Molecular analysis of multi-layered mechanisms of non-host resistance in Arabidopsis thaliana to bromoviruses

雀麦花叶病毒（BMV）和春美人潜伏病毒（SBLV）是两种关系密切的三重RNA植物病毒。BMV（M1株）显示有限的感染模式植物拟南芥，而SBLV有效地感染拟南芥。即使在原生质体中，BMV的积累水平也低于SBLV，并且通过cpr 5突变而增强。分别编码复制蛋白1a和2a的BMV RNA 1和2的5'和3'非编码区介导的翻译效率在cpr 5 -2突变体中增强。这表明至少与复制相关的病毒组分的增强的翻译允许BMV有效地感染cpr 5突变体。与BMV的M1株系相反，BMV的M2株系在没有cpr 5突变的拟南芥原生质体中有效地繁殖，而它们在BMV的系统宿主大麦原生质体中显示出类似的积累。利用BMV-M1和BMV-M2之间的杂交病毒和限制体进行的北方印迹分析表明，BMV-M2 RNA 2 3'端的tRNA样结构内的序列对BMV-M2在拟南芥细胞中的有效增殖贡献最大。荧光素酶报告基因分析表明，M2 RNA 2的非编码区在拟南芥细胞中比M1 RNA 2的非编码区更能促进BMV-M2的翻译，但在大麦细胞中的作用与M1 RNA 2的相似，表明RNA 2的2a蛋白的高水平表达支持BMV-M2在拟南芥细胞中的高效增殖。我们已经从模式植物本氏烟草中克隆了NbNACa 1基因，该基因编码的蛋白与来自各种生物体的初生多肽相关复合物的α链具有序列相似性，并在体内与BMV运动蛋白相互作用。NbNACa 1基因的下调减少了本氏烟草中BMV的细胞间运动。免疫荧光显微镜观察表明，NbNACa 1通过调节BMV MP定位于胞间连丝参与BMV细胞间运动。

Brome mosaic virus (BMV) and Spring beauty latent virus (SBLV) are closely related, tripartite RNA plant viruses. BMV (M1 strain) shows limited infection in a model plant Arabidopsis thaliana, while SBLV efficiently infects A.thaliana. Even in protoplasts, accumulation levels of BMV were lower than those of SBLV and were enhanced by the cpr5 mutations. Efficiency of translation mediated by the 5' and 3' noncoding regions of BMV RNAs 1 and 2 that encode replication proteins 1a and 2a, respectively, was enhanced in cpr5-2 mutant. This suggests that at least enhanced translation of viral components associated with replication allow BMV to efficiently infect cpr5 mutants. In contrast to the M1 strain of BMV, the M2 strain of BMV efficiently multiplied in A.thaliana protoplasts without cpr5 mutations, while they showed similar accumulations in barley protoplasts, a systemic host of BMV. Northern blot analyses using reassortants and hybrid viruses between BMV-M1 and -M2 demonstrated that sequences within the tRNA-like structure at the 3' end of BMV-M2 RNA2 most remarkably contributed to the efficient multiplication of BMV-M2 in A.thaliana cells. Luciferase reporter assay showed that the noncoding regions of M2 RNA2 enhanced translation more than those of M1 RNA2 in A.thaliana cells but similarly in barley cells, suggesting that the high level expression of 2a protein from RNA2 supported the efficient multiplication of BMV-M2 in A.thaliana cells. We have cloned the NbNACa1 gene from a model plant Nicotiana benthamiana that encoded a protein with sequence similarity to the alpha chain of nascent-polypeptide-associated complex from various organisms and interacted with BMV movement protein in vivo. Downregulation of the NbNACa1 gene reduced cell-to-cell movement of BMV in N.benthamiana. Immunofluorescence microscopy observation suggests that NbNACa1 is involved in BMV cell-to-cell movement through the regulation of BMV MP localization to the plasmodesmata.