Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-04-20T14:08:19Z No. of bitstreams: 1 texto completo.pdf: 472703 bytes, checksum: 809b2129de91ebfd2db6de72a8b436fa (MD5) / Made available in DSpace on 2017-04-20T14:08:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 472703 bytes, checksum: 809b2129de91ebfd2db6de72a8b436fa (MD5) Previous issue date: 2002-04-03 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / A família Geminiviridae, caracterizada pela morfologia de partículas icosaédricas geminadas e genoma composto por DNA de fita simples circular, é dividida em quatro gêneros, de acordo com o tipo de inseto vetor, gama de hospedeiros, organização do genoma e relacionamento filogenético. Os begomovírus possuem dois componentes genômicos, são transmitidos por mosca-branca e infectam dicotiledôneas. A partir de 1994 houve relatos sucessivos de begomovírus em tomateiro em diversos estados brasileiros. O sequenciamento parcial do genoma de alguns destes vírus revelou que há uma grande diversidade de espécies, provavelmente como resultado da disseminação de vírus nativos pelo biótipo B da mosca-branca Bemisia tabaci. O objetivo do presente trabalho foi a caracterização molecular de uma nova espécie de begomovírus detectada no município de Igarapé, MG, denominada Tomato chlorotic mottle virus, isolado MG-Ig1 (TCMV-[MG- Ig1]), e o estudo da ocorrência de eventos de pseudo-recombinação entre begomovírus que infetam tomateiro e Sida sp. Para a caracterização molecular, foram sintetizados dois pares de oligonucleotídeos sobrepostos com sítios de restrição que possibilitaram a amplificação do genoma completo (fragmentos de aproximadamente 2.600 nucleotídeos para os componentes A e B) do TCMV-[MG-Ig1]. Para o sequenciamento, o genoma completo foi divido em subclones sobrepostos e as seqüências foram montadas com o auxílio de programas apropriados. A análise comparativa das seqüências confirmou a identidade do TCMV-[MG- Ig1] como uma espécie do gênero Begomovirus. Para os estudos de pseudo-recombinação foram misturados componentes virais clonados em uma cópia e meia do TCMV (isolados BA-Se1 e MG-Be1), Tomato rugose mosaic virus (TRMV), Tomato yellow mottle virus (TYMoV) e Tomato golden mosaic virus (TGMV), provenientes de tomateiro, e Sida yellow mosaic virus (SYMV) e Sida mottle virus (SMoV), provenientes de Sida rhombifolia. A inoculação foi realizada via biobalística em plantas de Nicotiana benthamiana, e o diagnóstico da infecção foi confirmado via PCR, utilizando oligonucleotídeos universais para begomovírus. Foram observados sintomas sistêmicos nas plantas inoculadas com os pseudo- recombinantes recíprocos entre TRMV e TGMV, TCMV-[BA-Se1] e TGMV, além de TRMV-A + TYMoV-B, TRMV-B + TCMV-[MG-Be1]-A, TRMV-B + SYMV-A, TCMV- [BA-Se1]-A + TYMoV-B, TCMV-[BA-Se1]-B + SMoV e TCMV-[BA-Se1]-B + SYMV-A. As infecções foram confirmadas via PCR em todos os casos. Os sintomas observados variaram de acordo com a combinação, desde leve clorose entre as nervuras até mosaico amarelo e encarquilhamento severo das folhas. Não foram observados sintomas sistêmicos nas plantas inoculadas com os pseudo-recombinantes recíprocos entre TRMV e TCMV-[BA- Se1], além de TCMV-[BA-Se1]-B + TCMV-[MG-Be1]-A. Na combinação entre TRMV-B + SMoV-A foram observados sintomas sistêmicos, porém apenas o componente A de SMoV foi detectado via PCR. / The Geminiviridae family, characterized by a particle morphology of twinned, incomplete icosahedra and a single-stranded, circular DNA genome, is divided into four genera according to the type of insect vector, host range, genomic organization and phylogeny. The begomoviruses have two genomic components, are transmitted by whiteflies and infect dicot plants. Since 1994 there have been successive reports of begomovirus infection in tomato, in several states of Brazil. Partial sequencing of the genome of some of the isolates revealed a high degree of genetic diversity, probably due to the dissemination of indigenous viruses by the B biotype of the whitefly Bemisia tabaci. The objective of the present work was the molecular characterization of a new species of begomovirus detected at the city of Igarapé, MG, named Tomato chlorotic mottle virus, isolate MG-Ig1 (TCMV-[MG- Ig1]), and the study of pseudorecombination events among begomoviruses from tomato and Sida sp. for the molecular characterization, two sets of overlapping primers with a common restriction site were designed to amplify the full-length viral genome (approximately 2.600 nucleotide fragments for the DNA-A and DNA-B) of TCMV-[MG-Ig1]. The full-length clones were subcloned for sequencing, and the sequences were assembled using the appropriate computer programs. Comparative analysis confirmed the identity of TCMV-[MG- Ig1] as a species of the genus Begomovirus. For the pseudorecombination studies, cloned DNA components TCMV (isolates BA-Se1 and MG-Be1), Tomato rugose mosaic virus (TRMV), Tomato yellow mottle virus (TYMoV) and Tomato golden mosaic virus (TGMV), from tomato, and Sida yellow mosaic virus (SYMV) and Sida mottle virus (SMoV), from Sida rhombifolia were mixed and inoculated by particle bombardment into Nicotiana benthamiana plants. Viral infection was confirmed by PCR with general begomovirus primers. Sistemic symptoms were oberved in plants inoculated with the reciprocal pseudorcombinants between TRMV and TGMV, TCMV-[BA-Se1] and TGMV, and also with TRMV-A + TYMoV-B, TRMV-B + TCMV-[MG-Be1]-A, TRMV-B + SYMV-A, TCMV- [BA-Se1]-A + TYMoV-B, TCMV-[BA-Se1]-B + SMoV and TCMV-[BA-Se1]-B + SYMV- A. Infections were confirmed by PCR in all cases. The symptoms observed varied with each combination, from a mild internerval chlorosis to yellow mosaic and severe leak crumpling. Sistemic symptoms were not observed in plants inoculated with the reciprocal pseudorecombinants between TRMV and TCMV-[BA-Se1], and also with TCMV-[BA-Se1]- B + TCMV-[MG-Be1]-A. The combination of TRMV-B + SMoV-A yielded sistemic symptoms, but only the SMoV DNA-A was detected by PCR. / Dissertação importada do Alexandria

Chapter 3 Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this chapter, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. Further NSm was shown to be phosphorylated by N. benthamiana and tomato crude sap as observed in other movement proteins. Chapter 4 This chapter deals with localization of NSm to PD and identification of domain involved in localization. For this purpose NSm and its mutants were cloned in pEAQ:GFP vector and transiently expressed in N. benthamiana by infiltration of transformed Agrobacteria. The GFP tagged NSm was visualized by confocal microscopy. The results demonstrated that NSm forms punctate structures and localizes to PD as confirmed by colocalization of mCherry: PDLP1a, a PD marker which resides in PD, with GFP:NSm. To find out the domain involved in PD localization, sequential deletion mutants were made. It was found that C-terminal domain is involved in PD localization. On the other hand, N-terminal unfolded region was dispensable for PD localization. This is the first report of a coiled coil domain shown to be involved in PD localization. It has also been demonstrated that GBNV NSm interacts with NP. Further, membrane floatation assay carried in presence of NP suggested that interaction of NSm and NP affected membrane association of NSm. These results were further confirmed by localization studies of NSm in presence of NP. It was found that there was considerable relocalization of both NSm and NP. NSm was observed to be present in cytoplasm as well as on the membrane. At the same time, NP was observed on membrane apart from being present in the cytoplasm. When N-terminal 50 amino acids (unfolded) region of NSm was deleted and colocalization studies were carried out, it was found that NSm and NP do not colocalize, suggesting that NSm interacts with NP via the unfolded region and helps in the relocalization of NP to the membrane. Chapter 5 This chapter deals with the pathway of targeting NSm to PD. To decipher the pathway, followed by NSm, an inhibitor of endomembrane or vesicle mediated transport, Brefeldin A (BFA) was used. When GFP-NSm was expressed it was observed to form punctate structure at PD as before. Upon treatment with BFA, green islands were observed in the cytoplasm suggesting that ER was involved in targeting NSm to PD. Similarly, LatB, inhibitor of actin mediated targeting of protein to membrane, also abrogated the localization of NSm to PD. In order to further understand the role of ER in targeting NSm to PD, an ER marker, ER-GFP (GFP fused to HDEL peptide that directs it to ER) was coexpressed with GBNV NSm fused to mCherry. It was observed that NSm colocalizes with ER-GFP as yellow puncta on PD. The puncta appeared as patches and the whole ER-network was converted to vesicles. This was further confirmed by coexpressing ER-GFP with NSm without any tag. The green fluorescent vesicles were observed preferentially near cell membrane. To delineate the region of NSm involved in vesicle formation, point mutants and deletion mutants of NSm were generated without the tag and coexpressed with ER-GFP. When N-terminal 203 amino acids were deleted, NSm was able to transform ER membranes to vesicles suggesting that these residues are dispensable for vesicle formation. Interestingly, the deletion of coiled coil domain leads to cytosolic location of NSm. Furthermore, the C-terminal coiled coil domain when expressed alone was capable of inducing vesicle formation. This is the first report of involvement of such a domain in ER membrane association and vesicle formation.

The major obstacle many promising drugs struggle to overcome is the barrier imposed by the outer cell membrane. In addition to technologies such as liposomes and cell-penetrating peptides, more attention is being given to the class of proteins known as lectins to deliver therapeutic and antigenic proteins to the interiors of cells. Lectins bind to but do not modify sugars, and provide an efficient route to endocytosis. The galactose/N-acetyl-galactosamine specific lectin ricin B-chain (RTB) is especially attractive in possibly fulfilling a carrier role due to its well-characterized endocytotic trafficking and its efficacy over a wide range of cell types. By producing RTB recombinantly in plants it is possible to create a fully active, non-toxic carrier that does not rely on the processing of large amounts of toxic material (e.g. castor bean). Payload molecules such as small molecules and proteins can be attached to RTB via chemical conjugation at primary amine groups, without the loss of lectin or uptake activities. The biotin/streptavidin interaction and direct genetic fusion of polypeptides also provide efficient mechanisms for the attachment of payload proteins to RTB. An immunoglobulin domain-based scaffolding mechanism bridges modified RTB and payload proteins when co-expressed in Agrobacterium-infiltrated plant leaves. Carrier and payload proteins expressed in plants and E. coli, respectively, and purified independently are not able to assemble into an efficient carrier/payload arrangement. These findings show that plant cells are able to correctly produce the two components of the carrier/payload system and assemble them into an efficient and flexible capture and carry technology. / Ph. D.