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. 2011 Mar 17:8:123.
doi: 10.1186/1743-422X-8-123.

The nucleotide sequence and genome organization of Plasmopara halstedii virus

Marion Heller-Dohmen  1 , Jens C Göpfert, Jens Pfannstiel, Otmar Spring
Affiliations

The nucleotide sequence and genome organization of Plasmopara halstedii virus

Marion Heller-Dohmen et al. Virol J. .

Abstract

Background: Only very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking.

Methods: Viral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis.

Results: The complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2) were established. RNA1 consisted of 2793 nucleotides (nt) exclusive its 3' poly(A) tract and a single open-reading frame (ORF1) of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR) of 18 nt and a 3' untranslated region (3' UTR) of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp) and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A) and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A) tract and a second ORF (ORF2) of 1128 nt. ORF2 coded for the single viral coat protein (CP) and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb) and RNA2 (ca. 1.4 kb) were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl.

Conclusions: The results showed the presence of a single and new virus type in different P. halstedii isolates. Insignificant viral sequence variation indicated that the virus did not account for differences in pathogenicity of the oomycete P. halstedii.

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Figures

Figure 1
Figure 1
Mass spectroscopic analysis of the coat protein (CP) of Plasmopara halstedii virus (PhV). A Reflector ISD-MALDI-TOF spectrum. The N-terminal sequence of the intact PhV CP was analyzed by Top-Down MALDI-TOF mass spectrometry. The in source decay (ISD) spectrum shows two major ISD fragment ions of the PhV CP with masses of 1409.74 Da and 1765.93 Da, respectively. These ISD fragment ions masses fit to sequences D74-R85 and D74-R88 of the deduced amino acid sequence of the PhV CP, respectively. The corresponding amino acid sequences of the ISD fragment ions were shown. B MS/MS spectrum of the 1409.74 Da ISD-fragment. The N-terminal sequence of PhV CP DYTVQSNSIVQR deduced from the reflector ISD spectrum (Fig. 1A) was confirmed by MS/MS analysis of the 1409.74 Da ISD fragment ion. The observed b- and y-fragment ions are annotated in the spectrum and the corresponding amino acid sequence covered by b- and y-ions were shown.
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References

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