Estudio de la Variabilidad Genética del Sweet potato feathery mottle virus (SPFMV) y virus relacionados existentes en camote [Ipomea batatas (L.)]

En el Perú son varios los potyvirus que se encuentran infectando el camote (Ipomoea batatas), siendo el mas común el Sweet potato feathery mottle(SPFMV). Sin embargo, actualmente no existen datos de secuencia nucleotídica par estos virus. En el presente estudio, se determinaron y analizaron los datos de la secuencia nucleotídica de la región 3’ terminal (~1800 pb) de 17 aislamientos de potyvirus colectados de campos que se encuentran en las seis principales zonas de cultivo de camote en el Perú. Los resultados de la comparación de secuencias y análisis filogenéticos mostraron que tres de las cuatro cepas que son reconocidas dentro del SPFMV, se encuentran en el Perú, incluyendo la cepa Este de Africa. Del mismo modo, se encontraron aislamientos pertenecientes de otros dos potyvirus denominados sweet potato virus G(SPVG) y sweet potato virus 2 (SPV2). Análisis posteriores revelaron que SPFMV, SPVG y SPV2 son virus relacionados y forman una línea filogenética dentro del genero Potyvirus que tienen como hospederos a plantas del genero Ipomoea. El estudio también reporta la ocurrencia de eventos de recombinación entre aislamientos de diferentes cepas de SPFMV.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 ~1,800 nucleotide sequences of 17 potyvirus samples collected from the six main sweet potato-producing areas of Peru 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.Tesi

Molecular variability, genetic relatedness and a novel open reading frame (pispo) of sweet potato-infecting potyviruses

Sweet potato feathery mottle virus (SPFMV, genus Potyvirus) infects sweet potato wherever it is cultivated. In single infections, SPFMV often causes only mild symptoms a situation that changes dramatically when it co-infects the plants with Sweet potato chlorotic stunt virus (SPCSV, genus Crinivirus). Co-infection generates the sweet potato virus disease (SPVD), the most devastating viral disease of sweet potato that can cause total loss of yield. Previous studies have described RNase3 as the SPCSV protein responsible for the occurrence of SPVD. However, attempts to find resistance to SPVD based on this knowledge have failed, suggesting possible SPFMV determinants involved in the development of SPVD. This study aimed to contribute to the molecular understanding of SPFMV, in particular regarding its phylogenetic relationships, genomic structure, and ability to suppress RNA interference (RNAi). Deployment of adequate control measures requires proper characterization of the pathogen. Phylogenetic relationship of SPFMV isolates and closely related potyviruses was ambiguous due to misidentification of viruses often found in mixed infections, and lack of specific methods of detection. In this study, we sequenced the complete genome of SPFMV strain C and found phylogenetic evidence to support its reclassification as a different species, Sweet potato virus C (SPVC). Furthermore, we demonstrated that Sweet potato feathery mottle virus and Sweet potato virus C together with Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2) were found to cluster into one unique monophyletic subgroup among the potyviruses, the so-called SPFMV-group . Members of the SPFMV-group contain some unique genomic features. Based on pairwise comparisons of partial and complete genome sequences we found recombinant isolates in SPFMV and SPVC. We also identified, novel viral determinants characteristic for the SPFMV-group of potyviruses, mainly in the P1 cistron, a region known for its high variability among potyviruses. An additional domain at the N terminus of P1 (P1-N) which is not found in other potyvirus, but is found in sweet potato mild mottle virus (genus Ipomovirus) is invariably found in members of the SPFMV-group . The P1-N domain is followed by a hypervariable region which contains specific hallmarks for each member of the SPFMV-group , and is becoming a promising region for their rapid detection and characterization. However, perhaps the most remarkable finding was the identification of an extra open reading frame (ORF) overlapping the C-terminal part of P1, and which was designated as pispo (Pretty Interesting Sweet potato Potyvirus ORF). pispo is translated as a result of a transcriptional slippage mechanism occurring in the members of the SPFMV-group of potyviruses and results in a novel protein P1N-PISPO. Plants rely on RNAi, an antiviral defense machinery, to destroy viral ribonucleic acid (RNA) molecules recognized inside cells, and spread the alert signal to neighboring cells. To evade RNAi, viruses encode proteins termed suppressors. Our analyses revealed that both P1 and P1N-PISPO contain RNAi suppressor (RSS) activity. Hence, SPFMV utilizes novel suppressors expressed from the same P1 region in different reading frames. The protein P1N-PISPO suppresses cell-to-cell movement of silencing, probably by blocking the spread of signaling to neighboring cells. In contrast, P1 protein suppresses silencing only locally. In both cases, a conserved Glycine/Tryptophan (GW) motif located in the P1N part of P1 plays a crucial role in the RSS activity. On the other hand, HC-Pro, a widely known RSS protein of potyviruses was not able to suppress silencing in SPFMV. This particular arrangement, where the RSS activity resides on P1 region and not in HC-Pro, is not reported in potyviruses, but is known, e.g., in the members of the genus Ipomovirus of the Potyviridae family. Taxonomic, structural and functional findings of this study will contribute greatly to the understanding of the evolution of SPFMV, and characterization of diseases it causes.Bataatin höyhenlaikkavirus (SPFMV, suku Potyvirus) tartuttaa bataattia kaikkialla, missä bataattia viljellään. Yksinään SPFMV aiheuttaa useimmiten vain lieviä oireita, mutta jos kasveilla on samanaikaisesti bataatin kitukasvuvirus (SPCSV , suku Crinivirus) tartunta, tilanne muuttaa dramaattisesti. Kahden viruksen yhteistartunta johtaa ns. bataatin virustautiin (SPVD-tauti), joka on bataatin pahin virustauti, ja se voi johtaa jopa täydelliseen sadonmenetykseen. Aikaisemmat tutkimukset ovat osoittaneet, että SPCSV:n RNase3-proteiini yhdessä SPFMV:n kanssa on riittävä aiheuttumaan SPVD-taudin. Yritykset kehittää kestävyyttä bataatin SPVD-tautia vastaan tämän tiedon perusteella ovat kuitenkin epäonnistuneet, mikä voi johtua siitä, että myös SPFMV tarvitaan SPVD-taudin kehittymiseen. Tämän tutkimuksen tarkoituksena oli lisätä molekyylitason ymmärrystä SPFMV:sta ja erityisesti sen fylogeneettisistä sukulaissuhteista, genomirakenteesta ja kyvystä estää RNA-hiljennystä

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 GA(6) sequence. Here, we show that pispo is also expressed via polymerase slippage at a GA(6) sequence, with higher slippage efficiency (approximate to 5%) than at the pipo site (approximate to 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.Peer reviewe

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