| Summary: | 碩士 === 文化大學 === 生物科技研究所 === 85 === Bamboo mosaic virus (BaMV) has a genome consisting of single-stranded, positive-sense RNA. The virus is a member of the potexvirus group. Most susceptible bamboo species belong to the genera Bambusa and Dendrocalamus. Satellite BaMV (satBaMV) is the only one known to be associated with potexvirus group. Two satBaMVs, BSF4 and BSL6, with a 6.9 % difference in nucleotide sequence, exhibit distinct phenotypes in infected plants. BSF4 has little effect on BaMV RNA replication nor of symptom expression caused by BaMV. Moreover, BSF4 spreads in tobacco systemically and efficiently. In contrast, BSL6 not only reduces replication and delays systemic symptom development but also is inefficient for long distance transport in tobacco (Nicotiana benthamiana).
Immunogold-silver staining of the infected tissues was used for localization of BaMV capsid protein in systemically infected bamboo plants. The roll leaves and young stems of common bamboo (Bainbusa vulgaris) infected with BaMV-V (with satellite) and green bamboo {B. oldhamii) infected with BaMV-O (free of satellite) were embedded in paraffin, processed for immuno-staining, reacted with anti-BaMV-O virion serum and labelled with immunogolds followed by silver enhancement for visualization. The localization of silver aggregates was found to be limited in some areas showing a mosaic-like pattern. The virion or coat protein could be detected frequently in the leaf mesophyll, epidermal cells, bundle sheath extension fibers, fusoid cavities and bundle sheaths occasionally observed in the metaxylem, xylem parenchyma cells, xylem fibers, sieve elements and companion cells. However, silver aggregates in stem sections were detected in parenchyma cells surrounding vascular, protoxylem and epidermal cells. Moreover, sections of leaf sheath showed silver aggregates in epidermal cell, bundle sheath extension fibers, parenchyma cells, bundle sheaths, parenchyma cells of xylem, fiber of xylem, sieve elements and companion cells. Preimmune serum was used as control and no labelling was observed. The metaxylem, bundle sheath extension fibers, fusoid cavities, protoxylem and fiber of xylem are dead cells physiologically but labels were frequently observed in these structures.
To analyze the difference in systemic movement of the two isolates of satellite RNA, tobacco plants were infected with BaMV-S RNA, BaMV-S RNA with BSL6, BaMV-S RNA with BSF4, respectively. Inoculated and systemic leaves were harvested at different time intervals for the detection of viral RNA and satBaMV using dot blot hybridization. At 16 days post-inoculation (d.p.i.), viral RNA was detectable in systemic leaves of tobacco. Nine out of ten plants coinfected with BSF4 transcripts showed the presence with satBaMV whereas only 1 out of 10 plants coinfected with BSL6 was positive for satBaMV in the upper non-inoculated leaves. At second passage, all of the plants coinfected with BSF4 showed the presence of satellite at 20 d.p.i. wheresa 4 out of 10 plants coinfected with BSL6 were positive for satBaMV. After the third passage, 7 out of 7 and 10 out of 15 plants were positively detected with BSF4 and BSL6, respectively, in their coinfected systemic leaves at 14 d.p.i. . These results indicated that the third passage showed the fastest movement systemically for BSL6 satBaMV. To verify whether the satRNA in the systemic leaves is BSF4 or BSL6 in every passage, restriction enzymes were used to map the cDNA derived from reverse transcription-polymerase chain reaction of total RNAs from each samples. The results indicated that no evident changes in satRNA sequence was found during the whole process.
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