| Rice bacterial blight is a bacterial disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which is one of the most serious diseases of rice. Shenqinmycin is a microbial antibiotic secreted by Pseudomonas aeruginosa, and the main effective component is phenazine-1-carboxylic acid (PCA). It has high efficiency against various plant pathogens, low toxicity to humans, and environmental compatibility. Shenqinmycin is highly effective against Xoo both in vivo and in vitro, which makes it a potential bactericide against rice bacterial blight. We used proteomic method, biochemistry and molecular techniques to study the effects of shenqinmycin on the biology of Xoo in this study, which provided evidence for the field application on rice.1. Establishment of proteome reference database of XooWe used two-dimensional electrophoresis (2DE) to establish the proteome reference maps of Xoo, and identified 628 protein spots on 2DE gels with MALDI-TOF/TOF MS. These protein spots belong to 469 different protein species. The dataset has been submitted to World-2DPAGE website, and a database was constructed with the accession number of 0044. Bioinformatics analysis was carried out by searching KEGG and NCBI database, and the proteins were divided into 15 groups according to the function. Among them, carbohydrate metabolism, amino acid metabolism, transportation, and protein folding and degradation accounted for more than 10% of the protein spots. Carbohydrate metabolism, protein folding and degradation, transportation, translation, and energy metabolism related proteins are the most abundant proteins on the reference gels. In conclusion, we established a proteome reference database of Xoo, which laid the foundation for further comparative proteomic study.2. Biological effects and proteomic analysis of Xoo sensitivity to shenqinmycinWe tested the total carbohydrate, total protein, and extracellular polysaccharides content, and found that total carbohydrate and total protein were higher than control after treated with shenqinmycin for 2 d; extracellular polysaccharides content was higher after treated with less than 0.5μg/mL shenqinmycin for 5 d, but decreased significantly when treated with 1μg/mL shenqinmycin. ROS accumulation in Xoo was assayed using fluorescent probe method, and found that ROS accumulation in Xoo treated with 2μg/mL shenqinmycin was as high as positive control. CAT and SOD activities were tested when treated with shenqinmycin at different time points, the results showed that shenqinmycin can inhibit the activities of these two enzymes at multiple time points. Morphology of Xoo was also observed with transmission electron microscope, and particles and vacuoles were detected when treated with shenqinmycin.We compared H2O2 sensitivity of Xoo and Xanthomonas oryzae pv. oryzicola (Xoc), which indicated that Xoo was extremely sensitive to H2O2. In contrast, Xoc was much more tolerant to H2O2. qRT-PCR was used to compare the transcriptional level of three CAT encoding genes in Xoo and Xoc, the results showed that all three genes were up-regulated in Xoc. Two of them were 11 fold and 3 fold up-regulated, respectively.We used 2DE to analyze the proteome expression level, the results showed that carbohydrate metabolism, energy metabolism, and nutrient uptake of Xoo were inhibited by shenqinmycin. Gen III Microstation was used to analyze the carbon sources utilization ability of Xoo, which confirmed that shenqinmycin inhibited carbohydrate metabolism of Xoo. Spore germination experiment of Fusarium graminearum indicated that shenqinmycin inhibited energy metabolism. We knocked out the differentially expressed protein phosphoglucomutase, and tested phenotypes of the mutant. However, we didn’t find surprising differences in pathogenicity, sensitivity to shenqinmycin, or ROS accumulation, we also assayed the effect of shenqinmycin on the important differentially expressed protein pyruvate dehydrogenase, the results showed that shenqinmycin inhibited Xoo pyruvate dehydrogenase activity a little bit, but barely had any effect on pyruvate dehydrogenase of mice liver and rice leaves.In addition, we tested the reaction of shenqinmycin with NADH, which has a lower redox potential. We found that shenqinmycin could quickly react with NADH in vitro when provided with proton.In conclusion, shenqinmycin is a redox-cycling agent that can be reduced by some low redox potential electron donor in Xoo. Reduced form of shenqinmycin would constantly react with O2, resulting in ROS generation which would finally cause cell death. Shenqinmycin upsets the redox balance of Xoo, which in turn disturbs carbohydrate metabolism and energy production. Lower energy production caused by reduced carbohydrate metabolism also reduces nutrient uptake. Moreover, CAT and SOD activities were inhibited by shenqinmycin, which increased ROS accumulation. Xoo has a deficient H2O2 scavenging system, which made it impossible to clean out the accumulated ROS, thus made it extremely sensitive to shenqinmycin.3. Biological effects and proteomic analysis of Xoo resistance against shenqinmycinWe got four resistant Xoo strains by screening on plates containing shenqinmycin, which were later identified with Xoo specific primers. We assayed the pathogenicity of these strains and their sensitivity to shenqinmycin, and found that S-1 was the most resistant and pathogenic one; extracellular polysaccharides were also analyzed, but the results showed no relevance with the sensitivity to shenqinmycin; ROS accumulation in four resistant strains were determined using fluorescent probe method, and no ROS accumulation was found in S-1. We assayed CAT and SOD activities in both ZJ173 and S-1, and found that CAT activities in S-1 was significantly higher than in ZJ173; proteome expression level was analyzed using 2DE, and the results indicated that the enhancement of transportation system and energy metabolism was related with the resistance in S-1.In conclusion, the resistance against shenqinmycin in S-1 was caused by two aspects: one is the enhancement of multi-drug efflux system caused by enhancement of transportation system and energy metabolism; the other is the increase of CAT activities. |
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