| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 99 === Squash leaf curl virus (SqLCV) is a member of the family Geminiviriade, genus Begomovirus. In recent years, SqLCV causes severe economic loses of muskmelon in central and southern area of Taiwan. Previous studies of our laboratory revealed that SqLCV-infected Nicotiana benthamiana plants display various degrees of severity of downward leaf curling symptoms. Further studies showed that C4 protein might be the determinant of the directions of leaf curling in N. benthamiana. However, there is no previous evidence indicating that C4 protein is the pathogenecity determinant in muskmelon. Therefore, the objective of this study is to analyze the factors involved in SqLCV pathogenicity through sequence comparisons and infection assays using infectious clones of different isolates from different hosts and localities. The genomes of various SqLCV isolates from central and southern area of Taiwan were amplified by Rolling Circle Amplification (RCA), and cloned into pUC119 vector for full-length sequence analyses. Infectious clones were constructed using dimers of SqLCV DNA A and DNA B from partial restriction digestion of RCA products and subsequent cloning into pBin19 vector. Four different SqLCV infectious clones, designated SQ2, SQ3, SQ8, and SQ11, have been constructed. When N. benthamiana infected individually by SQ2, SQ8 or SQ3, the leaves display downward curling phenomenon. SqLCV DNAs were also detected in the upper uninoculated leaves, indicating successful systemic infections by these constructs. No obvious symptoms were observed when N. benthaimiana were inoculated with SQ11, but SqLCV DNAs were detected by PCR and RCA in systemic leaves, indicating that SQ11 is a mild strain of SqLCV. When N. benthamiana infected by DNA A of SQ11 plus DNA B of SQ8, the leaves displayed severe downward curling symptom in the pseudo-recombination experiment. SqLCV DNAs were also detected in the upper uninoculated leaves. On the other hand, no symptoms were observed when N. benthamiana were inoculated DNA A of SQ8 plus DNA B of SQ11. Only DNA A, but not DNA B, could be detected in the upper uninoculated leaves. It is speculated that SQ11 DNA B may cause mild symptom in N. benthamiana. In order to investigate whether C4 protein is the pathogenecity determinant in muskmelon, a new infectious clone, designated SQ8mAC4, was constructed using inverse PCR to mutate the C4 protein coding region of SqLCV. No symptoms were observed when muskmelons were inoculated with SQ8mAC4 and SqLCV DNA were not detect in the inoculated and systemic leaves, suggesting that C4 protein might be required for the accumulation of SqLCV DNAs. The feasibility of cross protection was tested using SQ11 as the protecting mild strain. When N. benthamiana was challenged by SQ8 after inoculation with SQ11 for 10 days, the upper uninoculated leaves displayed downward curling symptom in the cross protection assay. The lack of cross-protection may result from the recombination between the genomes of SQ8 and SQ11, or synergistic effects of the two isolates. In the future work, the detailed mechanisms controlling the severity of symptoms by SqLCV will be further analyzed using the infectious clones constructed in this study. It is expected that through the understanding of this mechanism, effective disease management measures for geminivirus infections will be established.
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