Molecular and cytological analyzes of bromovirus host specificity and infection spread

正链RNA溴病毒的非结构3a蛋白是植物感染传播所必需的，是全身性感染中宿主特异性的关键决定因素。为了确定3a突变体被捕获的步骤以及与3a基因相关的宿主特异性的性质，我使用原位杂交技术在豇豆叶表皮的单个细胞水平上检测了豇豆绿斑斑驳bromovirus （CCMV）及其衍生物的感染传播。3a移码衍生物在主要感染的表皮细胞中繁殖，但即使在4 dpi后也未能进入邻近细胞，这表明3a基因对细胞间传播至关重要。最有趣的是，一种带有3a基因的CCMV衍生物被一种不适合豇豆的溴化病毒取代，即溴化花叶病毒（BMV），最初在豇豆植物的细胞间传播，但在1至2 dpi之间停止传播，此时大多数感染灶包含40-80个表皮细胞。因此，BMV 3a蛋白施加的宿主特异性限制不是由于无法引导感染从最初感染的豇豆细胞中传播，而是由于更晚的阻断。在感染扩散的极限处明显没有任何预先存在的解剖边界和感染灶处的局部组织变化表明，诱导宿主反应可能是导致这种阻断的原因。溴病毒外壳蛋白是植物全身性感染所必需的。为了分析外壳蛋白缺乏突变体的感染传播，我试图构建表达绿色荧光蛋白（GFP）基因作为标记的CCMV衍生物。虽然已经产生了一些突变体，但我还没有在植物中检测到任何荧光信号。未来使用gfp融合蛋白系统进行分析可能有助于检测溴病毒感染在植物中的传播。

The nonstructural 3a protein of the positive-strand RNA bromoviruses is required for infection spead in plants and is a crucial determinant of host specificity in systemic infection. To determine the step at which 3a mutants are arrested and the nature of the host specificity associated with the 3a gene, I used in situ hybridization to examine infection spread by cowpea chlorotic mottle bromovirus (CCMV) and its derivatives at the level of individual cells in cowpea leaf epidermis. A 3a frameshift derivative multiplied in primarily infected epidermal cells but failed to move into neighboring cells even by 4 dpi, showing that the 3a gene is essential for cell-to-cell spread. Most interestingly, a CCMV derivative with the 3a gene replaced by that of a non-cowpea-adapted bromovirus, brome mosaic virus (BMV), initially spread from cell to cell in cowpea plants, but stopped spreading between 1 and 2 dpi, when most infection foci encompassed 40-80 epidermal cells. Thus, the host-specificity restriction imposed by BMV 3a protein did not result from an inability to direct the spread of infection out of initially-infected cowpea cells, but from a much later block. The apparent absence of any pre-existing anatomical boundary at the limit of infection spread and localized tissue changes at the infection foci suggested that induced host responses might have contributed to this block. The coat protein of bromoviruses is required for systemic infection in plants. To analyze the infection spread of the coat protein-defficient mutants, I tried to construct CCMV derivatives that express the green fluorescent protein (GFP) gene as a marker. Although several mutants have been made, I have not detected any fluorescence signals in planta. Future analyzes using the GFP-fusion protein system might be useful to detect bromovirus infection spread in plants.