New insights of Cowpea mild mottle virus (CPMMV) infection in soybean: the involvement of viral replicase in symptoms induction and its interaction with host factors

• Ano de defesa: 2017
• Autor(a) principal: Zanardo, Larissa Goulart
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa
• Palavras-chave em Português: Soja - Doenças e pragas; Carlavirus; Vírus - Genética; Recombinação (Genética); Genética Molecular e de Microorganismos
• Link de acesso: http://www.locus.ufv.br/handle/123456789/11660

Cowpea mild mottle virus (CPMMV, family Betaflexiviridae and genus Carlavirus) is an emerging virus in Brazil. The virus is transmitted by the whitefly Bemisia tabaci and it infects hosts preferentially of Fabaceae family, being found in different crops in almost all the continents in the world. In this work, several aspects about biology and genetics of CPMMV were dicussed and the CPMMV-host relationship was explored. CPMMV causes varied symptoms in plants in the fields and in greenhouse. The nature of this symptoms variation was determinate in this work using successive inoculations of a CPMMV Brazilian isolate causing necrosis in the CD206 soybean cultivar. Successive inoculations led to the isolate, which previously had caused necrosis to induce mild symptoms (mosaic and vein clearing). This was verified after six successive inoculations of a soybean sample from the field and also from a local lesion of a viral isolate, and thus submitted to a genetic bottleneck, in Nicotiana benthamiana leaves. We have shown that altering of symptoms pattern increased the fitness of the virus and vector, suggesting that the induction of different symptoms by viral variants is an adaptive advantage. The viral region involved in the induction of CPMMV symptoms was viral replicase (encoded by ORF1), different sites distributed throughout the recombinant blocks of ORF1 were associated with phenotype changes. In this work, also shought to the understanding about the replication of CPMMV in soybean identifying factors necessary for replication. To date, all that has been known about betaflexiviruses’s replication is that it occurs in the cytoplasm. None host factor was known. Here, two host proteins able to interact with the RNA-dependent RNA polymerase domain (RdRp) from a yeast two-hybrid vector-constructed soybean cDNA library were indentifyed. For the library construction, soybean plants cv. CD206 with CPMMV infection after 0, 3, 7 and 14 days of inoculation were used. The proteins identified were: the CSC1 type ERD4 (GmERD4) and an inositol methyltransferase (GmIMT). The interaction was confirmed by yeast two-hybrid assays and by Bimolecular Fluorescence Complementation (BiFC) assays. Prediction and subcellular localization of both proteins and the RdRp domain were also performed. GmERD4 was located in the endoplasmic reticulum (ER), GmIMT presented cytoplasmic location while the RdRp domain induced punctual structures in the plasma membrane and on the ER network. Analysis of the expression of both genes in soybean plants infected with CPMMV demonstrated that the they were induced after 3 and 7 days of inoculation. Overexpression of GmERD4 and GmIMT was performed on soybean protoplasts infected with CPMMV and it was found that overexpression of GmERD4 increased viral accumulation after 7 days of infection. Viral replicase is involved in different viral infection processes, acting not only on genome replication, but also on the induction of symptoms. This reinforces the multifunctional nature of viral proteins.