Analysis of the cell-to cell and long distant movement of a plant virus expressing GFP

苹果染色叶斑点三病毒（ACLSV）基因组的感染全长度cDNA克隆（pCLSF）是在金黄色病毒35 S启动子的控制下构建的。Mechanical inoculation of pCLSF onto Chenopodium quinoa induced systemic symptoms typical of ACLSV infection。基因组RNA和病毒蛋白在pCLSF-inoculated Leaves中的存在是通过北印迹和免疫印迹分析进行的，因此，相应地。由免疫电子显微镜观察到的与ACLSV反病毒反应的病毒颗粒。这些结果证实了pCLSF可以在相同的方式中启动感染过程，就像整个野生类型的小精灵一样。颗粒轰炸和机械注射之间的感染效率比较显示了奎诺阿植物对炸弹的50%感染的pCLSF对炸弹的影响约为0.02 ng /植物和机械注射的400 ng /植物的影响，表明轰炸过程接近10.D14.D1-比我更有效 ... More chanical inoculation。50 KDa蛋白（50 KP）由苹果氯叶斑点病毒（ACLSV）编码的ORF 2引起的绿色荧光蛋白（GFP）表现出了在Nicotiana occidentalis和Chenopodium quinoa叶子细胞中的瞬态。它的内部分布，细胞到细胞在叶表皮菌中的贩运，以及原塑料表面的管状形成已经分析了。50 KP-GFP荧光分布在外围细胞和两种植物物种的原型塑料上的微小红外点或纤维网络结构。在N的叶子流行病中。occidentalis，蛋白质从细胞中扩散，使其成为邻近细胞中的每一个年轻或成熟的叶子，并在这些细胞中靶向的等离子体。相比之下， GFP被限制在完全叶子中的单个细胞中。当50 KP和GFP在N的叶子流行病中共同表达。具体情况， GFP从初始细胞产生的范围更广，因为GFP是单独表达的，所以建议50 KP导致GFP的细胞对细胞贩运。50 KP-GFP可以用来补充在C. quinoa的叶子流行病中临时表达的50 KP-deficient病毒的局部传播。50 KP-GFP在原型塑料中的结果是在表面制造的管状结构中产生的。显示了C终端区域（aa位置287和475之间）的相互分析，并不需要对等离子体数据、细胞对细胞贩运、补充50 KP缺陷病毒运动或原型塑料的导管形成进行本地化。在对比中，N-终端区域的损失在所有这些活动的完全破坏中。Less（低）

An infectious full-length cDNA clone (pCLSF) of the apple chlorotic leaf spot trichovirus (ACLSV) genome was constructed under the control of the cauliflower mosaic virus 35S promoter. Mechanical inoculation of pCLSF onto Chenopodium quinoa induced systemic symptoms typical of ACLSV infection. The presence of genomic RNA and viral proteins in pCLSF-inoculated leaves was demonstrated by northern blot and immunoblot analyses, respectively. Virus particles that reacted with ACLSV antiserum were also observed by immunoelectron microscopy. These results confirmed that pCLSF could initiate the infection process in the same way as whole, wild type virions. Comparison of infection efficiency between particle bombardment and mechanical inoculation showed that doses of pCLSF for 50% infection in C.quinoa plants were about 0.02 ng /plant for bombardment and 400 ng /plant for mechanical inoculation, indicating that the bombardment procedure is approximately 10ィイD14ィエD1- fold more effective than me … More chanical inoculation.The 50KDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) fused to green fluorescence protein (GFP) was expressed transiently in cells of Nicotiana occidentalis and Chenopodium quinoa leaves. Its intracellular distribution, cell-to-cell trafficking in leaf epidermis, and tubular formation on the surface of protoplasts were analysed. The 50KP-GFP fluorescence distributed as small irregular spots or a fibrous network structure on the periphery of epidermal cells and on protoplasts of both plant species. In leaf epidermis of N. occidentalis, the protein spread from cells producing it into neighboring cells in either young or mature leaves and targeted plasmodesmata in these cells. In contrast, GFP was restricted to single cells in most cases in mature leaves. When 50KP and GFP were co-expressed in leaf epidermis of N. occidentalis, GFP spread more widely from initial cells producing it than that when GFP was expressed alone, suggesting that 50KP facilitated the cell-to-cell trafficking of GFP. The 50KP-GFP was able to complement local spread of 50KP-deficient virus when transiently expressed in leaf epidermis of C.quinoa. Expression of 50KP-GFP in protoplasts resulted in the production of tubular structures protruding from the surface. Mutational analyses showed that the C-terminal regions (between aa positions 287 and 475) were not essential for localization to plasmodesmata, cell-to-cell trafficking, complementation of movement of 50KP-deficient virus, or tubule formation on protoplasts. In contrast, deletions in the N-terminal regions resulted in the complete disruption of all these activities. Less