Isolation And Characterization Of A Recessive Resistance Gene, Xa13, For Bacterial Blight In Rice

Studies on plant resistant genes are one of the hotspots in the field of plant molecular biology. Until now, among the over 50 separated resistant genes, only 6 are recessive genes. The mechanisms of recessive genes can not be explained by those of dominant resistant genes, which have been universally accepted. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide. Thirty-two bacterial blight resistance gene (23 dominant genes and 9 recessive genes) in rice have been identified. However the xa13, located on the distal end of the long arm of the chromosome 8, is a typical recessive R gene in rice, which is fully recessive and specifically confers resistance to the Philippine Xoo race 6 (PXO99). The xal3 gene also interacts with other dominant or recessive R genes which indicated the specialty of disease resistant mechanism of xa13.To clone xa13 gene, a normalized whole-life-cycle cDNA library was firstly constructed with different tissues from 9 develomental stages using rice cultivar Minghui 63, based on the strategy of saturation hybridization with genomic DNA. Then a physical map that covers the region flanked by markers E6a and S14003 was constructed by chromosome walking using Minghui 63 BAC clones. And finally we fine-mapped xa13 to a DNA fragment of 9.2 kb using a series of sequence-based molecular markers. Sequence analysis indicated that this region contain only one candidate gene, which was annotated by full length EST sequence that isolated from the constructed cDNA library.Transformation of IRBB13 with the DNA fragment encompassing the Xa13 candidate that based on the optimised tissue cultural system, including the promoter region isolated from IR64, produced 45 independent transformants. Thirty of the 45 T₀ transgenic plants were susceptible upon infection by PXO99. PCR analysis demonstrated that all of the susceptible transgenic plants had the band derived from the Xa13-candidate fragment, while none of the resistant plants amplified this band. T₁ families derived from four of the single-copy To transgenic plants were further investigated. And the susceptibility cosegregated perfectly with the transgene in all four families, indicated that the clone of xa13 gene by map based cloning strategy was succeed, and the candidate gene in this fragment was indeed Xa13, and the allelic gene in recessive parent was xa13. At the same time, we suppresssed the expression of Xa13 or xa13 in different rice lines using the RNA interference (RNAi) strategy, we found that suppression of Xa13 expression in rice line Zhonghua 11 and MH63 significantly increased the resistance to PXO99, and the level of increased resistance was associated with the reduced accumulation of Xa13 transcripts. Suppression the expression of xa13 in rice line IRBB13 further enhanced xa13-mediated resistance.Interestingly, the expression of Xa13 or xa13 were very low in leaves but quite high in panicles and anther. And we also found that some RNAi transgenic plants with significantly reduced expression of xa13 or Xal3 also had reduced spikelet fertility. The reduced fertility could be ascribed to male sterility as the xa13- and Xa13-suppressed plants had smaller anthers than the respective wild types and produced mostly abortive pollen. Histological analysis showed that the development of the most of microspores stopped at the unicellular...