| Summary: | 博士 === 國立中興大學 === 生物科技學研究所 === 96 === Infectious clones of viruses are essential tools in both the basic and applied researches in virology. Most infectious clones of geminiviruses consist of (partial) tandem repeats of viral genomes in the vectors, which usually involve tedious, multi-step assemblies of genomic fragments during the constructions. To enhance the efficiency, a simplified procedure was devised in this study, which employed limited restriction digestions of the multimeric viral genomes produced by rolling circle amplification (RCA), followed by direct cloning into the proper vectors. The efficiency of the procedure and the infectivity of the dimeric constructs were demonstrated using three different geminiviruses, Ageratum yellow vein virus Nan-Tou isolate (AYVV-NT equal to AYVTV-NT), Tomato leaf curl virus Tainan isolate (TLCV-TN), and Squash leaf curl virus Yunlin isolate (SqLCV-YL). This procedure simplifies significantly the construction of multimeric infectious clones of geminiviruses, and may further be applied in both basic and practical research to viruses with circular genomes.
Viruses with segmented genomes suffer from the constraint that all segments must enter the same infection foci to initiate a successful infection. However, there is no apparent preference for monopartite genomes in the evolutionary history of begomoviruses. To compare the infection efficiency among geminiviruses with monopartite or bipartite entities, construction of a monopartite infectious clone of bipartite Squash leaf curl virus (SqLCV) was conducted. By limited digestion of rolling circle amplification (RCA) products, the tandem DNA A and B repeats of SqLCV was generated, pre-ligated, and further cloned into pBin19 vector to give the monopartite chimera named as pBinSqLAB10 Agroinfection of equivalent inoculums harboring mono- or bi-partite infectious clones with the decimal series dilutions (101~103), the infection efficiency of bipartite construct dropped drastically, but the high infection rates maintained on pBinSqLAB10-inoculated plants. Moreover, small defective DNAs derived from pBinSqLAB10-inoculated plants were detected by inverse PCR and accumulated stably dependent on the machineries of host plants. Through the cloning and sequencing analyses, all inverse PCR products reveled the constancy on consisting of partial chimeric sequence of DNA A and B compassing with a cognate replication origin. These data illustrated that the unit-length genomes of geminiviruses released from tandem-repeated construct were through the RCR mechanism rather than homologous recombination. This report firstly demonstrated that the construction of artificial monopartite chimera enhanced the infectivity of bipartite geminiviruses and presented the feasibility on creation of efficient viral vectors for multipartite viruses with segmented genomes.
Replication of genomic DNAs of plant-pathogenic begomoviruses has been demonstrated in prokaryotes, which supported the possibility of analyzing DNA replication process of begomoviruses in bacteria. However, previous studies indicated that the replication of begomovirus DNAs in prokaryotes requires tandem constructs of viral genomes with at least two copies of the origin of replication (ori). Since the rolling circle replication (RCR) systems of geminiviruses and phage M13 share substantial similarities, it is possible that the machinery of phage M13 may assist the replication of geminiviruses in bacteria. In this study, phage M13 vector harboring the unit-length genome with only a single copy of ori of a monopartite begomovirus, Ping-Dong strain of Ageratum yellow vein virus (AYVV-PD), was constructed and used to investigate the replication of AYVV-PD DNAs in Escherichia coli JM101. The generation of single-stranded, circular DNAs (sscDNAs) corresponding to the unit-length AYVV-PD genome of both polarity was observed and verified by Southern blot analyses, nuclease digestions and two-dimensional neutral/alkaline agarose gel electrophoreses (2D-AGE). Replication-associated protein (Rep) of AYVV-PD was detected only in bacteria generating the corresponding sscDNAs, whereas disruption of the Rep gene abolished the phenomenon. The results suggested that a single copy of ori is sufficient for the prokaryotes to support the generation of unit-length, genomic sscDNAs of begomoviruses, which requires the presence of functional Rep protein.
Initiation of replication in differentiated host cells that have already exited S-phase of cell cycle has been an important subject of researches for geminiviruses. As known the prokaryotic-type machineries supported the replication of begomoviruses, we proposed that self-proliferating, prokaryotic-type organelle, chloroplasts, might be the proper location for initiating the geminiviruses replication when viruses invaded the non-dividing cells. In this study, the replicative forms of viral DNA could be detected by strand-specific PCR (Sts-PCR) in purified chloroplasts of AYVV-infected plants. This implies that geminiviruses possible to do replicate in the bacterial-descended chloroplasts. Alternatively, the infectious recombinants (Rep-eGFP or Cp-eGFP) expressed the viral proteins fused with enhanced green fluorescence protein were constructed. After co-agroinfected the Nicotiana benthamiana, small portion of mesophyll cells of the symptomatic leaf (epinasty) expressed the eGFP proteins visualized by fluorescence microscope. In the future, this system will apply to observe the viral proteins subcellular localizations in vivo by using confocal. The observations of the viral proteins associated with the replication of geminivirus DNAs in chloroplasts may provide additional information on geminivirus life cycles and evolution.
Taken together, these studies suggested a common origin of evolution among different types of DNA replication machineries and indicated that the geminiviruses might have maintained the ability to utilize diverse DNA replication systems including eukaryotic- and prokaryotic-type machineries during the evolutionary process.
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