Dissertations / Theses on the topic 'Bean common mosaic virus'

Survey of crops of common bean (Phaseolus vulgaris L.) in Sonora, Mexico revealed the presence of two isometric viruses and one flexuous rod virus on the basis of host reaction, particle morphology, serology and physico-chemical properties. The isometric viruses were identified as Bean Southern Mosaic Virus (BSMV) and Cowpea Chlorotic Mottle Virus (CCMV); the flexuous rod virus was identified as Bean Common Mosaic Virus (BCMV). Using bean cultivar differentials, two strains of the potyvirus BCMV were identified, NY-15 and a previously undescribed strain designated YV-1. Host range, serological tests, and RNA electrophoresis indicated that the Sonoran BSMV cultures are similar to BSMV-strain A. Serology and RNA-electrophoresis indicated that the Sonoran CCMV isolates are identical to CCMV-strain A. BSMV and CCMV were always isolated as a mixture from seed lots and from field collected bean tissue. BCMV occurred alone or in mixed infections with BSMV and CCMV. BCMV was seed transmitted with an average efficiency of 58 percent. The BSMV-CCMV mixture was transmitted with an efficiency of 6 percent. BSMV and CCMV were seed transmitted together, but separate transmission of BSMV or CCMV was not detected. Commercial seed lots from two major bean growing regions of Sonora (Hermosillo Coast, Sonora River) were contaminated with the BSMV-CCMV mixture but not with BCMV. The average contamination level was 13 percent. Two common weeds present in Sonoran agricultural areas were found to be potential alternate hosts of CCMV. Both Sisymbrium irio L. and Melilotus indica L. were infected systemically, although the infection in M. indica was latent. Potential losses due to Sonoran bean viruses were measured in greenhouse experiments with the cultivar Pinto 111. BCMV strains caused a 29.4 to 60.1% reduction, whereas BSMV-CCMV mixtures induced a 22.5 to 74.6% yield reduction. A synergism occurred between the BSMV-CCMV mixture and BCMV resulting in more severe symptoms and a yield reduction of 92.7%. Synergistic effects were also observed between BSMV and CCMV. Actual yield reduction resulted from impaired flower production and, consequently, reduced pod production. Significant effects on plant tissue production, flower fertilization and seed quality were not observed. Cowpea chlorotic mottle virus infected mung bean (Vigna radiata (L.) Wilczek) a previously unreported host. Infection of mung bean by BSMV was only possible when CCMV was present in the inoculum. Both BSMV and CCMV could be isolated from symptomatic plants infected with the BSMV-CCMV mixture, however, symptoms on mung bean were unchanged from infection by CCMV alone.

A technique involving Restriction Fragment Length Polymorphism (RFLP) was used to observe the DNA fragment polymorphism between a bean cultivar with I/I genotype and a bean cultivar with i/i genotype. The I gene encodes immunity to bean common mosaic virus (BCMV).

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Abstract: In 2013, common bean plants of the cultivar Pérola were found in an experimental field belonging to Embrapa Arroz e Feijão Lat. 16 ° 28 '00 "(S); Long. 49 ° 17 '00 "(W); (GO) presenting leaf distortion, mosaic and blistering. The sample analysis by electron microscopy detected the presence of typical Comovirus genus viral particles, thus, the identification of the virus species through sequencing became essential and the search for control alternatives, due to the damage potential of Bean rugose mosaic virus (BRMV) to bean production fields. Therefore, the present work had as objectives: (1) the molecular characterization of BRMV-GO, an isolate from common bean and (2) the search for bean accessions resistant to this viral species. For germplasm selection, 172 accessions were analyzed by means of mechanical inoculation and visualization of symptoms at 5, 21 and 30 days after inoculation. The range of hosts was analyzed by inoculation of 15 typical indicator species. Soya plants (Cv. Savana, Cv. Cristalina, Cv. Doko and Cv. Conquista) and pea (Cv. Mikado and Cv. Triofin) were also analyzed for reactions to BRMV-GO. Leaves of the infected plants were used for detection of BRMV-GO through RT-PCR. Of the 172 analyzed accessions, 168 behaved as susceptible, 3 accessions: BGF0011750 (cv. Mulatinho), BGF0000880 (cv. Rico 23) and BGF0001083 (cv Rico 23) reacted to BRMV-GO with vein and petioles necrosis followed by death; 1 access, BGF0003174 cv. IPA5047, showed a hypersensitivity reaction. The two species of the indicator Chenopodium amaranticolor and C. quinoa reacted with local necrotic lesions and no systemic infection confirmed by RT-PCR. Soybean plants reacted as susceptible and all pea plants showed tip burning. For molecular characterization, complete genome sequencing was performed by Sanger method using the primer walking strategy, the 5 'and 3' ends were obtained by RACE method. Genome sizes were 5906 nucleotides with a 1856 amino acid polyprotein for RNA 1 and 3688 nucleotides with a polypeptide of 1096 amino acids for RNA 2. The nucleotide identity between BRMV-GO and BRMV-Paraná isolates was 92.7% for RNA 1 and 90.5% for RNA 2. The highest percentage of identity obtained by amino acids sequence alignment of the polymerase (RdRp) and the capsid protein (CP) was 63% (RdRp) and 66% (CPs) with Bean pod mottle virus (BPMV), however, the ICTV delimits 80% (RdRp) and 75% (CP) identity to be part of the Comovirus genus, however, these results agree with the results obtained in another work with a Paraná isolate, corroborating that BRMV is a distinct species among this genus. Phylogenetic analysis of regions RdRp and CPs showed that BRMV-GO and BRMV-Paraná do not have significant differences and revealed higher indexes of identity with BPMV, both for RNA 1 and RNA 2. The complete sequences of RNA 1 and RNA 2 were deposited on Genbank under accession numbers KY622124, KY622125, respectively.
Resumo: Em 2013, plantas de feijão-comum da cultivar Pérola foram observadas em um campo experimental da Embrapa Arroz e Feijão Lat. 16° 28’ 00”(S); Long. 49° 17’ 00”(W); (GO) apresentando deformação foliar, mosaico em desenho e bolhosidade. A análise das amostras por microscopia eletrônica detectou a presença de partículas virais típicas do gênero Comovirus, sendo assim necessária a identificação da espécie do vírus através de sequenciamento e a busca por alternativas para o controle, devido ao potencial de dano da espécie Bean rugose mosaic virus (BRMV) para a cultura do feijão. Por isso, o presente trabalho teve como objetivos: (1) a caracterização molecular do isolado BRMV-GO, proveniente de feijoeiro e (2) a busca por acessos de feijoeiro resistentes à essa espécie viral. Para a seleção de germoplasma, 172 acessos foram analisados por meio de inoculação mecânica e visualização dos sintomas aos 5, 21 e 30 dias após a inoculação. A gama de hospedeiras foi analisada mediante inoculação de 15 espécies indicadoras típicas. Plantas de soja (cv. Savana, cv. Cristalina, cv. Doko e cv. Conquista) e ervilha (cv. Mikado e cv. Triofin) também foram analisadas quanto à reação ao BRMV-GO. Folhas das plantas infectadas foram usadas para detecção de BRMV-GO via RT-PCR. Dos 172 acessos analisados, 168 se comportaram como suscetíveis, 3 acessos: BGF0011750 (cv. Mulatinho), BGF0000880 (cv. Rico 23) e BGF0001083 (cv. Rico 23) reagiram ao BRMV-GO com necroses nas nervuras e pecíolos seguida de morte; 1 acesso, BGF0003174 cv. IPA5047, apresentou reação de hipersensibilidade. As duas espécies de indicadoras Chenopodium amaranticolor e C. quinoa reagiram com lesões locais necróticas não ocorrendo infecção sistêmica confirmada por meio de RT-PCR. As plantas de soja foram suscetíveis e todas as plantas de ervilha apresentaram queima do topo. Para a caracterização molecular, o sequenciamento completo do genoma foi realizado pelo método de Sanger usando a estratégia primer walking, as extremidades 5’ e 3’ foram obtidas pelo método RACE. Os tamanhos dos genomas foram de 5906 nucleotídeos com uma poliproteína de 1856 aminoácidos para RNA 1 e para RNA 2 foi de 3688 nucleotídeos com uma poliproteína de 1096 aminoácidos. A identidade de nucleotídeos entre os isolados BRMV-GO e BRMV-Paraná foi de 92,7 % para RNA 1 e 90,5% para RNA 2. A maior porcentagem de identidade obtida através do alinhamento de sequências de aminoácidos da polimerase (RdRp) e da proteína do capsídeo (CP) foi de 63% (RdRp) e 66% (CPs) com Bean pod mottle virus (BPMV), entretanto, o ICTV delimita para membros do gênero Comovirus uma identidade de 75% para aminoácidos (CPs) e 80% para a RdRp, no entanto, esses resultados estão de acordo com os resultados obtidos em outro trabalho com um isolado do Paraná, corroborando que BRMV é uma espécie distinta dentro do gênero. As análises filogenéticas das regiões RdRp e CPs mostraram que BRMV-GO e BRMV-Paraná não possuem diferenças significativas e revelou maiores índices de identidade com BPMV, tanto para o RNA 1 como para o RNA 2. As sequências completas do RNA 1 e RNA 2 foram depositadas no Genbank com os números de acesso KY622124, KY622125, respectivamente.

PSbMV in field pea has resulted in substantial yield and seed quality losses world-wide and has recently been reported in North Dakota. Traditional management of this virus includes preventative measures such as removal of alternate hosts, planting virus free seed and the use of cultivar resistance. The objectives of this research were to screen field pea cultivars commonly grown in North Dakota for a response to North Dakota PSbMV isolate ND14-1 and ascertain the effect on plant symptoms, seed size and weight, the number of pods and seeds and seed transmission. Two cultivars were identified as highly resistant and one as partially resistant. The results from this study were combined into a risk assessment. Cultivars were categorized based on inherent risk of PSbMV infection, transmission and reduction in total seed weight. Common bacterial blight (CBB) in dry bean is capable of causing substantial yield losses and has been reported in up to 75% of fields in the Northarvest region in the last five years. Current management practices include the use of planting clean seed, crop rotation, partial host resistance and the application of cupric bactericides, although inconsistent for the management of CBB. Growers in this Northarvest region have recently shifted to growing upright (Type II) dry beans rather than prostrate (Type III) dry beans for ease of harvest. The objectives of this research were to evaluate copper products, surface sanitizers and growth promoters for the management of CBB and to discern if Type II dry beans experienced greater yield losses under CBB disease pressure than Type III dry beans. Numerous products were identified that significantly reduced CBB disease severity and spread; however, no significant yield benefit was observed. Across a wide range of disease severity (0-46%), no significant yield losses were observed between high and low disease severity any of the cultivars screened.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2011.
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Os tospovírus (família Bunyaviridae) são fitopatógenos que possuem genoma de RNA fita simples tripartido, denominados S (Small), M (Medium) e L (Large). Os dois primeiros possuem polaridade ambisenso e o último polaridade negativa. São vírus envelopados e transmitidos por tripes, insetos da ordem Thysanoptera, de maneira circulativa e propagativa. Além disso, acarretam significantes perdas na produção e qualidade de vegetais de interesse econômico em diversas partes do mundo. Neste trabalho um novo e distinto tospovírus foi isolado em plantas de feijão apresentando mosaico com áreas necróticas. Após experimentos de caráter biológico, sorológico e molecular, este foi caracterizado e nomeado tentativamente de Bean necrotic mosaic virus (BeNMV). No processo de caracterização biológica o BeNMV apresentou um estreito espectro de hospedeiros, replicando-se sistemicamente somente em três plantas indicadoras (Datura stramonium L, Physalis pubescens L. e Phaseolus vulgaris L. cv. Santana) após transmissão por inoculação mecânica. Diferentemente do esperado, os sintomas visualizados no campo não foram reproduzidos em Phaseolus vulgaris em casa de vegetação. BeNMV mostrou-se sorologicamente diferente quando comparado com outras espécies brasileiras em ensaio de diferenciação sorológica realizado após produção de anticorpo policlonal anti-BeNMV. Entretanto, uma fraca reação cruzada foi observada entre Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV) e este novo tospovírus. Na caracterização molecular duas proteínas estruturais foram elucidadas, o precursor das glicoproteínas do envelope Gn e Gc e a RNA polimerase dependente de RNA ou proteína L. Ambas apresentaram o maior tamanho entre as já caracterizadas, tendo 1141aa e 2932aa, respectivamente. Também, apresentaram uma baixa identidade com as demais, indicando, após estudos filogenéticos, a descoberta de uma provável nova ramificação evolutiva do gênero Tospovírus. Curiosamente, talvez por suas características intrínsecas e significativamente divergentes quando comparado as demais espécies do gênero, ainda não foi possível caracterizar a proteína estrutural do nucleocapsídeo (N) viral. Trabalhos estão em andamento para concluir a caracterização deste novo e distinto tospovírus. _________________________________________________________________________________________ ABSTRACT
Tospoviruses (family Bunyaviridae) are plant-infecting pathogens with a tripartite RNA genome, named S (Small), M (Medium) and L (Large). The latter has a negative polarity, while the other two have ambisense polarity. These viruses have enveloped particles and are transmitted by thrips insects (order Thysanoptera) in a circulative-propagative manner. Moreover, tospoviruses cause significant quality and yielding losses to economically important cultures worldwide. In this study a new and distinct tospovirus was isolated from bean plants (Phaseolus vulgaris L.) showing necrotic mosaic symptoms. After biological, serological and molecular assays, the new virus was characterized and tentatively named Bean necrotic mosaic virus (BeNMV). BeNMV showed a narrow host-range, presenting systemic infection, after mechanical inoculation, on three different indicator host-species (Datura stramonium L,. Physalis Pubescens L. and Phaseolus vulgaris L., cv. Santana). Alternate from what could be expected, field-observed symptoms were not reproducible on greenhouse grown beans. As demonstrated in serological differentiation assays utilizing specific polyclonal anti-sera, BeNMV was distinct from other known tospoviruses common in Brazil. Weak cross-reaction was detected between Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV) and the new tospovirus. Two viral structural proteins were resolved, being the largest ones known among the tospoviruses so far - the glycoprotein precursor of Gn and Gc envelope proteins and the RNA-dependent RNA polymerase (L protein) with 1141aa and 2932aa, respectively. Both proteins showed little identity with available sequences from other tospovirus species in phylogeny assays, indicating that BeNMV constitutes a new evolutionary branch in the genus Tospovirus. Due to BeNMV's discrete genome coding and significant divergence from other tospovirus species, the nucleocapsid structural protein (N) could not be characterized. Further efforts are being made to achieve complete characterization of this new and distinct tospovirus.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Plantas de feijão-vagem do cultivar Novirex apresentando sintomas de mosaico e enrolamento de vagens, sem deformação foliar evidente, foram coletadas em 2002 no município de Cordisburgo, MG. Estudos anteriores de microscopia eletrônica, eletroforese da proteína e do RNA viral e sorologia identificaram o isolado como o comovírus Bean rugose mosaic virus (BRMV). Neste trabalho deu-se prosseguimento à caracterização do isolado, por meio de análises em laboratório e casa de vegetação envolvendo produção e avaliação de anti- soro policlonal, determinação da gama de hospedeiros, estudo da transmissão do vírus por besouros crisomelídeos e avaliação de perdas em feijoeiro como resultado de infecção isolada ou em conjunto com o potyvírus Bean common mosaic virus (BCMV). O procedimento adotado para purificação possibilitou a obtenção de vírus purificado em rendimento satisfatório para a obtenção de anti-soro. A titulação dos anti-soros obtidos foi realizada por ELISA indireto, obtendo-se reações positivas com a diluição máxima testada (1:70.000), e nenhuma reação com extrato de planta sadia. Das 22 espécies vegetais testadas, Chenopodium quinoa apresentou inicialmente lesões locais cloróticas e posteriormente infecção sistêmica com mosaico e distorção foliar. Nos cultivares de feijão e soja observou-se sintomas de mosaico e bolhosidade, em conformidade com os resultados esperados para o BRMV. O isolado de BRMV foi transmitido pelo besouro crisomelídeo Cerotoma arcuata a uma taxa de 33,3%. Este isolado foi inoculado em plantas de feijoeiro ‘Ouro Negro’ e de feijão-vagem ‘Novirex’, levando a uma redução do peso de vagens por planta de 44,6% e 65,7%, respectivamente. O Bean common mosaic virus (BCMV) ocasionou porcentagens de redução de peso de vagens menores em ‘Novirex’ (13,1%) em comparação a ‘Ouro Negro’ (50,4%). Quando o BRMV foi inoculado inicialmente, seguido do BCMV, verificou-se redução do peso de vagens por planta de até 69,3% para ‘Novirex’ e de 91,5% para ‘Ouro Negro’. Não se observou diferença significativa em peso ou número de vagens por planta quando estas foram inoculadas seqüencialmente com o BCMV seguido do BRMV.
Bean plants of the cultivar Novirex, showing an atypical pod curling symptom without mosaic or leaf distortion, were collected in 2002 at Cordisburgo, MG. Previous studies involving electron microscopy and electrophoretic analysis of viral protein and RNA identified the isolate as the comovirus Bean rugose mosaic virus (BRMV). The present work continued the characterization of the isolate, and included its purification and production of a polyclonal antiserum, determination of a partial host range, vector transmission studies, and estimates of yield losses in beans due to single or mixed infection with the potyvirus Bean common mosaic virus (BCMV). The protocol adopted for virus purification led to purified preparations with high yield, and the antisera obtained after rabbit immunization reacted with the maximum dilution tested (1:70.000) in indirect ELISA, without any reactions with sap from healthy plants. Out of the 22 plant species tested as hosts, Chenopodium quinoa reacted with chlorotic local lesions which evolved to mosaic and leaf distortion in non-inoculated leaves. Bean and soybean cultivars reacted with mosaic of varied intensities, as expected for BRMV. The isolate was transmitted by Ceratoma arcuata to 33,3% of the inoculated plants. Upon inoculation onto ‘Ouro Negro’ and ‘Novirex’ beans, the total weight of pods per plant was reduced by 44,6% and 65,7%, respectively. Single infection by BCMV led to a smaller reduction of pod weight in ‘Novirex’ (13,1%) compared to ‘Ouro Negro’ (50,4%). When BRMV was inoculated first, followed by BCMV, total pod weight was reduced by up to 69,3% in ‘Novirex’ and 91,5% in ‘Ouro Negro’. No statistically significant differences were observed in total weight or number of pods per plant after inoculation with BCMV followed by BRMV.
Dissertação importada do Alexandria

Los virus producen graves pérdidas económicas en la agricultura. Esta problemática es muy dinámica ya que cada año aparecen nuevas virosis y es frecuente los fenómenos de emergencia con una rápida expansión de los virus. El control de las enfermedades víricas resulta poco eficaz en muchos casos porque la población viral es capaz de evolucionar y superar dichas estrategias. Por ello es clave entender la dinámica de las poblaciones y los factores implicados en la evolución de los virus con respecto a distintos aspectos de su biología del ciclo viral: replicación, movimiento dentro de la planta, respuesta a los mecanismos de defensa de la planta, transmisión a otras plantas, etc. El objetivo de esta tesis ha sido el estudio de los factores implicados en la evolución de dos virus que difieren en su variabilidad genética y gama de huéspedes: i) el Virus 1 del marchitamiento del haba (Broad bean wilt virus 1, BBWV-1), del género Fabavirus; y ii) el Virus del mosaico del tabaco (Tobacco mosaic virus, TMV) del género Tobamovirus. Primero se han desarrollado una serie de herramientas metodológicas que han permitido la detección rápida de BBWV-1 mediante hibridación molecular de improntas, la detección y cuantificación de BBWV-1 y TMV y su diferenciación de otras virosis del mismo género mediante RT-PCR cuantitativa a tiempo real. Se ha llevado a cabo la construcción de clones de cDNA del genoma completo de BBWV-1 para obtener transcritos infecciosos que puedan ser usados para estudiar la biología molecular, evolución y epidemiología. Una vez desarrollado esta metodología se ha usado para evaluar la eficacia biológica de BBWV-1 en el huésped y el efecto de algunos factores: concentración del inóculo, estado de desarrollo de la planta, tipo de huésped, aplicación de un activador de la defensa de la planta, y la infección con otro virus. Así mismo se han estudiado los factores relacionados con la eficacia biológica del virus durante su transmisión por pulgones: título viral
Ferriol Safont, I. (2012). FACTORS INVOLVED IN THE EVOLUTION OF BROAD BEAN WILT VIRUS 1 AND TOBACCO MOSAIC VIRUS [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16000
Palancia

The second and largest open reading frame within the southern bean mosaic virus (SBMV) genome encodes a 105 kDa polyprotein. Following translation, the polyprotein is cleaved to liberate various proteins necessary for SBMV replication. The elements within polyproteins of the picornavirus superfamily members have a conserved order: Vpg (viral protein, genome-linked)-protease-replicase. Amino acid sequence homologies indicate that the 105 kDa protein of SBMV contains a replicase very similar to those identified in polyproteins of the picorna-like viruses. The presence of a VPg covalently attached to the 5' end of the SBMV genome further suggests that SBMV may be considered a member of the picornavirus superfamily-Serine 558 within the SBMV polyprotein has been proposed to be a catalytic residue of a serine protease. Site-directed mutagenesis was used to create a mutant with a glycine at this position, and coupled in vitro transcription/translation was used to prepare 3H labeled translation products. SDS-PAGE and fluorography were then used to assay for the presence or absence of polyprotein cleavage. Although site-directed mutagenesis was successful in creating the mutant, a possible deletion that complicated the interpretation of the results was identified.

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2007.
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A produção mundial de feijão, compreendendo os gêneros Phaseolus e Vigna, é superior a 18 milhões de toneladas e o Brasil ocupa o segundo lugar na produção mundial e o primeiro quando se trata apenas do gênero Phaseolus. Entretanto, sua produção ainda está aquém do necessário para suprir a demanda interna. Dentre os principais problemas relacionados com a baixa produção de feijão no Brasil estão a competição com plantas daninhas, estresse hídrico e o ataque de pragas e doenças como mosaico dourado do feijoeiro causado por um geminivírus. O Bean golden mosaic virus (BGMV), é transmitido por mosca-branca (Bemicia tabaci) de uma forma persistente, circulativa, causando mosaico dourado em feijão comum (Phaseolus vulgaris). Os sintomas característicos são: mosaico verde amarelado nas folhas, nanismo e vagens distorcidas. A doença é largamente disseminada nas regiões de produção de feijão no Brasil e América Latina causando perdas severas (40 -100%). Neste trabalho, a tecnologia de RNA interferente foi explorada usando uma seqüência do gene viral AC1 para gerar plantas transgênicas de feijão comum altamente resistentes a geminivírus. Desta maneira o gene viral (AC1) foi escolhido para a construção do vetor de transformação uma vez que a proteína Rep exerce uma função essencial no ciclo de infecção viral. Rep não é uma replicase porém é a única proteína requerida para a replicação do genoma viral. O vetor pBGMVRNAiAHAS foi construído a partir de um fragmento de DNA de 411 pb do gene AC1 do BGMV (_1836-2247, acesso no GeneBank No. M88686). Dezoito linhagens de feijão foram obtidas com a construção de interferência, as quais poderão induzir o silenciamento pós-transcricional do gene AC1. Uma linhagem (denominada 5.1) apresentou alta resistência (aproximadamente 93% das plantas não apresentaram sintomas) após alta pressão de inoculação (mais de 300 moscas-brancas virulíferas por planta). Os siRNAs específicos foram detectados nas plantas transgênicas, ambas inoculadas e não inoculadas. Uma análise de PCR semiquantitativo revelou a presença do DNA viral nas plantas transgênicas expostas a moscas-brancas virulíferas por um período de 6 dias. Entretanto, após a retirada dos insetos, não foi detectado DNA viral após um período adicional de seis dias. Devido à importância social e econômica do feijão na América Latina e à falta de genes para resistência a doenças nos bancos de germoplasma, faz-se necessário o desenvolvimento de um programa de melhoramento associado à engenharia genética para o lançamento de novas variedades, que possibilitará a diminuição dos principais problemas relacionados a esta cultura. ____________________________________________________________________________________ ABSTRACT
Production of beans, including the genus Phaseolus and Vigna, is superior to 18 million tons. Brazil is the secondlargest producer of both species genus and the major world producer of Phaseolus. However, its production is still below the necessary to supply the internal demand. Competition with weeds, abiotic stress and the occurrence of diseases as bean golden mosaic are some of the main problems related to its low production. Bean golden mosaic virus (BGMV), is transmitted by the whitefly Bemisia tabaci in a persistent, circulative manner, and causes the golden mosaic of common bean (Phaseolus vulgaris) which characteristic symptoms are yellow-green mosaic of leaves, stunting, and fruit distortion. The disease is the largest constraint on bean production in Brazil and Latin America and causes severe yield losses (40 to 100%). Here we explored the RNAi concept to silence the AC1 viral gene and generate high resistant transgenic common bean plants. Since the Rep protein performs an essential function in the viral infecting cycle, the AC1 gene was chosen to construct the transformation vector. Rep is not the replicase, but the only protein required for the replication of the viral genome. The vector pBGMVRNAiAHAS was constructed using the 411 bp fragment from the AC1 gene (_1836-2247, GeneBank accession No. M88686). Eighteen transgenic common bean lines were obtained with the construction to induce post-transcriptional gene silencing against AC1 gene. One line (named 5.1) presented high resistance (approximately 93% of plants free of symptoms) upon inoculation at high pressure (more than 300 viruliferous whiteflies per plant). Transgene-specific siRNA were detected in both inoculated and noninoculated transgenic plants. A semi-quantitative PCR analysis revealed the presence of virus DNA in transgenic plants exposed to viruliferous whiteflies for a period of six days. However, when insects were removed, no virus DNA could be detected after an additional period of six days. Due to its social and economical importance in Latin America and to the lack of genes for resistence, it is necessary to develop an improving program associated to genetic engineering in order to generate new varieties. It Shall allow the decreasing to the production of beans

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A expressão de proteínas recombinantes em Escherichia coli produz proteínas que se acumulam como agregados insolúveis conhecidos como corpos de inclusão (CI). As proteínas na forma de CI são biologicamente ativas e com estrutura semelhante à proteína nativa quando a expressão é induzida a baixa temperatura. Estas características e a facilidade em purificar os CIs podem representar uma possibilidade de utilização dos CIs como antígenos para a produção de anticorpos policlonais. O gene da proteína capsidial (CP) do Southern bean mosaic virus (SBMV), um Sobemovirus, foi clonado no vetor pET 28a e a proteína expressa em E. coli a 25 º C. Uma análise comparativa entre a CP recombinante presente na forma de corpos de inclusão e a proteína nativa presente nas partículas virais purificadas foi realizada utilizando a técnica de espectroscopia de infravermelho (FT-IR). Os conteúdos de estruturas secundárias identificados diferiram nos percentuais de folha-β enquanto os percentuais hélices-α foram preservados. Uma estrutura tridimensional foi gerada por modelagem molecular por homologia e comparada com os dados obtidos nas análises de FT-IR. Os percentuais obtidos por FT-IR da CP nativa foram condizentes com os dados gerados por modelagem molecular por homologia. Mesmo com algumas diferenças nos conteúdos de folha- β da CP recombinante foi possível verificar um estado conformacional semelhante à proteína nativa. Os corpos de inclusão contendo a proteína recombinante expressa foram utilizados como antígenos para a imunização de coelhos. O antissoro obtido mostrou ser específico e compatível a um antissoro previamente preparado com partículas virais purificadas do SBMV na detecção do vírus em extratos de feijoeiros infectados utilizando imunoensaios por Western blot, PTA-ELISA, Dot Blot e Tissue Printing...
The production of recombinant proteins in Escherichia coli yields proteins that accumulate as insoluble aggregates known as inclusion bodies (IBs). Biologically active with native-like structure is trapped inside IBs when the expression is induced at low temperature. These features, and the facility to purify the IBs, can represent a possibility to use the IBs as an antigen for the production of polyclonal antibodies. The coat protein gene of Southern bean mosaic virus, a Sobemovirus, was cloned in the vector pET 28a and expressed in Escherichia coli at 25 ºC. The inclusion bodies containing the recombinant protein were isolated and purified. A comparative analysis between recombinant protein present in the form of inclusion bodies and native coat protein present in the purified virus particles was performed using Fourier transform infrared spectroscopy (FT-IR). The secondary structure contents identified differences in the percentage of β-sheets while α-helices content was much preserved. The three-dimensional structure was generated using molecular modeling and was compared to the data obtained from FT-IR analysis. The percentages obtained from FT-IR analyses of the native CP were consistent with the structure generated through homology molecular modeling. Nevertheless, some differences were present on the content of β-sheets structure of the recombinant CP indicating that the protein conformational state is found in a native like condition. The IBs containing the expressed protein were used as an antigen to immunize rabbits. The antiserum obtained showed to be specific and comparable to one previously prepared with purified SBMV particles, in the virus detection in leaf extracts of infected bean plants using Western blot, PTA-ELISA, Dot Blot and Tissue Printing assays. So, the use of IBs containing recombinant viral proteins to immunize rabbits is a new, easy and very... (Complete abstract click electronic access below)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris ‘Jalo’, obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão...
In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below)

Orientador: José Osmar Gaspar
Banca: Mário Tyago Murakami
Banca: Marinônio Lopes Cornélio
Resumo: No Brasil, foram descritas mais de dez viroses em feijoeiro, citando-se aquela causada pelo Southern bean mosaic virus (SBMV) do gênero Sobemovirus. Este vírus tem uma restrita gama de hospedeiras, confinada quase exclusivamente a espécies da família das leguminosas, sendo algumas de interesse econômico como o feijoeiro comum e a soja. O SBMV é a espécie tipo do gênero, possui partículas isométricas (28-30nm) contendo RNA genômico de 4-4,5kb com polaridade positiva e proteína capsidial com massa molecular de 29-30 kDa. O genoma do SBMV é constituído por quatro Open Reading Frames (ORFs) com sobreposição entre elas. A ORF 1 codifica uma possível proteína do movimento, a ORF 2 uma poliproteína, que por clivagem, origina três produtos funcionais: uma serino protease, uma proteína ligadora do genoma viral (VPg) e uma polimerase com atividade relacionada à replicação do RNA viral (RNA polimerase RNA-dependente, RpRd). Dentro da ORF 2 encontra-se a ORF 3, que codifica uma proteína de função desconhecida. A ORF 4 codifica a proteína capsidial, traduzida a partir de um RNA subgenômico. O presente trabalho consiste na expressão da proteína P1 do SBMV-SP em Escherichia coli, sua purificação e a posterior utilização para a produção de antissoro policlonal. As atividades realizadas compreenderam a purificação das partículas virais do SBMV isolado São Paulo (SBMV-SP) a partir de folhas infectadas de Phaseolus vulgaris 'Jalo', obtenção do RNA viral a partir de partículas purificadas, produção de cDNA utilizando primer anti-senso, amplificação do gene da proteína do movimento, purificação do fragmento amplificado, ligação em vetor de multiplicação pGEM-T, transformação em células competentes de Escherichia coli linhagem TOP 10, purificação do vetor, corte enzimático com as enzimas Bam HI e Hind III, subclonagem nos vetores de expressão... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: In Brazil, more than ten bean viroses were described, including the one caused by Southern bean mosaic virus (SBMV) from genus Sobemovirus. This virus has a strict host range, confined almost exclusively to species from leguminosas family, and some of them have economic importance, like the common bean and soya. SBMV is the type species from this genus, has isometric particles (28-30nm) that contains genomic RNA of 4-4,5kb with positive polarity and a capsidal protein with molecular mass of 29-30kDa. SBMV genome consistis of four Open Reading Frames (ORFs) with overlap between them. ORF 1 encodes a possible movement protein, ORF 2 a poliprotein, which origin three functional products by clivage: a serin protease, a viral genomic biding protein (VPg) and a polimerase. Inside ORF 2 is situated the ORF 3, that encodes a protein with unknow function. ORF 4 encodes the capsidal protein, translated by a subgenomic RNA. The activities performed until this moment were the purification of SBMV isolated São Paulo(SBMV-SP) viral particles, obtention of viral RNA from SBMV purified partcles, cDNA production using anti-sense primer, amplification of the supposed gene of movement protein, purification of the amplified fragment and later biding on expression vector pGEM-T. The recombinat vector were transformed in Escherichia coli cells competent and than purified and digested with Bam HI and Hind III enzime. The released fragment was subcloned in pMAL and pET28a, which were used on competent cells for expression tests. Analysis of gel poliacrialamida showed the efficiency of tests of expression. Using pET28a was observed an expression of a protein of approximately 22 kDa (17 kDa relating to MP + 5 kDa relating to the tail of histidine) which was purified in resin Ni-NTA by affinitive chromatography.Using pMAL was observed an expression of a protein of approximately... (Complete abstract click electronic access below)
Mestre

Orientador: José Osmar Gaspar
Banca: Júlio César Borges
Banca: Jorge Alberto Marques Rezende
Banca: Marinônio Lopez Cornelio
Banca: Roberto da Silva
Resumo: A expressão de proteínas recombinantes em Escherichia coli produz proteínas que se acumulam como agregados insolúveis conhecidos como corpos de inclusão (CI). As proteínas na forma de CI são biologicamente ativas e com estrutura semelhante à proteína nativa quando a expressão é induzida a baixa temperatura. Estas características e a facilidade em purificar os CIs podem representar uma possibilidade de utilização dos CIs como antígenos para a produção de anticorpos policlonais. O gene da proteína capsidial (CP) do Southern bean mosaic virus (SBMV), um Sobemovirus, foi clonado no vetor pET 28a e a proteína expressa em E. coli a 25 º C. Uma análise comparativa entre a CP recombinante presente na forma de corpos de inclusão e a proteína nativa presente nas partículas virais purificadas foi realizada utilizando a técnica de espectroscopia de infravermelho (FT-IR). Os conteúdos de estruturas secundárias identificados diferiram nos percentuais de folha-β enquanto os percentuais hélices-α foram preservados. Uma estrutura tridimensional foi gerada por modelagem molecular por homologia e comparada com os dados obtidos nas análises de FT-IR. Os percentuais obtidos por FT-IR da CP nativa foram condizentes com os dados gerados por modelagem molecular por homologia. Mesmo com algumas diferenças nos conteúdos de folha- β da CP recombinante foi possível verificar um estado conformacional semelhante à proteína nativa. Os corpos de inclusão contendo a proteína recombinante expressa foram utilizados como antígenos para a imunização de coelhos. O antissoro obtido mostrou ser específico e compatível a um antissoro previamente preparado com partículas virais purificadas do SBMV na detecção do vírus em extratos de feijoeiros infectados utilizando imunoensaios por Western blot, PTA-ELISA, Dot Blot e Tissue Printing... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The production of recombinant proteins in Escherichia coli yields proteins that accumulate as insoluble aggregates known as inclusion bodies (IBs). Biologically active with native-like structure is trapped inside IBs when the expression is induced at low temperature. These features, and the facility to purify the IBs, can represent a possibility to use the IBs as an antigen for the production of polyclonal antibodies. The coat protein gene of Southern bean mosaic virus, a Sobemovirus, was cloned in the vector pET 28a and expressed in Escherichia coli at 25 ºC. The inclusion bodies containing the recombinant protein were isolated and purified. A comparative analysis between recombinant protein present in the form of inclusion bodies and native coat protein present in the purified virus particles was performed using Fourier transform infrared spectroscopy (FT-IR). The secondary structure contents identified differences in the percentage of β-sheets while α-helices content was much preserved. The three-dimensional structure was generated using molecular modeling and was compared to the data obtained from FT-IR analysis. The percentages obtained from FT-IR analyses of the native CP were consistent with the structure generated through homology molecular modeling. Nevertheless, some differences were present on the content of β-sheets structure of the recombinant CP indicating that the protein conformational state is found in a native like condition. The IBs containing the expressed protein were used as an antigen to immunize rabbits. The antiserum obtained showed to be specific and comparable to one previously prepared with purified SBMV particles, in the virus detection in leaf extracts of infected bean plants using Western blot, PTA-ELISA, Dot Blot and Tissue Printing assays. So, the use of IBs containing recombinant viral proteins to immunize rabbits is a new, easy and very... (Complete abstract click electronic access below)
Doutor

Orientador: Jorge Vega
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo:A aplicação de determinados reguladores de crescimento em plantas pode resultar na indução de resistência contra ataques subseqüentes de patógenos. Este fenômeno é conhecido como resistência sistêmica adquirida (SAR) e vários mecanismos estão envolvidos, entre eles a resposta de hipersensibilidade (HR), o aumento na atividade das enzimas peroxidases (POX) e da fenilalanina amônia-liase (PAL) nos tecidos resistentes. A fim de se detenninar o efeito do ácido salicílico (AS) e de um brassinolídeo (BR 16) na resposta hipersensível, plantas de feijoeiro (Phaseolus vulgaris L.), cultivar Moruna (nc) foram tratadas com estes reguladores de crescimento e inoculadas com o Southern bean mosaic virus (SBMV). Detenninou-se o número de lesões necróticas das folhas primárias e a atividade das enzimas guaiacol peroxidases (g-POX) e PAL. Houve uma redução significativa no número de lesões necróticas nas plantas tratadas com ambos os reguladores de crescimento e um aumento significativo na atividade das g-POX, mas nenhum efeito na atividade da PAL. O efeito da aplicação de AS e BR 16 na resposta suscetível foi verificado através da aplicação destes reguladores de crescimento em plantas de feijoeiro da cultivar lalo, inoculadas com o SBMV. Os tratamentos promoveram uma redução significativa na concentração viral na sa folha trifoliolada, 28 dias após a inoculação das folhas primárias. Os resultados indicam que as aplicações de ácido salicílico e do brassinolídeo BR 16 podem ter promovido uma indução de resistência tanto local quanto sistêmica, em plantas de feijoeiro. O aumento na atividade das enzimas g-POX, envolvidas nos processos de detoxificação de espécies reativas de oxigênio, promovido pelo tratamento com ambos os reguladores de crescimento, sugere uma participação destas na redução do número de lesões necróticas das plantas inoculadas com o SBMV
Abstract: The use of certain growth regulators in plants can result in the induction of resistance against attacks of some plant pathogens. This phenomenon is known as Systemic Acquired Resistance (SAR) and several mechanisms are involved in the induction of SAR.,among them are the Hypersensitive Reaction (HR), the increase in the activity of some enzymes like peroxidases (POX) and Phenylalanine ammonia-Iyase (PAL). Two different growth regulators (salicylic acid "SA" and a brassinosteroid "BR 16") were used in order to determine the induction of local resistance in bean plants (Phaseolus vulgaris L.) of cultivar Moruna (nc). Afier the application, the plants were mechanically inoculated with Southern bean mosaic virus (SBMV), genus Sobemovirus. The number of necrotic lesion was determined in the the primary leaves and the activity of the enzymes guaiacol peroxidases (g-POX) and Phenylalanine ammonia-Iyase (pAL) were also measured. There was a significant reduction in the number of necrotic lesions in the treated plants with both growth regulators and a significant increase in the activity ofthe g-POX, but there was no effect in the PAL activity. The effect ofthe application of SA and BR 16 in the induction of systemic resistance was verified through the application of these growth regulators in bean plants cultivar Jalo, inoculated with SBMV. The treatments promoted a significant reduction in the viral concentration in the SIDleaf, 28 days afier the inoculation of the primary leaves. The results indicated that the applications of SA and of the BR 16 promoted both local and systemic resistances. The increase in the activity of the guaiacol peroxidases, enzymes involved in the scavenning of reactive oxygen species, caused by the treatment with both growth regulators, suggested a contribution in the reduction of the number of necrotic lesions in the plants inoculated with SBMV. The use of SA and BR 16 did not change the activity of PAL, enzyme involved in the lignification process and in the mechanisms of plant defense against infections
Doutorado
Doutor em Biologia Vegetal

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
A cultura do feijoeiro comum no Brasil, além do imenso valor que representa na cadeia econômica e para milhares de agricultores no país, é fundamental devido à contribuição que possui na segurança alimentar da população. Cultivares com evento de resistência ao Bean golden mosaic virus (begomovirus), vírus responsável por causar uma das doenças que mais afeta a produtividade da cultura no país, foram desenvolvidas após vários anos de pesquisas. Infecções durante testes em campo desses materiais por outro vírus, o Cowpea mild mottle virus (carlavirus), gerou novas preocupações tanto aos pesquisadores envolvidos no projeto do feijoeiro resistente ao mosaico dourado quanto aos produtores que aguardavam a liberação comercial dessas cultivares. Apesar de alguns trabalhos já terem sido desenvolvidos a fim de se avaliar os prejuízos produtivos que o CPMMV causa sobre as isolinhas transgênicas de feijoeiro, assim como sua distribuição, nenhum conhecimento se tem sobre a diversidade desse vírus em feijão comum ou transgênico no Brasil, e poucos trabalhos dessa natureza são encontrados na literatura até hoje. Nesse trabalho, buscou-se avaliar a variabilidade de populações do CPMMV para cada uma de quinze cultivares de feijoeiro comum, sendo dez transgênicas (resistentes ao BGMV) e cinco convencionais, em um campo experimental com ocorrência e transmissão natural do CPMMV. Também foram quantificados os níveis virais em cada cultivar a partir de três repetições. Para cada uma das quinze plantas representando 15 diferentes genótipos de feijoeiro comum, o genoma completo de cinco isolados de CPMMV foi sequenciado pela montagem de sequenciamentos de blocos de PCR. Diferenças foram encontradas na variabilidade dos cinco isolados de CPMMV em plantas transgênicas e em plantas convencionais. Os valores dos descritores de variabilidade π, S, K e Θ foram geralmente maiores nos grupos de isolados de plantas transgênicas. Isso se repetiu para todas as ORF’s virais analisadas. As ORF’s 2, 3 e 4 foram as que tiveram a maior diversidade registrada, enquanto que a diferenças entre os grupos já citados foi mais perceptível nas regiões das ORF’s 2, 5 e 6. Eventos de recombinação foram encontrados na ORF 1 viral, quase sempre ocorrendo em isolados de plantas transgênicas, assim como alguns na ORF 2 e 6. Analisando as sequencias da ORF 1, nota-se que os cinco isolados de cada planta se agrupam e tendem a formar clados próximos a grupos de isolados de genótipos hospedeiros similares, o que pode decorrer da interação entre a replicase viral e a planta. Para a região 3’ do genoma, houve a separação do conjunto de 75 isolados em dois grupos de variantes. A identidade nucleotídica par a par entre isolados de grupos distintos variou entre 75 e 85%. Pelos testes de seleção, existe evidência significativa de que vária populações virais estão sobre processo de seleção não neutra. O acúmulo viral não teve diferença significativa entre plantas transgênicas e convencionais. A quantificação também não revelou diferenças em níveis virais em plantas transgênicas originadas de retrocruzamentos com a cultivar Pérola em comparação aos níveis naquelas retrocruzadas com a cultivar BRS Pontal. Os resultados desse trabalho reforçam resultados anteriores de que dois grupos de estirpes de CPMMV estão distribuídos pelas regiões produtoras brasileiras, provavelmente pela presença em plantas daninhas (onde a variabilidade desse vírus nunca foi analisada) e em hospedeiros cultivados como o próprio feijoeiro. Também comprova a alta variabilidade desse vírus de RNA, principalmente nas novas cultivares de feijoeiro resistente ao mosaico dourado por transgenia. É provável que a presença de BGMV nas cultivares convencionais e consequentemente a infecção mista dos dois vírus tenha algum efeito sobre os valores de variabilidade apresentados nesse estudo. Os mecanismos moleculares dessa interação, porém, não são conhecidos. Os resultados apresentados e o fato de que hospedeiros não cultivados estão distribuídos por grandes áreas de produção e que estes podem atuar como reservatório viral, além da grande distribuição da mosca branca pelo Brasil, fazem com que novos trabalhos com esse patógeno sejam de extrema importância.
The common bean crop in Brazil, besides its economic importance, represents a major source of what is daily consumed by Brazilian population in terms of proteins and carbohydrates, contributing to food security. Cultivars with a transgenic resistance event to Bean golden mosaic virus (begomovirus), a virus that causes one of the most important diseases of common bean, were developed after many years of research. The release of these cultivars immune to BGMV is undergoing difficulties because of the re-emergence of Cowpea mild mottle virus (carlavirus) in common bean, which has raised some concerns for the researchers and the growers. Although works to access the damage potential into different genotypes of these resistant isolines and to investigate the virus distribution are being reported, no study is found evaluating CPMMV molecular characteristics and diversity in transgenic as well as conventional common bean cultivars in Brazil. In fact, there are very few studies of this kind globally. The objective of this work was to evaluate the variability on CPMMV populations from each of the fifteen common bean cultivars, ten transgenic and resistant to BGMV, and five conventional cultivars, from a field experiment with natural CPMMV transmission by whitefly. CPMMV was also quantified on the three plant replicates of each genotype. Five CPMMV isolates were completely sequenced on all fifteen plants with different genotypes, providing 75 full virus genomes after assembly of PCR sequence blocks. Differences in variability were found between those groups of isolates from transgenic plants to those from conventional ones. With the π, S, K, and Θ-W descriptors, we detected a considerable higher CPMMV variability within transgenic plants in comparison to the virus variability within conventional cultivars in most of the cases. This was the case for all analyzed ORF’s. The ORF’s 2, 3 and 4 were the ones with the highest variability in the genome; at ORF’s 2, 5, and 6, the differences in variability mentioned above are most discernible. Recombination events between isolates happening at the ORF 1 region were detected, as well as at ORF 2 and at ORF 6. Mostly of these were between isolates from transgenic plants. The phylogenetic analysis with ORF 1 sequences of all seventy-five isolates reveals the formation of groups based on host genotypes, and that these groups are most likely grouping near a cluster of isolates from a similar host plant genotype. These could be the result of the direct and specific interactions needed between the viral replicase and the plant. The results of phylogenetic analysis and sequence comparisons with the 3’ region of the viral genome (ORF 2-6), divided the 75 isolates of this study into two groups of CPMMV variants. The pairwise nucleotide differences between isolates from distinct groups ranged from 75 to 85%. The selection tests at some ORF’s give significant evidence that some populations are evolving under a non- random process. The viral accumulation on conventional cultivars did not differ statistically to the accumulation at transgenic plants. In addition, there is no evidence of differences between CPMMV levels at transgenic cultivars that have Pérola as the reccurent parent to those that have the BRS Pontal. The results from this work corroborate with previous studies that indicate the existence of two CPMMV strains naturally distributed in Brazilian production areas. It also confirms the expected high variability potential of this RNA virus; the high variability registered on the newly developed BGMV-resistant transgenic common bean cultivars is also troublesome. The presence of BGMV in mixed infections with CPMMV at conventional cultivars is probably influencing the results of CPMMV variability, but the molecular properties of this interaction is still unknown. These results, in addition to the fact that non-cultivated host plants are distributed along major production areas and may act as viral reservoirs and the known widespread of whiteflies in growing regions of Brazil, make further studies with this pathogen of fundamental importance.

There are four open reading frames (ORF) in three positive-sense translational phases of Southern Bean Mosaic Virus (SBMV). ORF2 codes for a 105 KDa polyprotein which is proposed to be autocatalytically cleaved into smaller functional proteins. Amino acid sequence homology between the SBMV and poliovirus, foot and mouth disease virus, and cowpea mosaic virus reveal that SBMV has a similar genomic organization to picornaviruses with a conserved order: Vpg (Viral protein, genome-linked )-protease-replicase. Two Gln-Ser (QS) amino acid pairs within the 105 KDa polyprotein, C75= 930-936, C60= 1305-1310, are proposed to be the cleavage sites based on 1) similar structural arrangement around the two QS pairs to that of known cleavage sites of picornaviruses and potyviruses; 2) computer prediction of the cleavage products from the SBMV RNA sequence; 3) observed in vitro translation products. In order to investigate the role of the C60 QS pair in the proteolytic processing of the 105 KDa polyprotein, a mutagenic oligo was designed to create various amino acid substitutions in the QS pair. A coupled in vitro transcription/translation system was used to produce the protein products of both wildtype and mutants. During the procedure, tritiated leucine was incorporated into the protein products. The protein products were separated by SDS-PAGE and the cleavage patterns were detected after autoradiography. Two substitutions of the C60 QS pair-Pro-Val (PS) in mutant6, Gin-Pro (QP) in mutant19-reduced the yield of the 60 KDa protein by different levels. This result supports the hypothesis that the C60 QS is a cleavage site.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Common bean is susceptible to several viral diseases including Bean golden mosaic virus (BGMV) and Cowpea mild mottle virus (CpMMV). Since both viruses are whitefly transmitted, double infection can be easily overlooked due to the much intense BGMV symptomatology. The development of Embrapa 5.1 event of common bean resistant to BGMV (BGM), and near isogenic lines of two commercial cultivars (Pérola and BRS Pontal) allows the evaluation of the damage caused only by CpMMV, since these transgenic isolines do not get infected by BGMV. This study was conducted with the BGM to determine: 1) the damage caused by nymphs of B. tabaci in three phases of plant development; 2) the incidence and damage of CpMMV in correlation with the population of B. tabaci. The damage caused by six different densities of whitefly nymphs on BGM and conventional bean (CB) in the stages of primary leaves (V2), vegetative (V3-V4) and flowering (R6) were assessed in the greenhouse of Embrapa Rice and Beans. At the V2 stage were evaluated a mean number of 0, 20, 40, 60, 100 and 200 nymphs leaf-1; V3-V4 stage a mean number of 0, 40, 80, 130, 250 and 480 nymphs leaf-1 and at stage R6 a mean number of 0, 20, 50, 120, 280 and 760 nymphs leaf-1. The experimental design was completely randomized factorial of 6 x 2 (population levels of nymphs whitefly x BGM and FC), with 12 repetitions, except for the experiment in R6 with nine replicates. The experimental unit was represented by two bean plants. The incidence and severity of BGMV, the presence of sooty mold and yield components (number of pods, seeds per plant, seeds per pod, weight of grains) were determined. Field experiments were conducted in two experimental farms of Embrapa Rice and Beans in Santo Antônio de Goiás (16°30’24,57” S; 49°17’06,53” W) and Brazabrantes (16°26’09, 20” S, 49°24’05,80” W), State of Goiás. Four isolines derived from cv. Pérola, and six from cv. BRS Pontal, the two parental commercial cultivars, and three other entries named IPR Eldorado, BRB 169, and CNFC 15882 were compared. The incidence of BGMV, CpMMV, number of whitefly eggs, nymphs and adults were assessed as yield and yield components. The infestation of ≤ 200 nymphs of B. tabaci biotype B per leaf on primary leaves (V2), ≤ 480 nymphs per leaf in vegetative growth stage (V3-V4) and ≤ 760 nymphs per leaf in flowering stage (R6) not reduced grain yield per plant, number of pods per plant, and seeds per pod of CBGM. The BGMV affect the CB production at V2 and V3-V4 stages but not for R6 stage. The growth of the fungus Capnodium sp. (sooty mold) on the honeydew excreted by nymphs, was only observed on plants at V3-V4 stage. At field, the adult population of whiteflies was significantly lower in the GM lines derived from cv. Pérola (CNFCT 16201 and CNCF 16203) and from cv. BRS Pontal (CNFCT 16205), as well as on the commercial cv. IPR Eldorado, access BRB 169 and line CNFP 15882. It was not observed significant differences in whitely eggs and nymphs among the GM isolines and other bean genotypes. Despite the low incidence of CpMMV in the GM isolines derived from cv. Pérola, yield of these isolines was lower than that for some of the isolines derived from cv. BRS Pontal (CNFCT 16205, CNFCT 16206, CNFCT 16209 and CNFCT 16210) which had a higher incidence of CpMMV. At the Brazabrantes field a higher disease incidence was observed. The yield for cv. Pérola and BRS Pontal reached only 81 and 299 kg ha-1, respectively, and were significantly lower than the GM lines derived from cv. Pérola (711 kg ha-1) and cv. BRS Pontal (1073 kg ha-1). The low yield of the conventional common bean parental cultivars as compared to the GM isolines was due to the severe occurrence of BGMV. The GM isolines derived from cv. Pérola and from cv. BRS Pontal yielded an average of 878% and 358% higher, respectively, than the conventional accesses. The GM isolines have yield potential even at conditions of high incidence of B. tabaci and the CpMMV if a management program for whiteflies including cultural practices and insecticides is established.
O feijão é suscetível a várias viroses, incluindo o Bean golden mosaic virus (BGMV) e o Cowpea mild mottle virus (CpMMV). Como estes dois vírus são transmitidos pela moscabranca, Bemisia tabaci e a sintomatologia do BGMV é muito mais intensa em comparação a do CpMMV, é difícil separar a ocorrência das duas doenças em plantas de feijão. O desenvolvimento do feijoeiro evento Embrapa 5.1, resistente ao BGMV e de linhagens isogênicas transgênicas de dois cultivares comerciais (Pérola e BRS Pontal) permite a avaliação dos danos causados somente por CpMMV, uma vez que estas isolinhas não são infectadas por BGMV. Este estudo foi conduzido com o feijoeiro geneticamente modificado (FGM) para determinar: 1) os danos causados por ninfas de B. tabaci em três fases de desenvolvimento das plantas; 2) a incidência e os danos causados pelo CpMMV e correlacionar com o nível populacional de B. tabaci. Em casa de vegetação da Embrapa Arroz e Feijão foram avaliados os danos causados por seis diferentes densidades populacionais de ninfas de mosca-branca no FGM e feijoeiro convencional (FC) em três fases de desenvolvimento. No estádio V2 foram avaliadas médias de 0, 20, 40, 60, 100 e 200 ninfas folha-1; no estádio V3-V4 médias de 0, 40, 80, 130, 250 e 480 ninfas folha-1 e no estádio R6 médias de 0, 20, 50, 120, 280 e 760 ninfas folha-1. O delineamento experimental utilizado foi inteiramente casualizado, em esquema fatorial 6 x 2 (níveis populacionais x variedades de feijoeiro), com 12 repetições, exceto para o experimento infestado em R6, com nove repetições. A unidade experimental foi representada por duas plantas de feijoeiro. Foram avaliadas a incidência e severidade de BGMV, a presença de fumagina e os componentes de produção (número de vagens, grãos por planta, grãos por vagem e massa de grãos). Os experimentos de campo foram conduzidos em duas áreas experimentais da Embrapa Arroz e Feijão em Santo Antônio de Goiás (16°30’24,57” S; 49°17’06,53” W) e Brazabrantes (16°26’09, 20” S, 49°24’05,80” W), Goiás. Três isolinhas de FGM oriundas da cv. Pérola e seis oriundas da cv. BRS Pontal, as duas cultivares comerciais parentais e três outras cultivares convencionais IPR Eldorado, BRB 169, e CNFC 15882 foram comparadas. Foi avaliada a incidência de BGMV, CpMMV, número de ovos, ninfas e adultos de mosca-branca e os componentes de produção (massa de cem grãos e produtividade). A infestação de até 200 ninfas de B. tabaci biótipo B por folha em fase de folhas primárias (V2), 480 ninfas folha-1 em fase de crescimento vegetativo (V3-V4) e 760 ninfas folha-1 em fase de florescimento (R6) não reduziu a massa de grãos por planta, o número de vagens por planta, grãos por planta e grãos por vagem do FGM. O BGMV afetou a produção do FC em estádio entre V2 e V3-V4, mas não para o R6. O crescimento do fungo Capnodium sp. (fumagina), na substância açucarada excretada pelas ninfas, foi observado somente em plantas no estádio V3-V4. A população de adultos de B. tabaci biótipo B foi significativamente menor nas linhagens geneticamente modificadas provenientes da cv. Pérola (CNFCT 16201 e CNFCT 16203) e da BRS Pontal (CNFCT 16205) e nas cultivares convencionais IPR Eldorado, BRB 169 e CNFP 15882. Não foi observada diferença significativa na população de ninfas e ovos de moscabranca entre as linhagens de FGM e os outros genótipos de feijão. Apesar da baixa incidência de CpMMV nas linhagens de FGM provenientes da cv. Pérola (CNFCT 16201, CNFCT 16203 e CNFCT 16204), a produtividade destas linhagens foi significativamente menor em comparação as linhagens geneticamente modificadas da BRS Pontal (CNFCT 16205, CNFCT 16206, CNFCT 16209 e CNFCT 16210), que apresentaram alta incidência de CpMMV. No experimento em Brazabrantes foi observada maior incidência de doenças. A produtividade da cv. Pérola e BRS Pontal foi somente 81 e 299 kg ha-1, respectivamente, e significativamente menor que as linhagens FGM derivadas de cv. Pérola (711 kg ha-1) e cv. BRS Pontal (1073 kg ha-1). A baixa produtividade do FC em comparação ao FGM foi devido à ocorrência do BMGV que não se expressa nas linhagens geneticamente modificadas. As isolinhas do FGM derivadas da cv. Pérola e BRS Pontal produziram, em média, 878% e 358%, respectivamente, a mais de grãos em comparação ao FC. O feijoeiro geneticamente modificado, apesar da incidência do CpMMV, tem potencial para produzir em épocas de alta incidência de mosca-branca e de plantas infectadas por vírus se estabelecido programas de manejo de populações de adultos da mosca-branca com práticas culturais e inseticidas químicos.

Les interactions plante-virus diffèrent des autres interactions plante-pathogènes du fait de la nature des virus qui sont des parasites intracellulaires obligatoires. Plus spécifiquement, l’interaction haricot commun (Phaseolus vulgaris L.)-Bean pod mottle virus (BPMV) a été étudiée en mettant l’accent à la fois sur la résistance de la plante mais aussi sur la virulence du virus dans l’objectif de mieux comprendre et d’identifier les facteurs intervenant dans le dialogue moléculaire entre plante et virus. Ces deux partenaires interagissent selon le modèle «gène-à-gène» de Flor. 1) Côté plante, nous avons identifié un gène de résistance dominant vis-à-vis du BPMV chez BAT93, le gène R-BPMV. Ce gène est localisé à l’extrémité du chromosome Pv02, dans la région du locus I, un locus de résistance multi-parasitaire vis-à-vis de différents virus, bactérie et champignon. La cartographie fine du gène R-BPMV suivie du séquençage de la région à partir d’un contig de clones BACs chez BAT93 a permis d’identifier des séquences codant pour des protéines NB-LRR qui pourraient correspondre au gène R-BPMV. Des études de microsynténie et de phylogénie ont été réalisées afin de mieux comprendre l’évolution des gènes présents dans cette région. L’étude au niveau cellulaire du phénotype associé à la résistance a permis de montrer que le gène R-BPMV bloque le mouvement de cellule à cellule du virus et que le phénotype associé est température-dépendant. 2) Côté virus, le clonage de toutes les ORFs du BPMV associé à des expériences d’agroinfiltration sur P. vulgaris et Nicotiana benthamiana ont permis d’identifier deux facteurs viraux importants dans le dialogue moléculaire plante-virus : la protéine VPg du BPMV correspond à la protéine d’avirulence agissant en interaction avec le produit du gène R-BPMV dans le cadre du modèle «gène-à-gène», et l’ARN polymérase ARN-dépendante virale correspond à un suppresseur de silencing à effet faible. 3) A ce jour, la transformation génétique stable n’est pas applicable en routine chez les légumineuses. Un objectif de la thèse est de développer des outils de validation fonctionnelle pouvant s’appliquer à des gènes d’intérêt agronomique, dont des gènes de résistance aux maladies. L’approche VIGS basée sur un vecteur viral dérivé du BPMV, déjà utilisée chez le soja, a ainsi été adaptée sur haricot et pois (Pisum sativum), une légumineuse économiquement importante en Europe
Plant-virus interactions differ from other plant-pathogen interactions because viruses are obligate intracellular parasites. More specifically, common bean (Phaseolus vulgaris L.)-Bean pod mottle virus (BPMV) interaction was studied by focusing both on the plant resistance and on the virus virulence in order to highlight and identify factors involved in the molecular dialog between plant and virus. These two partners interact according to the “gene-for-gene” model described by Flor. 1) On the plant side, we identified a dominant resistance gene against BPMV in cv. BAT93, the R-BPMV gene. This gene is located at one end of chromosome Pv02 in the I locus region, a multi-parasitic resistance locus involved in resistance to different viruses, bacteria and fungi. Fine mapping of R-BPMV followed by sequencing of the region from a BACs contig in BAT93 allowed us to identify sequences encoding NB-LRR proteins that could correspond to R-BPMV. Microsynteny and phylogeny studies were performed to understand the evolution of genes present in this region. When resistance phenotype was studied at the cellular level, we found that R-BPMV blocks BPMV cell-to-cell movement and that resistance phenotype is temperature-dependent. 2) On the virus side, cloning of all BPMV ORFs in association with agroinfiltration assays in P. vulgaris and Nicotiana benthamiana allowed us to identify two important factors involved in plant-virus molecular dialog: the BPMV VPg acting as an avirulence factor in interaction with the product of R-BPMV in the “gene-for-gene” model, and the viral RNA-dependent RNA polymerase that corresponds to a weak RNA silencing suppressor. 3) To date, stable genetic transformation is not routinely feasible in legumes. One objective of this thesis was to develop news tools for functional validation studies for genes of agronomic interest, including disease resistance genes. The VIGS approach based on the viral BPMV vector, first used in soybean, was adapted to common bean and pea (Pisum sativum), a legume species of high economic importance in Europe

Bean pod mottle virus (BPMV, Genus Comovirus, Family: Comoviridae)is an important virus in soybean (Glycine max (L.) Merrill), causing quality and yield loss due to seed coat mottling and seed weight reduction. Although BPMV has been known in Virginia since 1958 and has always been regarded as causing negligible losses, its impact is changing as BPMV incidence has increased in many soybean growing areas of Virginia and the USA in general. From 1997 to 2001, a total of five BPMV isolates (V-W1, V-W2, V-S98-1, V-S98-15 and V-S01-10) were collected in Virginia and characterized. In this study, the effects of these isolates were studied, alone or with Soybean mosaic virus (SMV, Genus Potyvirus, Family Potyviridae) strain SMV G1, and isolates S98-51 and S98-52, on selected soybean cultivars. Individual isolates of BPMV showed variable symptom severity, and resulted in yield loss of between 40.4 to 58.1%, while SMV caused 23.7% in the most severe interactions. Up to 100% yield loss was realized from double inoculations of selected BPMV and SMV isolates, BPMV V-S98-1 + SMV S98-52 and BPMV S98-15 + SMV S98-52 on Hutcheson and Hutcheson Roundup Ready® (BC5) soybeans, respectively. Time of inoculation, a critical factor in the impact of many virus diseases, affected seed coat mottling in four cultivars and seed weight in two cultivars, in tests with four BPMV isolates and three stages of soybean development. All BPMV isolates inoculated to plants at vegetative stage V1-V3 severely increased seed coat mottling and reduced seed weight than those inoculated at V4-V6 and reproductive stage R1-R3. Seedlings grown from non-mottled seeds germinated more uniformly had fewer thin-stemmed seedlings and grew faster than those grown from mottled seeds. Inoculation of various cultivars and breeding lines showed that there was no correlation between the severity of virus-induced foliar symptoms, relative accumulation of SMV, and extent of seed coat mottling. Thus, by avoiding the presence of BPMV at an early growth stage through proper timing of planting to avoid vectors, proper cultural practices like weed control, use of SMV free seeds, and chemical control, it is possible to greatly improve seed quality and reduce yield losses in soybean.
Ph. D.

Southern Bean Mosaic Virus (SBMV) codes for a large polyprotein which is subsequently cleaved into smaller functional proteins. A virally encoded protease is suspected of mediating the cleavages. SBMV, picornaviruses and picornavirus-like viruses appear to have similar genomic organizations, which would place the protease region of the genome between the VPg and RNA dependen,, RNA polymerase regions. Protein sequence comparisons revealed homology between SBMV putative protease and the known proteases from foot and mouth disease virus, encephlamyocarditis virus, poliovirus, and cowpea mosaic virus. Experimental evidence provides little information on the exact location of the protease; however, protein sequence analysis suggests that the protease is indeed located between the VPg and RNA polymerase regions of SBMV. Comparisons of known cleavages sites to SBMV suggests that the QS amino acid pairs, at postions 539-540 and 664-665, are the most likely site for cleavage. The QS pair is present on the surface, in a region with high flexibility, which is in a turn. This evidence, although circumstantial, is supported by the deletion of the C-terminal end of the protein.

Bean pod mottle virus (BPMV), a member of the genus Comovirus in the family Comoviridae, is widespread in the major soybean-growing areas in the United States. The complete nucleotide sequences of the genomic RNAs of the naturally occurring partial diploid strain IL-Cb1 were determined. Intermolecular RNA1 recombinants were isolated from strain IL-Cb1 and characterized at the molecular level. Structurally similar recombinant RNA1 was also generated after four passages in soybean derived from plants previously inoculated with a mixture of infectious RNA1 transcripts from two distinct strains. BPMV was developed as a plant viral vector that is appropriate for gene expression and virus-induced gene silencing (VIGS) in soybean. The foreign gene was inserted between the movement protein (MP) and the large coat protein (L-CP) coding regions. The recombinant BPMV constructs were stable following several serial passages in soybean and relatively high levels of protein expression were attained. Successful expression of several proteins with different biological activities was demonstrated from the BPMV vector. Double infection of soybean by BPMV and SMV triggers a synergistic interaction leading to a serious disease. To investigate the underlying mechanism, helper componentprotease (HC-Pro) genes from several SMV strains and TEV were expressed from BPMV vectors. The recombinant BPMV vectors carrying the HC-Pro genes from SMV strain G7 or TEV induced very severe symptoms on soybean whereas constructs containing the HC-Pro gene from SMV isolate P10, a mild strain with an apparent defect in synergism, induced only very mild symptoms. Transient agroinfiltration assays using GFP-transgenic Nicotiana benthamiana showed that HC-Pro from SMV isolate P10 was not a RNA silencing suppressor, whereas those of SMV strain G7 and TEV exhibited strong suppressor activities. Analysis of chimeric HC-Pro genes and point mutations indicated that a positively charged amino acid at position 144 is critical for the suppressor function of not only SMV HC-Pro but also other potyvirus HC-Pro proteins. Although amino acid substitution at position 144 resulted in changes in small RNA profile, it did not affect HC-Pro stability.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
O presente trabalho consistiu no seqüenciamento e caracterização molecular das Cadeias Abertas de Leitura (Open Reading Frames - ORFs) 2 e 3 do genoma do isolado São Paulo do Southern bean mosaic virus (SBMV-SP), completando-se o seqüenciamento de todo o genoma desse isolado. A ORF 2 codifica uma poliproteína (serino protease – VPg – RNA polimerase RNA-dependente) e a ORF 3 um produto com função desconhecida. O seqüenciamento da ORF 2 apresentou 2889 nucleotídeos, incluindo-se o códon de terminação UGA, com 962 aminoácidos deduzidos e massa molecular estimada de aproximadamente 105 kDa. Dentro da ORF 2, localiza-se a ORF 3 contendo 398 nucleotídeos, incluindo-se o códon de terminação UAA, com 132 aminoácidos e massa molecular estimada de aproximadamente 15 KDa. A análise feita a partir das seqüências das ORFS 2 e 3 do genoma do SBMV-SP, quando comparadas com outras espécies do mesmo gênero e isolados do SBMV, depositadas no GenBank, mostrou que a ORF 2 apresenta maior identidade (91,4% na seqüência de nucleotídeos e 95,0% na seqüência de aminoácidos deduzidos) com o isolado de Arkansas. Resultado similar foi obtido em relação à ORF 3 com valores de identidade de 97,0% tanto para as seqüências de nucleotídeos e aminoácidos deduzidos. Dados de filogenia corroboram os dados de identidade. Regiões conservadas do gênero Sobemovirus também foram identificadas, tais como Sítio de Ligação à Capa Protéica (CBPS), a tríade catalítica da serino protease (H-D-S) e a seqüência de heptanucleotídeos (TTTAAAC). A expressão em Escherichia coli da porção C-terminal da RNA Polimerase RNA Dependente (RpRd) produziu uma proteína de fusão de aproximadamente 67 kDa no sistema pMAL c2-x e de 30 kDa no sistema pET 28a. Quando a proteína de fusão foi injetada em coelhos houve a produção de anti-soro específico para a proteína recombinante.
The present work consisted of the sequencing and molecular characterization of the Open Reading Frames (ORFs) 2 and 3 from the São Paulo isolate genome of Southern bean mosaic virus (SBMV-SP), completing the sequencing of all the genome of this isolate. The ORF 2 encodes a polyprotein (serine protease - VPg - RNA dependent RNA polimarase) and the ORF 3 one product with unknown function. The sequencing of the ORF 2 reveals 2889 nucleotides, including the stop codon (UGA), with 962 deduced amino acids e and estimated molecular weight of approximately 105 kDa. Nested in the ORF 2 were found the ORF 3 with 398 nucleotides, including the stop codon (UAA), with 132 deduced amino acids and estimated molecular weight of approximately 15 kDa. The analysis made from the sequences of the ORFs 2 and 3 from the SBMV-SP genome, when compared with other species of the same gender and isolates of SBMV, deposited in the GenBank, showed that the ORF 2 presents higher identity (91,4% in the nucleotide sequence and 95,0% in the deduced amino acids sequence) with Arkansas isolate. Similar result was obtained in relation to the ORF 3 with identity values of 97,0% for the nucleotides and deduced amino acids sequences. Phylogeny data corroborate the identity data. Conserved regions of the Sobemovirus gender had also been identified such as Coat Protein Binding Site (CPBS), serine protease catalytic triad (H-D-S) and heptanucleotide sequence (TTTAAAC). The Expression in Escherichia coli of the C-terminal region of the RNA dependent RNA polimerase produced a fusion protein of approximately 67 kDa in pMAL c2-x system and a 30 kDa protein in pET 28a system. When the fusion protein ...(Complete abstract click electronic access below)

Orientador: José Osmar Gaspar
Banca: Claudia Regina Bonini Domingos
Banca: Eliezer Rodrigues de Souto
Resumo: O presente trabalho consistiu no seqüenciamento e caracterização molecular das Cadeias Abertas de Leitura (Open Reading Frames - ORFs) 2 e 3 do genoma do isolado São Paulo do Southern bean mosaic virus (SBMV-SP), completando-se o seqüenciamento de todo o genoma desse isolado. A ORF 2 codifica uma poliproteína (serino protease - VPg - RNA polimerase RNA-dependente) e a ORF 3 um produto com função desconhecida. O seqüenciamento da ORF 2 apresentou 2889 nucleotídeos, incluindo-se o códon de terminação UGA, com 962 aminoácidos deduzidos e massa molecular estimada de aproximadamente 105 kDa. Dentro da ORF 2, localiza-se a ORF 3 contendo 398 nucleotídeos, incluindo-se o códon de terminação UAA, com 132 aminoácidos e massa molecular estimada de aproximadamente 15 KDa. A análise feita a partir das seqüências das ORFS 2 e 3 do genoma do SBMV-SP, quando comparadas com outras espécies do mesmo gênero e isolados do SBMV, depositadas no GenBank, mostrou que a ORF 2 apresenta maior identidade (91,4% na seqüência de nucleotídeos e 95,0% na seqüência de aminoácidos deduzidos) com o isolado de Arkansas. Resultado similar foi obtido em relação à ORF 3 com valores de identidade de 97,0% tanto para as seqüências de nucleotídeos e aminoácidos deduzidos. Dados de filogenia corroboram os dados de identidade. Regiões conservadas do gênero Sobemovirus também foram identificadas, tais como Sítio de Ligação à Capa Protéica (CBPS), a tríade catalítica da serino protease (H-D-S) e a seqüência de heptanucleotídeos (TTTAAAC). A expressão em Escherichia coli da porção C-terminal da RNA Polimerase RNA Dependente (RpRd) produziu uma proteína de fusão de aproximadamente 67 kDa no sistema pMAL c2-x e de 30 kDa no sistema pET 28a. Quando a proteína de fusão foi injetada em coelhos houve a produção de anti-soro específico para a proteína recombinante.
Abstract: The present work consisted of the sequencing and molecular characterization of the Open Reading Frames (ORFs) 2 and 3 from the São Paulo isolate genome of Southern bean mosaic virus (SBMV-SP), completing the sequencing of all the genome of this isolate. The ORF 2 encodes a polyprotein (serine protease - VPg - RNA dependent RNA polimarase) and the ORF 3 one product with unknown function. The sequencing of the ORF 2 reveals 2889 nucleotides, including the stop codon (UGA), with 962 deduced amino acids e and estimated molecular weight of approximately 105 kDa. Nested in the ORF 2 were found the ORF 3 with 398 nucleotides, including the stop codon (UAA), with 132 deduced amino acids and estimated molecular weight of approximately 15 kDa. The analysis made from the sequences of the ORFs 2 and 3 from the SBMV-SP genome, when compared with other species of the same gender and isolates of SBMV, deposited in the GenBank, showed that the ORF 2 presents higher identity (91,4% in the nucleotide sequence and 95,0% in the deduced amino acids sequence) with Arkansas isolate. Similar result was obtained in relation to the ORF 3 with identity values of 97,0% for the nucleotides and deduced amino acids sequences. Phylogeny data corroborate the identity data. Conserved regions of the Sobemovirus gender had also been identified such as Coat Protein Binding Site (CPBS), serine protease catalytic triad (H-D-S) and heptanucleotide sequence (TTTAAAC). The Expression in Escherichia coli of the C-terminal region of the RNA dependent RNA polimerase produced a fusion protein of approximately 67 kDa in pMAL c2-x system and a 30 kDa protein in pET 28a system. When the fusion protein ...(Complete abstract click electronic access below)
Mestre

Les plantes sont des organismes immobiles qui doivent répondre et s’adapter à des contraintes abiotiques et biotiques. Parmi les stress biotiques, les maladies virales, établies ou émergentes, peuvent être responsables de pertes de rendement majeures aux conséquences économiques importantes. Face aux phytovirus la lutte génétique constitue le moyen de lutte le plus efficace, le plus respectueux de l’environnement et du consommateur. Comprendre l’interaction entre les plantes et les virus reste indispensable pour rechercher de nouvelles sources de résistances. Ce travail de thèse s’intéresse à l’étude du pathosystème naturel Arabidopis thaliana/Turnip mosaic virus (TuMV). Les essais ont été menés majoritairement en conditions extérieures permettant une analyse de l’interaction dans un environnement multistress. La réponse d’A. thaliana a été explorée par l’étude de traits liés à la maladie et par la variation en métabolites primaires et secondaires. Ce travail a permis i) de caractériser de façon fine la réponse d’A. thaliana au TuMV en conditionsmultistress en exploitant la diversité naturelle d’une population mondiale et française ii) de déterminer l’architecture génétique de cette interaction par des approches de génétique d’association et de QTL mapping. Plusieurs nouveaux loci potentiellement impliqués dans la réponse ont été identifiés iii) de montrer l’intérêt du phénotypage métabolique pour discriminer les accessions en fonction de leur sensibilité au TuMV. La multidisciplinarité des approches constitue la richesse de ce travail de thèse qui contribue à une meilleure caractérisation et compréhension de la réponse des plantes lors d’une infection virale
Plants are immobile organisms which have to adapt to abiotic and biotic constraints. Among bioticstress, established or emerging viral diseases, may be responsible for major yield losses withsignificant consequences. Genetic control is the most effective, environmentally and consumerfriendlyway to control viral infections. Understanding plant/virus interactions remains essential tosearch for new sources of resistance. This work, focuses on the study of the natural pathosystemArabidopsis thaliana/Turnip mosaic virus (TuMV). Most of the trials were conducted in commongarden conditions allowing the analysis of the interaction in a multistress environment. A. thaliana’sresponse was explored through the study of disease-related traits and the variations in primary andsecondary metabolites. This work allows i) the fine characterization of A. thaliana’s response toTuMV in multistress conditions through the exploration of the natural diversity of a world and Frenchpopulation ii) to determine the genetic architecture of this interaction by genome wide associationsand QTL mapping. Several new loci potentially involved in the response have been identified iii) tohighlight the interest of metabolic phenotyping to discriminate accessions according to theirsusceptibility to TuMV. The multidisciplinary approaches contribute to a better characterization andunderstanding of plant-virus interaction

Polyclonal antisera were raised against isolates of bean common mosaic virus (BCMV) and bean common mosaic necrosis virus (BCMNV) using conventional serological methods. Infected tissues containing, respectively, 22 recognized BCMV and BCMNV isolates were tested against the two antisera by antigen-coated plate (ACP) ELISA and double antibody sandwich (DAS) ELISA. Results indicated that each immunoglobulin was virus-specific by DAS-ELISA, providing clear distinction between BCMV and BCMNV. A reverse transcription, polymerase chain reaction (RT-PCR)-based assay in combination with restriction endonuclease analyses, was developed for molecular detection of BCMV, BCMNV and their pathogroups. Specific detection of the two viruses was accomplished by constructing two virus-specific primer pairs that amplified a PCR product specific for each virus. Distinction of two BCMNV pathogroups (PG-III and PG-VI) was achieved by restriction enzyme XbaI digestion of BCMNV PCR products. However, none of the tested restriction enzymes clearly differentiated the five recognized BCMV pathogroups. A primer pair Dts/Uny15 specific for BCMV pathogroup V was also developed. By its RT-PCR application, four BCMV-PG-V isolates were differentiated from the other known variants of BCMV pathogroup I, II, IV and VII. Thus, by a combination of RT-PCR and restriction enzyme analyses, it was possible to differentiate both viruses, and two pathogroups of BCMNV, and one pathogroup of BCMV.
Graduation date: 1996

碩士
國立高雄師範大學
生物科技系
101
Abstract Soybean leaves with mosaic symptoms were collected from Wantan, Pintong in 2010. A virus isolate B2-1 was obtained via three successive single lesion isolations on Chanopodium quinoa. The purified preparation of the virus isolate B2-1 contained filamentous particles with the length of 883-866 nm and the wide of 11-15 nm. The virus isolate B2-1 could react with the antisera against Peanut stripe virus and potyviruses. Therefore, the RT-PCR was carried out using conserved degenerate primers for potyviruses and a specific primer designed in this study. The capsid protein gene sequence alignment showed the virus isolate B2-1 belongs to the genus Potyvirus. A total of 10,096 near full-length nucleotide sequences of B2-1 was amplified and determined by Vector NTI 19 program. The genome of virus isolate B2-1 consists of a 5ˈuntranslated region (5ˈ UTR) of 174 nucleotides, a single open reading frame (ORF) of 9,666 nucleotides encoding 3,221 amino acids, and a 3ˈuntranslated region of 256 nucleotides. As other potyviruses, the polyprotein of B2-1 contains nine cleavage sites for virus proteinases with the capacity to produce ten characteristic functional proteins of potyviruses. Among them, CP gene had a DAG motif, and the stop codon is TAA; NIb gene had a GDD motif; CI gene had a nucleotide binding motif, HC-Pro gene had a C-X8-C-X18-C-X2-C motif, a FRNK motif and a KLSC motif that associated with aphid transmission. The FRNK motif was also associated with the symptom appearance of B2-1. Comparison of the nucleotide and amino acid sequences of B2-1 virus with other potyviruses deposited in the GenBank of NCBI was carried out. The nucleotide or amino acid sequences of CP of B2-1 showed 90-91% identity with Bean common mosaic virus (BCMV) strain NL1. Furthermore, the CP nucleotide and amino acid sequences of virus isolate B2-1 showed a high identity (90-91%) with other BCMV strains, including MS1, NL1, NL2 ,US1 and BCMNV. The bootstrap consensus tree obtained from the phylogenetic analysis of CP nucleotide sequences between virus isolate B2-1 and foreign Potyvirus isolates showed the virus isolate B2-1 is a new strain in the BCMV family. The B2-1 antiserum was then generated using New Zealand white rabbit and the IgG was fractioned from the antiserum. The results of indirect ELISA demonstrated the virus isolate B2-1 antiserum had a high titer. Therefore, the IgG was applied in indirect ELISA, SDS-immunodiffusion and immunocapture RT-PCR for detecting and identifying BCMV isolate B2-1. These results will be useful for plant quarantine and for the management of BCMV virus disease in the future.

One of he economically important diseases of beans in 3outh Africa is caused by bean common mosaic virus. The virus occurs as a number z t .iso1ates> world-wide and breeding for resistance is aided by the id ’fixation of the prevalent strain. The isolate prevalent in South Africa was found to belong to virus pathogenicity group V. It was identified by inoculating a set of bean differential hosts recommended by Drijfhout (1978). Samples collected from the Transvaal and parts of Natal did not differ significantly in symptom development in response to the tests. The South African isolate causes a temperature-dependant systemic necrosis in plants with the I-gene and without the recessive gene resistance. It was still capable of inducing necrosis in "Nep 2", host resistance group 8, 60 hours post inoculation and 84 hours for "Peru 0257", host resistance group 9, when it transferred from a cold (20°C day) to a warm glasshouse (30°C). An hour at 30°C was sufficient to induce necrosis in "Nep 2". The results of a speckled sugar bean breeding programme are included and further suggestions are made for breeding for resistance and the elimination of bean common mosaic virus as a problem in South Africa.

碩士
國立屏東科技大學
植物保護系所
96
Canna spp. is an annual ornamental plant. A filamentous virus of 750-830 nm in particle size, was successfully isolated by mechanical inoculation from canna with mosaic symptom in 2005, temporarily and named CA-FT. The thermal inactivation point of the virus was 55 ℃, dilution end point was 10-4 and the longevity in vitro was 1 day at 24 ℃ and more than 6 months at -80 ℃. It had a very narrow host range restricted to three chenopodiaceous plant species among 68 tested plant species belonging to 17 families. Pinwheel and Laminated aggregates inclusion bodies typical of potyvirus infection were found in local lesion of Chenopodium quinoa and mosaic leaf of canna. A purifred virus preparation was obtain with a yield of 1.2 mg/ 100 g from diseased leaf. A virion protein with molecular weight of 32 kDa was detected where purified virus was denatured with sodium dodecyl sulfate (SDS) and analyzed by electrophoresis in polyacrylamide gels (SDS-PAGE) and by Western blotting. An antiserum with a titer of 1024 was obtained by immunizing a white rabbit with the purified virions, the antibody can effectively apply to detect virus in the field and in SDS-agar gel double diffusion test. The antiserum against the canna virus isolate reacted strongly with both its homologous antigen and Bean yellow mosaic virus (BYMV) antigen . A sequence of 1263 nucleotides (nts) was found and corresponding to BYMV infecting canna in China, in percent identities of the CP gene and the 3′-non coding regions (3′-NCR) by 93％ and 95％, respectively, and in percent identities of CP amino acid sequence of 93％. Accumulated data mentioned above indicate that the studied virus isolated from mosaic canna is a strain of BYMV . It is a new record of BYMV that infecting canna in Taiwan.

Thesis (M.S.)--University of Wisconsin--Madison, 1991.
Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 50-60).

碩士
國立臺灣大學
植物病理與微生物學研究所
95
Phaius tankervilliae is an indigenous orchid of Taiwan. Owing to be an almost extinct species, the plant needs to be protected extremely. Virus-like mosaic symptoms were observed on the leaves of P. tankervilliae which were found in mountain area of Chung-ho, Taipei County. Systemic symptom and chlorotic lesions appeared when we mechanically inoculated the plant saps onto the leaves of Nicotiana benthamiana and Chenopodium quinoa, respectively. After three successive single lesion isolations in C. quinoa, one virus isolate was obtained and named PT. The host range test of PT isolate was performed. We observed the filamentous virus particles with length of 750 nm in crude plant extract via the transmission electron microscope (TEM). Indirect ELISA test showed that the originally infected and PT-inoculated plants reacted with anti-potyvirus monoclonal antibody positively. Furthermore, RT-PCR was performed by using degenerate primers for potyviruses. A 1.8-kb fragment was amplified and cloned. The sequence of the fragment was compared with the NCBI GenBank database. The result indicated that the 1.8-kb fragment of PT isolate containing the 3’-terminal region of potyvirus including partial NIb gene, CP gene and 3’ untranslated region (UTR). Moreover, it had 97% nucleotide identity with Bean yellow mosaic virus (BYMV). Therefore, the virus obtained from P. tankervilliae is an isolate of BYMV, and designated as BYMV-PT isolate. RT-PCR and 5’RACE techniques were employed to analyze the remaining sequence of BYMV-PT by utilizing potyvirus degenerate primers and BYMV specific primers. After cloning, sequencing and analysis, the genomic sequence of BYMV-PT has 9547 nucleotides in length excluding poly(A) tail. Besides the 205-nt 5’UTR and 174-nt 3’UTR, there is one large open reading frame which encodes a polyprotein of 3046 amino acids with an M(r) of 347 kDa. According to the proteinase cleavage sites, the polyprotein can be processed into P1, HC-Pro, P3, 6K1 and 6K2, CI, NIa-VPg, NIA-Pro, NIb, and CP. Through the whole genome comparisons of BYMV-PT with other 44 potyviruses, BYMV isolates clustered together, and the most closely related virus to BYMV is Clover yellow mosaic virus (ClYVV). In order to study the function of virus genes during infection process, we used positional cloning and RT-PCR amplification to construct BYMV-PT full-length cDNA clone and acquired 23 clones with T7 promoter. The biological activity test of the cDNA clones of BYMV-PT is still going on. By back inoculation of BYMV-PT to healthy P. tankervilliae, the ELISA value was positive for the inoculated plants 7 days after inoculation. It demonstrated that P. tankervilliae could be infected by BYMV-PT. However, no obvious symptom was observed yet.

Four bean cultivars (“Bountiful”, “Pinto”, “Top Crop”, and “Tendergreen”) allowed only subliminal replication of the cowpea strain of southern bean mosaic virus (SBMV-C). Bean protoplasts, on the other hand, sustained replication of SBMV-C upon in vitro inoculation. Antigen accumulation of the bean strain of southern bean mosaic virus (SBMV-B) and SBMV-C in bean protoplasts was similar, indicating that the replicating capacity of both viruses does not differ in bean cells. When the four bean cultivars were inoculated with a mixture of sunn hemp mosaic virus (SH1VIV), a tobamovirus, and SBMV-C, the latter was readily detected in the inoculated primary leaves. The rate of spread of SBMV-C in the presence of SHMV was compared to the rate of spread of SBMV-B in bean co inoculated with SHMV. Virus accumulation in leaf blades, lateral veins, mid-ribs, petioles, stems and roots was similar for both strains in the non-vascular tissue of the inoculated leaf; a sharp decline in SBMV-C accumulation was observed starting from the lateral veins towards the mid and distal parts of the petiole, where virtually no virus could be found. These results contrasted with the uniform presence of SBMV-B throughout infected bean plants. Leaf strips blotted on nitrocellulose paper and developed as for Western blotting confirmed these results, with SBMV-C antigen being detected in mesophyll tissue and in epidermal cells of the lateral veins of the inoculated primary leaves. Electron micrographs of immunogold-labelled sections revealed the absence of uniform SBMV-C particles in the mesophyll cells; instead, heavily labelled, amorphous protein clumps in the vacuole were found. SBMV-C coat protein from infected cowpea and bean plants showed no difference in its mobility during electrophoresis in denaturing polyacrylamide gels. These results indicate that SHMV facilitates cell to cell spread of SBMV-C in inoculated bean leaves but does not allow for the movement of the latter through the vascular system. Lack of efficient assembly of SBMV-C does not impede cell-to-cell movement of the virus in the doubly-infected leaves, yet it is probably an important factor involved in determining the inability of SBMV-C to move into and/or through the vascular system.