Discovery of Ethyl 2-Nitro-3-Arylacrylates Molecules as T3SS Inhibitor Reducing the Virulence of Plant Pathogenic Bacteria Xanthomonas

Abstract

Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative pathogen which causes leaf blight disease. Known traditional bactericides are not much more effective in inhibiting this bacteria than before. Selecting the virulence factor of the bacteria as the target without affecting their growth has been considered as a novel method for developing new anti-microbial drugs. Type III secretion systems (T3SS) are one of the important and highly conserved virulence factors in most gram-negative pathogens, which has been considered as an effective target to develop new anti-microbial drugs. In order to discover potential anti-microbial drugs against Xoo pathogens, a series of ethyl 2-nitro-3-arylacrylates compounds were screened. Among them, the compounds I-9, I-12, and I-13 could highly inhibit the promoter activity of a harpin gene hpa1, which were used to further check for the influence on bacterial growth and on the hypersensitive response (HR) caused by Xoo bacteria on non-host plants. The results showed that above compounds could reduce HR without affecting bacterial growth and survival. Moreover, qRT-PCR analysis indicated that treatment with the three inhibitors (I-9, I-12, and I-13) could suppress the expression of the Xoo T3SS in different extent. The mRNA levels of representative genes in the hrp cluster, including the key regulatory genes hrpG and hrpX, were decreased. Last but not least, in vivo test ensured that the above compounds reduced the disease symptoms of Xoo on the rice and Xcc on the Chinese radish.

Keywords: Xanthomonas campestris pv. campestris (Xcc); Xanthomonas oryzae pv. oryzae (Xoo); anti-virulence compounds; ethyl 2-nitro-3-arylacrylates molecules; type III secretion system (T3SS).

FIGURE 1

The chemical structures of PCA, OCA, and SA_1–3.

FIGURE 2

The chemical structures of compounds I-9, I-12, and I-13.

FIGURE 3

Effects of three compounds (I-9, I-12, and I-13) on bacterial growth. (A) The growth rate of Xanthomonas oryzae pv. oryzae (Xoo) PXO99^A in rich medium (M210) supplemented with DMSO (dimethylsulfoxide) or respectively 100 μM of I-9, I-12, and I-13. (B) The growth rate of Xoo PXO99^A in hrp-inducing medium (XOM2 plus 0.5% sucrose) supplemented with DMSO or 100 μM of I-9, I-12, and I-13 respectively. The optical density at 600 nm (OD₆₀₀) of the culture suspensions was recorded every 2 h during the 48 h period. (C) Effects of three compounds on bacterial growth. (D) Quantify the number of colonies on bacterial growth.

FIGURE 4

Effects of three compounds (I-9, I-12, and I-13) on the HR induced by Xoo on tobacco leaves. Three compounds suppressed HR induced by Xoo. TS006 was used as the control. The similar results are representative of at least three independent experiments. HR symptoms appeared at 24 h after inoculation, then the symptoms were recorded by photographing. CK represented DMSO.

FIGURE 5

Relative mRNA levels of representative genes in the hrp cluster in Xoo PXO99^A incubated with compound I-9, I-12, and I-13 were measured by qRT-PCR. (A) Compared with the DMSO control, the mRNA levels of hap1, hrpE and hrpF was reduced significantly after treatment with these inhibitors. (B) Compared with the DMSO control, the mRNA levels of two hrc genes (hrcC and hrcU) were also reduced in different extents after treatment with these inhibitors. The DNA gyrase subunit B (gyrB) gene was used as the internal control for data analysis. Each experiment had three replicates. ^∗∗P < 0.01, ^∗∗∗P < 0.0001.

FIGURE 6

The effects of the three compounds (I-9, I-12, and I-13) on the promoter activity of hrpG (A), hrpX (B) and hrcT (C) in Xoo grown in XOM2 medium supplement with 100 μM of tested compounds. DMSO was used as a solvent control. GFP mean fluorescence intensity (MFI) was determined by flow cytometry. The calculated method was following the formula: DMSO% = 100 × MFI (XOM2 with tested compounds)/MFI (XOM2 with DMSO). Each experiment had three replicates. ^∗∗P < 0.01, ^∗∗∗P < 0.0001.

FIGURE 7

(A) Secretion of exopolysaccharide (EPS) in Xoo wild-type and that of treatment with I-9, I-12, or I-13 compounds had no significant difference. From left to right, it provided WT, WT + DMSO, WT + I-9, WT + I-12, and WT + I-13. Error bars represent standard deviation from three repeats. (B) Detection of cellulase secreted by Xoo grown in PSA containing 0.5% carboxymethyl cellulose for 24 h. Pale-yellow clear zones showed no difference between Xoo wild-type and that of treatment with I-9, I-12, or I-13 compounds. (C) Detection of xylanase secreted by Xoo grown in PSA 0.2% RBB-xylan for 48 h. Both Xoo wild-type and that of treatment with I-9, I-12, or I-13 compounds were appeared as the white clear zones among a blue background. (D) Detection of swimming motility on 0.3% soft agar plates supplemented with I-9, I-12, or I-13 compounds at 28°C after 72 h.

FIGURE 8

(A) The effect of I-9, I-12, and I-13 on the water-soaking symptoms caused by Xoo wild-type on IR24 seedling. From left to right, it provided pure DMSO, WT, WT + DMSO, WT + I-9, WT + I-12, and WT + I-13.The disease symptoms (B) and lesion lengths (C) of Xoo wild-type on adult plants of rice cultivar IR24 were reduced after supplement with 100 μM of I-9, I-12, and I-13. At least three experiment tests had similar results. Asterisks indicate statistically significant differences. ^∗∗∗P < 0.0001.

FIGURE 9

(A) Effects of three compounds (I-9, I-12, and I-13) on the HR induced by Xcc on tobacco leaves. Three compounds suppressed HR induced by Xcc. The similar results are representative of at least three independent experiments. (B) Effects of three compounds on bacterial growth. (C) Quantify the number of colonies on bacterial growth.

FIGURE 10

Relative mRNA levels of representative genes in the hrp cluster in Xcc8004 incubated with compound I-9, I-12, and I-13 were measured by qRT-PCR. (A) Compared with the DMSO control, the mRNA levels of hap1, hrpG, and hrpX were dropped remarkably after adding these inhibitors. (B) Compared with the DMSO control, the mRNA levels of hrpE and hrpF genes were dropped considerably after adding these inhibitors. (C) Compared with the DMSO control, the mRNA levels of three hrc genes (hrcC, hrcT, and hrcU) were also dropped in different degree after adding these inhibitors. The DNA gyrase subunit B (gyrB) gene was used as the internal control for data analysis. Each experiment had three replicates. ^∗∗P < 0.01, ^∗∗∗P < 0.0001.

FIGURE 11

The effect of I-9, I-12, and I-13 on the “V” shaped yellow disease symptoms caused by Xcc 8004 wild-type on Chinese radish. (A) From left to right, it provided WT, WT + DMSO, WT + I-9, WT + I-12, and WT + I-13. (B) The percentage of diseased leaf area (%DLA) was quantification of the reduction in lesion area by the selected compounds. %DLA = area covered by lesions/whole leaf area × 100.