The Methyltransferase HemK Regulates the Virulence and Nutrient Utilization of the Phytopathogenic Bacterium Xanthomonas citri Subsp. citri

Abstract

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (Xcc), seriously affects fruit quality and yield, leading to significant economic losses around the world. Understanding the mechanism of Xcc virulence is important for the effective control of Xcc infection. In this report, we investigate the role of a protein named HemK in the regulation of the virulence traits of Xcc. The hemK gene was deleted in the Xcc jx-6 background, and the ΔhemK mutant phenotypically displayed significantly decreased motility, biofilm formation, extracellular enzymes, and polysaccharides production, as well as increased sensitivity to oxidative stress and high temperatures. In accordance with the role of HemK in the regulation of a variety of virulence-associated phenotypes, the deletion of hemK resulted in reduced virulence on citrus plants as well as a compromised hypersensitive response on a non-host plant, Nicotiana benthamiana. These results indicated that HemK is required for the virulence of Xcc. To characterize the regulatory effect of hemK deletion on gene expression, RNA sequencing analysis was conducted using the wild-type Xcc jx-6 strain and its isogenic ΔhemK mutant strain, grown in XVM2 medium. Comparative transcriptome analysis of these two strains revealed that hemK deletion specifically changed the expression of several virulence-related genes associated with the bacterial secretion system, chemotaxis, and quorum sensing, and the expression of various genes related to nutrient utilization including amino acid metabolism, carbohydrate metabolism, and energy metabolism. In conclusion, our results indicate that HemK plays an essential role in virulence, the regulation of virulence factor synthesis, and the nutrient utilization of Xcc.

Keywords: HemK; RNA-seq; Xanthomonas citri subsp. citri; biofilm; exoenzyme; motility; stress tolerance; virulence.

Figure 1

HemK influences the production of cell motility, biofilm formation, extracellular polysaccharides, and enzymes in Xcc jx-6. (A) The growth rates (OD₆₀₀ values) of the wild-type (WT) strain Xcc jx-6, ΔhemK, and C-∆hemK on YEB at 28 °C were measured at 4 h intervals. (B) Swimming and swarming motility for the WT strain, ΔhemK, and C-∆hemK were detected on a 0.28% agar swimming plate and 0.6% agar swarming plate, respectively. A 2 µL aliquot of the bacterial suspension was inoculated onto the swimming plate or swarming plate and incubated for 60 h at 28 °C to observe the bacterial motility. (C) The level of motility was determined by measuring the area of the colony with ImageJ. (D) The biofilm formation of the WT strain, ∆hemK, and C-∆hemK in glass tubes was detected by crystal violet staining and quantified by measuring the optical density at 590 nm, after dissolution in 33% acetic acid. (E) The production of extracellular polysaccharides (EPS) of the WT strain, ∆hemK, and C-∆hemK was assessed on 2% glucose NYGA, and the production was measured with ethanol precipitation. (F–H) A 20 µL (for proteases) or 2 µL (for cellulases and amylases) aliquot of bacterial supernatant was added to the exoenzymes’ test plates and incubated at 28 °C for 48 h. The production of hydrolysis circles by cellulases, amylases, and proteases was measured on plates containing 1% (m/v) skimmed milk (F), 1% (m/v) sodium carboxymethyl ethyl cellulose (G), and 1% (m/v) potato starch (H), respectively. All experiments were repeated three times, with three repetitions for each strain. Only one representative result is presented. A significant difference between the WT and ∆hemK was demonstrated with the respective treatments: **** p < 0.0001, *** p < 0.001, ** p < 0.01, ns: no significance (Student’s t-test).

Figure 2

HemK contributes to stress tolerance. (A) The tolerance of wild-type Xcc jx-6 strain, ∆hemK, and C-∆hemK was performed under H₂O₂-induced oxidative stress and heat shock. (B) Bacterial cell viability was estimated by measuring the value of OD₆₀₀ on the YEB medium before (T0) and after (T1) treatment. The survival rate was calculated as the ratio of the cell count at T1 to that at T0. Each test was repeated five times, and these five times had similar results. The data shown are the means and standard errors of five replicates. The significant difference between the WT and ∆hemK with respective treatments: *** p < 0.001.

Figure 3

HemK regulates the virulence and hypersensitive response of plants. (A) Pathogenicity assay for wild-type, ∆hemK, and C-∆hemK was performed on citrus leaves. The bacterial suspensions (approximately 10⁵ CFU/mL in XVM2) were inoculated into the young leaves of sweet orange by pressure infiltration with a needleless syringe. Sterile H₂O was used as a negative control. A representative leaf from three replicates was photographed 10 days post-inoculation. (B) The area of the lesions was determined by ImageJ. The means and standard errors of three replicates from one representative result are shown. The significant difference between the WT and ∆hemK with respective treatments: *** p < 0.001 (Student’s t-test). (C) The symptoms of hypersensitive response were photographed 3 days post-inoculation on the leaf surface of N. benthamiana. (D) The area of the infection spot was measured using ImageJ. Sterile H₂O was used as a negative control. The means and standard errors of three replicates from one representative result are shown. The significant difference between the WT and ∆hemK, with respective treatments: * p < 0.05, ns: no significance (Student’s t-test).

Figure 4

HemK regulates multiple clusters of orthologous genes (COG) functional categories. The x-axis represents the functional classification of each COG category. The y-axis represents the relative abundance (%) of DEGs in each COG category. The number of genes (presented above each category) in different functional classes reflects the metabolic and physiological bias in a certain period or environment.

Figure 5

The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of pathways regulated by HemK. The number of annotated genes (x-axis) versus KEGG categories (y-axis).

Figure 6

HemK regulates the expression of T3SS- and T2SS-associated genes. (A) Heat map of gene expression of T2SS and T3SS by RNA-seq data. (B) The relative expression of genes associated with the virulence identified by RNA-seq analysis was determined by qRT-PCR analysis. The target genes included hrpB2, hpaA, hrcC and hrpE (T3SS regulators), virK, avrXacE1 and avrBs2 (virulence protein), XAC3545 (protease), XAC0612 (cellulase), XAC3490 (amylosucrase or alpha-amylase) and XAC2853 (cysteine protease).