The cDNA cloning of a novel bacterial blight-resistance gene ME137

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the most serious diseases of rice worldwide [1]. Use of resistant varieties has become the most economic and effective method to control the disease. To date, more than 30 genes have been found and characterized. Five dominant R genes, Xa1, Xa3, Xa21, Xa26, Xa27, and two recessive R genes, Xa5, Xa13, have been cloned [2–8]. However, because of the differentiation of physiological race and the continually emerging of new toxic bacterial flora, cultivated varieties with narrower resistance spectrum will lose their resistance ability to BB in a short period [9]. Therefore, excavating and identification of new resistance genes are of significance to control BB. Wild rice species have long been used as genetic resources in rice breeding. Some genes that confer resistance to blight disease have been identified in wild rice. Xa21, which was first reported by Khush et al. [10], was the first gene found in wild rice (Oryza longistaminata). After cloning and application in breeding resistant varieties, resistance genes were identified successively in O. rufipogon, O. minuta, O. officinalis, and O. australiensis [11– 14]. A previous study revealed that some of O. meyeriana materials are immune to the BB disease [15]. So far the genome of O. meyeriana has not been demonstrated, thus it is difficult to investigate the resistance gene. Furthermore, because hybridization could not happen normally between O. meyeriana (GG genome 2n 1⁄4 24) and cultivated rice (AA genome), it is hard to use the resistance gene of O. meyeriana by conventional hybridization approaches for improving cultivated rice. All of these indicate that we should try some other approaches to find and identify novel disease-resistance genes. It is interesting why O. meyeriana can be immune to BB disease and what kind of genes is related to BB disease resistance in O. meyeriana. In this study, a novel BB resistance-related gene, named temporarily ME137, was cloned from O. meyeriana. This study would lay the foundation for finding, identification and usage of new resistance genes, and breeding resistant varieties. The main materials used in this study were as follows: X. oryzae pv. oryzae C1, O. meyeriana planted in a vinyl house (Biotechnology & Genetic Resources Institute, Yunnan Academy of Agricultural Sciences, Kunming, China) at natural temperature and light, RNAiso Plus (TaKaRa, Dalian, China), M-MLV-reverse transcriptase (TIANGEN, Beijing, China), 50-Full RACE kit (TaKaRa), SanPrep DNA gel extraction kit (Sangon, Shanghai, China), and pMD18-T Vector (TaKaRa). O. meyeriana was inoculated with C1 by the leafclipping method in the flowering stage. Fresh leaves inoculated were cut and collected for cloning cDNA. The fresh leaves at 1 d before Xoo induction and 1, 2, 3, 4, 5 d after Xoo induction were used for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. All the materials were frozen in liquid nitrogen immediately after collection and stored at 2808C for further use. Total RNA was extracted from collected leaf tissues and amplified into first-strand cDNA. The Expressed Sequence Tag sequence that has low homology with previously cloned ones to obtain a novel resistance gene was chosen from suppression subtractive hybridization (SSH) library and confirmed by a pair of primers 137-1 (50-GACCCTGCC CAACA-30) and 137-2 (50-ACCGCTGCCTGAT-30). The SSH library was constructed by the conventional method, after O. meyeriana was inoculated with Xoo as tester, and non-inoculated materials as driver [16]. The primers used for amplification of actin gene were actin-F (50-GTATGG TGAAGGCTGGATTTGC-30) and actin-R (50-ATTTCCCG CTCTGCTGTTGTGG-30). To obtain 30 end of ME137, the primer AP-dT (50-CG CGGATCCAAGCTTATCGATTTTTTTTTTTTTTTTTT-30) instead of Oligo dT was used when amplified into first-strand cDNA. Then, a pair of primers AP (50-CGCGGATCCAA GCTTATCGA-30) and 137-1 (50-GACCCTGCCCAACA-30) were designed to get the 30 end of cDNA sequence. 50 RACE was performed as recommended by the manufactures. To obtain specific band, nested PCR was used. In the outer PCR, a pair of primers outer-F (50-CATGGCTACATGCTG Acta Biochim Biophys Sin 2013, 45: 422–424 | a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt029. Advance Access Publication 4 April 2013