The Enzyme Glucose-1-Phosphate Thymidylyltransferase RmlA Plays a Crucial Role in the Pathogenesis of Pectobacterium actinidiae GX1

Abstract

Pectobacterium actinidiae is one of the primary pathogens that causes summer canker disease in kiwifruit, yet its pathogenic mechanisms remain unknown. The exopolysaccharide PCAP-1a, isolated from the fermentation broth of P. actinidiae strain GX1, exhibits notable cytotoxicity and acts as a virulence factor facilitating host infection. Genome-wide analysis revealed a 21-gene cluster responsible for the biosynthesis of exopolysaccharides in GX1. Homologous recombination was used to systematically knock out these genes, which led to the identification of RmlA as a key protein in the synthesis of the PCAP-1a precursor. The deletion of the rmlA gene significantly affected the yield of PCAP-1a and resulted in a direct reduction in GX1 pathogenicity. Further studies revealed that mutations in the substrate binding site of RmlA weakened its capacity to bind G-1-P and dTTP, which led to markedly reduced pathogenicity in the corresponding complemented strains. This study indicates that the exopolysaccharide PCAP-1a serves as a virulence factor in the pathogenesis of GX1, and its biosynthesis depends on the polysaccharide synthesis gene rmlA and the substrate binding activity of its encoded protein.

Keywords: Pectobacterium actinidiae; biosynthesis‐related gene; genome; pathogenicity; polysaccharide.

FIGURE 1

Characterisation of the complete genome map and exopolysaccharide (EPS) biosynthesis gene clusters of Pectobacterium actinidiae GX1. (A) Whole‐genome map of P. actinidiae GX1. The outer ring represents various functional annotations based on COG categories, with colour coding for different categories of biological processes. The centre of the map shows a graphical representation of the genomic features and annotations. (B) Analysis of EPS biosynthesis gene clusters in P. actinidiae GX1. The gene cluster diagram shows the specific genes involved in EPS biosynthesis. Each gene in the cluster is annotated with its corresponding function.

FIGURE 2

Analysis of exopolysaccharide (EPS) biosynthesis‐related gene clusters in the Pectobacterium genus. (A) Comparative analysis of EPS biosynthesis‐related gene clusters in the Pectobacterium genus. The clusters represent genes involved in the biosynthesis of EPS in various Pectobacterium species, key genes such as glycosyltransferases (GTs). These clusters have been aligned to show their relative relatedness, highlighting conserved and divergent regions across species. (B) Biological activity tests of the exopolysaccharides of GX1, P. brasiliense 1692, P. carotovorum WPP14 and Pseudomonas syringae Susan2139. The resulting EPSs were extracted and prepared as 0.5 mg/mL solutions, which were then infiltrated into the same Nicotiana benthamiana leaf. Cell death in each leaf was observed after 72 h, and the observed phenotypes correlate with the presence or absence of specific biosynthesis‐related genes in (A).

FIGURE 3

Impact of the rmlA gene on the yield of exopolysaccharides (EPSs) from and the morphology of Pectobacterium actinidiae GX1. (A) Both the ∆rmlA strain and its EPSs had a reduced ability to induce Nicotiana benthamiana cell death. (B) The yield of EPSs produced by the ∆rmlA strain decreased. The error bars represent the standard deviation; n = 3 bacterial samples. Significant differences from ANOVA are shown: ** = p  < 0.01. The control is the amount of EPS produced by the wild‐type strain of GX1 (WT). The experiment was repeated three times, and the results all showed the same trend. (C) The colony morphology phenotypes of the WT and ∆rmlA strains, as well as the surface morphology of the WT and ∆rmlA strains, were observed through scanning electron microscopy.

FIGURE 4

Impact of the rmlA gene on the pathogenicity of Pectobacterium actinidiae GX1. (A) To assess the pathogenicity of the ∆rmlA strain, ∆rmlA, ∆rmlA‐C and wild‐type (WT) strains were cultured to logarithmic growth stage, and the OD₆₀₀ was adjusted to 0.4 with sterile water, and 6 μM PCAP‐1a was used for the treatment. Then, a 1.5 μL bacterial suspension or mixed solution of bacterial suspension and PCAP‐1a was inoculated on a Chinese cabbage core approximately 2 × 2 cm in size, and the lesion diameter and degree of bacterial colonisation were measured after 18 h at 28°C. (B) Lesion diameters corresponding to the assay data in (A). (C) Bacterial colonisation corresponding to the assay data in (A). The error bars represent the standard deviation; n = 12 stems. Significant differences from ANOVA are shown: ** = p < 0.01. The control consisted of Chinese cabbage stems incubated with WT GX1. The experiment was repeated three times, and the results all showed the same trend.

FIGURE 5

Heterologous expression of RmlA and its binding affinity for its substrates glucose‐1‐phosphate (G‐1‐P) and dTTP. (A) Molecular docking simulation of the RmlA binding site with G‐1‐P. (B) Molecular docking simulation of the RmlA binding site with dTTP. (C) Microscale thermophoresis (MST) was used to assess the binding affinity between RmlA and G‐1‐P. (D) MST was used to assess the binding affinity between RmlA and dTTP. (E–H) Binding affinity assays for RmlA mutants with substrates (G‐1‐P, dTTP). (E) Molecular docking simulations of RmlA mutant binding sites with G‐1‐P. (F) Molecular docking simulations of RmlA mutant binding sites with dTTP. (G) MST assays of the binding affinity between the RmlA mutant and G‐1‐P. (H) MST assays of the binding affinity between the RmlA mutant and dTTP.

FIGURE 6

Induction of cell death by Pectobacterium actinidiae GX1 and determination of its growth curve. (A) To assess the ability of the ∆rmlA, wild‐type (WT), ∆rmlA‐C and ∆rmlA‐C‐mut strains to induce Nicotiana benthamiana cell death, the strains were grown to the logarithmic growth phase, sterile water was used to resuspend the bacteria to OD₆₀₀ = 0.3, and 7‐week‐old N. benthamiana leaves were infiltrated with the bacterial suspensions. After 16 h, cell death was observed and recorded. (B) For growth curve analysis of the ∆rmlA, WT, ∆rmlA‐C and ∆rmlA‐C‐mut strains, the strains were grown to the logarithmic growth phase and then inoculated into 200 mL of Luria Bertani liquid medium at a ratio of 1:200. The culture was incubated in a shaker at 28°C or 16°C, and the culture was taken every 2 h to measure the OD₆₀₀ value.

FIGURE 7

Changes in the motility of different Pectobacterium actinidiae GX1 mutants. (A) Assessment of the mobility of the ∆rmlA, wild‐type (WT), ∆rmlA‐C and ∆rmlA‐C‐mut strains on agar plates. (B) Mobility area analysed by ImageJ in (A). (C) Bacterial mobility area corresponding to the assay data in (B). The error bars represent the standard deviation; n = 3 plates. Significant differences from ANOVA are shown: **** = p < 0.0001. The control is the motility area of WT GX1. The experiment was repeated three times, and the results all showed the same trend.

FIGURE 8

Effects of RmlA substrate‐binding capacity on the pathogenicity of Pectobacterium actinidiae GX1. (A) Assessment of the pathogenicity of the ∆rmlA‐C‐mut strain in Chinese cabbage. (B) Lesion diameters corresponding to the assay data in (A). (C) Bacterial colonisation corresponding to the assay data in (A). The error bars represent the standard deviation; n = 5 cabbage stems. Significant differences from ANOVA are shown: ** = p < 0.01; ns indicates no significant difference. The control consisted of Chinese cabbages incubated with wild‐type GX1 (WT). The experiment was repeated three times, and the results all showed the same trend. (D) Pathogenicity testing of the WT and mutant strains on kiwifruit branches.