Molecular and cytological analyzes of bromovirus host specificity and infection spread

点击翻译按钮获取中文摘要

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.