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

溴雀花花叶病毒（BMV）和春美潜伏病毒（SBLV）是密切相关的三联RNA植物病毒。BMV （M1菌株）在拟南芥中表现出有限的感染，而SBLV在拟南芥中表现出有效的感染。即使在原生质体中，BMV的积累水平也低于SBLV，并因cpr5突变而增强。在cpr5-2突变体中，分别编码复制蛋白1a和2a的BMV RNAs 1和2的5‘和3’非编码区介导的翻译效率得到了提高。这表明至少与复制相关的病毒成分的增强翻译允许BMV有效地感染cpr5突变体。与BMV M1菌株相比，BMV M2菌株在没有cpr5突变的拟南芥原生质体中繁殖效率较高，而它们在BMV的系统宿主大麦原生质体中也表现出类似的积累。利用BMV-M1和-M2之间的重组病毒和杂交病毒进行的Northern blot分析表明，BMV-M2 RNA2 3'端trna样结构内的序列对拟南芥细胞中BMV-M2的高效增殖贡献最大。荧光素酶报告基因分析显示，在拟南芥细胞中，M2 RNA2的非编码区比M1 RNA2的非编码区更能促进BMV-M2的翻译，而在大麦细胞中也类似，这表明RNA2高水平表达2a蛋白支持BMV-M2在拟南芥细胞中的高效增殖。我们从模式植物benthamiana Nicotiana中克隆了NbNACa1基因，该基因编码的蛋白质序列与来自多种生物的新生多肽相关复合物α链相似，并在体内与BMV运动蛋白相互作用。NbNACa1基因的下调减少了benthamiana细胞间BMV的运动。免疫荧光显微镜观察表明，NbNACa1通过调节BMV MP向胞间连丝的定位参与了BMV细胞间的运动。

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.