Identificação e caracterização de um potyvírus isolado de Zinnia elegans

O presente trabalho teve como objetivo a identificação e caracterização de um potyvírus isolado de Zinnia elegans, na Região Noroeste do Estado de São Paulo. O potyvírus foi transmitido por inoculação mecânica e apresentou uma gama restrita de hospedeiras sendo que as espécies mais afetadas pertencem à família Asteraceae. Em SDS-PAGE, a massa molecular da proteína capsidial (CP) foi estimada em 33 kDa e, em "Western-blot", reagiu com anti-soro para o Bidens mosaic virus (BiMV). Um fragmento de aproximadamente 820 pb foi amplificado por RT/PCR, clonado e seqüenciado. O fragmento, que inclui o gene da proteína capsidial, mostrou similaridade de aminoácidos do "core" da CP variando de 55% (Tobacco vein mottling virus, TVMV) a 95% (Sunflower chlorotic mottle virus, SuCMoV) e da CP completa de 55% (TVMV) a 91% (SuCMoV). Na região N-terminal, o potyvírus de Zinnia tem uma deleção de quatro aminoácidos (posições 9 a 12 após o sítio de clivagem entre a proteína NIb e a CP) quando comparada com a seqüência do SuCMoV. A análise filogenética agrupou o potyvírus de Zinnia e o SuCMoV em um mesmo ramo em 100% das réplicas, mostrando uma relação de parentesco muito próxima entre esses dois vírus. Os resultados obtidos no presente trabalho demonstraram que o potyvírus de Zinnia e o SuCMoV são estirpes do mesmo vírus. Sugere-se o nome Sunflower chlorotic mottle virus, isolado Zinnia (SuCMoV-Zi), ao potyvírus encontrado em Z. elegans no Brasil.

Fluorescent labelling reveals spatial separation of potyvirus populations in mixed infected Nicotiana benthamiana plants

The distribution of potyviruses in mixed infected Nicotiana benthamiana plants was investigated by using green and red fluorescent proteins (GFP, DsRed). Full-length cDNA clones of Plum pox virus (PPV-NAT-AgfpS; PPV-NAT-red), Tobacco vein mottling virus (TVMV-gfp; TVMV-red) and Clover yellow vein virus (ClYVV-GFP) expressing fluorescent proteins, referred to here as labelled viruses, were used to characterize the distribution of different potyviral populations (e.g. TVMV-gfp/PPV-NAT-red), as well as populations of identical, but differently labelled potyviruses (e.g. PPV-NAT-AgfpS/PPV-NAT-red) or in mixed infections of potyviruses with labelled Potato virus X (PVX). Plants infected by any of the PVX/potyvirus combinations exhibited synergistic symptoms and large numbers of cells were doubly infected. In contrast, co-infections of differently labelled potyvirus populations appeared non-synergistic and remained predominantly separate in the infected plants, independent of whether different viruses or identical but differently labelled viruses were co-infecting. Contact of differently labelled virus populations that exhibited spatial separation was restricted to a small number of cells at the border of different fluorescent cell clusters.

Suppressor activity of potyviral and cucumoviral infections in potyvirus-induced transgene silencing

The process known as ‘recovery’ by which virus-infected plants become resistant to the infection is an interesting phenomenon where both RNA silencing and virus resistance fully converge. In a previous study, we showed that transgenic Nicotiana benthamiana NIbV3 plants, transformed with a mutated NIb coding sequence from Plum pox virus (PPV), showed a delayed, very specific, resistance phenotype, which was induced by the initial infection. This recovery was the consequence of the activation of an RNA silencing mechanism in the PPV-infected plant, which took place even though PPV encodes a silencing suppressor (HCPro). Making use of plants regenerated from the recovered tissue, which maintained the transgene silencing/virus resistance phenotype, we have demonstrated that both Cucumber mosaic virus (CMV) and Tobacco vein mottling virus (TVMV), expressing the silencing suppressor 2b and HCPro, respectively, were able to reactivate transgene expression. Surprisingly, only the silencing suppression caused by CMV, but not that originating from TVMV, was able to revert the recovered NIbV3 plants to a PPV-susceptible phenotype.

Effect of Aphid Behavior on Efficiency of Transmission of Soybean mosaic virus by the Soybean-Colonizing Aphid, Aphis glycines

The soybean aphid (Aphis glycines) was a poor vector (0.83% transmission) when the aphids were allowed overnight acquisition feed on Soybean mosaic virus (SMV)-infected soybean leaves. However, A. glycines was shown to be a very efficient vector (34.72% transmission) when individual aphids were allowed a 1-min acquisition probe on the same infected leaves used for the feeding treatment. Similar results were obtained with Myzus persicae and tobacco in transmission experiments of the potyviruses Tobacco etch virus (feeding: 1.36%; probing: 45.5%) and Tobacco vein mottling virus (feeding: 2.0%; probing: 47.5%). A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect SMV in single soybean aphids using a pair of primers designed to amplify a 469-bp PCR fragment in the coding region of SMV coat protein. In contrast to the low transmission rate obtained with the soybean aphids that acquired virus through overnight feeding, RT-PCR detected SMV in 100% of these aphids. Interestingly, the rate of SMV detection by RT-PCR in aphids that were allowed a 1-min acquisition probe (31.67%) coincided with percent transmission (34.72%). The practical application of RT-PCR in detecting nonpersistently transmitted viruses and its implications for virus epidemiology are discussed.