| Bacterial blight caused by Xanthomonas oryzae pv. Oryzae (Xoo) is one of the most serious diseases of rice. The development of novel rice cultivars with broad-spectrum resistance genes to bacterial blight is the most efficient strategy to defence the disease. Oryza meyeriana L. shows high resistance to bacterial blight, but the resistance genes were hardly to be utilized because of the hybrid incompatibility. To take advantage of these resistance genes, a cultivar (Y73) is developed from asymmetric somatic hybridization between an elite japonica rice cultivar (Dalixiang) and Oryza meyeriana, and it was found resistance to bacterial blight and Agrobacterial tumefaciens. To analyze the resistance of Y73 to Agrobacterial tumefaciens, the transformation efficiency of Y73 was compared with Dalixiang with different AAMs and co-cultivation temperatures.Under normal conditions, the transformation efficiency of Y73 was 35.7%, which was lower than Dalixiang (71.2%). This result indicated that the feature of resistance to bacterial blight in Y73 also has an effect on Agrobacterium-mediated transformation. By reducing the transformation temperature and using AAM3, the transformation efficiency of Y73 was improved from 29.9% to 66.5%. These results also indicated that the resistance of Y73 to Agrobacterium tumefaciens can be weakened under lower temperature.To further analyze the molecular mechanisms of Y73 response to Agrobacterium tumefaciens under different conditions, the expression levels of OsMPKs, OsVIP1s and OsPR1s are studied by qRT-PCR. In Arabidopsis, it has been reported that Agrobacterium can induce the activation of MAPK3. As a direct target of MAPK3, the VirE-interacting protein 1 (VIP1) is phosphorylated and localized to nuclei, and then regulates the expression of PRls. The VIP1 encoded by host cell is related to the nuclear import of Agrobacterium T-DNA complex. According to the results of quantitative real-time PCR (qRT-PCR) analysis, the expression levels of 2 OsVIPls and almost all OsPR1s were higher when infected by Agrobacterium at 20℃than 25℃, while the expression levels of OsMPKs remained unchanged at different temperatures. The induced OsVIPl proteins probably interacted with VirE2 and facilitated the import of the T-DNA complex, and led to a higher transformation efficiency in Y73. This study suggests that rice Y73 has similar MAPK/VTP1 defense signaling pathway as in Arabidopsis, which involved in Agrobacterium-mediated transformation. The disease resistance gene play an important role in plant defense system. The products coded by most disease resistance genes belong to NBS-LRR protein family, which is related with protein-protein interactions and defence signal transduction. According to gene chip analysis, the expression of a NBS-LRR gene in Y73 was induced notably after inoculation with Xanthomonas oryzae pv.oryzae (PXO124). The CDS (2631 bp) of the gene was cloned by RT-PCR and was named OsNLR1 (Rice NBS-LRR Resistance gene 1). Sequence analysis shown that the gene is from cultivated rice. Possibly, the expression of this gene is influenced by the genes from wild rice and exhibited a disease-resistant function. To analyze the function of OsNLRl, over-expression and RNA interference vectors were constructed with pCAMBIA1300 and the transgenic rice lines were developed by Agrobacterium-mediated transformation. Results of transgenic analysis revealed that RNA interference lines were more sensitive to rice bacterial blight compared with wild type plants, while the over-expressing lines shown more resistance to bacterial blight. These results suggested that OsNLR1 was involved in the rice bacterial blight resistance. Furthermore, the subcellular localization of OsNLRl were also analized, the sGFP fusion analysis shown that it localized in cell plasma membrane and nuclear. This result indicated that OsNLRl was probably involved in the signal transduction and the regulation of the transcription of downstream genes. |
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