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.

A cyclic di‐GMP‐binding adaptor protein interacts with a N5‐glutamine methyltransferase to regulate the pathogenesis in Xanthomonas citri subsp. citri

“…Taken together, the observed differences in motility, virulence factor production and gene expression support that N5MapZ interacts directly HemK to mediate gene expression and virulence factor production, and that c‐di‐GMP is likely to exert its regulatory effect on gene expression and virulence factor production via the N5MapZ–HemK interaction. Our data further suggest that N5MapZ interacts with HemK to suppress the methylation of its substrate PrfA, which corroborates our previous finding about the role of HemK in virulence factor production and gene expression (Shi et al., 2022 ).…”

“…Extracellular enzyme activity assay on NYGA was examined as described by Shi et al. ( 2022 ) with modification. Briefly, Xcc strains were grown in XVM2 medium at 28°C with shaking for about 18 h, adjusted to an OD 600 of 0.8 and an aliquot of 1.5‐μL bacterial cultures were spotted onto the surface in the centre of NYG plates supplemented with 1% (wt/vol) sodium carboxymethyl ethyl cellulose (for cellulase detection) or 1% (wt/vol) potato starch (for amylase detection).…”

“…Extracellular polysaccharides (EPS) in bacterial culture supernatants were determined, as described previously (Shi et al., 2022 ). Bacterial strain was grown in XVM2 medium until the OD 600 reached 0.8.…”

“…The stress tolerance assay was performed as described previously (Shi et al., 2022 ) with modification. Cells were cultured in the LB medium to the early exponential stage (10 8 cfu/mL) and collected for stress treatment.…”

Chemotactic Responses of Xanthomonas with Different Host Ranges

“…Chemotaxis plays a key and early role in bacterial attachment, biofilm development, and bacterial regulation in response to the environment [ 21 , 41 , 42 , 43 , 44 ]. Moreover, in previous studies, chemotaxis in Xcc has been described as a central plant colonization factor at the early stages of the microbe–plant interaction [ 44 , 45 ]. In addition, biofilm formation has been reported as an important step for citrus canker establishment and for Xcc to survive on the plant surface [ 25 , 46 ].…”