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

在花椰菜花叶病毒 35S 启动子的控制下构建了苹果褪绿叶斑毛病毒 (ACLSV) 基因组的感染性全长 cDNA 克隆 ​​(pCLSF)，将 pCLSF 机械接种到藜麦上诱导出现 ACLSV 感染的全身典型症状。分别通过 Northern 印迹和免疫印迹分析证明了 pCLSF 接种的叶子中基因组 RNA 和病毒蛋白的变化。还通过免疫电子显微镜观察了 ACLSV 抗血清，这些结果证实 pCLSF 可以以与完整野生型病毒体相同的方式启动感染过程，颗粒轰击和机械接种之间的感染效率比较表明，pCLSF 的剂量可实现 50% 的感染。轰击藜麦植株约为 0.02 ng/株，机械接种约为 400 ng/株，表明轰击程序约为比我有效10D14D1-倍……更多机械接种。苹果褪绿叶斑病毒（ACLSV）ORF2编码的50KDa蛋白（50KP）与绿色荧光蛋白（GFP）融合，在西烟草和藜麦叶子细胞中瞬时表达其细胞内分布、叶表皮中的细胞间运输以及表面的管状形成。对原生质体进行了分析，在两种植物的表皮细胞和原生质体上，50KP-GFP 荧光分布为不规则的小斑点或纤维网络结构，蛋白质从产生它的细胞扩散到邻近的细胞中。相比之下，当 50KP 和 50KP 时，GFP 仅限于单个细胞。 GFP 在 N. occidentalis 的叶表皮中共表达，与单独表达 GFP 时相比，GFP 从产生它的初始细胞中传播得更广泛，这表明 50KP 促进了 GFP 的细胞间运输。当在藜麦叶表皮中瞬时表达时，补充 50KP 缺陷病毒的局部传播。原生质体中 50KP-GFP 的表达导致管状结构的产生。突变分析表明，C 末端区域（氨基酸位置 287 和 475 之间）对于胞连丝定位、细胞间运输、50KP 缺陷病毒运动的补充或小管形成并不重要。相反，N 末端区域的缺失导致所有这些活性完全破坏。

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)。

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。

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）

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）