Summary auto-generated
Researchers successfully created infectious full-length cDNA clones of PVY-N605, a Swiss necrotic isolate of potato virus Y, overcoming previous cloning difficulties in E. coli. The main problem was toxicity caused by expression of the CI gene from cryptic prokaryotic promoter elements within the viral genome. To resolve this, the researchers maintained the viral cDNA as two overlapping subclones that were ligated before infection. The complete cDNA could be propagated as separate fragments or, with lower stability, as a single piece in certain E. coli strains. By replacing the 35S promoter with SP6 and capping RNA transcripts with m7G(5')ppp(5')G, the researchers obtained infectious transcripts. Both the cDNA and RNA transcripts proved infectious when delivered via biolistic (particle gun) or mechanical inoculation to three Nicotiana species and potato plants, though with varying efficiency. In vitro transcripts generally showed better infectivity than DNA inoculations, particularly on some plant species. Viruses derived from the clones developed typical symptoms with a 2-5 day delay compared to wild-type virus, but progeny viruses produced symptoms indistinguishable from wild-type PVY-N605.
Key findings
- Full-length PVY cDNA toxicity in bacteria stems from the CI gene region, which was overcome by splitting the genome into two stable subclones that could be ligated after propagation
- In vitro transcripts capped with m7G(5')ppp(5')G were more consistently infectious than cDNA via mechanical inoculation
- Biolistic delivery enabled successful infection on potato plants where mechanical inoculation failed
- Progeny viruses from infected plants produced normal symptoms and viral proteins indistinguishable from wild-type PVY-N605
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Abstract
A full-length cDNA clone of the potato virus Y (PVY) genome was obtained after cloning difficulties in Escherichia coli were overcome. These difficulties were mainly due to the expression of the CI gene from upstream prokaryotic promoter-like elements within the PVY genome. To overcome this problem, PVY cDNA was maintained in two subclones which were ligated before infection. A plasmid in which these two fragments were contained could be propagated in some E. coli strains but was unstable and yielded only low levels of plasmid DNA. Replacement of the 35S promoter by the SP6 promoter slightly increased the stability of the plasmid and its RNA transcripts were infectious when capped with m7G(5')ppp(5')G. Using two inoculation methods (mechanical or biolistic) the cDNA and its RNA transcript proved infectious on three Nicotiana species and on Solanum tuberosum.