a conserved domain of DUF2169. TseG can directly interact with the chaperone TecG for delivery and with a downstream immunity protein TsiG for protection from its toxicity. TseG, highly conserved in the Pantoea genus, is involved in virulence in maize, potato, and onion. Additionally, P. ananatis uses TseG to target Escherichia coli, gaining a competitive advantage. This study provides the first report on the T6SS‐1‐secreted effector from P. ananatis, thereby enriching our understanding of the various types and functions of type VI effector proteins.

A type VI secretion system‐secreted effector, TseG, exhibits dual functionality, acting both as a bactericidal agent and as a pathogenicity factor.

1. INTRODUCTION

The type VI secretion system (T6SS) was discovered and proposed in 2006 in Vibrio cholerae, and bioinformatics analysis revealed that the T6SS is present in more than 25% of gram‐negative bacterial species (Pukatzki et al., 2006). The T6SS is a novel needle‐like contractile nanomachine that can destroy and manipulate eukaryotic or prokaryotic cells by injecting effector proteins (Ma & Mekalanos, 2010). It acts as a pathogenicity factor, directly attacking host cells, mediating interspecific competition among bacteria, and potentially influencing the environmental adaptability of bacteria (Peterson et al., 2020). For instance, the T6SS of Salmonella enterica facilitates competition with host microbiota and intestinal colonization (Sibinelli‐Sousa et al., 2020). The Pseudomonas putida T6SS contributes to fighting against plant pathogens and protects plants from pathogen attack (Bernal et al., 2017).

In 2007, Shalom proposed a nomenclature for the T6SS gene clusters containing at least 13 core genes (type VI secretion [tss], tssA–tssM), and a variable number of non‐conserved accessory elements that encode the T6SS ‘injectisome’ (Shalom et al., 2007). The T6SS consists of a cytoplasmic tube (Hcp), enclosed in a TssB‐TssC contractile sheath, and topped by a membrane‐puncturing trimeric VgrG spike (Basler et al., 2013). The needle tube is assembled on the cytoplasmic baseplate structure with the membrane complex as a docking site. Proline–alanine–alanine–arginine repeats (PAAR) cover the VgrG spike and assemble into a triangular shape (Shneider et al., 2013). As the sheath contracts, the tube and spike penetrate through the membrane complex, gaining access to the target cells. To avoid bacterial self‐intoxication, the bacterium expresses an immunity protein to inactivate its cognate effector (Dong et al., 2013; Hood et al., 2010). Notably, certain structural proteins can also function as effectors, such as Hcp, VgrG, and PAAR (Ma, Pan, et al., 2017; Ma, Sun, et al., 2017; Rigard et al., 2016; Whitney et al., 2015). A dedicated chaperone or adaptor is required for the delivery of some effectors, for example, DUF4123 chaperones play an important role in the interaction between a C‐terminal VgrG β‐strand and the effector, while DUF1795 chaperones and DUF2169 chaperones assist PAAR proteins in extricating effectors from the cell (Alcoforado Diniz & Coulthurst, 2015; Bondage et al., 2016; Unterweger et al., 2017). To date, only a few T6SS‐dependent effectors have been characterized and reported to contribute to the bactericidal activity. Most studies have focused on human pathogens, such as Rhs2 from Serratia marcescens (Alcoforado Diniz & Coulthurst, 2015), TseT from Pseudomonas aeruginosa (Burkinshaw et al., 2018), PoNe from Vibrio parahaemolyticus (Jana et al., 2019), and Tce1 from Yersinia pseudotuberculosis (Song et al., 2021). However, there is a lack of research on type VI effector proteins in plant‐pathogenic bacteria, such as Tde1 and Tae from Agrobacterium tumefaciens (Ma et al., 2014), and RhsB from Acidovorax citrulli (Pei et al., 2022). The presence and pathogenic function of bacterial plant pathogen T6SS effectors remains largely obscure.

Pantoea ananatis is a gram‐negative, facultative anaerobic bacterium belonging to the family Enterobacteriaceae. It was first discovered as a pathogen in the Philippines in 1928. P. ananatis is a pathogen of many plants, including maize, rice, onion, and other crops, resulting in significant agricultural losses in several regions (Azad et al., 2000; De Maayer et al., 2012; Polidore et al., 2021). Upon plant infection, these bacteria cause internal rotting, dieback, and blight, especially in maize and onions (Goszczynska et al., 2007; Kistner et al., 2021; Shin et al., 2023). P. ananatis is widely distributed in nature, including in a variety of animals, plants, insects, the human body, soil, rivers, and refrigerated food; however, the majority of P. ananatis strains have been isolated from plants (Coutinho & Venter, 2009; Ercolini et al., 2006).

In 2014, Shyntum analysed the T6SS gene clusters of 46 strains of P. ananatis and found that all strains contained two or three T6SS gene clusters. The T6SS‐1 and T6SS‐3 gene clusters were located on the genome of all strains, while the T6SS‐2 gene cluster was located on a plasmid in one‐third of the strains (Shyntum et al., 2014). Another study found that T6SS‐1 plays an important role in the pathogenicity of P. ananatis LMG 2665^T in onion plants and bacterial competition (Shyntum et al., 2015). However, the presence and functions of T6SS effectors from P. ananatis remain elusive.

Here, we identified and characterized a previously unknown T6SS effector in P. ananatis that is delivered via the T6SS‐1, and TseG is not similar to the previously reported T6SS effector proteins and is highly conserved in the Pantoea genus. Remarkably, TseG exhibits dual functionality, acting both as a bactericidal agent and a pathogenicity factor. As the first report, our findings significantly enhance the understanding of the diversity and roles of type VI effector proteins. Furthermore, the bactericidal capability of type VI effectors suggests the potential for developing therapeutic strategies based on the T6SS mechanism.

2. RESULTS

2.1. P. ananatis DZ‐12 colonized in the vascular bundle of maize

The bacterium P. ananatis DZ‐12 causes brown stalk rot on maize, leading to significant economic losses (Zhao et al., 2021). The pathogenic process of DZ‐12 on maize leaves was observed by fluorescence microscopy, and found that DZ‐12 is localized in the vascular bundle of maize leaves, suggesting DZ‐12 might stunt the growth of the host plant by obstructing its ability to transfer nutrients. Furthermore, scanning electron microscopy (SEM) observations suggested that P. ananatis DZ‐12 may penetrate into maize leaves through stomata (Figure 1).

FIGURE 1.

The infection process of Pantoea ananatis in maize seedling leaves. (a) Confocal microscopy images showing the infection of maize seedling leaves by GFP‐tagged P. ananatis at 3, 10, and 12 days post‐inoculation (dpi). The transverse section of maize infected with P. ananatis is depicted in the first two rows, with a zoomed‐in view of the vascular bundle in the third row. GFP, green fluorescent protein; BF, bright field. (b) Scanning electron microscopy images of maize seedling leaves infected by P. ananatis wild‐type DZ‐12 at 12 h post‐inoculation.

2.2. The T6SS‐1 of P. ananatis DZ‐12 is required for pathogenesis and antibacterial activity

Previous sequence analyses (Zhao et al., 2021) demonstrated the presence of three gene clusters in the genome of P. ananatis DZ‐12 that contain genes homologous to those present in T6SSs (Shyntum et al., 2014). T6SS‐1 and T6SS‐3 are located on the chromosome, whereas T6SS‐2 is located on the pDZ‐12 plasmid (Figure 2). To understand whether three T6SS gene clusters are required for P. ananatis virulence, we generated deletion mutants of T6SS‐1, T6SS‐2, and T6SS‐3 (Figure S1). Deletion of T6SS‐1, T6SS‐2, and T6SS‐3 did not affect the growth of P. ananatis in vitro (Figure S2); however, ∆T6SS‐1 and ∆T6SS‐2 mutants exhibited significantly reduced virulence on maize, potato, and onion plants compared with the wild‐type (WT) (Figure 3). Next, we generated the double‐deletion mutants of T6SS‐1 and T6SS‐2 (∆T6SS‐1∆T6SS‐2), the ∆T6SS‐1∆T6SS‐2 mutant showed no attenuated virulence on maize, potato, and onion plants compared with ∆T6SS‐1, ∆T6SS‐2 mutants. These findings indicate that T6SS‐1 and T6SS‐2 are important for pathogenesis in maize, potato, and onion plants, and T6SS‐1 and T6SS‐2 have no synergistic effect. Conversely, T6SS‐3 has no effect on pathogenicity, possibly due to the lack of core genes.

FIGURE 2.

Type VI secretion system (T6SS) gene cluster of Pantoea ananatis DZ‐12. Genes with homology to conserved core T6SS components are designated as type VI secretion (tss) and indicated in red, while genes associated with T6SSs are designated as type VI secretion associated gene (tag) and indicated in different colours; the rhsD protein from bacterial polymorphic toxin systems is indicated in blue. The nomenclature is based on that proposed by Shalom et al. (2007).

FIGURE 3.

T6SS‐1 and T6SS‐2 are required for pathogenicity of Pantoea ananatis DZ‐12. (a, b) Representative pictures were taken at 7 days post‐inoculation (dpi) and the lesion length was measured. PBS, phosphate‐buffered saline. (c, d) Disease symptoms of potato plants inoculated with the P. ananatis strains. Representative pictures were taken at 14 dpi and the lesion length was measured. (e, f) Disease symptoms of onion plants inoculated with the P. ananatis strains. Representative photographs were taken at 7 dpi to indicate representative results and the lesion areas were measured. In (b, d, f), the values are the means ± SD (n = 15 biologically independent samples). Different letters indicate significant differences (p < 0.05, one‐way analysis of variance).

An increasing number of T6SSs have been associated with the interbacterial killing of gram‐negative bacteria through the delivery of different toxins targeting the peptidoglycan of susceptible bacterial species (Basler et al., 2013; Li et al., 2022; Ma et al., 2014). To determine whether the T6SS gene cluster plays a role in the antibacterial activity of DZ‐12, we investigated the in vitro competition assay of WT, ∆T6SS‐1, ∆T6SS‐2, and ∆T6SS‐3 strains. The results revealed that the WT DZ‐12, ∆T6SS‐2, and ∆T6SS‐3 strains exerted a strong inhibition on the gram‐negative bacterium Escherichia coli. However, the ∆T6SS‐1 strain was impaired in killing E. coli (Figure 4). The overall result indicates that only T6SS‐1 is crucial for the pathogenicity and antibacterial activity of DZ‐12 against E. coli.

FIGURE 4.

Type VI secretion system (T6SS)‐dependent killing of Escherichia coli by Pantoea ananatis. Killing cells are wild‐type (WT) DZ‐12 and its mutant strains ∆T6SS‐1, ∆T6SS‐2, and ∆T6SST‐3. Prey cells are E. coli Top10. LB, Luria Bertani broth.

2.3. P. ananatis DZ‐12 gene 4287 encodes a putative T6SS effector TseG

The contextual genes of the known adaptor proteins (DUF1795, DUF2169, or DUF4123) all exhibited a high proportion of encoding T6SS effector proteins (Alcoforado Diniz & Coulthurst, 2015; Bondage et al., 2016; Burkinshaw et al., 2018). To understand the molecular mechanisms of the pathogenicity and antimicrobial activity of T6SS‐1, we identified DUF2169 domains in DZ‐12004286 within T6SS‐1 through a BLASTP search of the NCBI database. We discovered a small cluster of genes 4286–4290 located on the T6SS‐1 of P. ananatis. The small gene cluster encodes a DUF2169 chaperone protein (4286), a putative T6SS effector (4287), an immunity protein (4288), a PAAR protein (4289), and a VgrG protein (4290) as shown in Figure 5a.

FIGURE 5.

TseG effector is secreted by T6SS‐1. (a) Organization of the PA004286–PA004290 operon. The corresponding Pantoea ananatis gene number is indicated above the proposed gene names. Predicted domains are indicated below the proposed gene names. (b) Secretion of TseG is T6SS1‐dependent. Total and secreted proteins were extracted from wild‐type (WT) DZ‐12 and the mutant strains ∆VgrG1, ∆Hcp1, ∆TseG, and ∆VgrG1‐c carrying ptseG‐FLAG, phcp‐FLAG, or an empty vector. HCP, known T6SS‐dependent secreted protein. RecA, known intracellular protein, as a loading control.

2.4. TseG is secreted by the T6SS‐1

To investigate whether TseG functions as a substrate of the T6SS‐1, the TseG‐FLAG protein was electroporated into various P. ananatis DZ‐12 strains, including WT, ∆VgrG1, ∆Hcp1, ∆TseG, and the VgrG1 complemented strain (∆VgrG1‐c). The results demonstrated that TseG was easily detected in the supernatant of WT DZ‐12 bacterial culture, which proved that TseG was an exogenous protein. The disruption of T6SS‐1 through the deletion of key components, such as the needle protein VgrG1 or the inner sheath protein Hcp1, resulted in a significant reduction in TseG excretion. When VgrG1 was reintroduced into the mutant ∆VgrG1, the secretion deficiency of TseG returned to normal level. The intracellular protein RecA could be detected in the total protein extract but not in the exogenous protein, which validates the integrity of the protein extraction procedure and rules out the possibility of intracellular protein cleavage (Figure 5b). In conclusion, TseG is a substrate secreted mainly by the T6SS‐1.

2.5. TseG interacts directly with the immunity protein TsiG and chaperone TecG

Bacteria express a cognate immunity protein that inactivates the cognate effector, thus neutralizing the toxic effects of these effectors and safeguarding themselves. Certain effectors require specialized molecular chaperones (adaptor proteins) for facilitating the extracellular translocation. Our investigation revealed that the effector TseG could interact with both the immunity protein TsiG and chaperone protein TecG via double‐bacterial assay, co‐immunoprecipitation (Co‐IP), and pull‐down assay (Figure 6). We investigated the phylogenetic distribution of TseG in organisms with published genomes. Interestingly, TseG was found to be exclusively present in bacteria, and orthologues were highly conserved in the Pantoea genus (Figure 7).

FIGURE 6.

Protein–protein interaction study between TseG and TecG or TsiG. (a, b) Bacterial two‐hybrid assay. BTH101 reporter cells producing the indicated proteins fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on Luria Bertani plates containing X‐gal. The blue colour of the colony reflects the interaction between the two proteins. Zip and Zip are two proteins known to interact. (c, d) Co‐immunoprecipitation (Co‐IP) assay. TseG, tagged with VSV, was co‐expressed with FLAG‐tagged TecG or TsiG in Pantoea ananatis DZ‐12. Co‐IP assays were performed using FLAG‐trap A beads, and the indicated proteins were immunoblotted with anti‐FLAG and anti‐VSV antibodies. (e, f) Pull‐down assay. Protein complexes involved in interactions were immunoprecipitated with anti‐GST beads, and the indicated proteins were detected by immunoblotting. VSV, vesicular stomatitis virus glycoprotein; GST, glutathione S‐transferase.

FIGURE 7.

TseG is highly conserved in Pantoea genus. The phylogenetic tree was constructed based on the amino acid sequences of RPB2 from 16 prokaryotic species with MEGA 7.0 using the maximum‐likelihood method. The bootstrap values from 1000 replications are indicated on the branches. On the right side, the purple colour indicates the level of homology with TseG.

2.6. TseG is an important virulence factor of DZ‐12 in host plants

To explore the function of TseG in the strain DZ‐12, we generated a mutant strain ∆TseG (Figure S3); deletion of TseG did not affect the growth and swimming motility of P. ananatis (Figures S4 and S5). Subsequently, the maize leaves were inoculated with WT, ∆TseG, and a complemented strain ∆TseG‐c. The WT and complemented strains exhibited comparable levels of pathogenicity whereas the mutant strains showed significantly reduced pathogenicity compared with the WT (Figure 8a,b).

FIGURE 8.

TseG is required for pathogenicity of Pantoea ananatis DZ‐12. (a, b) Disease symptoms of maize plants inoculated with the indicated P. ananatis strains. Representative pictures were photographed at 7 days post‐inoculation (dpi) and the lesion length was measured. PBS, phosphate‐buffered saline. (c, d) Disease symptoms of potato plants inoculated with the indicated P. ananatis strains. Representative pictures were photographed at 14 dpi and the lesion length was measured. (e, f) Disease symptoms of onion plants inoculated with the indicated P. ananatis strains. Representative photographs were taken at 7 dpi and the lesion areas were measured. In (b, d, f), the values are the means ± SD (n = 15 biologically independent samples). Different letters indicate significant differences (p < 0.05, one‐way analysis of variance).

Potatoes were injected and inoculated with WT, ∆TseG, and ∆TseG‐c. The potato tubers infected by WT and ∆TseG‐c exhibited extensive lesion lengths at the injection site, along with outward spreading. In contrast, the potato tubers injected with ∆TseG displayed milder symptoms, manifesting only slight rot at the injection site without the development of large areas of disease spots (Figure 8c,d). The same inoculation approach was employed for onions. There was a reduction in the severity of onion disease when injected with the ∆TseG strain compared with the WT (Figure 8e,f). The overall pathogenicity outcomes revealed that TseG is an important virulence factor of DZ‐12 in host plants.

2.7. TseG exhibits antagonistic activity against E. coli and TsiG is the immunity protein of TseG

To investigate whether the T6SS‐1 effector TseG possesses antagonistic activity against E. coli, the growth of E. coli strains expressing pME6032‐TseG, pME6032‐TseG‐TsiG, and empty vector pME6032 were assessed. The growth of E. coli harbouring the effector TseG slowed down after 6 h of incubation and stopped when the OD₆₀₀ reached about 0.9. Notably, the survival rate of E. coli was restored upon the introduction of immunity protein TsiG (Figure 9). In summary, these findings demonstrate that the effector TseG indeed exhibits bactericidal activity against E. coli, with TsiG serving as the corresponding immunity protein for TseG.

FIGURE 9.

Bacteriostatic activity of TseG in Escherichia coli. The growth of E. coli expressing harbouring TseG or TseT‐TsiT was monitored by measuring OD₆₀₀. The values are the means ± SD (n = 3 biological replicates). IPTG, isopropyl β‐D‐1‐thiogalactopyranoside.

3. DISCUSSION

Protein secretion systems often play a crucial role in the virulence and interactions with hosts in gram‐negative bacterial pathogens (Costa et al., 2021; Ho et al., 2014). Among the various secretion systems, the T6SS has been widely found in various bacteria since it was formally proposed in 2006 (Pukatzki et al., 2006). The T6SS interferes with the life activities of other bacteria by secreting effectors to gain a competitive advantage, modulating the activities of eukaryotic host cells to contribute to bacterial infection, and enhancing bacterial adaptability within their environments (Boyer et al., 2009; Burkinshaw et al., 2018; Shyntum et al., 2015; Stietz et al., 2020). Historically, most of the past studies focused on the T6SS function in human‐pathogenic bacteria, suggesting that different sources of T6SS show different killing effects against other bacteria and fungi. In contrast, there are fewer reports on the systematic characterization of T6SS function in plant pathogens. A. tumefaciens (Wu et al., 2008) and Pseudomonas syringae (Haapalainen et al., 2012) harbour a T6SS, which acts against members of the plant microbiota. Notably, a mutation in the Hcp gene of A. tumefaciens C58 decreases the tumour formation in potato tuber slices (Wu et al., 2008). P. syringae DC3000 antagonizes plant‐associated bacteria like Dickeya dadantii, Pseudomonas savastanoi, and Xanthomonas euvescatoria through its T6SS clusters, especially HSI‐II (Chien et al., 2020). The plant pathogen Burkholderia glumae, causing bacterial panicle blight in rice, has four T6SS gene clusters (T6SS group_1, T6SS group_2, T6SS group_4, and T6SS group_5). The T6SS group_1 confers bacterial competition ability in rice plants, whereas T6SS group_4 and T6SS group_5 contribute to virulence towards rice plants (Kim et al., 2020). Here, we found the plant pathogen P. ananatis DZ‐12, which colonizes the vascular bundle of maize, harbours three T6SS gene clusters, T6SS‐1, T6SS‐2, and T6SS‐3. Only T6SS‐1 of P. ananatis DZ‐12 plays an important role in both pathogenesis and antibacterial activities.

Following the contraction of the T6SS tubular injection structure, the injection needle body passes through the bacterial inner–outer membrane, allowing effectors to be delivered into eukaryotic or prokaryotic cells, where the effectors perform their functions (Bondage et al., 2016; Yun & Lai, 2017). The ability to secrete multiple effectors makes the T6SS a versatile system that helps bacteria compete with prokaryotic and eukaryotic competitors. A. tumefaciens can deliver two DNase effectors, Tde1 and Tde2, providing a competitive advantage to A. tumefaciens during host colonization (Bondage et al., 2016). Hcp2, a secreted protein of P. syringae DC3000, is required for fitness for competition against bacteria and yeasts (Haapalainen et al., 2012). Burkholderia gladioli deploys certain T6SS effectors (TseTBg), having both DNase and RNase activities to kill target bacteria (Yadav et al., 2021). Effectors generally possess their own cognate immunity proteins to neutralize the toxicity of the effectors and thus protect themselves (Song et al., 2021; Yang et al., 2018), while chaperone proteins with typical structural domains contribute to better secretion of effector proteins (Unterweger et al., 2017). For example, a single DUF2169 domain‐containing protein encoded by atu3641 in A. tumefaciens C58 is a T6SS adapter associated with Tde2, an effector of T6SS, and contributes to the secretion of Tde2 (Bondage et al., 2016). T6SS accessory proteins, including DUF2169 domain‐containing protein, contribute to bacterial virulence in T6SS group_5 of B. glumae BGR1 (Kim et al., 2021). In our investigation, we identified a protein containing the typical chaperone domain DUF2169 within T6SS‐1 of P. ananatis DZ‐12, subsequently named TecG. According to the research of Salomon et al. (2014), effectors are generally located in adjacent locations to chaperones, and immunity proteins are often located downstream of effectors. The gene TseG, located downstream of TecG, is predicted to be an effector, while the gene TsiG located downstream of TseG, is predicted to serve as the cognate immunity protein for the effector. Importantly, our findings establish that TseG directly interacts with both the immunity protein TsiG and the chaperone TecG. Moreover, TseG was confirmed to be an exogenous protein, predominantly secreted via the T6SS‐1 of DZ‐12.

The pathogenicity results showed that TseG is an important pathogenicity factor of DZ‐12 in host plants. The deletion of the chaperone TecG also affected the pathogenicity of the strain (data not shown). Chaperones potentially assist in the process of exogenous effector delivery, resulting in reduced pathogenicity; however, the molecular mechanisms need to be further studied. The effector TseG has a toxic activity to E. coli, which indicates that TseG plays a toxic role in the competition between P. ananatis and E. coli, thus enabling P. ananatis DZ‐12 to be in a dominant position. The toxicity of TseG to E. coli was almost lost upon introducing the downstream protein TsiG, which proved that TsiG is the immunity protein of TseG. After entering the receptor target cells, the immune protein binds to the effector and inactivates it, thus neutralizing the toxicity of the effector and protecting the target cells from toxicity (Burkinshaw et al., 2018; Yadav et al., 2021), which offers a robust strategy to enable bacterial adaptations to host microenvironmental changes.

Next, we investigated the phylogenetic distribution of TseG in organisms with published genomes using the alignment of amino acid sequences. The analysis revealed that TseG is highly conserved in the Pantoea genus and has no similarity to previously validated effector proteins, which suggests that diverse effectors are involved in targeting different species. The identification of type VI effectors contributes to the exploration of the novel avenues for Pantoea disease therapy. However, the mode of action of TseG remains elusive and necessitates further investigation. Interestingly, the YjbI domain, pentapeptide repeats of TseG, was identified through a BLASTP search of the NCBI database. TseG, belonging to the His–Me finger superfamily, occurs in a large and diverse superfamily of endonuclease proteins present in all forms of life and involved in various cellular processes (Jablonska et al., 2017). In Drosophila, salto, containing the YjbI domain, is required for sperm head morphogenesis (Augière et al., 2019). Hence, the functions of TseG may be related to this domain, and future studies should explore whether the YjbI domain influences the virulence and antibacterial activity of TseG. The presence of a T6SS provides a significant advantage to many bacteria, as it delivers toxins to neighbouring pathogens for competitive survival. Additionally, the T6SS translocates protein effectors into host cells, leading to the disruption of lipid membranes, cell walls, cytoskeletons, and evasion of host innate immune responses. Our future work will focus on understanding the molecular mechanism of TseG. In conclusion, our study illustrates an unknown effector delivered by the T6SS, showing its antagonism activity against E. coli. This advancement will further our knowledge of how P. ananatis interacts with plants and competes with other species in a polymicrobial environment, and then provide a theoretical basis for the prevention and control of diseases caused by P. ananatis.

4. EXPERIMENTAL PROCEDURES

4.1. Bacterial strains and growth conditions

The bacterial strains and plasmids used in this study are shown in Table S1. P. ananatis strains were derived from strain DZ‐12 (NCBI accession number: PRJNA506327) (Zhao et al., 2021). P. ananatis was grown in Luria Bertani (LB) broth or on LB plates supplemented with 1.5% agar at 30°C or 37°C. The LB medium was also supplemented with 50 μg/mL kanamycin and 50 μg/mL spectinomycin as required. The growth curves were obtained by measuring OD₆₀₀ values of cultures hourly for 24 h in LB broth (n = 3). Swimming motility was characterized using LB medium with 0.3% agar.

4.2. Generation of variants of P. ananatis DZ‐12

The plasmid pBBR‐GFP (Table S1) containing green fluorescent protein (GFP) (Obranic et al., 2013) was introduced into DZ‐12. The DZ‐12 genes were knocked out with the λ‐Red recombinant system of pCas plasmid (Table S1), which guides the recombination and substitution of linear fragments with homologous regions as previously described (Jiang et al., 2015; Zhao et al., 2021). Firstly, the pCas was electroporated into DZ‐12. Subsequently, the fusion PCR product was electroporated into the DZ‐12 strain containing pCas plasmid. The PCR product included the selectable spectinomycin resistance gene (Table S1) with flanking regions homologous to those upstream and downstream of the target gene of DZ‐12. Primers used for amplifying are listed in Table S2. Finally, the resulting strains were identified (Figure S1) and pCas plasmid was eliminated at high temperature (37°C).

4.3. Protein purification

The full‐length open reading frames (ORFs) of TecG, TseG, TsiG were cloned into the pET28a and pET41a vectors, and were transformed into E. coli BL21 (DE3). Subsequently, bacteria were inoculated on 20 mL LB medium at 37°C and 180 rpm for 12 h. Following this, 1% of the bacterial volume was transferred to 200 mL LB medium and cultured at 37°C and 180 rpm for 2–3 h until OD₆₀₀ reached 0.6. At this point, isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 0.1 mM at 16°C and 180 rpm with shaking overnight. The bacteria were centrifuged at 5000 g at 4°C for 30 min and resuspended in Buffer A or phosphate‐buffered saline (PBS). After ultrasonic crushing, the supernatant was harvested at 4°C at 10,000 g for 20 min, and the protein was purified using the AKTA avant 25 (GE Healthcare). The expression of proteins was evaluated using SDS‐PAGE.

4.4. GST pull‐down assay

To test the interaction relationship of TseG with TsiG or TecG, the above purified proteins were mixed in pairs in a 1:1 ratio and glutathione Sepharose 4B suspension was added to the protein mixture, followed by incubating on a rotator for 4–12 h at 4°C. Subsequently, the mixture was centrifuged at 800 g for 5 min, and the resulting pellet was washed with PBS containing Triton X‐100 for five or six times. Finally, the eluted samples were detected with anti‐GST tag and anti‐His tag (Sigma‐Aldrich).

4.5. Protein secretion assay

The secreted protein was analysed as described (Ma et al., 2009). To detect the secreted protein, P. ananatis strains expressing pVSP61‐tseG‐FLAG or pVSP61‐hcp1‐FLAG were cultured in LB medium at 28°C with shaking overnight. On the second day, 1% of the bacterial solution was transferred to LB liquid medium and cultured to OD₆₀₀ = 0.6. Subsequently, the bacterial solution was centrifuged at 15,000 g for 20 min, and 3 mL of cold trichloroacetic acid was added to the supernatant and incubated for 3 h at 4°C. Finally, the secreted protein was harvested by centrifugation and resuspended in PBS (200 μL).

4.6. Bacterial two‐hybrid assay

The bacterial two‐hybrid assay was performed as previously described (Battesti & Bouveret, 2012). Briefly, the TecG and TseG proteins were fused to the isolated PKT25 and PUT18C catalytic domains of the Bordetella adenylate cyclase, respectively. The TecG and TseG fragments were amplified from DZ‐12 genomic DNA by tecG‐FP/RP (PKT25) and tseG‐FP/RP (PUT18C) primers (Table S2). The TecG fragment and PKT25 vector were digested with BamHI and EcoRI enzymes, while the TseG fragment and PUT18C vector were digested with XbaI and EcoRI enzymes. The resulting vectors were transformed into the reporter BTH101 strain, then the transformed strains were cultured on LB plates containing suitable antibiotics (0.5 mM IPTG and 4 μg/mL X‐Gal). After incubating at 28°C for 48 h, the colour reaction of the colonies on LB‐X‐Gal plates was observed to demonstrate the protein interaction between TseG and TecG. The same method was used to confirm protein interactions between TseG and TsiG.

4.7. Co‐IP assay

TseG, TecG, and TsiG were individually amplified and cloned into pUCP26 or pVSP61 plasmid. A starter culture of P. ananatis DZ‐12 containing the indicated plasmids was diluted 100‐fold in LB broth. When the OD₆₀₀ reached 1.2, cells were collected at 10,000 g for 20 min and resuspended in 2 mL of PBS. Resuspended cells were sonicated (10 × 3 s) with a microtip, followed by centrifugation at 10,000 g, and 150 μL of supernatant was boiled for 5 min as input. For Co‐IP, FLAG beads were added to the supernatant and incubated at 4°C for 12 h. The beads were washed with Tris‐buffered saline three times, and protein–protein interactions were detected by immunoblotting using anti‐FLAG and anti‐VSV (Sigma‐Aldrich) antibodies.

4.8. Bacterial competition assays

The competition assays were performed according to MacIntyre et al. (2010). Briefly, cultures were mixed together at a ratio of 10:1 (predator to prey), spotted on LB medium for 3 h at 37°C. Subsequently, the cultures were resuspended in 1 mL of LB medium. To assess the survival of prey cells, serial dilutions were performed in LB medium, followed by plating on specific agar medium.

4.9. Bacterial cell killing assay

TseG and TseG‐TsiG were individually amplified and cloned into pME6032 vector, and the resulting vectors were transformed into E. coli. After growing at 37°C for 12 h, the cells were retransferred to LB at a proportion of 1% and cultured until OD₆₀₀ = 0.4. Subsequently, 0.1 mM IPTG was added to induce protein expression. OD₆₀₀ was measured every 2 h, and the experiment was repeated three times.

4.10. Pathogenicity assays

For maize experiments, the initial cultures of corresponding P. ananatis strains were diluted 100‐fold in LB broth and incubated at 30°C. When the OD₆₀₀ reached 1, the cultures were washed twice in PBS and resuspended in the same buffer to an OD₆₀₀ of 1. In each test, all leaves of 7‐day‐old maize seedlings (cultivar B73) were sprayed with a suspension containing 10⁷ cfu/mL, PBS was used as negative controls. The treated plants were maintained in a greenhouse at 30°C under natural light conditions. The development of disease symptoms was monitored, and pictures were taken 7 days post‐inoculation. Pathogenicity assays were repeated in triplicate.

For red onions and potatoes, the starter cultures of corresponding P. ananatis strains were diluted 100‐fold in LB broth and incubated at 30°C. Once the OD₆₀₀ reached 1, the cultures were resuspended in PBS to achieve an OD₆₀₀ of 1. For red onions, the cultures were resuspended in PBS to achieve an OD₆₀₀ of 0.3 (Shin et al., 2023). Subsequently, 1 mL of bacterial suspension was injected into onion and potato tubers, while PBS was injected as a control. The experiment was repeated three times.

4.11. Phylogenetic analysis

All the genomes used in the study are publicly available at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/). Accession numbers for proteins used in comparison are shown in Table S3. Phylogenetic trees were constructed using the MEGA X software (https://www.megasoftware.net/) with the maximum‐likelihood method (bootstrap = 1000, p‐distance, pairwise deletion).

4.12. Statistical analysis

Statistical analysis of the data was performed using SPSS software v. 26 (SPSS). Each experiment was repeated thrice, and data were evaluated using one‐way analysis of variance.

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no competing interests.

Supporting information

Figure S1. PCR identification of the wild‐type PH‐1 and the indicated mutants. M, Marker; −, negative control.

Figure S2. Growth of Pantoea ananatis strains DZ‐12, ∆T6SS‐1, ∆T6SS‐2, ∆T6SS‐3, and ∆T6SS‐1∆T6SS‐2 in Luria Bertani broth at 30°C. The values are the means ± SD (n = 3 biological replicates).

Figure S3. PCR identification of the wild‐type PH‐1 and the indicated mutant. M, Marker; −, negative control.

Figure S4. Growth of Pantoea ananatis strains DZ‐12 and ∆TseG in Luria Bertani broth at 30°C. The values are the means ± SD (n = 3 biological replicates).

Figure S5. Swimming motility of Pantoea ananatis strains DZ‐12 and ∆TseG on a semisolid agarose plate. Swimming motility was characterized using Luria Bertani medium with 0.3% agar.

Table S1. Bacterial strains and plasmid used in this study.

Table S2. Oligonucleotide primers used in this study.

Table S3. Accession numbers for proteins used in comparison.

Zhao, X. , Gao, L. , Ali, Q. , Yu, C. , Yuan, B. , Huang, H. et al. (2024) A type VI secretion system effector TseG of Pantoea ananatis is involved in virulence and antibacterial activity. Molecular Plant Pathology, 25, e13442. Available from: 10.1111/mpp.13442

DATA AVAILABILITY STATEMENT

The data that support the findings of this article are available from the corresponding author upon request.