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

表达 GFP 的植物病毒的细胞间和长距离运动分析

• 批准号: 10660041
• 负责人: YOSHIKAWA Nobuyuki
• 金额: $ 0.51万
• 依托单位: IWATE UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660041/
• 关键词: ACLSV; infectious cDNA clone; cell-to-cell movement; GFP; 50K movement protein; ACLSV

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

以花椰菜花叶病毒35 S启动子为控制基因，构建了苹果褪绿叶斑病毒（ACLSV）感染性全长cDNA克隆（pCLSF）。机械接种pCLSF到藜上诱导ACLSV感染的典型全身症状。分别通过北方印迹和免疫印迹分析证明了pCLSF接种叶片中基因组RNA和病毒蛋白的存在。用免疫电镜观察到与ACLSV抗血清反应的病毒颗粒。这些结果证实pCLSF可以以与完整的野生型病毒粒子相同的方式启动感染过程。基因枪和机械接种的侵染效率比较表明，pCLSF对昆诺阿藜植株的50%侵染剂量，基因枪为0.02 ng /株，机械接种为400 ng /株，表明基因枪的侵染效率比机械接种高10倍左右。 ...更多信息 将苹果褪绿叶斑病毒（Apple chlorotic leaf spot virus，ACLSV）ORF 2编码的50 KDa蛋白与绿色荧光蛋白（green fluorescence protein，GFP）融合，在烟草（Nicotiana occurentalis）和藜（Chenopodium quinoa）叶片中进行瞬时表达。分析了其在细胞内的分布、叶表皮细胞间的运输和原生质体表面管状结构的形成。50 KP-GFP荧光在两种植物的表皮细胞和原生质体上呈不规则的小斑点或纤维状网状结构分布。在叶表皮中，N.在occupientalis中，该蛋白质从产生它的细胞扩散到幼叶或成熟叶中的邻近细胞中，并靶向这些细胞中的胞间连丝。相反，在大多数情况下，GFP仅限于成熟叶片中的单细胞。当50 KP和GFP共表达于N.与单独表达GFP相比，50 KP促进了GFP在细胞间的运输。当在藜麦叶表皮中瞬时表达时，50 KP-GFP能够补充50 KP缺陷病毒的局部传播。50 KP-GFP在原生质体中的表达导致从表面突出的管状结构的产生。突变分析表明，C-末端区域（氨基酸位置287和475之间）是不必要的本地化到胞间连丝，细胞到细胞的贩运，50 KP缺陷病毒的运动的互补，或在原生质体上的小管形成。相反，N-末端区域的缺失导致所有这些活动的完全中断。少

项目成果

寺田道一、佐藤寛、寺内英貴、吉川信幸、高橋壮: "カピロおよびトリコウイルスのORF2がコードするタンパク質の細胞間移行"日本植物病理学会報. 64. 605 (1998)

Michiichi Terada、Hiroshi Sato、Hidetaka Terauchi、Nobuyuki Yoshikawa 和 So Takahashi：“毛状病毒和毛状病毒 ORF2 编码的蛋白质的细胞间运动”，日本植物病理学会通报 64. 605 (1998)。

佐藤寛、松田浩徳、寺田道一、吉川信幸、高橋壮: "リンゴクロロティックリーフスポットウイルス(ACLSV)50Kタンパク質の細胞間移行能と管状構造形成能"日本植物病理学会報. 65・3. 336 (1999)

Hiroshi Sato、Hronori Matsuda、Michiichi Terada、Nobuyuki Yoshikawa、So Takahashi：“苹果褪绿叶斑病毒（ACLSV）50K蛋白的细胞间迁移能力和管状结构形成能力”日本植物病理学会通报65。・3.336（1999）

H.Satoh,N.Yoshikawa and T.Takahashi: "Construction and Biolistic Inoculation of an infections cDNA Clone of Apple Chlorotic Leaf Spot Trichovirus" Ann.Phytopathol.Soc.Japan. 65・3(印刷中). (1999)

H. Satoh、N. Yoshikawa 和 T. Takahashi：“苹果退绿叶斑毛病毒感染 cDNA 克隆的构建和生物射弹接种”，Ann. Phytopathol，1999 年。

Satoh, H., Matsuda, H., Terada, M., Yoshikawa, N., and Takahashi,T.: "Cell-to-cell movement and tubule-forming capacity of apple chlorotic leaf spot trichovirus 50K protein."Annals of the Phytopathological Society of Japan. 65. 336-336 (1999)

Satoh, H.、Matsuda, H.、Terada, M.、Yoshikawa, N. 和 Takahashi,T.：“苹果褪绿叶斑毛病毒 50K 蛋白的细胞间运动和小管形成能力。”

吉川信幸、寺田道一、池田泰子、脇葉順、高橋壮: "リンゴクロロティックリーフスポットウイルス50Kタンパク質とGFPの融合タンパク質のプラスモデスマータへの局在と師管への集積"日本植物病理学会報. 65・6. 669 (1999)

Nobuyuki Yoshikawa、Michiichi Terada、Yasuko Ikeda、Jun Wakiba、So Takahashi：“苹果褪绿叶斑病毒 50K 蛋白和 GFP 的融合蛋白定位于胞间连丝并在韧皮部中积累”日本植物病理学学术通报 65・6。 1999）