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

雀麦花叶病毒(BMV)和春美潜伏病毒(SBLV)是关系密切的三元RNA植物病毒。BMV(M1株)在模式植物拟南芥上表现出有限的侵染，而SBLV则有效地侵染拟南芥。即使在原生质体中，BMV的积累水平也低于SBLV，并且cpr5突变增强了BMV的积累水平。在cpr5-2突变体中，编码复制蛋白1a和2a的BMV RNAs1和2的5‘和3’非编码区分别介导的翻译效率提高。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株相比，M2株BMV在没有cpr5突变的拟南芥原生质体中有效繁殖，而在BMV系统寄主大麦原生质体中表现出相似的积累。利用BMV-M1和BMV-M2之间的重组体和杂交病毒进行的Northern杂交分析表明，BMV-M2 RNA2 3‘端的tRNA样结构中的序列对BMV-M2在拟南芥细胞中的有效增殖具有显著的促进作用。荧光素酶报告基因分析表明，M2RNA2的非编码区在拟南芥细胞中的翻译效率高于M1RNA2，但在大麦细胞中的作用相似，这表明RNA2中2a蛋白的高水平表达支持了BMV-M2在拟南芥细胞中的高效增殖。我们从模式植物本氏烟草中克隆了NbNACa1基因，该基因编码的蛋白与多种生物中新生的多肽相关复合体的α链序列相似，并在体内与BMV运动蛋白相互作用。NbNACa1基因的下调减少了BMV在N.benthamiana中的细胞间移动。免疫荧光显微镜观察表明，NbNACa1通过调节BMV MP在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.