Reaearch On Promoters Of HrpX Regulation Genes In Xanthomonas Campestris Pv.campestris

Xanthomopv.campestrisampests(Xcc)is gram-negative bacteria that cause disease in a lot of plant hosts,including many economically important crops.Xcc is a model strain for research on interaction between pathogen and plant.Type III secretion system and type III effectors are closely related to pathogen virulence.Type III secretion systems are encoded by hrp cluster in phytopathogenic bacteria.hrpG and hrpX are the most key regulatory genes,and are located outside of the hrp gene cluster in Xanthomonas.The HrpG protein controls the expression of hrpX.The AraC-type transcriptional activator HrpX regulates the expression of the operons hrpB to hrpF and most members of the hrpX regulon.There are two cis-elements in promoter region of many HrpX-regulated genes:one is Plant-inducible promoter box(PIP-box),another is-10 box located 30-32 bp downstream of PIP-box.HrpX regulates gene expression via binding to the PIP-box,indicting this cis-element plays important role in genes transcription.The aims of this research is dedicated to investigate the different mechanism of PIP-box and no PIP-box in the promoter region in hrpX regulation.According to the perfect PIP-box,inperfect PIP-box and non PIP-box,9 genes were selected to identify their transcriptional start sites through 5' RACE.The site-directed mutations of-10 box were generated using fusion PCR to construct the GUS fusion report strains.The results demonstrated that the transcription initiation sites were always A56 or T33,-10 box was-10 region,and the countdown second base of-10 box was C in 6 of 9 genes.Deviate from the-10 box of YANNDT of Ralstonia solanacearum and X.oryzae pv.oryzae,Xcc harbors the-10 region of YANNNT,Y(C/T),D(A/G/T),N(A/G/T/C).We selected XC0268,XC0052,XC1553,and XC3160,in which the countdown second base of-10 box YANNNT is C,T A,S respectively.We introduced base substitutions at the countdown second base of-10 box YANNNT,constructing 48 GUS rerorter strains with fusion PCR.The GUS activities were mensurated in the plant-induced medium XCM2.As a result,the GUS activities of XC0052(except for C-G),XC1553 and XC3160 were generally reduced by base substitutions.On the whole,XC1553 and XC3160 were all regulated by hrpX and hrpG in medium XCM2.The trend of XC0052 C-G GUS enzyme activity in 8004AhrpX and 8004?AhrpG is not consistent.In XCM2 medium,the XC0268 wild-type reporter strain turned blue,but three different bases substitutions in Xcc 8004,8004AhrpX and 8004AhrpG did not turned blue.The bacterial one-hybrid results failed to determine whether HrpX was combined with promoter region.This result indicated that-10 box plays an important role in the promoter transcriptional activity,but the specific mechanisms need to study further.