Role of Intron-Mediated Enhancement on Accumulation of an Arabidopsis NB-LRR Class R-protein that Confers Resistance to Cucumber mosaic virus

The accumulation of RCY1 protein, which is encoded by RESISTANCE TO CMV(Y) (RCY1), a CC-NB-LRR class R-gene, is tightly correlated with the strength of the resistance to a yellow strain of Cucumber mosaic virus [CMV(Y)] in Arabidopsis thaliana. In order to enhance resistance to CMV by overexpression of RCY1, A. thaliana was transformed with intron-less RCY1 cDNA construct under the control of strong CaMV35S promoter. Remarkably, a relative amount of RCY1 protein accumulation in the transformants was much lower than that in plants expressing genomic RCY1 under the control of its native promoter. To identify a regulatory element of RCY1 that could cause such differential levels of RCY1 accumulation, a series of RCY1 cDNA and genomic RCY1 constructs were transiently expressed in Nicotiana benthamiana leaves by the Agrobacterium-mediated infiltration method. Comparative analysis of the level of RCY1 accumulation in the leaf tissues transiently expressing each construct indicated that the intron located in the RCY1-coding region of genomic RCY1, but not the native RCY1 genomic promoter or the 5′-and 3′-untranslated regions of RCY1, was indispensable for high level RCY1 accumulation. The increased levels of RCY1 accelerated plant disease defense reactions. Interestingly, such intron-mediated enhancement of RCY1 accumulation depended neither on the abundance of the RCY1 transcript nor on the RCY1 specific-intron sequence. Taken together, intron-mediated RCY1 expression seems to play a key role in the expression of complete resistance to CMV(Y) by maintaining RCY1 accumulation at high levels.

CC-NB-LRR类抗病基因（CC-NB-LRR class R-gene）RESISTANCE TO CMV(Y)（简称RCY1）编码的RCY1蛋白积累量，与拟南芥（Arabidopsis thaliana）抗黄瓜花叶病毒Y株（Cucumber mosaic virus [CMV(Y)]）的抗性强度紧密相关。为通过过表达RCY1提升对CMV的抗性，研究人员采用由强CaMV35S启动子（CaMV35S promoter）调控的无内含子RCY1 cDNA构建体转化拟南芥。值得注意的是，转化株中RCY1蛋白的相对积累量远低于以天然启动子调控基因组RCY1表达的植株。为鉴定导致RCY1积累水平出现此类差异的RCY1调控元件，研究人员通过农杆菌介导的浸润法（Agrobacterium-mediated infiltration），在本氏烟（Nicotiana benthamiana）叶片中瞬时表达一系列RCY1 cDNA及基因组RCY1构建体。对各构建体瞬时表达的叶片组织中RCY1积累水平进行比较分析后发现，基因组RCY1编码区所含的内含子——而非RCY1天然基因组启动子或其5'、3'非翻译区（untranslated region, UTR）——对RCY1的高水平积累不可或缺。RCY1积累量的提升会加速植物的病害防御反应。有趣的是，这种内含子介导的RCY1积累增强效应，既不依赖RCY1转录本的丰度，也不依赖RCY1的特异性内含子序列。综上，内含子介导的RCY1表达似乎通过将RCY1蛋白维持在高水平积累状态，在介导植物对CMV(Y)的完全抗性中发挥关键作用。