Mechanism Analysis Of Major Disease Resistance Gene Xa3/Xa26 In Rice-Xoo Interaction

Rice is one of the most important staple food around the world.Disease-resistant breeding is crucial for rice production worldwide.Rice bacterial blight caused by Xanthomonas oryzae pv.oryzae（Xoo）is the most damaging bacterial disease,affecting rice yield seriously.Utilization of major disease resistance（MR）gene is an effective way to control the damage of bacterial blight during agriculture production.Therefore,investigation on the mechanism of MR genes can provide important theoretical guidance for disease-resistant breeding.Xa3/Xa26,which has been used in rice production for a long time as a MR gene,encodes a plasma membrane localized LRR receptor like kinase protein and confers a broad-spectrum and durable resistance to Xoo at both seedling and adult stages.Rice varieties carrying Xa3/Xa26 have been planted across a large area of China.However,the molecular mechanism of the Xa3/Xa26-initiated defense pathway against Xoo is still largely unknown.Here,we first use the rice genome microarray to analysis the gene expression profile in the Xa3/Xa26 transgenic plants（Rb49）after inoculation of Xoo.There are large numbers of genes have been showed differential expression after inoculation of Xoo in the Xa3/Xa26 transgenic plants,comparing with no-inoculated plants and inoculated wild-type plants.These genes may be involved in rice-pathogen interaction or Xa3/Xa26-mediated resistance pathway.Secondly,we use a yeast two-hybrid assy to identify the XA3/XA26-interacting proteins.There are two interacting proteins,named Os TPI1.1 and K35,have been proved to positively or negatively regulate Xa3/Xa26-initiated defense pathway respectively by genetic technique.Os TPI1.1 encodes a cytoplasm-localized triosephosphate isomerase（TPI）.Bimolecular fluorescence complementation and co-immunoprecipitation experiments verified the interaction between XA3/XA26 and Os TPI1.1 in vivo.Transcriptional suppression of Os TPI1.1 markedly impaired XA3/XA26-mediated resistance to Xoo.Constitutive expression of Os TPI1.1 in a susceptible rice variety resulted in plants having slightly decreased susceptibility to Xoo.We expressed and purified the Os TPI1.1 protein in Escherichia coli and examined its enzyme activity in vitro.The recombinant Os TPI1.1protein could catalyze the interconversion between glyceraldehyde-3-phosphate（GAP）and dihydroxyacetone phosphate（DHAP）fastly.Os TPI1.1 also has the ability to perfect complement the yeastΔtpi1 mutant.These results indicate that Os TPI1.1 is a functional triosephosphate isomerase and the reaction catalyzed by Os TPI1.1 is a part of cytosolic glycolysis pathway.Suppression of Os TPI1.1 in rice plant results in less TPI enzyme activity,high DHAP/GAP and reduced/oxidized nicotinamide adenine dinucleotide phosphate（NADPH/NADP⁺）ratios,and low hydrogen peroxide content.Overexpression of Os TPI1.1 results in increased TPI enzyme activity,low DHAP/GAP and NADPH/NADP⁺ratios,and high hydrogen peroxide content.Reactive oxygen species（ROS）play a crucial role in plant resistance to pathogens,and hydrogen peroxide is the major and most stable type of ROS.These results suggest that in rice-Xoo interaction,Os TPI1.1 may modulate ROS at a certain level against Xoo.Suppression of Os TPI1.1reduces TPI activity and,in turn,probablely through the pentose phosphate pathway to increase reduction of NADP⁺to NADPH,which may act as redox cofactor to scavenge ROS,resulting in resistant Xa3/Xa26-carrying rice plants becoming susceptible to Xoo.K35 is another XA3/XA26-interacting protein identified by yeast-two hybrid screening.Here,we found that there are two different transcripts of K35 in rice variety Mudanjiang 8,named K35orf1 and K35orf3 respectively.Only the full-length protein of K35ORF3 could interact with XA3/XA26 in yeast-two hybrid assay.Constitutive expression of K35orf1 and K35orf3 in Rb49（Xa3/Xa26 carrying）impaired Xa3/Xa26-mediated resistance to Xoo.Constitutive expression of K35orf1 and K35orf3 in the susceptible rice variety Mudanjiang 8 leads to accelerated senescence and even death at the seedling stage.Transcriptional suppression of K35 in rice variety Mudanjiang 8results in difficulty to obtain resistant callus during transgenic process,and stunted growth,increased leaf angle,infertility of the positive transgenic T₀plants.These results suggest that K35 may be involved in the Xa3/Xa26-mediated desease resistance as well as the growth and development of rice plants.