Cloning And Functional Characterization Of A Dominant Resistance Gene,Xa14,for Bacterial Blight In Rice

Bacterial blight of rice,caused by Xanthomonas oryzae pv.oryzae（Xoo）,jeopardizs the stability of rice production.An effective way is to use the host resistance genes in agricultural production.The investigation on functions and mechanisms of disease resistance genes have an important effect on science and ameliorate the diseases on rice.Xa14,a typical dominant R gene in rice,which is fully dominant and specifically resistant to the Philippine Xoo race5（PXO112）.To isolate Xa14,we used an F2population of 599 individuals derived from the cross of rice cultivar IRBB14（carrying Xa14）and IR24（lacking Xa14）,then inoculated with Xoo strain PXO112,which could trigger Xa14-mediated resistance in rice.After lesion length and genotype analysis,Xa14gene was further localized between maker 52940 and RM5473.A candidate gene encoding BED-NLR protein,named RGAf-BB14,was found in the localization interval through bioinformatics.To validate our results,the RGAf-BB14 gene driven by a natural promoter was transformed into IR24.Three different T₁ families from the positive T₀plants were inoculated with PXO112.Similarly,the positive T₁ individuals were significantly resistant to Xoo,comparing to the negative T₁ individuals or wild type.These results strongly suggested that candidate RGAf-BB14 was Xa14.Comparative sequencing revealed that Xa14 and Xa1 are alleles.Homologous cloning and transgenic complementing experiments showed that Xa1-2 and Xa31（t）are the same gene and alleles of Xa1 as well.Furthermore,we analyzed the differences in the domains of these proteins encoded by these alleles.We found a specific and highly conserved repeating structure in the LRR region,called central tandem repeat（CTR）,and named multiple domains that may affect rice resistance.For example,XA14 contained an additional motif between Initial LRR and CTRs,named as Linker,compared with XA1 and XA1-2.RGAF contained an identical Linker to that of XA14,but it also contained a unique motif that was not found in all the three R proteins—named,intervening motif.In order to study the relationships among Xa1,Xa1-2,and Xa14 genes,we made several crosses within them.After inoculation with bacterial blight,we found that the disease resistance of Xa1/Xa14 and Xa1-2/Xa14 hybrid plants was lower than that of their parents,while the disease resistance of Xa1/Xa1-2 hybrid plants was similar to that of their parents.These results suggested that interactions between Xa14 and Xa1 or Xa1-2could impair their resistance,but the interaction between Xa1 and Xa1-2 did not.Further analysis revealed that BED finger domains of XA1 and XA14,called XA1-BED and XA14-BED respectively,could interact with themselves or each other.In addition,an evolutionary analysis of 100 homologous proteins from the gramineae showed that RGAf firstly originated from an ancestor gene in a wild rice.Then Xa14 originated from RGAf.Xa1 and Xa1-2 might be generated from Xa14,as the deletion of the Linker-encoding sequence suggested.But it was difficult to tell which one between Xa1 and Xa1-2 was the ancestral gene.It was found that the m RNA expression of Os WRKY45 gene in disease-resistant parent IRBB14 significantly increased compared to susceptible parents IR24 after inoculation with Xoo strain PXO112.Then we overexpressed Nah G gene in the carrying Xa14 materia encoding a hydroxylase of salicylic acid,which can degrade salicylic acid.Interestingly,we found the disease resistance was inhibited.In this study,Xa14,Xa1-2 and Xa31（t）were cloned,and found that them had complex relationships.If they were aggregated in the same rice plant,the antagonism will be had different effects.Our study also conducted a preliminary exploration of the disease resistance mechanism of Xa14 and its alleles,and we found that the Nah G gene can inhibit Xa14 resistance.Our study provides genetic resources and theoretical basis for rice disease resistance breeding.