Molecular and cytological analyzes of bromovirus host specificity and infection spread

正链RNA溴病毒的非结构蛋白3a是侵染植物所必需的，是系统侵染中宿主特异性的关键决定因子。要确定的步骤，其中3A突变体被逮捕和与3A基因的宿主特异性的性质，我用原位杂交检查感染豇豆褪绿斑驳溴病毒（CCMV）及其衍生物在豇豆叶表皮的单个细胞的水平传播。3a移码衍生物在主要感染的表皮细胞中繁殖，但即使在4dpi也未能进入相邻细胞，这表明3a基因对于细胞间传播至关重要。最有趣的是，CCMV衍生物的3a基因被非豇豆适应的溴病毒雀麦花叶病毒（BMV）的3a基因所取代，最初在豇豆植物中从一个细胞传播到另一个细胞，但在1和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，由BMV 3a蛋白施加的宿主特异性限制不是由于不能直接将感染扩散出最初感染的豇豆细胞，而是由于更晚的阻断。在感染扩散的极限处明显不存在任何预先存在的解剖学边界，并且在感染病灶处存在局部组织变化，这表明诱导的宿主反应可能促成了这种阻滞。溴病毒的外壳蛋白是系统侵染植物所必需的。为了分析外壳蛋白缺失突变体的感染传播，我试图构建表达作为标记的绿色荧光蛋白（GFP）基因的CCMV衍生物。虽然已经产生了几个突变体，但我没有在植物中检测到任何荧光信号。使用GFP融合蛋白系统的未来分析可能有助于检测植物中溴病毒感染传播。

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.