Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis.

Baculoviruses have a biphasic replication cycle producing two types of virions, budded virus (BV) and occlusion derived virus (ODV) which are required for the systemic spread or oral infection with the insect host respectively. Little is known about the events of the BV pathway and the mechanism by which nucleocapsids are selected and directed from the nucleus to plasma membrane to form BV. The Autographa californica Multiple Nucleopolyhedrovirus (AcMNPV) gene exon0 (orf141) is known to be required for the efficient production of BV and in this study the function and mechanism by which EXON0 affects BV production was investigated. Confocal microscopic analysis showed that EXON0 localized in the nucleus in the ring zone of virogenic stroma where nucleocapsids are assembled. In addition EXON0 also concentrated in the cytoplasm at the plasma membrane. Analysis of virions revealed that EXON0 copurified with nucleocapsid fractions of both BV and ODV. In support of this yeast 2-hybrid screening, co-immunoprecipitation, and confocal microscopy revealed that EXON0 interacted with the known nucleocapsid proteins FP25 and BV/ODV-C42. Transmission electron microscopy showed that deletion of exon0 results in nucleocapsids being unable to efficiently egress from the nucleus to the cytoplasm. Cellular protein interaction analyzed by tandem affinity purification and co-immunoprecipitation showed that beta-tubulin co-purified with EXON0. Immunofluorescence also showed that EXON0 and microtubules co-localized during virus infection. The microtubule inhibitors colchicine and nocodazole affected the localization of EXON0 and significantly reduced BV production. These data support the conclusion that egress of AcMNPV nucleocapsids is facilitated by interaction of EXON0 with beta-tubulin and microtubules. Deletion and point mutation analysis mapped domains of EXON0 required for efficient budding, dimer formation and association with FP25, BV/ODV-C42 and beta-tubulin. The Leucine zipper domain was required for dimer formation, beta-tubulin and BV/ODV-C42 interaction and also reduced interaction with FP25. Multiple domains were also shown to affect BV production. This study provides a detailed analysis of EXON0 which is one of the first baculovirus genes shown to be specific for the BV pathway. The results extend our understanding of the BV pathway which is a major determinant of baculovirus pathogenesis. / Land and Food Systems, Faculty of / Graduate

Orientador: Mara Cristina Pessôa da Cruz / Resumo: Nas áreas de cultivo de cana-de-açúcar em que se empregam mudas pré-brotadas, o transplantio é um momento crítico, e há necessidade de adoção de práticas de manejo que levem ao enraizamento rápido, de modo que irrigações suplementares possam ser reduzidas ou dispensadas. A adubação, sobretudo o uso de fósforo e de nitrogênio, pode ser um fator de aceleração do enraizamento. Deste modo, objetivou-se, com o presente estudo, avaliar o enraizamento de mudas pré-brotadas de cana-de-açúcar em função do fornecimento de nitrogênio e fósforo, buscando a redução de tempo para o início da emissão radicular após o transplantio. O experimento foi conduzido em casa de vegetação, utilizando amostra da camada superficial (0-20 cm) de solo de textura média, com baixo teor de fósforo. Os tratamentos foram arranjados em esquema fatorial com três fatores (doses de nitrogênio, doses de fósforo e tempos de crescimento), em delineamento inteiramente ao caso com cinco repetições. As doses de nitrogênio e fósforo foram equivalentes a 0 e 100 mg dm-3, e os tempos de crescimento foram 7, 14, 21 e 28 dias. O experimento foi conduzido empregando mudas do cultivar RB97 5201, produzidas em condições comerciais. Em cada data de avaliação foram determinados comprimento, área, diâmetro médio e densidade de raízes, matéria seca de raízes e da parte aérea e concentrações e acúmulos de nitrogênio e de fósforo na parte aérea. O aumento da concentração de nitrogênio no solo promoveu diminuição no diâmetro das raíz... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In sugarcane cultivation areas where pre-sprout seedlings are used, transplanting is a critical moment and there is a need to adopt methods that lead to fast rooting, so that additional irrigations can be minimized or dispensed. Fertilization, especially the use of phosphorus and nitrogen, can be a factor of rooting acceleration. Thus, the objective of the present study was to evaluate the rooting of pre-sprouted sugarcane seedlings due the supply of nitrogen and phosphorus, aiming to reduce the time to start root emission after transplanting. The experiment was carried out in a greenhouse, using a sample of sandy loam topsoil layer (0-20 cm) with low phosphorus content. The treatments were arranged in a factorial scheme with three factors (nitrogen doses, phosphorus doses and growth times), in a full design with five replicates. The nitrogen and phosphorus doses were equivalent to 0 and 100 mg dm-3 N or P, and growth times were 7, 14, 21 and 28 days. The experiment was conducted using seedlings of cultivar RB97 5201, produced under commercial conditions. At each evaluation date, length, area, root average diameter, root density, dry matter in roots and shoot, and concentration and accumulation of nitrogen and phosphorus in shoot were determined. The increase in concentration of nitrogen in the soil promoted the reduction of root diameter in the sugarcane seedlings at 28 days after transplantation. The increase in concentration of phosphorus in the soil decreased the area, di... (Complete abstract click electronic access below) / Mestre