A thiadiazole reduces the virulence of Xanthomonas oryzae pv. oryzae by inhibiting the histidine utilization pathway and quorum sensing

Abstract

Thiazole, isothiazole, thiadiazole and their derivatives are widely thought to induce host defences against plant pathogens. In this article, we report that bismerthiazol, a thiadiazole molecule, reduces disease by inhibiting the histidine utilization (Hut) pathway and quorum sensing (QS). Bismerthiazol provides excellent control of bacterial rice leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo), but does not greatly inhibit Xoo growth in vitro. According to RNA-sequencing analysis, the transcription of the Hut pathway genes of Xoo ZJ173 was inhibited after 4.5 and 9.0 h of bismerthiazol treatment. Functional studies of hutG and hutU indicated that the Hut pathway had little effect on the growth and bismerthiazol sensitivity of Xoo in vitro, but significantly reduced the aggregation of Xoo cells. Deletion mutants of hutG or hutU were more motile, produced less biofilm and were less virulent than the wild-type, indicating that the Hut pathway is involved in QS and contributes to virulence. The overexpression of the hutG-U operons in ZJ173 reduced Xoo control by bismerthiazol. Bismerthiazol did not inhibit the transcription of Hut pathway genes, QS or virulence of the bismerthiazol-resistant strain 2-1-1. The results indicate that bismerthiazol reduces Xoo virulence by inhibiting the Hut pathway and QS.

Keywords: Hut pathway; QS; Xanthomonas oryzae pv. oryzae; bismerthiazol; rice; thiadiazole; virulence.

Figure 1

RNA‐sequencing (RNA‐seq) profiles of bismerthiazol‐treated and untreated Xanthomonas oryzae pv. oryzae (Xoo) in nutrient broth (NB) medium. The three treatments were as follows: CK, treated with N,N‐dimethylformamide for 4.5 h; 1, treated with 30 mg/L bismerthiazol for 4.5 h; 2, treated with 30 mg/L bismerthiazol for 9 h. (A) Heat map and hierarchical cluster analysis of expression of the most differentially expressed mRNAs in the presence and absence of bismerthiazol (q value < 0.005). (B) Volcano plot of overall gene‐based differential expression results for bismerthiazol‐treated Xoo compared with the control (each dot corresponds to a gene). The y‐axis corresponds to the negative logarithm (base 10) of the q value, whereas the x‐axis corresponds to the negative logarithm (base 2) of the fold change for the difference in expression following bismerthiazol treatment. Red dots and green dots represent the differentially expressed genes (DEGs) with high and low expression, respectively.

Figure 2

Expression of hut operons in Xanthomonas oryzae pv. oryzae (Xoo) as affected by bismerthiazol. Effects of 30 mg/L bismerthiazol, 0.5 mg/L streptomycin or 1 mg/L shenqinmycin on the transcription level of the six hut genes of Xoo in nutrient broth (NB) medium (A) and XOM3 medium (B). Gene expression was determined when treated and untreated bacteria grew to population densities of 0.6 × optical density (OD). Values are the means and standard errors (SEs) of three independent experiments. In each group of five means, means with an asterisk are significantly different from the control according to Student's t‐test: *P < 0.05.

Figure 3

Expression of hut operons in Xanthomonas oryzae pv. oryzae (Xoo) affected by 2‐amino‐5‐mercapto‐1,3,4‐thiadiazole (AMT), 5‐amino‐1,3,4‐thiadiazole (ATDA) and benzothiadiazole (BTH). (A) Chemical structures of AMT, ATDA, BTH and bismerthiazol. (B) Effects of AMT, ATDA and BTH on the transcription level of the hut operons of Xoo. Xoo ZJ173 was treated with the same concentrations (30 mg/L) of AMT, ATDA or BTH for 4.5 h. Gene expression was determined when treated and untreated bacteria grew to population densities of 0.6 × optical density (OD) in nutrient broth (NB) medium. Values are means and standard errors of three independent experiments (results were similar for each experiment). In each group of four means, means with an asterisk are significantly different from the control according to Student's t‐test: *P < 0.05.

Figure 4

Genetic structure of hut operons and the Hut pathway of Xanthomonas oryzae pv. oryzae (Xoo) and of three reference bacteria. (A) hut operons. Genes with the same name and same colour are homologous (≥ 60% identity). The length of the box and the direction of the associated arrow indicate transcription units and direction. The illustration was drawn approximately to scale. (B) The Hut pathway of Xoo (http://www.genome.jp/kegg/). The gene product involved in each individual step is in bold type.

Figure 5

Effects of the Hut pathway on the growth and bismerthiazol sensitivities of Xanthomonas oryzae pv. oryzae (Xoo). (A) The growth rates of the ΔhutG and ΔhutU mutants and their complemented strains in nutrient broth (NB) and XOM3 media. (B) The colony morphologies of the ΔhutG and ΔhutU mutants and their complemented strains on nutrient agar (NA) medium. (C) The bismerthiazol sensitivities of ΔhutG and ΔhutU mutants and their complemented strains in NB medium. Values in (A) and (C) are means and standard errors of three independent experiments (results were similar for each experiment).

Figure 6

The expression of hut operons and the effect of the Hut pathway on the aggregation of Xanthomonas oryzae pv. oryzae (Xoo) cells at the late growth stage. (A) The expression of hut operons at different growth stages [0.5, 1 and 2 × optical density (OD)] of Xoo. Values are the means and standard errors of three independent experiments. (B) Cells of the hutG and hutU deletion mutants of Xoo, but not those of the wild‐type or complemented strains, tended to aggregate in nutrient broth (NB) medium. The aggregation phenotype was assessed after saturated cultures of the Xoo strains had been grown in NB medium at room temperature for 4 h.

Figure 7

The effects of the Hut pathway on the quorum sensing (QS) and pathogenicity of Xanthomonas oryzae pv. oryzae (Xoo). (A) Cellulase activity, protease activity, motility and biofilm production of the ΔhutG and ΔhutU mutants, complemented strains and their overexpression strains. (B) The pathogenicity of the hut mutants. The lengths of lesions on clip‐inoculated leaves were measured on day 5 and day 10. Representative leaves were photographed on day 10. Values are means and standard errors of three independent experiments. Means with asterisks are significantly different from the control according to Student's t‐test: *P < 0.05.

Figure 8

The effects of bismerthiazol on the quorum sensing (QS) of the bismerthiazol‐resistant mutant 2‐1‐1 of Xanthomonas oryzae pv. oryzae (Xoo). (A) Effects of bismerthiazol on the transcription level of the hut genes in 2‐1‐1. 2‐1‐1 was treated with 30 mg/L bismerthiazol in XOM3 medium. Gene expression was determined when treated and untreated bacteria grew to population densities of 0.6 × optical density (OD). The motility (B) and biofilm production (C) of the wild‐type ZJ173 and 2‐1‐1 were measured in the presence of 2 mg/L bismerthiazol, 0.02 mg/L streptomycin or 0.04 mg/L shenqinmycin. For CK, Both ZJ173 and 2‐1‐1 were not treated with any of these three bactericides. In a pathogenicity assay (D), rice plants were treated with 100 mL of 100 mg/L bismerthiazol and inoculated with ZJ173, ZJ173(hutG‐U) or 2‐1‐1, 1 day later. The lengths of the lesions on the clipped leaves were measured on day 10, and the inhibition rate was calculated. Values are means and standard errors of three independent experiments (results were similar for each experiment). Means with different letters are significantly different according to Fisher's least‐significant difference test: *P < 0.05. Means with asterisks are significantly different from the control according to Student's t‐test: *P < 0.05.