Identification and functional characterization of P1N-PISPO, a new gene product present in sweet potato potyviruses
[eng] Sweet potato feathery mottle virus (SPFMV) (Potyvirus genus, Potyviridae family) causes important yield losses in sweet potato crops, in particular in co-infections with the unrelated crinivirus Sweet potato chlorotic stunt virus (SPCSV). This thesis addresses the characterization of some nove...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2016 |
| País: | España |
| Institución: | Universidad de Barcelona |
| Repositorio: | Dipòsit Digital de la UB |
| OAI Identifier: | oai:diposit.ub.edu:2445/103390 |
| Acceso en línea: | https://hdl.handle.net/2445/103390 http://hdl.handle.net/10803/396653 |
| Access Level: | acceso abierto |
| Palabra clave: | Virologia molecular Pèptids Plantes Moniatos Molecular virology Peptides Plants Sweet potatoes |
| Sumario: | [eng] Sweet potato feathery mottle virus (SPFMV) (Potyvirus genus, Potyviridae family) causes important yield losses in sweet potato crops, in particular in co-infections with the unrelated crinivirus Sweet potato chlorotic stunt virus (SPCSV). This thesis addresses the characterization of some novel aspects in the infectious cycle of SPFMV, such as the expression, production and function of a new gene product named P1N-PISPO. A better understanding of SPFMV genome organization and the functions of their gene products might be relevant to improve the control strategies against this virus and the associated diseases in sweet potato crops. The positive-sense RNA genome of SPFMV contains a large ORF, translatable as a polyprotein yielding a set of functional mature gene products (P1, HCPro, P3, 6K1, CI, 6K2, VPg-Nla, Nlb and CP), and a short ORF named PIPO in the -1 frame, embedded within the P3 region. In addition to this organization, common to all the members of the Potyvirus genus, another ORF named PISPO was predicted in the genome. PISPO is in the -1 frame within the P1 region of SPFMV and other related potyviruses, starting at a conserved G1_2A6_7 motif, similar to the motif found upstream of PIPO. The expression of PISPO during SPFMV viral infection could result in the production of a putative new gene product P1N-PISPO. In the present work, the presence of the PISPO frame has been investigated in a Spanish isolate of SPFMV infecting Ipomoea batata plants. The genome sequence of this isolate has been assembled from NGS data, showing that the expected trans-framed PISPO sequences is present, preceded by a G2A6 motif. A specific analysis of the NGS data has revealed a significant proportion of transcripts with an extra A in the motif at the beginning of PISPO, as well as a lower proportion of transcripts with an extra in the corresponding conserved motif preceding the PIPO region. These results have demonstrated that a polymerase slippage mechanism could generate transcripts containing extra A residues (G2A7) to allow the translation of P1N-PISPO and P3N-PIPO gene products. Analysis of the viral gene products present in SPFMV infected plant tissues has been performed using LC-MS/MS after separation in SDS- PAGE, focusing in products > 50KDa. Peptides corresponding to the P1 protein have been detected from both the N-terminal portion (11 different peptides, 39% coverage), before the frameshifting signal and therefore common for P1 and P1N-PISPO, and in the C- terminal part (2 peptides exclusive for P1, 10% coverage). Interestingly, four peptides exclusive of PISPO, in its unique ORF (21.3% coverage), have been also found. These results have confirmed that both products P1 and P1N-PISPO are expressed and coexisted during SPFMV infection. Furthermore, transient expression of SPFMV gene products coagroinfiltrated with a reporter gene in Nicotiana benthamiana have revealed that P1N-PISPO acts as an RNA silencing suppressor, a role normally associated with HCPro in other potyviruses. Moreover, mutation of WG/GW motifs present in P1N-PISPO abolished its silencing suppression activity, suggesting that the function might require interaction with Argonaute components of the silencing machinery, as was shown for other viral suppressors. Altogether, the results of this thesis have confirmed the expression of P1N-PISPO during SPFMV infection and they have revealed a polymerase slippage mechanism as the responsible of P1N-PISPO production. Our results also have demonstrated the role of P1N-PISPO as a RNA silencing suppressor. |
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