Identification Of The Micromolecule Compounds Controlling The Hrp Type Ⅲ Secretion System In Xanthomonas Campestris Pv. Campestris And Their Interaction Mechanism

In agricultural production,the annual crop diseases cause huge economic losses.The main method of prevention and cure of crop diseases were breeding and cultivating resistant varieties or used of chemical pesticide at present,but the resistance varieties always losed of resistance to crop diseases mainly due to the insufficient source,geographical restriction and the rapid evolutionary variation of pathogens.The widespread use of chemicals caused serious environmental pollution and ecological destruction,even increased the resistance of pathogenic bacteria.Therefore,it was necessary to research and developed a new method for controlling diseases.Xcc known as wild rape xanthomonas wild rape pathogenic variety,was one of the top ten plant pathogens.As a typical model species of xanthomonas genus,Xcc can infect almost all cruciferous plants and cause black rot that caused huge agricultural economic losses every year.According to the new disease resistance strategy,it was possible to resist the invasion of host plant by restricting the pathogenic factors.Type III secretion system（T3SS）was a crucial pathogenic factor for many plant and animal pathogens which could transports effector proteins to host plants for causing diseases.Therefore,many new antibacterial drugs took T3SS as an important target of disease resistance to achieve the purpose of inhibiting T3SS to prevent diseases without killing pathogens.At present,lots of some type molecules that inhibition of typeⅢsecretion system in animal and plant pathogens had been identified,but there was still no reports about the compound inhibition of Xcc T3SS.The discovery and identification of compounds inducing T3SS expression were full of theoretical and practical value in revealing the regulatory mechanism of T3SS,and provided a new seed clues for us to discover new lead compounds and establish new disease-resistant strategies.At present,a small amount of compounds inducing T3SS of animal and plant pathogens had been reported.In this study,an induction medium named XZM and an efficient and rapid luciferase screening system were established.XZM medium has the advantages of the previously medium XVM₂and XCM₁that had been reported.First,two compounds Xylose and Imidocca dipropionate that inhibiting Xcc T3SS were obtained by screening the common compounds in the basic medium and type III inhibitors reported in other pathogens.These provided that this high-throughput method is feasible.In this study,a total of 17045 small-molecule compounds were screened from the national small molecule compound resource center.There were 64 compounds that inducing T3SS,118 compounds that inhibiting T3SS and 630 compounds that showing sterilization activity in the preliminary screening.We got rid of the 630compounds that had significant influence on Xcc 8004 growth of and bought the 64inducers and the 118 inhibition compounds for second screening.After further screening we got 11 inducers and 24 inhibitors compounds,and then we screened the35 compounds with different medium,finally 6 inducing compounds and 12inhibiting compounds that influencing Xcc T3SS were identified,plus the two inhibitors had been identified,there were a total of 20 small molecule compounds.Luciferase assays were performed on the 20 compounds at various concentrations to determine their optimum concentration for use.We found that Pentetic acid and Splitomycin have the best inducing effect in the inducers and Xerophilusin G and Excisanin B have the best inhibiting effect in the inhibitors.We also found that these 20 compounds could significantly affect the expression of Xcc type III effector gene and the hrp gene by q RT-PCR,as well as the expression of other genes such as hrp X,hrp G,col R,hpa R.But they had no significant effect on the expression of other regulatory genes and other secretory systems such as type I,II,and IV.It revealed that these compounds were specific to the type III secretory system.By the detection of virulence,we found that a number of inhibitors could reduce virulence.These inhibitory compounds reduced about 20%to 40%virulence of Xcc8004 on host plants.In this study,the leaf growth curve of Xcc 8004 was also detected,and it was found that the compound did not affect the growth of Xcc 8004 in the leaves of plants.By the quantitative detection of HR,we found that some compounds could affect the hypersensitive response of Xcc 8004 in non-host plants ECW-10R pepper.In order to make these inhibitors have better inhibition effect,we jointed the different inhibitors together to screen some combination that had more prominent inhibitory on T3SS,finally we got the combination of Xerophilusin G and Excisanin B and Xylose,which can achieve the result that make T3SS almost closed.To further study the genes of Xcc 8004 that are sensitive to these compounds,we screened the Tn5 mutant library of 8004/p Lgus3011 on a plate with the inhibitor WB64,and finally found a candidate gene XC1690.This gene is annotated as a quaternary ammonium compound-resistance protein also as small multidrug resistance pump.The deletion of XC1690 and XC1691 could weaken or even lose the original inhibitory effect of the inhibitor.We found that both XC1690 and XC1691 can positively regulate T3SS genes and hrp X,hrp G,col R by q RT-PCR.And only XC1691 had effect on pathogenicity and HR by detection of pathogenicity and HR.We searched for the signal compound which inducing T3SS in host plants used the method of metabonomics.By screened the difference compounds,we found5-O-Methylembelin had significant induction of Xcc T3SS,its inducing effect even was better than Pententic acid.In conclusion,this study identified some small molecular compounds that inducing or inhibiting Xcc T3SS by high-throughput screening.These compounds have specific inhibitory effects on hrp III secretion system of Xcc,and we also conducted a series of studies on their mechanism of interaction.The results provided a theoretical basis for further research on the interaction mechanism between small molecules compounds and the type III secretion system and the development of T3SS inhibitors.