Role of Formate Chemoreceptor in Pseudomonas syringae pv. tabaci 6605 in Tobacco Infection

Abstract

Chemotaxis is essential for infection by plant pathogenic bacteria. The causal agent of tobacco wildfire disease, Pseudomonas syringae pv. tabaci 6605 (Pta6605), is known to cause severe leaf disease and is highly motile. The requirement of chemotaxis for infection has been demonstrated through the inoculation of mutant strains lacking chemotaxis sensory component proteins. Pta6605 possesses 54 genes that encode chemoreceptors (known as methyl-accepting chemotaxis proteins, MCPs). Chemoreceptors are classified into several groups based on the type and localization of ligand-binding domains (LBD). Cache LBD-type chemoreceptors have been reported to recognize formate in several bacterial species. In the present study, we identified Cache_3 Cache_2 LBD-type Mcp26 encoded by Pta6605_RS00335 as a chemoreceptor for formate using a quantitative capillary assay, and named it McpF. Although the deletion mutant of mcpF (ΔmcpF) retained attraction to 1% yeast extract, its chemotactic response to formate was markedly reduced. Swimming and swarming motilities were also impaired in the mutant. To investigate the effects of McpF on bacterial virulence, we conducted inoculations on tobacco plants using several methods. The ΔmcpF mutant exhibited weaker virulence in flood and spray assays than wild-type and complemented strains, highlighting not only the involvement of McpF in formate recognition, but also its critical role in leaf entry during the early stages of infection.

Pseudomonas syringae pv. tabaci strain 6605 (Pta6605) is a Gram-negative hemibiotrophic pathogen that causes wildfire disease in tobacco (Ichinose et al., 2003). The infection cycle of Pta6605 begins with an epiphytic phase on the leaf surface. During the early stages of infection, bacteria that have adapted to environmental conditions enter the leaf interior through natural openings, such as stomata or wounds. Pta6605 then resides within the leaf apoplast and deploys its virulence factors to evade or suppress the plant’s immune system (Melotto et al., 2006; Xin et al., 2018). The ability of bacteria to enter plant leaves is a critical factor for successful infection. Plant-derived compounds may play a crucial role in facilitating bacterial movement in leaf tissue (Watanabe et al., 2023). Chemotaxis is an ability to move towards an attractant or away from a repellent (Adler, 1966). Therefore, chemotaxis appears to contribute to this stage; bacteria move towards the plant apoplastic fluid where they find and enter stomata. Pta6605 is attracted by the apoplastic fluid extracted from both host and non-host plants, indicating that chemotaxis to common plant apoplastic compounds facilitates the entry of bacteria into leaves (Watanabe et al., 2023).

Chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs) play a critical role in the regulation of chemotaxis. A typical chemoreceptor contains a periplasmic ligand-binding domain (LBD), transmembrane domain, histidine kinases, adenylate cyclases, methyl-accepting chemotaxis proteins, and phosphatases (HAMP) domain, and signaling domain. The periplasmic sensing domain may be a LBD, which binds directly with chemoeffectors, or a domain that binds with a chemoeffector-loaded periplasmic protein (Ortega et al., 2017). Fifty-four putative chemoreceptor genes have been identified in Pta6605, and their deduced protein structures were classified into four classes based on their amino acid sequence, topology, and localization (Ichinose et al., 2023).

Formic acid (HCOOH), a one-carbon (C1) organic acid, plays an essential role in plants. It may be synthesized through various pathways, including photorespiration, direct CO₂ reduction, and the decarboxylation of glyoxylate in peroxisomes, chloroplasts, or mitochondria in C3 plants (Hanson and Roje, 2001; Igamberdie and Bykovaa, 1999). Formate appears to contribute to plant metabolism by incorporating TCA cycle-associated organic acids (Zbinovsky and Burris, 1952; Tolbert, 1955; Prabhu and King, 1996). The rapid conversion of formic acid to malic acid and other organic acids has been reported in tobacco leaves, indicating that this is the normal metabolic process for formate in tobacco leaves (Zbinovsky and Burris, 1952).

MCPs for formate have been identified in several animal- and plant-associated bacteria. Atu0526, a single Calcium channels and chemotaxis receptors_3_2 (sCache_3_2) LBD-type MCP in the soil-borne bacterium Agrobacterium fabrum C58, binds specifically to formate, but not to acetate, propionate, butyrate, citrate, malate, succinate, fumarate, or salicylate (Wang et al., 2021). PacF, a Cache_3 Cache_2 LBD-type MCP in the phytopathogenic bacterium Pectobacterium atrosepticum SCRI1043, also binds to formate at a distal module (Monteagudo-Cascales et al., 2025). A sCache_2 LBD containing the chemoreceptor McpV in Sinorhizobium meliloti binds to four carboxylates: acetate, propionate, butyrate, and formate; however, among these carboxylates, formate exhibited the lowest affinity to McpV (Compton et al., 2018). A dCache_1 LBD-type chemoreceptor to formate, Tlp1, was also found in the pathogenic bacterium Campylobacter jejuni in poultry. Tlp1 is critical for formate sensing because deletion mutants lost the full chemotactic response to formate (Duan et al., 2023). Despite these findings, the relationship between formate chemotaxis and bacterial virulence has not yet been elucidated. Therefore, we herein aimed to provide insights into the identification of formate chemoreceptors and how they contribute to the motility and virulence of Pta6605.

Materials and Methods

Bacterial strains and growth conditions

The bacterial strains used in the present study are listed in Table 1. Escherichia coli DH5α and S17-1 strains were grown in Luria-Bertani (LB) medium with the appropriate antibiotics at 37°C. Pta6605 was cultured in King’s B (KB) medium supplemented with 50‍ ‍μg mL^–1 nalidixic acid (Nal) at 27°C and minimal medium (MM; 50‍ ‍mM K₂SO₄, 7.6‍ ‍mM [NH₄]₂SO₄, 1.7‍ ‍mM MgCl₂, and 1.7‍ ‍mM NaCl) (Huynh et al., 1989) supplemented with 10‍ ‍mM mannitol and fructose (MMMF). To measure bacterial growth overnight, cultured Pta6605 in KB medium was washed, resuspended in fresh medium, and growth was monitored as previously described (Tumewu et al., 2020).

Table 1.

Bacteria strains and plasmids used in the present study

Bacterial strain, plasmid	Relevant characteristics	Reference or source
Escherichia coli
DH5α	F–λ–ϕ80dLacZ ΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–mK+) supE44 thi-1 gyrA relA1	Nippon Gene
S17-1	thi pro hsdR hsdR hsdM+ recA (chr::RP4-2-Tc::Mu-Km::Tn7)	Schäfer et al., 1994
Pseudomonas syringae pv. tabaci
Isolate 6605	Wild type isolated from tobacco, Nalr	Ichinose et al., 2003
ΔpscA	Isolate 6605 ΔRS16340, Nalr	Tumewu et al., 2021a
ΔpscB	Isolate 6605 ΔRS23495, Nalr	Tumewu et al., 2021a
ΔpscC1	Isolate 6605 ΔRS16480, Nalr	Tumewu et al., 2021a
ΔpscC2	Isolate 6605 ΔRS11960, Nalr	Tumewu et al., 2021a
ΔmcpG	Isolate 6605 ΔRS09525, Nalr	Tumewu et al., 2020
Δmcp24	Isolate 6605 ΔRS19225, Nalr	This study
Δmcp34	Isolate 6605 ΔRS11260, Nalr	This study
Δmcp26	Isolate 6605 ΔRS00335, Nalr	This study
Δmcp26-C	Isolate 6605 Δmcp26, pD-mcp26 complemented, Nalr, Kmr	This study
Plasmid
pUC118	Cloning vector, Ampr	Takara Bio
pUC-mcp26	mcp26 fragment containing pUC118, Ampr	This study
pGEM-T Easy	Cloning vector, Ampr	Promega
pG-mcp24	mcp24 fragment containing pGEM-T Easy, Ampr	This study
pG-mcp34	mcp34 fragment containing pGEM-T Easy, Ampr	This study
pK18mobSacB	Small mobilizable vector, Kmr, sucrose sensitive (sacB)	Schäfer et al., 1994
pK18-Δmcp26	mcp26 deleted DNA-containing pK18mobsacB, Kmr	This study
pK18-Δmcp24	mcp24 deleted DNA-containing pK18mobsacB, Kmr	This study
pK18-Δmcp34	mcp34 deleted DNA-containing pK18mobsacB, Kmr	This study
pDSK519	Broad host range cloning vector	Keen et al., 1988
pD-mcp26	pDSK519 possessing expressible mcp26, Kmr	This study

Nal^r: nalidixic acid resistant; Amp^r: ampicillin resistant; Km^r: kanamycin resistant.

Generation of mutant and complemented strains

To generate the Δmcp26 strain, the mcp26 gene (Pta6605_RS00335) was amplified using the primer pairs listed in Table S1 and inserted into a pUC118-HincII/BAP vector (Mighty Cloning Reagent Set; Takara Bio). Inverse PCR to delete the open reading frame (ORF) of mcp26 was followed by digestion with BamHI and then self-ligation and transformation into E. coli DH5α cells. The target fragment was subcloned into the mobilizable cloning vector pK18mobsacB via the EcoRI and SphI sites (Schäfer et al., 1994). The deletion mutant was generated by double homologous recombination as previously described (Tumewu et al., 2022). Recombinant pK18mobsacB was introduced into E. coli S17-1 and subsequently used for conjugation with the Pta6605 wild type (WT). Colonies were selected on KB medium supplemented with 10% sucrose to isolate deletion mutants by second homologous recombination. Δmcp26 deletion mutant strains were confirmed by PCR and sequencing. The complemented strain was generated by introducing recombinant pDSK519 (Keen et al., 1988) with the ORF of mcp26. Similarly, mcp24 (RS19225) and mcp34 (RS11260) were amplified and inserted into a pGEM-T Easy vector (Promega). Inverse PCR to delete the ORFs of mcp24 and mcp34 was conducted similarly to mcp26, and Δmcp24 and Δmcp34 were generated. Mutant strains of ΔmcpG, ΔpscA, ΔpscB, ΔpscC1, and ΔpscC2 were previously described (Tumewu et al., 2020, 2021a).

Quantitative capillary assay

The quantitative capillary method was described in a previous study (Tumewu et al., 2020). Glass capillaries (Drummond Scientific) were flame-sealed at one end and filled with 5‍ ‍μL of either a control or chemoattractant. The negative control was 10‍ ‍mM HEPES (pH 7.4) buffer, and 1% yeast extract (YE) served as the positive control. Formate solution was prepared by diluting formic acid (Wako Chemicals) to the indicated concentration in 10‍ ‍mM HEPES. Capillaries were incubated in 200‍ ‍μL of a bacterial suspension (OD₆₀₀ of 0.05 in 10‍ ‍mM HEPES) at room temperature for 40‍ ‍min. After the incubation, bacteria inside the capillary were suspended in 45‍ ‍μL of 0.9% NaCl, serially diluted, and plated for colony counting.

Plant growth and virulence assays

Three inoculation methods were used to assess bacterial virulence in tobacco (Nicotiana tabacum L. var. Xanthi NC). The flood inoculation method was modified for tobacco plants (Ishiga et al., 2017; Tumewu et al., 2020). Tobacco seedlings were cultivated in Murashige-Skoog (MS) plates containing 0.8% agar, 0.1% sucrose, and vitamins (3‍ ‍mg L^–1 thiamin hydrochloride, 5‍ ‍mg L^–1 nicotinic acid, and 0.5‍ ‍mg L^–1 pyridoxine hydrochloride). Seedlings were inoculated for 10‍ ‍s with 10‍ ‍mL of a bacterial suspension at OD₆₀₀ of 0.004 (8×10⁶ CFU mL^–1) in 10‍ ‍mM MgSO₄ and 0.025% Silwet L-77 (OSI Specialties). After decanting the excess inoculum and drying for 30‍ ‍min in a cabinet, plants were incubated at 22°C under 16/8‍ ‍h light/dark conditions. To quantify bacterial growth, two leaves per seedling were sampled; leaf disks were obtained, ground, diluted, and plated on KB-Nal medium. Bacterial colonies were counted to examine bacterial populations in the leaves.

In the spray inoculation method, 8- to 9-week-old plants grown in soil at 25°C under 12/12‍ ‍h light/dark conditions were sprayed with a bacterial suspension (4×10⁸ CFU mL^–1 in 10‍ ‍mM MgSO₄ with 0.04% Silwet L-77) and were then incubated under high humidity conditions for 7 days. In the infiltration inoculation method, attached leaves were infiltrated with bacterial suspensions (2×10⁵ CFU mL^–1 in 10‍ ‍mM MgSO₄) using a needleless 1-ml syringe. Plants were incubated at 22°C under 16/8‍ ‍h light/dark conditions and symptoms were monitored for 7 days.

Surface motility assay

Swarming and swimming assays were performed to assess bacterial surface motility (Taguchi and Ichinose, 2011). MMMF medium with 0.25% agar (Bacto agar; Becton, Dickinson and Company) was used for the swimming assay, while 0.4% agar (Bacto agar) SWM medium (0.5% peptone, 0.3% YE) was used for the swarming assay. Bacteria were grown at 27°C overnight in LB containing 10‍ ‍mM MgCl₂, resuspended in 10‍ ‍mM MgSO₄, and adjusted to an OD₆₀₀ of 0.3. Two microliters of a bacterial suspension was injected and spotted at the center of MMMF or SWM plates for the swimming and swarming assays, respectively. MMMF plates were incubated at 23°C for 72 h, while SWM plates were incubated at 27°C for 24 h. The spread of the bacterial halo was measured using ImageJ software (Fiji Distribution, version 1.54f).

Bioinformatics

The protein domain architecture was predicted using the SMART tool (Letunic and Bork, 2018). Multiple alignment was performed using MAFFT software (version 7) (Katoh et al., 2019), and a phylogenetic tree was constructed using Mega software (version 11) (Tamura et al., 2021).

Statistical ana­lysis

All statistical ana­lyses were performed using GraphPad Prism (version 10.4.1, GraphPad Software). A one-way ana­lysis of variance (ANOVA) was followed by Dunnet’s post hoc test. The confidence level was set at 95%, and P<0.05 indicated a significant difference.

Results

Chemotaxis of Pta6605 to formate

To investigate whether formate is a chemoattractant of Pta6605, a quantitative capillary assay was performed with a range of formate concentrations (100, 10, 1, and 0.1‍ ‍mM). To evaluate chemoattraction, the number of bacteria in the capillaries containing the attractant was compared with that in capillaries containing 10‍ ‍mM HEPES buffer. Among the different concentrations tested, Pta6605 exhibited a significant chemotactic response to 1‍ ‍mM formate (Fig. 1).

Fig. 1.

Dose-dependent chemotactic response to formate in Pta6605

Chemotaxis to formate was measured by a quantitative capillary assay. As negative and positive controls, 10‍ ‍mM HEPES buffer and 1% yeast extract (YE), respectively, were used. Data represent the number of bacterial cells in each capillary from three independent experiments in triplicate. Asterisks indicate significant differences from the negative control by a one-way ANOVA followed by Dunnett’s multiple comparison test (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05, and ns=not significantly different).

Comparison of LBDs of formate chemoreceptors and Cache domain-containing MCPs in Pta6605

MCPs with various types of LBDs have been identified as bacterial formate chemoreceptors. Several formate chemoreceptors have been reported to date. Based on previous findings, MCPs with Cache domain-containing LBD appear to be formate chemoreceptors. Since eight Cache domain-containing MCPs are present in Pta6605 (Ichinose et al., 2023), they were selected as candidates for formate chemoreceptors and included Cache_3 Cache_2 domain-containing Mcp26 (encoded in RS00335 and tentatively numbered), sCache_2 domain-containing Mcp24 (encoded in RS19225) and Mcp34 (encoded in RS11260), and dCache domain-containing McpG, PscA, PscB, PscC1, and PscC2 (Tumewu et al., 2020, 2021a). The amino acid sequences of the LBD described above were compared using a maximum likelihood tree (Fig. 2). Based on the results obtained, all of the candidates were considered to be possible formate chemoreceptors.

Fig. 2.

Maximum likelihood tree based on the ligand-binding domain (LBD) of previously known formate chemoreceptors and their homologs in Pta6605

Multiple alignment was performed using MAFFT software (version 7) (Katoh et al., 2019). A phylogenetic tree was created using Mega software (version 11) (Tamura et al., 2021).

Mcp26 is a specific chemoreceptor for formate in Pta6605

The chemotactic response of each deletion mutant to 1‍ ‍mM formate was assessed using quantitative capillary assays. As shown in Fig. 3, all mutant strains, except for the Δmcp26 strain, exhibited chemotaxis to formate. The Δmcp26 strain did not show a significant attraction to 1‍ ‍mM formate, but was still attracted to 1% YE. Although the chemotactic response of the Δmcp26 mutant to formate varied, only Δmcp26 exhibited a significant reduction in formate attraction among the mutants tested (Fig. 3). This variability may be attributed to the presence of other environmental factors, such as inorganic ions, a pH gradient (pH taxis), or oxygen levels (aerotaxis) (Matilla and Krell, 2018), which were difficult to fully control under experimental conditions and may have affected formate chemotaxis. To confirm the regulation of Mcp26 in chemotaxis to formate, a complemented strain, Δmcp26-C, carrying mcp26 on the plasmid was generated. As expected, formate sensing was restored in Δmcp26-C, and the attraction level was even higher than in WT (Fig. 4). Based on these results, we designated Mcp26 as the specific formate chemoreceptor, McpF, in Pta6605.

Fig. 3.

Chemotactic response to formate in deletion mutants for the Cache LBD-containing mcp gene in Pta6605

Chemotaxis to formate was measured by a quantitative capillary assay to 1‍ ‍mM formate. Data represent the number of bacteria in each capillary from three independent experiments in triplicate. Statistical ana­lyses and data presentation are the same as in Fig. 1.

Fig. 4.

Chemotactic responses to formate of WT, Δmcp26, and Δmcp26-C in Pta6605

Chemotaxis to formate was measured by a quantitative capillary assay to 1‍ ‍mM formate. Data represent the number of bacteria in each capillary from three independent experiments in triplicate. Statistical ana­lyses and data presentation are the same as in Fig. 1.

Surface motility

The Δmcp26 mutant exhibited reduced swimming and swarming motilities (Fig. 5). In the complemented strain, only swimming motility was restored. To investigate the effects of formate on swimming motility, we supplied 1‍ ‍mM formate to MMMF medium. The motilities of both WT and Δmcp26 were not affected by formate (Fig. S1). In addition to moving them towards a favorable environment, chemotaxis enables bacteria to detect and access a nutrient pool (Matilla et al., 2023). The expansion of bacterial halos may‍ ‍be affected by their ability to utilize carbon sources. Swimming tests were performed using MM medium with 1‍ ‍mM formate to evaluate the ability of Pta6605 to utilize formate as a carbon source. Neither WT nor Δmcp26 grew in MM medium without carbon sources other than formate (Fig. S2), indicating that Pta6605 cannot use formate as a unique carbon source for metabolism.

Fig. 5.

Surface motility tests

Bacterial motility was assessed after an incubation for 72‍ ‍h using a swimming assay (A) and 24‍ ‍h using a swarming assay (B). The spread areas of each strain were measured using ImageJ software for the swimming (C) and swarming (D) tests. Statistical ana­lyses and data presentation are the same as in Fig. 1.

Virulence of mutants on host tobacco leaves

The flood inoculation method was used to assess bacterial virulence by evaluating disease symptoms and bacterial populations inside the leaves (Fig. 6A and B). The population of the Δmcp26 mutant was significantly smaller than that of the WT strain at three days post inoculation (dpi), whereas the number of complemented strains recovered to WT levels (Fig. 6B). Since motility affects successful infection by pathogenic bacteria, reduced motility may hinder entry into the leaf apoplast, thereby markedly affecting bacterial multiplication. The deletion of mcp26 (mcpF) resulted in reductions in swimming and swarming motilities, suggesting the impaired leaf invasion ability of the mutant. Furthermore, no significant differences in growth were observed among three strains in the culture (Fig. S3), indicating that differences in the bacterial population inside the leaves were not driven by bacterial growth. Consistent with bacterial growth, disease symptoms in tobacco leaves were severe when inoculated with the WT and Δmcp26-C strains (Fig. 6A). The symptoms of the leaves inoculated with Δmcp26 were also severe; however, the onset of symptoms was slower than with the inoculation with the WT strain. In addition, disease symptoms following the spray inoculation were consistent with those observed after the flood inoculation, where leaves inoculated with the WT and complemented strains exhibited more severe symptoms than those inoculated with the mutant (Fig. 6C). These outcomes suggest that McpF (Mcp26) deficiency reduced bacterial virulence by limiting leaf entry, but not invasiveness. To further investigate this hypothesis, we conducted an infiltration inoculation by directly injecting a bacterial suspension into the leaves. Disease symptoms were observed in leaves infected with all strains at 7 dpi, indicating that the mutant remained virulent and capable of causing disease in the tobacco host plant (Fig. S4).

Fig. 6.

Inoculation of tobacco host plants

(A) Tobacco seedlings were flood-inoculated with WT, Δmcp26, or Δmcp26-C. Representative photos were taken at 3 and 7 dpi. (B) Bacterial populations in leaves were quantified at 3 hpi (0 dpi) and 3 dpi. Statistical ana­lyses and data presentation are the same as in Fig. 1. The inoculation was performed with three independent repeats (n=3 for 0 dpi, n=8 for 3 dpi). (C) Tobacco leaves were sprayed with a bacterial suspension at a density 4×10⁸ CFU mL^–1 in 10‍ ‍mM MgSO₄ and 0.04% Silwet L-77. Symptoms were observed at 7 dpi. The bar is 10‍ ‍mm.

Discussion

The Cache domain is the predominant extracellular LBD of chemoreceptors in prokaryotes (Upadhyay et al., 2016). Its structure is characterized by a Per/Arnt/Sim (PAS)-like module and long N-terminal α-helix (Ortega et al., 2017). McpF (Mcp26) was identified as a unique Cache_3 Cache_2 LBD-type chemoreceptor that responds to formate, while sCache_2 and dCache_1 LDB were not involved in this recognition in Pta6605. The amino acid sequence of LBD of McpF is homologous to that of PacF in P. atrosepticum SCRI1043 and Atu0526 in A. fabrum C58 (Fig. S5). A conserved arginine residue (R115) in Atu0526 was previously shown to directly bind to formate and function in the regulation of chemotaxis (Wang et al., 2021). Modifications to a conserved arginine residue (R142) and threonine residues (T145 and T158) in PacF was found to affect formate binding (Monteagudo-Cascales et al., 2025). A multi-alignment ana­lysis of McpF with Atu0526 and PacF revealed a conserved R139 in McpF; therefore, further studies on R139 binding with formate at the membrane distal module and its response to formate are warranted (Fig. S5).

Research on the role of formate in the plant apoplast is limited. Formate was previously detected at low concentrations in the apoplastic fluid of leaves from some Fragaceae plants (Gabriel and Kesselmeier, 1999). Small formate pools in plants were typically in the range of 0.1 to 1‍ ‍μmol g^–1 fresh weight (Hanson and Roje, 2001). The presence of formate may be associated with its incorporation into other‍ ‍organic acids involved in plant metabolism (Zbinovsky and Burris, 1952). Plants release formic acid into the atmosphere, with its emission from terrestrial vegetation accounting for 3% of global formic acid emissions (Hanson and Roje, 2001). The directional emission of gaseous formic acid via the stomata has been universally observed in higher plants, and the apoplast is the site at which formate is transformed from a liquid to its gas form (Gabriel et al., 1999; Seco et al., 2007; Mochizuki and Tani, 2021), apart from a few crops including Zea mays, Pisum sativum, Hordeum vulgare, and Avena sativa (Kesselmeier et al., 1998). These findings support the hypothesis that the presence of formate in stomata and apoplasts may be related to interactions between plants and phyllosphere bacteria.

The relationship between chemoreceptors and bacterial swimming ability is complex and has not yet been fully elucidated. In the case of Pta6605, reduced motility was observed in a mutant lacking amino acid chemoreceptor genes (Tumewu et al., 2021a). Chemotaxis systems are functionally classified into three groups: those involved in controlling flagellar motility (F), which includes 17 distinct classes (F1–F17), those regulating type IV pilus’ motility, and those associated with alternative cellular functions unrelated to motility (Wuichet and Zhulin, 2010). In addition, chemosensory signaling pathways typically involve a chemoreceptor and two-component histidine kinase complex, consisting of the kinase CheA and the response regulator CheY, which modulates bacterial flagellar rotation. In Pta6605, the deletion of cheY2 completely abolished swimming and swarming motilities, indicating the crucial role of CheY2 in chemotaxis and flagellar-mediated motility (Tumewu et al., 2021b). Since CheY2 in Pta6605 has been linked to the F6 chemosensory pathway (Ichinose et al., 2023), we hypothesized that Mcp26 may be associated with a flagellar-dependent chemotaxis pathway, contributing to the regulation of bacterial motility. Interestingly, while CheY2 is critical for swimming and swarming, we found that cheY2 expression levels were similar in the Δmcp26 mutant and WT strain (data not shown). Therefore, motility regulated by Mcp26 may not be solely dependent on cheY2 expression and may involve additional signaling mechanisms or unknown chemotaxis pathways. On the other hand, the complemented strain restored abilities for formate sensing and swimming motility, but not swarming motility, suggesting a polar effect caused by the deletion mutation.

The chemotaxis of pathogenic bacteria facilitates initial entry into the plant apoplast during the early stages of infection (Matilla and Krell, 2018). Among 54 MCPs, eight MCPs and their respective ligands have been investigated, with six being shown to contribute to the virulence of Pta6605 (Tumewu et al., 2020, 2021a, 2022). McpG, a specific chemoreceptor of γ-aminobutyric acid (GABA), has been reported to affect both the early and late stages of infection (Tumewu et al., 2020). Additionally, among the four other dCache_1 LBD-type MCPs, PscB, PscC1, and PscC2, which are responsible for chemotaxis towards proteinogenic amino acids, excluding tyrosine, are required for the full virulence of Pta6605. The deletion of these genes results in the abolishment of virulence due to the complete loss of motility (Tumewu et al., 2021a). Furthermore, AerA and AerB have been shown to meditate aerotaxis, with AerA playing an important role in host infection (Tumewu et al., 2022). Beyond the six MCPs previously associated with virulence, the present study revealed that McpF was also required for leaf entry in both the flood and spray inoculation methods. However, when bacteria were directly introduced into the apoplast via infiltration, the mutant maintained full pathogenicity. This phenotype may be linked to the decreased swimming and swarming motilities of the mutant, which affected its ability to enter host leaves. Moreover, the present results suggest that chemotaxis towards formate plays a crucial role in the early stage of infection in Pta6605.

In conclusion, the present study suggests that McpF (Mcp26), which possesses the Cache_3 Cache_2 type LBD, serves as a specific chemoreceptor for formate in Pta6605. The absence of McpF reduces virulence in tobacco plant leaves, which is attributed to diminished motility.

Citation

Nguyen, P. Q. T., Watanabe, Y., Matsui, H., Sakata, N., Noutoshi, Y., Toyoda, K., and Ichinose, Y. (2025) Role of Formate Chemoreceptor in Pseudomonas syringae pv. tabaci 6605 in Tobacco Infection. Microbes Environ 40: ME25019.

https://doi.org/10.1264/jsme2.ME25019

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Research (B) (No. 22H0234814) from the Ministry of Education, Culture, Sports, Science and Technology of Japan to YI.

Conflict of interest

All authors declare that there is no conflict of interest.

Ethical approval

This article does not include any experiments on animals or humans conducted by any of the authors.

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