Roles Of Three Type-Ⅲ Proteins In Xanthomonas Oryzae-rice Interactions And Induced Plant Defense

Xanthomonas oryzae pv.oryzae(Xoo) and pv.oryzicola(Xooc) cause leaf blight and leaf stripe,respectively,in rice.To provide a basis for studying mechanisms that underlie interactions between rice and the bacteria,virulence and race classification of Xoo isolates in China were determined.From more than 22 rice-growing provinces and districts in China,285 isolates of the pathogen were collected;these included 108 isolates collected during 1970-1992 and additional 177 in 2003-2004.To identify proper rice lines that can distinguish variations in virulence,24 near-isogenic rice lines,which contain 1-4 resistance genes,were determined for responses to inoculation with 91 of the 285 isolates.Most isolates were avirulent on the pyramided lines,except IRBB51,and hence,the pyramided lines can not be used as differentials for identifying Xoo races.The 13 rice lines with single gene were used further to test virulence of Xoo isolates.IR24 and IRBB 10 were susceptible to the isolates with several exceptions,whereas IRBB5,IRBB7,and IRBB21 were resistant. Based on these results,six single-gene rice cultivars(IRBB5,IRBB13,IRBB3,IRBB14, IRBB2,and IR24) were chosen as differentials,and the 285 tested isolates were classified into 9 races.The reaction patterns concerning responses of the rice lines to the 9 races sequentially were as follows:RRRRRR,RRRRRS,RRRRSS,RRRSSS,RRSSSS, RSRRRS,RSSRRS,RSSSSS,and SSSSSS,while "R" and "S" refer to resistant and susceptible,respectively.The race frequencies of the patterns were 10.18%,10.53%,4.91%, 10.18%,24.21%,5.96%,11.23%,22.46%,and 0.35%accordingly.So a system for identifying races of Xoo in China was established based on the results.The virulence of representative strains of 8 Philippine races on 6 differentials was determined and compared with the virulence of the Chinese races.The frequency distributions of Xoo races were described for different regions and different periods in China.HrpA_Xoo and HrpF_Xoo in Xoo,and HrpA_Xooc HrpF_Xooc in Xooc as well,are important type-Ⅲproteins.Mutagenesis in the hrpA and hrpF genes of the wild-type(WT) Xoo stain PXO99 was done by single recombinant,generating mutants PXO99/PMD-A(AOS) and PXO99/PMD-F(FOS),which both diminished pathogenicity on rice.Complementing AOS and FOS with the pUFR034 vector containing WT hrpA and hrpF,respectively,produced the conjugates AOS/pUFRO34::hrpA(cAOS) and FOS/pUFRO34::hrpF(cFOS),which partially restored pathogenicity of AOS and FOS on rice.The PXO99,AOS,FOS,cAOS, and cFOS were labeled by GFP,producing PXO99/pHM1::gfp(PXO99-GFP), AOS/pHMI::gfp(AOS-GFP),FOS/pHMI::gfp(FOS-GFP),AOS/pHM1::hrpA::gfp (cAOS-GFP),and FOS/pHM1::hrpF::gfp(cFOS-GFP).Inoculation tests showed that the virulence of strains with GFP and without GFP were similar in pathogenicity on rice.AOS was avirulent to IR24 and did not induce HR in tomato.Virulence of FOS to IR24 markedly decreased compared to WT,but the induction of HR was not affected evidently.The virulence and inducing HR of mutants AOS and FOS can not been restored entirely by pUFR034 with hrpA or hrpF gene.Fluorescence microscopy(FM) and Electron microscopy(EM) revealed that PXO99-GFP,FOS-GFP,cAOS-GFP,and cFOS-GFP multiplied and congregated on aquaporin and adjacent areas of IR24 but AOS-GFP cells dispersed over epidermal surfaces.This result indicates that hrpA plays an important role in localization of the bacterial colonies on flee.The hrpA and hrpF genes from Xoo and Xooc,which encode HrpA and HrpF proteins in both pathovars,were cloned in pET30a(+) containing an IPTG-inducible promoter and fused with gfp in the vector,generating recombinant plasmids pET30::hrpA_Xoo::gfp(pOAG), pET30::hrpA_Xooc::gfp(pRAG),pEY30::hrpF_Xoo::gfp(pOFG),and pET30::hrpF_Xooc::gfp (pRFG).They subsequently were transferred into Escherichia coli cells.After the bacterial cells were cultured under inducting by IPTG,suspected proteins were produced based on SDS-PAGE analysis.The molecular masses of the 4 proteins are 94.6 kD,94.8 kD,116.3 kD and 116.3 kD,in consistence with the prediction by a pertinent software program.This result is an essential basis for further studies on functions of the gene and proteins in plants.HpaG_Xooc,produced by Xooc,is a member of harpin group of proteins that stimulate plant growth,hypersensitive cell death(HCD),and pathogen defense.The protein contains two copies of the glyeine-rieh motif(GRM),a characteristic of harpins,and a eysteine, which is absent in other harpins.Genetic modification generated the protein mutants HpaG_XoocMG(MG) by deleting GRMs and HpaG_XoocC47T(C47T) by replacing cysteine with threonine.When applied to tobacco plants,C47T and MG were 1.2-fold and 1.7-fold stronger,respectively,than HpaG_Xooc in inducing HCD,which occurred consistently with expression of the marker genes hinl and hsr203.The proteins markedly alleviated infection of tobacco by tobacco mosaic virus and tomato by Pseudomonas syringae.Treating tobacco plants with HpaG_Xooc,C47T,and MG decreased the viral infection by 58%,81%, and 92%,respectively.In tomato plants treated with HpaG_Xooc,C47T,or MG,P.syringae multiplication was inhibited;bacterial population multiplied in 5 d in these plants were ca. 160-fold,1,260-fold,or 15,860-fold smaller than that in control plants.So pathogen defense was induced in both plants.Defense-related genes Chia5,NPR1,and PR-1αwere expressed consistently with resistance.In response to HpaG_Xooc,C47T,and MG,aerial parts and roots of tomato plants increased growth by 15%and 53%,25%and 77%,and 46%and 106%,relative to controls.The expansin gene,EXP2,involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG_Xooc represses the effects of the protein in plants.To determine interactions between the bacterial type-Ⅲproteins,yeast two-hybrid screening was conducted.Multiple combinations were between pGADT7::hrpA_Xooc and pGADT7::hrpF_Xooc as AD vectors,and pGBKT7::hpaG_Xooc,pGBKT7::MG and pGBKT7::C47T as DNA-BD vectors.An AD vector and a BD vector were cotransformed into the yeast strain Y190.Theβ-galactosidase assay was used to observe the relationships of pairs HrpA_Xooc-HpaG_Xooc,HrpA_Xooc-MG,HrpA_Xooc-C,47T,HrpF_Xooc-HpaG_Xooc,HrpF_Xooc-MG, and HrpF_Xooc-C47T by.Results indicated that there was no interaction between HrpA_Xooc and HpaG_Xooc,and between HrpF_Xooc and HpaG_Xooc.In conclusion,this study has characterized specificity in the popular interaction between Xoo races and near-isogenic rice lines with importance in evaluating virulence variations,studied function of the critical hrpA and hrpF genes as well as the effector protein HpaG_Xooc in rice and nonhost plants.The production of several constructs will facilitate further studies on functions of the genes and proteins in plants.