Functional Analysis Of Xa13in Rice-Xoo Interaction

The rice bacterial blight (BB), caused by Xanthmonas oryzae pv. Oryzae,(Xoo),was oneof the most devastating diseases of rice world wide and causes tremendous agricultural yieldloss each year. Utilization of host resistance genes during agricultural production is the mosteffective and economic way. Understanding the function and mechanism on disease resistancegenes may have important significance in science and application for improving rice.In the experiments of Xoo for copper stress response, high concentrations of copperwill inhibit the growth and reproduction of the bacterial blights. The strain of the Philippines6(PXO99) is the most sensitive to copper among all the Xoos tested. In order to study thecopper sensitive mechanism,firstly a genomic library constructed from PXO61which isresistant to copper and were transformed into PXO99.Secondly, anti-copper mutants whichgrow well in the medium containing1mM copper were screened. the cosmids of mutant wereextracted and sequenced. We found that the fragments contained two anti-copper gene copAand copB. Further studies showed that, pathogens of PXO99and PXO61have the same thecoding region of the copB. Compared with encoding region of copA genes in the PXO61,thecoding region of copA in the PXO99is lack of six amino acids which is “DGTTQG”.Byreplacing of the coding region of copA from pathogen of PXO99and PXO61,we found thesensitivity to copper of PXO99is caused by missing six amino acids of the coding region"DGTTQG"of copA gene.Secondly, in order to study the role of the copA and copB genes in the pathogenicity ofXoo, we obtained the fragmants of copA and copB from PXO61.Then it was ligated into thevector pHM1.Transformation was conducted by electroporation to PXO99. We choosed16mutant strains which were more resistant to copper, and inoculated susceptible varietiesIR24.The result shows that the copper-resistant clones have stronger virulence, but can notrestore the susceptibility to IRBB13. At the same time, copA and copB were knocked by usinga suicide vector pK18mobsacB. We obtained7copA and copB genes deletion mutants, andfound that the mutants were more susensitive to copper. Mutans and wild type wereinoculated IR24and IRBB13.These results showed that the pathogenicity of the mutants weredeclined in IR24and IRBB13comparing with wild typre after14days. The results indicatedthat the copA and copB were as pathogenic factors. In addition, anti-copper mutants can notfully restore susceptible, indicating that copper was only partially involved in xa13-mediatedresitance to bacteaial blight disease, and suggesting xa13has still other unknown functions. By homology analysis of rice XA13protein, we found that the gene frommaize(ZmXa13) was the highest homologous to OsXa13,which the homology of the N-terminal was up to90.5%and the homology of the C-terminal XA13was up to40.6%. Usingthe copper ions complementation experiments in S. cerevisiae ctr1Δctr3Δmutant MPY17indicated that combined with OsCOPT1and OsCOPT5, OsXA13(1-220)protein could beformed complex to play the role of transporting copper into cells from the extracellularenvironment. Yeast two-hybrid (Y2H)experiments showed that ZmXa13fragment (56-100aa)which is94valine to alanine impact the interaction with OsCOPT5protein.In order to furtherverify the role of C-terminal of OsXA13played in pathogenicity to rice, four trangenic plantsnamed PXa13: OsXA131-220、PXa13:ZmXa13、PXa13:NOs-CZM、PXa13:NZM-COswere inoculatedwith PXO99.We found that only the PXa13:NOs-CZMtrangenic plants restored susceptibility toPXO99. Thus, the C-terminus of the OsXa13is necessary and its function is conserved.In order to resolve the function and mechanism of C-terminal XA13, We found twoproteins CacyBP and HMG could interact with XA13by using the Y2H. The interactionsbetween XA13and CacyBP or HMG were further confirmed by bimolecular fluorescencecomplementation(BiFC) experiment. To determine the subcellular location of CacyBP andHMG in plant cells, a construct containing a GFP fusion was transient expressed intobacco,we found that CacyBP located in the cell membrane area, and HMG located inmembrane and nucleus. To identify to the biological function of CacyBP and HMG, we over-express or suppress CacyBP and HMG in rice varieties Zhonghua11. Transgenic plants atbooting stage were measured the resistance to PXO99. The results showed that, comparedwith the wild-type varieties, CacyBP over-expression lines were more sensitive to PXO99,while CacyBP suppression lines exhibited high resistance to PXO99, and HMG transgeniclines had similar results to CacyBP. These results demonstrated that CacyBP and HMG genesare negative factors involved in regulating xa13-mediated resitantce to bacteaial blight disease.