Resistance of Xanthomonas oryzae pv. oryzae to Lytic Phage X2 by Spontaneous Mutation of Lipopolysaccharide Synthesis-Related Glycosyltransferase

Abstract

Phage therapy is a promising biocontrol management on plant diseases caused by bacterial pathogens due to its specificity, efficiency and environmental friendliness. The emergence of natural phage-resistant bacteria hinders the application of phage therapy. Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of the devastating bacterial leaf blight disease of rice. Here, we obtained a spontaneous mutant C2R of an Xoo strain C2 showing strong resistance to the lytic phage X2. Analysis of the C2R genome found that the CDS2289 gene encoding glycosyltransferase acquired a frameshift mutation at the 180th nucleotide site, which also leads to a premature stop mutation at the 142nd amino acid. This mutation confers the inhibition of phage adsorption through the changes in lipopolysaccharide production and structure and bacterial surface morphology. Interestingly, glycosyltransferase-deficient C2R and an insertional mutant k2289 also showed reduced virulence, suggesting the trade-off costs of phage resistance. In summary, this study highlights the role of glycosyltransferase in interactions among pathogenic bacteria, phages and plant hosts, which provide insights into balanced coevolution from environmental perspectives.

Keywords: Xanthomonas oryzae pv. oryzae; glycosyltransferase; lipopolysaccharide; phage resistance; virulence.

Figure 1

Resistance of Xoo spontaneous mutant C2R to phage infection. (a) Spot assays on Xoo wild-type strain C2 and mutant C2R by spotting 2 μL of phage solutions onto freshly bacterial lawns and then incubating overnight at 30 °C. (b) Bacterial growth curve of Xoo wild-type strain C2 and mutant C2R in presence or absence of phage X2. (c) Live/dead cell staining of Xoo wild-type strain C2 and mutant C2R after 0, 1, 2, 4 and 6 h incubation with phage X2. Red and green fluorescent represents dead and live bacteria, respectively. (d) TEM images of Xoo wild-type strain C2 and mutant C2R after 0 and 6 h incubation with phage X2.

Figure 2

Information about the mutation site CDS2289 of phage-resistant mutant C2R of Xoo. (a) Frameshift mutation site and premature stop mutation in mutant C2R. (b) Annotation of CDS2289 protein.

Figure 3

CDS2289 is widely distributed in bacteria. Phylogenetic analysis of CDS2289 glycosyltransferase proteins from different bacteria. The phylogenetic tree was constructed by MEGA 6.0 using the neighbor-joining method. The other 21 glycosyltransferase sequences were collected from UniProtKB. CDS2289 in Xoo wild type C2 is shown in red.

Figure 4

Verification of glycosyltransferase gene in phage resistance by constructing insertional mutant k2289 and complement c2289. (a) PCR verification of k2289 and c2289. (b) Spot assays of phage X2 on k2289 and c2289. (c) Expression of glycosyltransferase gene by qRT-PCR. Data were presented as means ± standard errors (n = 3). Columns with different letters are significantly different according to LSD test (p < 0.05).

Figure 5

Phage adsorption to wild-type Xoo strain C2, spontaneous phage-resistant mutant C2R, insertional mutant k2289 and complement c2289 after 30 min of incubation. (a) Infection of tenfold dilution unabsorbed phage X2 to bacteria. (b) Adsorption efficiency of X2 binding to bacteria. Data were presented as means ± standard errors (n = 3). Columns with different letters are significantly different according to LSD test (p < 0.05). (c) Visualization of SYTO-labeled X2 phage adsorption to bacterial surfaces under a laser scanning confocal microscope.

Figure 6

Mutation of glycosyltransferase gene alters the bacterial morphology and motility. (a) SEM images about cell surface morphology of the C2, C2R, k2289 and c2289. (b) Motility of C2, C2R, k2289 and c2289. Colony diameter (cm) was presented as means ± standard errors (n = 3). Columns with different letters are significantly different according to LSD test (p < 0.05).

Figure 7

Mutation of glycosyltransferase gene inhibits phage infection by changing LPS structure of Xoo. (a) LPS concentration extracting from the bacterial culture at OD₆₀₀ = 0.8 was determined based on the standard curve of saccharide established using glucose and the anthrone-sulfuric acid method. (b) LPS profiles of C2, C2R, k2289 and c2289 displayed by SDS-PAGE and silver staining.

Figure 8

Role of glycosyltransferase gene in Xoo virulence. (a) HR lesions in tobacco leaves determined at 24 h after infiltration of Xoo strains. (b) Lesions in rice leaves at 14 d after inoculation of Xoo strains. Lesion lengths were presented as means ± standard errors (n = 3). Columns with different letters are significantly different according to LSD test (p < 0.05).

Figure 9

Mechanistic model for glycosyltransferase-mediated initial interaction between bacteria and phages. (a) After the phage binds to the host cell surface receptors including LPS, phage contracts and injects phage DNA into bacterial cytoplasm, leading to bacterial cell lysis. (b) The mutation of the glycosyltransferase gene reduces LPS receptors and alters bacterial surface structures, leading to the reduced adsorption of phages and the resistance to phages. Red T sign means the Barrier of phage adsorption to bacterial surface.