Pathological Roles Of The Sucrose Transporters And Three Xop Proteins Of Xanthomonas Oryzae Pv.Oryzicola

Xanthomonas oryzae pv.oryzae and Xanthomonas oryzae pv.oryzicola cause bacterial leaf blight and bacterial leaf streak in rice,which are important bacterial diseases of rice.The type III secretion system?T3SS?effectors are considered as disease determinants of Xanthomonas.and invoved in bacterial nutrient acquisition and host immune inducing.T3SS effectors are commonly divided into two groups:TAL?transcription activator-like?effectors and non-TAL?non transcription activator-like?effectors.The TALs regulate host susceptibility gene or resistance gene expression to promote disease or resistance by binding host gene promoter in a sequence-specific manner.Susceptibility gene induced by native Xoo strains are identified as three of 21 SWEET genes,which encoding the sucrose transporter.At least one of the three SWEET genes will be transcriptionally induced to mediate sucrose efflux from putative phloem parenchy main to the phloem apoplasm.Where the Xoo strain can uptake the sucrose as nutrition to enlarge the bacterial population,and then produce the disease.So acquisition and anabolism of sucrose is key to the pathogenisis of Xoo.A locus named Sux?sucrose utilization in Xanthomonas?conserved in Xanthomonas has been identified for sucrose uptake and metabolism.However,the role of Sux in bacterial virulence of Xoo and Xoc strain is remain to know.The non-TAL includes genes encoding the Xanthomonas outer protein?Xop?and other proteins,such as AvrRxol of Xoc secreted through T3SS.The result from research and bioinformation analysis indicats that some Xops have the activity of enzyme or regulation of host defence response.And that some Xop effectors show roles in host race specifity.So Xops are very important for bactria-host interaction.This thesis conducts researches on the virulence function of Sux gene cluster of Xoc in comparison with Xoo strain and the pathogenisis roles of 3 Xops special to Xoc strain.The Sux cluster resembles a non-PTS?non-phosphotransferase?sucrose pathway involved in pathogenic virulence and sucrose uptaking and metabolism.This cluster consists of two MFS transporter SuxA and SuxC,one sucrose hydrolase gene SuxB and one regulatory gene SuxR.These genes serve as autoregulatory device to keep sugar utilization at an appropriate level which make bacteria the ability of adaptation to their nutrient condition at any particular time.Gene PXO02417 adjoining neighbor of Sux gene cluster encodes protein homology to Thi/PfpI family,which is deduced to involve in thiamine biosynthesis and bacterial growth.If this gene correlated with bacterial virulenc is not know.Based on homology recombination the Sux gene mutants are constructed for RS105 and PXO99A.Strain of RS105_SuxR?RSI05_SuxC?RS105_SuxB?RS105_Sux cluster and PXO₀2417?PXO_SuxC?PXO_Sux cluster and C-RS105_SuxR are obtained.The result of growth curve for wild type RS105 and PX099A in sucrose,glucose indicates that sucrose and glucose are favorite carbonhytrate for these two bacteria.Colony phenotype on glucose and sucrose solid mediums show that sucrose is the best for EPS synthesis.The Sux gene expression of wild type is explored under different sugar medium.Observation suggests that sucrose enhance the expression of Sux gene and other sugar depress Sux gene transcription.The roles of virulence development and hypersensitive response?HR?of Sux are measured for Sux deletion mutants on rice cv nipponbare and on non-host tobacco,respectivly.Results show that SuxC and the Sux cluster deletion of PXO99A cause virulence decline.But those gene impairments in RS105 somehow enhance disease.We suppose that Xoc strain do not like Xoo uptaking sucrose as mainly carbon nutrient from host which may result from the differment disease tissue specifity of BB and BLS.The deletion of PXO02417 gives no difference in bacterial virulence and colony phenotype comparing to wild type PXO99A.These observations indicate that thiamine is not a factor significantly corelated to bacterial EPS produce and virulence.And the activity to induce hypersensitive response on tobacoo show that Sux gene defection do not impair the effectors secretion from T3SS for that muants and wild type strain produce HR with similar strength of cell death at the inoculation site on tobacco leaf.XopAF,XopAK and XopO present in the Xoc genome but vanish from Xoo genome.Two frames have been predicted automaticly for XopAF and XopAK in the published data.And no reports and protein structure clues can be used to infer the possible fuction for thses 3 genes.Firstly,the coding frames for the XopAF and XopAK are investigated by RT-PCR method,data indicate that different frame may be transcripted when bacteria growing in medium and interacting with host rice.Based on this observation,then,vectors of protein expression are constructed fusion with flag or his tag to detect the native translation products by western blot in the future.These 3 genes are knock-out respectively and the XopAF/XopAK double mutant is produced.The virulence on rice and HR on tobacco are measured for these mutants comparing with wild type strain RS105.Results indicate that the individual gene defections show no significant difference with RS105 in the virulence and HR,but the XopAF/XopAK double mutant show decreased virulence and stronger and faster HR on tobacco.This information suggests that XopAF and XopAK are redudancy and participation in supression the host immune response.The analysis of gene expression when bacteria in rich nutrient medium NB and inducible medium XOM3 and interaction with rice show that 3 gene expression are increased only in rice.Which implys that the transcription of XopAF and XopAK and XopO is induced by the rice.But no typical plant induced promoter sequence?PIP-box?presents in the 5'UTR region for these 3 genes.In order to confirm the possible promoter function driven by host,vectors for 5'UTR regions of these genes fused with the enhanced green fluorescence gene?eGFP?are prepared respectively to check if green fluorescence can be activated specially in rice.