Genetic And Functional Analysis Of Bacterial Blight Resistant Genes Xa26 And Xa4 In Rice

Rice bacterial blight is a devasting disease trigged by Xanthomonas oryza sative pv oryza, causing tremendous yield loss each year. Therefore, disease control is of great importance. A very effective way among such control is the utilization of host resistance genes. Till now, many genes were identified in rice, some of which were cloned. It will help us prompting our knowledge of molecular basis of disease resistance; facilitate genetic improving of rice disease resistance.Xa26 and Xa4 are two rice major resistance genes against bacterial blight. They are located on the terminal long arm of Chromosome 11. Xα4 was once widely used in rice breeding. Xα26 derived from Minghui 63 with wide-spectrum resistance. Based on that, cloning of those two genes makes sense.Candidate genes transformation method was used to identify genes. Previous study showed that MRKαand MRKb might be candidates of Xα26. Thereby those two genes with complete ORFs and promoters were introduced into susceptible japonica cultivar Mudanjiang 8 by agrobacteria-mediated transformation. After inoculation with Xoo, all TO transgeneic plants carrying MRKb exhibited resistance against bacterial blight. Further T1 analysis showed that such phenotype were cosegaregated with the ingressed MRKb, powerfully proving MRKb was Xα26. Intriguingly, transgenic plants diplayed a higher level of resistance after inoculation than its donor line Minghui 63. The same results were observed when Xα26 were introdued into other two susceptible japonica cultivar, Zhonghua 11 and 02428. This might suggest a vital rloe genetic background played in the resistance reaction. Further oversepressing Xα26 under ubiquitin promoter in Zhonghua 11 and Mudanjiang 8 endowed the transgenic plants with higher level of resistance, also exhibiting wide resistance-spectrum. Howerer, reducing its expression level using a pathogen induced promoter in the same genetic background caused a decresed resistance level in transgenic plants.The above results were confirmed by real-time quantitative PCR: expression level of Xα26 under origin pomotor in japonica bacgroud is markedly higher than that in the indica background. This might interpret why transgenic plants of japonica background immuned from more bacterial than those of indica background, probablely duing to greater amounts of Xα26 mRNA. When overexpressing Xα26 in its donor cultivar Minghui 63, higher resistance level was speculated in transgenic plants. Concluding from above results implies a potential mechanism Xα26 might act in the resistance reaction: the effect of Xα26 in disease resistance was directly correlated with its mRNA level, functioning with a dosage effect way.Another appealing result rose up when checking Xα26 expression profile in two genetic backgrounds after inoculation with Xoo. Xα26 transcribed remarkedly rise after being infected by incompatible bacterial strain in Mudanjiang 8. Such trend was not so evident when Xα26 expressed in another elite japonica cultivar-Zhonghua 11. However, analysis of gene expression in indica cultivar (Minghui 63 & IRBB3) showed a typical on and off pattern. Expression analysis revealed that OsWRKY13 and NH1, downstream members of resistance signal pathway, were positively regulated once there was an increased expression of Xα26.As Xα26 belongs to a gene family, therefore we checked whether other members of this RLK family were able to confer resistance. It was achieved by overexpressing MRKαand MRKc. Transgenic plants of MRKαexhibited moderate resistance to pathogen, while those of MRKc were susceptible. It seems that members of the resistance gene family were yet with residual effect against pathogen. We introduced a chimeric gene, Xα3/Xα26LT-MRKαK which could partialy enhance resistance to Xoo. MRKαand MRKc showed similar expression pattern as Xα26, which expressed only in the vascular systems of different tissues. The expressional characteristic of MRKαand MRKc perfectly fits the function of genes conferring resistance to Xoo, a vascular pathogen.RNAi method used to study the function of Xα26, all transgenic plants showed the enhanced susceptible phenotype after inoculated with Xoo, and showed no influence to the growth of plants indicated that Xα26 may had the unique function: disease resistance.Xα4 is currently under cloning. Several candidate genes such as B4-RKα, TQ-RKα, TQ-RKb, TQ-RKd were already excluded. Another possible candidate gene was believed to be Xα4, as transgenic plants overexpressing this gene showed moderate resistance. This gene weakly expressed in plants, with a slight rise exposing to pathogen. Comparing its sequence among IRBB4, Teqing, 93-11 indicated there was no diversity among these three cultivars which contain Xα4. Further analysis should be done to confirm its role as Xα4.