Abstract
BackgroundOnly 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.MethodsViral 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.ResultsThe 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.ConclusionsThe 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.
Highlights
IntroductionIsometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower
Very few viruses of Oomycetes have been studied in detail
Genome organization Based on the recent comparison of a partial nucleotide sequence of Plasmopara halstedii virus (PhV) in various samples of P. halstedii [1], the viral isolate Ph8-99 was selected for complete sequencing of the two singlestranded RNA (ssRNA) segments and genome analysis
Summary
Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. Only the virions of Sclerophthora macrospora and Plasmopara halstedii have been studied more in detail. Sclerophthora macrospora virus A (SmV A) virus B (SmV B) are the only virions of Oomycetes of which the genome has yet been fully characterized [2,3]. They were isolated from Japanese isolates of S. macrospora, the downy mildew pathogen of Oryza sativa and other species. P. halstedii is a worldwide distributed pathogen with a broad spectrum of pathotypes (physiological races) [6], causing sunflower downy mildew infections. A more recent screening of P. halstedii isolates from different countries showed the occurrence of morphologically and biochemically indistinguishable virions in all samples independent of their host’s origin or pathotype or fungicide tolerance [1]
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