Isolation and Characterization of a Recessive Resistance Gene, xa13, for Bacterial Blight in Rice 3%v)!dTa<^
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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 genes (23 dominant genes and 9 recessive genes) in rice have been identified. However the xa13, which located on the distal end of the long arm of the chromosome 8, was a typical recessive R gene in rice, that is fully recessive and specifically confers resistance to the Philippine Xoo race 6 (PXO99). The xa13 gene also interacts with other dominant or recessive R genes which indicated the specialty of disease resistant mechanism of xa13. ZTmy } @l
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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 have 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. b6k`R4S3
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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 T0 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 have 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. \Qml~?$@lH
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Interestingly, the expression of Xa13 or xa13 was 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 Xa13 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 pollen grain stage or bicellular pollen grain stage and gradually degenerated afterwards in those transgenic plants. Coincided with the development stage of abnormal pollen appearance, the expression of Xa13 in anther was identified by in situ hybridization. Results show that no transcripts were detectable at early stage and Xa13 transcripts accumulated to high level at unicellular pollen grain stage. The coincidence in time between the accumulation of Xa13-transcripts in wild type plants and the appearance of pollen abortion in Xa13-suppressed plants during pollen development suggests an indispensable role of Xa13 in pollen development. uq5?t
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Further research data show that the expression of Xa13 in leaves was modulated by pathogen inoculation. The core mutation resulted in loss-of those functions in xa13 genes would happened on a 18 bp region in the promoter region, rather than on gene code region. And this mutation wouldn’t influence the expression of xa13 in anther. XA13 is a unknown function plasma membrane protein, belong to the MtN3 gene family. It acted as important functions both during in the process of PXO99 aggression and anther development. Therefore it would mediate a new pathway of resistance independent with those by activated expression of pathogenesis-related genes or by increased thicken of secondary cell-wall in vascular bundle element of leaves. Above all, the cloned xa13 and its dominant allele Xa13 gene in this study, would not only display a new resistance mechanism: suppressing the expression level of resistance gene is the foundation of plant for resistant to pathogen with high efficiency, but aslo provide a new jumping-off point for the study on the relationship between resistance to disease and anther development in plant. /HIyQW\Ki-
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Key words: Rice; Bacterial blight; Xanthomonas; Resistance gene; cDNA library; ?
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Map based cloning
