Abstract
Geminiviruses are a group of small single-stranded DNA viruses that replicate in the host cell nucleus. It has been reported that the viral replication initiator protein (Rep) and the conserved common region (CR) are required for rolling circle replication (RCR)-dependent geminivirus replication, but the detailed mechanisms of geminivirus replication are still obscure owing to a lack of a eukaryotic model system. In this study, we constructed a bacterial–yeast shuttle plasmid with the autonomous replication sequence (ARS) deleted, which failed to replicate in Saccharomyces cerevisiae cells and could not survive in selective media either. Tandemly repeated copies of 10 geminivirus genomic DNAs were inserted into this deficient plasmid to test whether they were able to replace the ARS to execute genomic DNA replication in yeast cells. We found that yeast cells consisting of the recombinant plasmid with 1.9 tandemly repeated copies of tomato leaf curl Yunnan virus isolate Y194 (TLCYnV-Y194, hereafter referred to as Y194) can replicate well and survive in selective plates. Furthermore, we showed that the recombinant plasmid harboring the Y194 genome with the mutation of the viral Rep or CR was still able to replicate in yeast cells, indicating the existence of a non-canonic RCR model. By a series of mutations, we mapped a short fragment of 174 nucleotides (nts) between the V1 and C3 open reading frames (ORFs), including an ARS-like element that can substitute the function of the ARS responsible for stable replication of extrachromosomal DNAs in yeast. The results of this study established a geminivirus replication system in yeast cells and revealed that Y194 consisting of an ARS-like element was able to support the replication a bacterial–yeast shuttle plasmid in yeast cells.
Highlights
Geminiviruses are plants single-stranded DNA viruses that are characterized by unique twinned icosahedral particles
The mutation of viral replication-related proteins or elements in the recombinant plasmid (a bacterial–yeast shuttle plasmid deleted of an autonomous replication sequence (ARS)) with the Y194 genome was still able to replicate in yeast cells, suggesting the existence of a non-canonic
The resultant plasmids were transformed into yeast strain W303-1B cells, and yeast cells transformed with the original plasmid pRS316 and the ARS-deficient plasmid deficient yeast shuttle vector pRS316 (dpRS316) were used as positive controls and negative controls, respectively
Summary
Geminiviruses are plants single-stranded DNA viruses that are characterized by unique twinned icosahedral particles. The genome is composed of covalently closed circular single-stranded DNA of about 2.5–3.0 kb for monopartite geminiviruses, and about 4.8–5.6 kb for bipartite geminiviruses. Begomovirus represents the Viruses 2018, 10, 536; doi:10.3390/v10100536 www.mdpi.com/journal/viruses. Viruses 2018, 10, 536 largest genus, in which viruses are transmitted by the whitefly (Bemisia tabaci). The monopartite begomoviruses contain the genome, just like the DNA-A component of bipartite begomoviruses, and are often associated with betasatellites, which are very important as they induce severe symptoms in crops [4]. The unique βC1 open reading frame (ORF), encoded by the betasatellite complementary strand, is a pathogenicity determinant, and was reported to be a suppressor of both transcriptional gene silencing and post-transcriptional gene silencing [4,5,6]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
