| Plant diseases could cause significant agricultural economic losses.The prevention and control of crop diseases at this stage mainly relies on selecting and cultivating resistant varieties or the use of chemical pesticides to kill bacteria.However,the growth cycle of pathogenic bacteria is short,the mutation speed is fast,and resistant varieties or chemical pesticides are easy to lose their effect.Therefore,a new strategy for plant disease control is urgently needed.It is a good new strategy to target a certain virulence factor of bacteria,by screening or designing and synthesizing the related toxicity inhibitors.This strategy does not aim to kill bacteria,but only targets specific virulence mechanisms,resulting in low survival selection pressure on pathogenic bacteria and no harm to beneficial microbial populations in the environment.The type III secretion system(T3SS)of pathogenic plant bacteria can transport type III secretion effectors(T3SEs)to the host plant of pathogenic bacteria,thereby causing the host plant to produce disease.T3 SS is a significantly important virulence factor for pathogenic bacteria.Therefore,T3 SS can be used as a novel disease resistance target of the strategy.A series of small molecule inhibitors of T3 SS of Xanthomonas campestris pv.campestris(Xcc)have been identified in our laboratory before.T3 SS is also the virulence determinant of Xanthomonas oryzae pv.oryzicola(Xoc).Whether these inhibitors have the same effect on T3 SS of Xoc is worth investigating.In this study,the promoter regions of xop N,hrp B1,hrp X,and hrp G genes in Xoc were fused with luciferase(Lux)or β-Glucuronidase(GUS)reporter gene respectively to construct the fusion reporter systems.A total of 8 reporter strains were obtained.The Lux reporter strain with the promoter region of the xop N gene was used to screen the laboratory existing T3 SS inhibitors of Xcc.As a result,Carmofur,5-Fluorocytosine,A-3,and Dealkaline lignin were obtained that inhibit the promoter activity of xop N significantly.The effect of four compounds on Xoc T3 SS was further confirmed by other reporter strains and real-time quantitative PCR(q PCR).The results showed that four compounds could significantly inhibit Xoc T3 SS,among which Dealkaline lignin may inhibit Xoc T3 SS specifically.However,none of the four compounds affect the virulence of Xoc on the host plant rice Oryza sativa L.cv.Nipponbare.In order to accurately detect the hypersensitive response(HR)of Xoc and the biocontrol effect of small molecule inhibitors,a reporter strain GX01/avr Bs1 was constructed in this study through fusion PCR and homologous double-crossover principle.The results showed that GX01/avr Bs1 can rapidly stimulate HR on non-host plant pepper(Capsicum annuum cv.ECW-10R)which has the cognate resistant gene Bs1.Dealkaline lignin can delay the HR of Xoc on pepper ECW-10 R.To further identify the genes of Xoc that sense these compounds,six deletion mutants of Xoc T3 SS regulatory genes(hpa S(XOC＿3909),trh(XOC＿3790),hrp G(XOC＿3441),hrp X(XOC＿3439),xrv A(XOC＿2077)and zur(XOC＿1607))wre constructed in this study.The effects of four compounds on T3 SS gene expression in each mutant was then detected.The results showed that the effects of the four compounds were not significantly different between the mutants and wild type.In conclusion,in this study,a system for screening T3 SS inhibitors was established in Xoc,and four compounds include Carmofur,5-Fluorocytosine,A-3,and Dealkaline lignin were obtained that inhibit T3 SS of Xoc screened from T3 SS inhibitors of Xcc,and the mechanism of action of the inhibitors was preliminarily explored.In addition,a reporter strain GX01/avr Bs1 was also constructed,which could detect HR mediated by type III secretion system in pepper ECW-10 R.These works provide a certain theoretical basis and technical support for the subsequent identification and study of Xoc T3 SS inhibitors,as well as the further development of new plant-derived biosafety pesticides that resist bacterial leaf streak of rice. |
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