| Summary: | 博士 === 國立中興大學 === 植物病理學系所 === 98 === The virus-host interaction is one of the most interesting topics for virologists. Often time that challenges virologists to embark on projects that are unprecedented. In this dissertation, two unprecedented projects were explored: (1) to develop a reverse genetics system for a plant negative-strand RNA virus and (2) to apply infectious DNA clones of a circular single-strand DNA virus for its functional genomics study.
The term, reverse genetics, is used in molecular virology to describe the generation of viruses owning its genome derived from cDNA clones. Since the first development of a negative-strand RNA virus entirely from cDNA clones in 1994, similar reverse genetics systems have been established for several members of the Mononegavirales Order. A breakthrough of the reverse genetics development occurred in 1996 when a segmented virus, with three genomic segments, of the Bunyaviridae Family was generated from cloned cDNAs. Following that, a more complex influenza virus, whose genome contains eight RNA segments, was generated from cloned cDNAs in 1999. Whether the negative-strand RNA viruses are segmented or non-segmented, similar strategies of reverse genetics system were used by virologists. Thus, the reverse genetics system has become a popular, reliable and powerful tool to dissect the virus life cycle, the role of viral proteins and the interaction between viruses and hosts. To date, there is still no efficient routine reverse genetics system for detailed molecular investigation and functional genomics elucidation of any member of the plant negative-strand RNA viruses including tospoviruses. Tospoviruses, in the Family Bunyaviridae, are a group of enveloped plant viruses containing a tripartite single-stranded negative-sense RNA genome. In theory, we can follow the model of the reverse genetics applied in animal negative-strand RNA virus to develop a reverse genetics system for a tospovirus in conjunction with the eu-/pro-karyote RNA expression system and the transgenic plants that express the essential viral proteins. In this dissertation, we explored the development of minireplicon or reverse genetics systems for the Watermelon silver mottle tospovirus (WSMoV) via the development of the T7 RNA polymerase and RNA polymerase II RNA expression systems. In T7 RNA polymerase RNA expression system, the genomic RNAs of WSMoV or green fluorescence gene (GFP)-containing S-like RNA were in vitro expressed from DNA clones. The RNA transcripts, genomic RNAs mixture or S-like RNA alone, were mechanically inoculated onto L protein-expressed N transgenic plants or WSMoV-infected plant. There was no replicated RNAs detected in inoculated leaves and systemic leaves and no GFP expressed in WSMoV-infected plants. Although the in vitro RNA transcription and L and N proteins expression were functional, the efficiency of RNAs inoculation was indeed a stumbling block in T7 RNA polymerase expression system. For RNA polymerase II RNA expression system, GFP-containing S-like RNA were cloned and driven by 35S promoter. When RNAs transiently expressed from cDNA clones with agro-infiltration in plant tissue, the full-length S-like RNA was not detectable. Results of northern blot showed that the intergenic region (IGR) of viral RNAs might disrupt the transcription of RNA polymerase II. Although the construct of IGR deletion was made and full-length RNA was detected, the expression of GFP is not detected. It may be due to that there is no interaction or recognition between viral proteins and virus-like RNAs. The efficiency of protein-RNA interaction is the major and the most important barrier to both strategies for developing a reverse genetics system for plant negative-strand RNA viruses.
Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus that causes extensive damage in tomato and cucurbitaceous plants. An isolate of ToLCNDV was isolated from an oriental melon plant exhibiting leaf curl and puckering symptoms, and was designated as Tomato leaf curl New Delhi virus oriental melon isolate (ToLCNDV-OM). The pathogenicity and mechanical transmissibility of ToLCNDV-OM was confirmed by mechanical inoculation of the progeny virions from the agroinfected Nicotiana benthamiana onto oriental melon which developed symptoms similar to those observed in the fields. The most interesting and significant discovery was that there were no symptoms observed and no virus detected on ToLCNDV-OM-inoculated tomato plants. Detailed sequence comparison showed that the N terminus of nuclear shuttle protein (NSP) of ToLCNDV-OM is shorter than those of the tomato isolate (ToLCNDV-Svr, NCBI GenBank accession No.: U15017) and cucumber isolate (ToLCNDV-Cuc, NCBI GenBank accession No.: AB330080) by 13 and 28 amino acids, respectively. A mutant, with the addition of 13 amino acids sequences to the N-terminal, was made to the NSP gene of ToLCNDV-OM. The progeny virions of these two mutants were individually mechanically inoculated from the agroinfected N. benthamiana to tomato plants. Three weeks post-inoculation, the systemic symptoms of mosaic and leaf curling on tomato were observed and the mutated viruses were detected by polymerase chain reaction. These results indicate that the N-terminal sequences, loss on the NSP of ToLCNDV-OM, may be involved in its ability to systemically infect tomato plants.
Although it was difficult and only partially successful so far, the eventual development of a reverse genetics system for tospoviruses will be the efficient technique to manipulate viral genomes. On the contrary, the infectious DNA clones of begomoviruses are easier to establish and will contribute enormously for functional genomics study.
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