Development Of Molecular Markers And Identification Of Vir Ulence Factors In Xanthomonas Oryzae

Rice bacterial blight and bacterial streak, caused by Xanthomonas oryzae pv. oryzae （Xoo） and X. oryzae pv. oryzicola （Xoc） respectively, are two most important bacterial diseases on rice in China and even in some areas of Southeast Asia. Xoo and Xoc belong to the same rice Xanthomonas spp, they have a very close relationship, shared the same characteriscs in the shape, morphology, and physiological and biochemical properties. Thus, they often caused some difficulties during the disease management, such as survey for the initial amount of pathogens during primarily infection and thereafter infections, and seeds daily inspection and quarantine. So it really requires more convenient, fast and accurate identification methods in the future.In this study, based on computational analysis of several Xanthomonas spp complete sequenced genomes, a series of specific e-PCR primers were designed to distinguish Xanthomonas oryzae pv. oryzae from Xanthomonas oryzae pv. oryzicola which contain917pairs of molecular markers. They were divided into three types, included Xoo specific molecular markers, Xoc specific molecular markers, and Xoo-Xoc codominant markers. The total of130pairs of primers were randomly selected for PCR testing on40strains, including25Xoo strains,10Xoc strains and5other reference strains.As a result,68molecular markers, including30pairs Xoo specific molecular markers,24pairs Xoc specific molecular markers and14pairs of Xoo-Xoc codominant molecular markers were validated to remarkably separate Xoo and Xoc by PCR.These specific molecular markers can be directly applied to the inspection and quarantine work, and help to develop the more accurate detection method with multiple markers worked in coordination with each other.Meanwhile, we also developed a Xoc specific markers, Xoo-OKF2, based on avrRxol gene which encodes a specific type III secretion effectors in Xoc. AvrRxo1belongs to the non-transcription activator-like （non-TAL） effectors without characterized pathogenic functions. By using transient expression system in nicotiana benthamiana, we found that AvrRxo1has cytotoxic function to plant cell. Surprisingly, the two AvrRxo1alleles from five different Xoc strains has no toxic functions, and they share the same amino acid substitution at residue344. We further concluded that the amino acid substitution at residue 344of two AvrRxol is abolished for toxin function. Aslo, a series of truncations from the carboxyl terminus （C-terminus） were conducted.They are suggested that the complete C-terminus of AvrRxo1plays important functional roles as a suppressor or cytotoxic protein. The amino terminus （N-terminus） was unessential for toxicity.In addition, the previously studies was indicated that Xoo may metabolize IAA and may use IAA as virulence factors. In this study, mutants were generated for each four putative genes predicted to involve in IAA synthesis with homozygous recombination method in PXO99A, named P△pxo₀4004,P△pxo₀4823,P△pxo₀0867and P△pxo₀3584respectively. After analysis of metabolic products by HPLC, we identified that PXO99A could produce the IAA at about2.6ug/ml, and the four mutants deficiently produced IAA. Contrary to the prediction, four mutants enhanced the susceptibility than wild strain PXO99a in rice. They were found to produce more EPS, a kind of important virulence factor than PXO99A. And the amounts and species of secretion proteins were identified to increase expression in four mutants. These results concluded that PXO99Acould produce IAA which negatively regulated the synthesis of other pathogenic virulence factors.Totally, we have developed a lot of PCR markers to distinguish Xoo from Xoc, and have analyzed the pathogenesis function of one marker target gene avrRxol, and have proved that Xoo has the synthesis pathway to produce the IAA to negatively regulate the pathogen virulence. These results would be beneficial on plant quarantine and disease management in the future...