A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing.

The single-stranded, positive-sense RNA genome of viruses in the genus Potyvirus encodes a large polyprotein that is cleaved to yield 10 mature proteins. The first three cleavage products are P1, HCpro and P3. An additional short open reading frame (ORF), called pipo, overlaps the P3 region of the polyprotein ORF. Four related potyviruses infecting sweet potato (Ipomoea batatas) are predicted to contain a third ORF, called pispo, which overlaps the 3' third of the P1 region. Recently, pipo has been shown to be expressed via polymerase slippage at a conserved GA6 sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA6 sequence, with higher slippage efficiency (∼5%) than at the pipo site (∼1%). Transient expression of recombinant P1 or the 'transframe' product, P1N-PISPO, in Nicotiana benthamiana suppressed local RNA silencing (RNAi), but only P1N-PISPO inhibited short-distance movement of the silencing signal. These results reveal that polymerase slippage in potyviruses is not limited to pipo expression, but can be co-opted for the evolution and expression of further novel gene products.

Sequence characterization of a Peruvian isolate of Sweet potato chlorotic stunt virus: further variability and a model for p22 acquisition.

Sweet potato chlorotic stunt virus (SPCSV) is probably the most important virus infecting sweetpotato worldwide, causing severe synergistic disease complexes with several co-infecting viruses. To date only one isolate (Ug), corresponding to the EA strain has been completely sequenced. It was later shown to be unusual in that, in contrast to most isolates, it encoded an additional p22 protein at the 3' end of RNA1. We report the complete sequence and genome organization of a Peruvian isolate of SPCSV (m2-47) as determined by siRNA deep sequencing. We confirm that the ORF encoding p22 is lacking from m2-47 and all tested Peruvian and South American isolates, whereas additional isolates containing p22 were identified from Uganda. Other potentially important genomic differences such as two small ORFs encoding putative small hydrophobic proteins instead of one, upstream the hsp70h gene and a more divergent sequence at its RNA1 3'-UTR in contrast to SPCSV isolates that contain p22 are discussed and a model for recent acquisition of p22 in Uganda is proposed. A role for p22 as a pathogenicity enhancer of SPCSV is also provided by complementary expression of p22 in transgenic sweetpotato plants.

Analysis of complete genomic sequences of isolates of the Sweet potato feathery mottle virus strains C and EA: molecular evidence for two distinct potyvirus species and two P1 protein domains.

The complete nucleotide sequence of the isolate C1 of Sweet potato feathery mottle virus (SPFMV) strain C and the 5' region of several other strains were determined and analyzed together with the sequences of isolates representing the EA, RC and O strains. This provided molecular evidence for the reclassification of SPFMV strains into two species and the occurrence of a complex recombinant isolate. Analysis also revealed a hypervariable domain in the P1 protein, which separates an N-terminal region unique to SPFMV and members of the ipomovirus species Sweet potato mild mottle virus from the C-terminal protease domain, which is conserved among all potyviruses.

PCR assays for the detection of members of the genus Ilarvirus and family Bromoviridae.

A PCR assay was developed for the universal detection of ilarviruses using primers designed to the RNA-dependent RNA polymerase gene in RNA2. The assay detected 32 isolates of 15 definite and 2 tentative ilarvirus species using a one-step RT-PCR. The assay was more specific, and at least as sensitive as a commercial assay, and allowed direct sequencing of amplicons. No cross-reaction was observed with neither healthy plants of 15 host species nor from isolates in other genera of the Bromoviridae. A further PCR assay targeting the helicase motif of RNA1 was able to detect all species tested within the family Bromoviridae, including members of the Alfamovirus, Anulavirus, Bromovirus, Cucumovirus and Ilarvirus. The assays provide a sensitive and cost-effective way for detecting and characterising members of the Bromoviridae and can be used for quarantine and certification programmes.

Elimination of antiviral defense by viral RNase III.

Sweet potato (Ipomoea batatas) is an important subsistence and famine reserve crop grown in developing countries where Sweet potato chlorotic stunt virus (SPCSV; Closteroviridae), a single-stranded RNA (ssRNA) crinivirus, synergizes unrelated viruses in co-infected sweet potato plants. The most severe disease and yield losses are caused by co-infection with SPCSV and a potyvirus, Sweet potato feathery mottle virus (SPFMV; Potyviridae). Potyviruses synergize unrelated viruses by suppression of RNA silencing with the P1/HC-Pro polyprotein; however, the SPCSV-SPFMV synergism is unusual in that the potyvirus is the beneficiary. Our data show that transformation of an SPFMV-resistant sweet potato variety with the double-stranded RNA (dsRNA)-specific class 1 RNA endoribonuclease III (RNase3) of SPCSV broke down resistance to SPFMV, leading to high accumulation of SPFMV antigen and severe disease symptoms similar to the synergism in plants co-infected with SPCSV and SPFMV. RNase3-transgenic sweet potatoes also accumulated higher concentrations of 2 other unrelated viruses and developed more severe symptoms than non-transgenic plants. In leaves, RNase3 suppressed ssRNA-induced gene silencing (RNAi) in an endonuclease activity-dependent manner. It cleaved synthetic double-stranded small interfering RNAs (siRNAs) of 21, 22, and 24 bp in vitro to products of approximately 14 bp that are inactive in RNAi. It also affected total siRNA isolated from SPFMV-infected sweet potato plants, suggesting a viral mechanism for suppression of RNAi by cleavage of siRNA. Results implicate RNase3 in suppression of antiviral defense in sweet potato plants and reveal RNase3 as a protein that mediates viral synergism with several unrelated viruses, a function previously described only for P1/HC-Pro.

Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses.

We report the first identification of novel viruses, and sequence of an entire viral genome, by a single step of high-throughput parallel sequencing of small RNAs from diseased, as well as symptomless plants. Contigs were assembled from sequenced total siRNA from plants using small sequence assembly software and could positively identify RNA, ssDNA and dsDNA reverse transcribing viruses and in one case spanned the entire genome. The results present a novel approach which cannot only identify known viral pathogens, occurring at extremely low titers, but also novel viruses, without the necessity of any prior knowledge.

Molecular variability of sweet potato feathery mottle virus and other potyviruses infecting sweet potato in Peru.

Several potyviruses are found infecting sweet potato (Ipomoea batatas) in Peru, of which sweet potato feathery mottle virus (SPFMV, genus Potyvirus) is the most common. However, sequence data for these viruses are not available from Peru. In this study, the 3'-terminal approximately 1,800 nucleotide sequences of 17 potyvirus samples collected from the six main sweet potato-producing areas of Peru over the past 20 years were determined and analyzed. Results of sequence comparisons and phylogenetic analysis showed that three of the four recognized SPFMV strain groups, including the East African strain, are established in Peru as well as two other potyviruses: sweet potato virus G (SPVG) and sweet potato virus 2 (SPV2). The analysis further revealed that SPFMV, SPVG and SPV2 are related and form an Ipomoea-specific phylogenetic lineage within the genus Potyvirus and identified for the first time recombination events between viruses from different strain groups of SPFMV.

Synergistic Interaction of Sweet potato chlorotic stunt virus (Crinivirus) with Carla-, Cucumo-, Ipomo-, and Potyviruses Infecting Sweet Potato.

Co-infection of Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus) with Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) results in sweet potato virus disease (SPVD), a synergistic disease that is widely distributed in the sweet potato (Ipomoea batatas) growing regions of the world. Since both SPCSV and SPFMV are common and often detected as part of multiple co-infections of severely diseased plants, the occurrence of synergistic interactions with other viruses was investigated. Data from this study show that SPCSV, but not SPFMV, can cause synergistic diseases in sweet potato with all viruses tested, including members of the genus Potyvirus (Sweet potato latent virus, Sweet potato mild speckling virus), Ipomovirus (Sweet potato mild mottle virus), Cucumovirus (Cucumber mosaic virus), and putative members of the genus Carlavirus (Sweet potato chlorotic fleck virus and C-6 virus). The synergism was expressed as an increase in the severity of symptoms, virus accumulation, viral movement in plants, and as an effect on yield of storage roots. The presence of a third different virus in plants affected with SPVD increased the severity of symptoms even further compared with SPVD alone. There was a positive correlation between increase in virus accumulation and symptom expression in double and triple SPCSV-associated co-infections. The epidemiological implications of the results are discussed.