PhD / In South Africa, tomato (Solanum /ycopersicum) is an important vegetable crop with considerable nutritional and economic value. Over the last decade, begomovirus (family Geminiviridae) infections associated with an upsurge of the whitefly vector, Bemisia tabaci, on tomato crops has become a serious threat to sustainable tomato production in South Africa. Begomovirus disease control in tomato is challenging and requires an integrated "pest" and "vector" management strategy, primarily based on the use of chemical and cultural practices aimed at reducing the virus vector as well as the use of resistant cultivars. Development of effective disease management practices for South Africa therefore requires detailed information on the complex vector-virus-host cropping system interactions. The aim of the study presented in this thesis was to investigate the South African whitefly vector/begomovirus/tomato-host pathosystem, with emphasis on the virus and vector diversity and distribution, and the identification of possible resistance sources. A survey of tomato-infecting begomoviruses was conducted during a six-year period (2006-2011 ). Techniques used to determine begomoviruses diversity included whole genome amplification using PCR, RCA (rolling circle amplification), conventional as well as next generation sequencing and development of a RCA-RFLP (restriction fragment length polymorphism) for rapid assessment of diversity. Sequence comparisons and phylogenetic analyses revealed the presence of three new monopartite begomovirus species, in addition to ToCSV, all of which belong to the African/South West Indian Ocean (SWIO) begomovirus clade. Recombination analysis indicated that all four tomato-infecting begomovirus species appear to be complex recombinants and suggests that they have evolved within the sub-Saharan Africa region, along with other African begomoviruses and that they are most likely indigenous to the region. Several weed species were also confirmed as symptomless begomovirus reservoirs, supporting their role in the emerging begomovirus epidemics in South Africa.

Vegetables play a major role in the livelihoods of the rural poor in Africa. Among major constraints to vegetable production worldwide are diseases caused by a group of viruses belonging to the genus Begomovirus, family Geminiviridae. Begomoviruses are plant-infecting viruses, which are transmitted by the whitefly vector Bemisia tabaci and have been known to cause extreme yield reduction in a number of economically important vegetables around the world. Several begomoviruses have been detected infecting vegetable crops in West and Central Africa (WCA). Small single stranded circular molecules, alphasatellites and betasatellites, which are about half the size of their helper begomovirus genome, have also been detected in plants infected by begomoviruses. In WCA, B. tabaci has been associated with suspected begomovirus infections in many vegetable crops and weed species. Sequencing of viral genomes from crops such as okra resulted in the identification of two previously known begomovirus species (Cotton leaf curl Gezira virus and Okra yellow crinkle virus) as well as a new recombinant begomovirus species (Okra leaf curl Cameroon virus), a betasatellite (Cotton leaf curl Gezira betasatellite) and new alphasatellites. Tomato and pepper plants with leaf curling were shown to contain isolates of new begomoviruses, collectively referred to as West African tomato-infecting begomoviruses (WATIBs), new alphasatellites and betasatellites. To study the potential of weeds serving as begomovirus reservoirs, begomoviruses and satellites in the weed Ageratum conyzoides were characterized. Sequence analyses showed that they were infected by isolates of a new begomovirus (Ageratum leaf curl Cameroon virus) that belong to the WATIBs group, a new betasatellite (Ageratum leaf curl Cameroon betasatellite), an alphasatellite and two types of defective recombinants between a begomovirus and an alphasatellite. Putative recombinations were detected in begomovirus genomes for all four plant species studied, indicating that recombination is an important mechanism for their evolution. A close relationship between the begomoviruses infecting pepper and tomato and A. conyzoides and the detection of the same alphasatellite in them support the idea that weeds are important reservoirs for begomoviruses and their satellites. With this high diversity, recombination potential and transmission by B. tabaci, begomoviruses and ssDNA satellites pose a serious threat to crop production in West and Central Africa.

Pathogen-derived resistance (PDR) strategies, particularly those based on post-transcriptional gene silencing, have been used with great success for the generation of transgenic plants with resistance to RNA viruses. In contrast, a suitable strategy for transgenic resistance to ssDNA plant viruses, including those viruses belonging to the Geminiviridae, has remained elusive. Further, there is no convincing evidence that either post-transcriptional gene silencing, or pathogen-derived resistance in general, would be broadly applicable to ssDNA plant viruses. Researchers at QUT have been developing a novel resistance strategy against ssDNA viruses based on virus-activated expression of a stably integrated suicide gene. The strategy, based on InPAct (In Plant Activation) technology, relies on a &quotsplit" suicide gene cassette being arranged in such a way that expression of a lethal ribonuclease (barnase) is dependent on the virus-encoded replication-associated protein (Rep). Upon infection, Rep mediates the release of the construct resulting in the reconstitution of a transcribable and translatable episomal suicide gene expression cassette. The research for this PhD describes the development of an InPAct vector designed to confer resistance to Tomato leaf curl begomovirus (ToLCV), a major cause of disease in Solanaceous crops in the tropics and subtropics. ToLCV-based InPAct vectors were constructed based upon two ToLCV isolates from Australia and North Vietnam. Prior to the generation of InPAct cassettes, the entire ToLCV-[Au] and ToLCV-Vie intergenic regions (IRs) were embedded within the castorbean catalase intron of a β-glucuronidase expression vector to determine the effect of the IR upon transcript processing. Using transient reporter gene assays in tobacco NT-1 cells, it was demonstrated that the ToLCV IRs both contained cryptic intron splice sites which interfered with efficient transcript processing and GUS expression. A series of truncations to the IRs were subsequently made to identify the potential cryptic intron splice sites and/or interfering sequences in both the ToLCV-[Au] and ToLCV-Vie IRs. The final truncated IRs, which were used in the construction the InPAct cassettes, comprised approximately 100 bp and appeared to contain all the necessary cis-acting elements required for efficient rolling circle replication (RCR). Using histochemical GUS assays and Southern analyses, the InPAct cassettes were shown to be activated and replicated only in the presence of the cognate viral Rep. GUS expression levels were shown to be further enhanced in the presence of the ToLCV replication-enhancer protein (REn) and by the addition of the Tobacco yellow dwarf mastrevirus origin of second strand synthesis into the cassette. Under these conditions, Rep-activated GUS expression from the InPAct vectors was found to reach levels similar to that of the benchmark CaMV 35S promoter. Fifteen independent transgenic lines containing the ToLCV-[Au] and -Vie InPAct-GUS cassettes were generated by Agrobacterium-mediated transformation of tobacco leaf discs. Using agroinfiltration and histochemical assays, Rep-mediated activation of the InPAct cassettes and subsequent GUS expression was demonstrated in 11 out of the 15 lines tested; six of which showed expression levels equivalent to, or higher than, that obtained using a CaMV 35S promoter control. Evidence for activation of the integrated InPAct cassettes at the molecular level was provided by Southern analyses, with showed both linear and open circular forms of the replicating InPAct episome in genomic DNA extracted from infiltrated leaf tissue. Following the demonstration of Rep-activatable reporter gene expression and episomal replication of the ToLCV-based InPAct-GUS vectors using transient and stable tobacco transformation assays, new ToLCV-based InPAct vectors were designed to express the lethal RNase, barnase, in an attempt to generate virus resistant plants. Although transient assays in NT-1 cells demonstrated some &quotleaky" expression of barnase from the InPAct vectors, the level of barnase-mediated cell death from the InPAct vectors was found to be significantly increased in the presence of the cognate Rep and REn. Thirteen independently transformed tobacco lines containing the ToLCV-[Au] InPAct-barnase cassette were generated by Agrobacterium-mediated transformation of tobacco leaf discs. However, agroinfiltration of these plants with ToLCV Rep and REn failed to activate a barnase response. Subsequent molecular analyses on two transgenic lines revealed that both contained mutations in the barnase-coding gene in a region known to encode the active site. These mutations were presumed to result from the leaky barnase expression during initial stages of the Agrobacterium transformation which would favour the selection of barnase mutant InPAct plants. To overcome the problems associated with leaky expression of barnase, a barstar-expression cassette was included in the ToLCV-[Au] InPAct-barnase cassette. Transient assays in non-transgenic tobacco leaves demonstrated that the basal levels of barstar expressed from the modified InPAct vector were sufficient to negate the effects of leaky barnase expression. Importantly, however, the level of barnase expression in the presence of Rep and REn was shown to be sufficient to overcome the basal levels of barstar. Seventeen independently transformed lines were generated with the ToLCV-[Au] InPAct-barnase/barstar cassette, and analysis of one line revealed the presence of an uncorrupted barnase-coding region. Using transient agroinfiltration assays, seven of the transgenic lines showed varying levels of cognate Rep and REn-activated, barnase-induced cell death. Fifteen transgenic lines were challenged with ToLCV-[Au] by injection of recombinant Agrobacteria containing an infectious ToLCV clone. Unfortunately, all lines displayed typical ToLCV symptoms and tested positive for virus by PCR at 28 days post-inoculation. The inability of the InPAct cassette to confer resistance to ToLCV may have been due to one or a combination of factors, including (i) a delay in barnase-induced cell death, (ii) homology-dependent silencing of the integrated cassette, (iii) generally low-level, Rep-activated barnase expression or (iv) excessive virus load due to the artifical method of inoculation. This study details the first report of a ToLCV-based InPAct system for Rep-induced transgene expression in planta. Despite failing to generate ToLCV-resistant plants, the research findings will provide a solid foundation to develop a more effective InPAct vector and ultimately assist in the generation of transgenic plants with resistance to ToLCV and potentially other ssDNA plant viruses, particularly the begomoviruses.

Begomoviruses are whitefly-transmitted, single-stranded DNA viruses which are often associated with weed plants, which may act as natural reservoirs of viruses that cause epidemics in crop plants. The aim of this study was to characterize the diversity of begomoviruses infecting weeds in Brazil, as a step to access their importance as natural reservoirs of these viruses. Total DNA was extracted from weeds collected in Viçosa (Minas Gerais state) and some municipalities of Alagoas state in 2009 and 2010. The viral genomes were amplified by RCA, cloned and sequenced. The sequences were used for comparisons with other begomoviruses, phylogenetic and recombination analyses. A total of 26 DNA-A clones were obtained. Sequence analysis indicated the presence of ten different begomoviruses. All isolates originating from samples of Blainvillea rhomboidea belonged to a single viral species, Blainvillea yellow spot virus (BlYSV), suggesting that BlYSV may be the only begomovirus present in this ubiquitous weed species. Four viruses represent new species, for which the following names are proposed: Sida yellow net virus (SiYNV) obtained from Sida micrantha, Sida mottle Alagoas virus (SiMoAV) obtained from Sida urens and Sida sp., Sida yellow blotch virus (SiYBV) and Sida yellow mosaic Alagoas virus (SiYMAV) obtained from S. urens. Recombination events were detected in the SiYBV isolates BR:Rla1:10 and BR:Rla2:10, with BR:CPH1:10 (also SiYBV) and an unknown virus as putative parents, and in the SiYNV isolate BR:Vic2:10, with BR:Vsa2:10 (SiMoAV) and Sida mottle virus (SiMoV) as putative parents. These results consitute further evidence that recombination is an important evolutionary process on the generation of genetic variability and on the evolution of begomoviruses. / Fundação de Amparo a Pesquisa do Estado de Alagoas / Begomovírus são vírus de DNA circular fita simples transmitidos por mosca branca, os quais são frequentemente associados com plantas daninhas, as quais podem servir como reservatórios naturais de vírus que causam epidemias em plantas cultivadas. O objetivo deste trabalho foi caracterizar a diversidade de begomovírus infectando plantas daninhas no Brasil, como um passo para acessar sua importância como reservatórios naturais desses vírus. DNA total foi extraído a partir de plantas daninhas coletadas em Viçosa (Minas Gerais) e alguns municípios do estado de Alagoas em 2009 e 2010. Os genomas virais foram amplificados por RCA, clonados e sequenciados. As sequências foram usadas para comparações com outros begomovírus e análises filogenética e de recombinação. Um total de 26 clones de DNA-A foram obtidos. Análise de sequência indicou a presença de dez diferentes begomovírus. Todos os isolados originários de Blainvillea rhomboidea pertenciam a uma única espécie viral, Blainvillea yellow spot vírus (BlYSV), sugerindo que BlYSV pode ser o único begomovírus presente nesta espécie de planta invasora. Quatro vírus representam espécies novas, para as quais os seguintes nomes são propostos: Sida yellow net virus (SiYNV) obtido de Sida micrantha, Sida mottle Alagoas vírus (SiMoAV) obtido de Sida urens e Sida sp., Sida yellow blotch virus (SiYBV) e Sida yellow mosaic Alagoas virus (SiYMAV) obtidos de S. urens. Eventos de recombinação foram detectados no SiYBV, isolados BR:Rla1:10 e BR:Rla2:10, com BR:CPH1:10 (também SiYBV) e um vírus desconhecido como possíveis parentais, e no SiYNV, isolado BR:Vic2:10, com BR:Vsa2:10 (SiMoAV) e Sida mottle virus (SiMoV) como possíveis parentais. Estes resultados constituem uma evidência adicional de que recombinação é um importante processo evolutivo na geração de variabilidade genética e evolução de begomovírus.

Made available in DSpace on 2015-03-26T12:42:05Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1674702 bytes, checksum: ab48c56e0fe4a5a3fc7dfa63a5d9e325 (MD5) Previous issue date: 2011-02-28 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Begomoviruses (family Geminiviridae) have one or two genomic components, infect dicotyledonous plants and are naturally transmitted by the whitefly Bemisia tabaci (Homoptera: Aleyrodidae). Begomoviruses cause diseases of major economic importance in many crops, especially in tropical and subtropical regions. In Brazil, a viral complex composed of at least eight species, including Tomato rugose mosaic virus (ToRMV), Tomato severe rugose virus (ToSRV) and Tomato yellow spot virus (ToYSV), is responsible for losses in tomato. The complete DNA-B sequences of ToRMV and ToSRV show an identity of 98.2% and the high identity of their common region sequences (96.2%) indicates that the two viruses may share the same DNA-B. For the successful establishment of a viral infection, several interactions are required between begomovirus and host proteins. In this work, experiments were conducted with the following objectives: (1) to analyze the importance of recombination and pseudorecombination in the generation of variability and adaptation to the host of ToRMV and ToSRV; (2) to examine the involvement of two components of the anaphase-promoting complex (APC7 and APC10) in begomovirus replication; (3) to detect and characterize host proteins which interact with the ToYSV movement protein, MP. For the first objective, Nicotiana benthamiana plants were inoculated with all possible combinations of ToRMV and ToSRV DNA-A and DNA-B. Additionally, recombination and nucleotide/amino acid sequence comparisons between ToRMV and ToSRV were performed. The results indicate that ToRMV has both a recombinant and pseudorecombinant origin, and that ToRMV exerts a negative interference over ToSRV. For the second objective, Arabidopsis thaliana plants overexpressing APC7, APC10 and different APC7 mutants were inoculated with Cabbage leaf curl virus (CaLCuV), and viral DNA accumulation was estimated in plants at 14 and 28 days post-inoculation (dpi). The results indicate that APC7 and APC10 negatively affect CaLCuV DNA accumulation, and that both the N-terminal and C-terminal portions of the APC7 protein are involved in this effect. For the third objective, leaves of N. benthamiana were agroinfiltrated with the "bait" construct NTAPi-MP, and 48 hours after agroinfiltration the leaves were collected for extraction and purification of protein heterocomplexes. However, this transient expression assay in leaves of N. benthamiana failed to detect interactions between ToYSV MP and host proteins. / Os begomovírus (família Geminiviridae) possuem um ou dois componentes genômicos, infectam plantas dicotiledôneas e são transmitidos naturalmente pela mosca- branca Bemisia tabaci (Homoptera:Aleyrodidae). Os begomovírus causam doenças de grande importância econômica em diversas culturas, principalmente em regiões tropicais e subtropicais. No Brasil, um complexo viral composto por pelo menos oito espécies, incluindo o Tomato rugose mosaic virus (ToRMV), Tomato severe rugose virus (ToSRV) e Tomato yellow spot virus (ToYSV), é responsável por grandes perdas na cultura do tomateiro. A sequência completa dos DNAs-B do ToSRV e ToRMV apresenta identidade de 98%, e a elevada identidade de sequência da região comum (96,2%) indica que os dois vírus podem compartilhar o mesmo DNA-B. Para o estabelecimento de uma infecção viral é necessária uma série de interações entre proteínas dos begomovírus e do hospedeiro. Neste trabalho, foram conduzidos experimentos com os objetivos de: (1) analisar a importância da recombinação e da pseudo-recombinação na geração de variabilidade e na adaptação ao hospedeiro dos begomovírus ToRMV e ToSRV; (2) analisar o envolvimento de APC7 e APC10, dois componentes do complexo promotor da anáfase, na replicação de begomovírus; (3) detectar e caracterizar proteína(s) do hospedeiro que interagem com a MP do begomovírus ToYSV. Para o primeiro objetivo, plantas de N. benthamiana foram inoculadas com todas as combinações possíveis entre o DNA-A e o DNA-B do ToRMV e do ToSRV. Análises de recombinação e das sequências de nucleotídeos e aminoácidos entre ToRMV e ToSRV também foram realizadas. Os resultados indicaram que o ToRMV apresenta uma origem recombinante e pseudo-recombinante, e que o ToRMV exerce uma interferência negativa sobre o ToSRV. Para o segundo objetivo, plantas de Arabidopsis thaliana superexpressando APC7 ou APC10 e diferentes mutantes para APC7 foram inoculadas com o begomovírus Cabbage leaf curl virus (CaLCuV) e o acúmulo de DNA viral foi estimado em plantas infectadas, aos 14 e 28 dias pós-inoculação (dpi). Os resultados indicaram que APC7 e APC10 afetam o acúmulo do CaLCuV, e que tanto a região N-terminal quanto a C-terminal de APC7 estão envolvidas neste efeito. Para o terceiro objetivo, folhas de N. benthamiana foram agroinfiltradas com a construção "isca" NTAPi-MP, e 48 horas após a agroinfiltração estas folhas foram coletadas para extração e purificação do heterocomplexo protéico. Entretanto, esse ensaio de expressão transiente em folhas de N. benthamiana não permitiu detectar interações entre a proteína MP do ToYSV e proteína(s) do hospedeiro.

Made available in DSpace on 2015-03-26T13:37:39Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2474594 bytes, checksum: 6cdab11f886d77e7d0c8a3d024dde318 (MD5) Previous issue date: 2008-07-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The genus Begomovirus (family Geminiviridae) includes viruses with a genome comprised of one or two molecules of circular, single-stranded DNA, transmitted to dicot species by the whitefly Bemisia tabaci. In Brazil, after the introduction of the B biotype of B. tabaci in the early 1990s, the incidence of begomoviruses in tomato has become frequent, with several reports of new viral species. Some of these species have become prevalent under field conditions, including Tomato severe rugose virus (ToSRV) and Tomato yellow vein streak virus (ToYVSV). The purpose of this study was to characterize two isolates of these species through the cloning of their whole genomes followed by molecular analysis, and the production of infectious clones for determination of their host ranges. Total DNA extracts of infected plants were used for whole genome amplification using the phi29 phage DNA polymerase. The amplification products were cloned into plasmids and completely sequenced. Sequence analysis indicated that the DNA-A and DNA-B of ToSRV-[BR:Pir1:05] and ToYVSV- [BR:Pda30:05] isolates had greater than 90% identities with other isolates of ToSRV and ToYVSV, respectively. The molecular analysis indicated that the DNA-A of the BR:Pir1:05 isolate may be the result of a recombination event, in which the virus acquired the rep ORF of Tomato rugose mosaic virus (ToRMV) and the cp ORF of an unidentified virus. Analysis of the DNA-B of the same isolate indicated the existence of a relationship with viruses that infect weed/wild hosts in Brazil, corroborating the hypothesis that viruses present in wild hosts led to the virus currently found in tomatoes. Additionally, because of the high identity observed between the DNA-B of ToSRV-[BR:Pir1:05] and ToRMV, it is possible that this genomic component is shared by the two viruses. In host range assays, plants showing latent infections were observed for both isolates. Such plants can act as natural reservoirs and serve as a primary source of inoculum for host plants of economic importance such as the tomato. The infectious clones of ToYVSV- [BR:Pda30:05] had low infectivity in the host range assays. It is possible that the inoculation method was not effective in the transmission of this isolate, or one or both clones could contain mutations that reduce the efficiency of their replication. / O gênero Begomovirus (família Geminiviridae) inclui vírus com genoma composto por uma ou duas moléculas de DNA fita simples, transmitidos a espécies de plantas dicotiledôneas pela mosca-branca Bemisia tabaci. No Brasil, após a introdução do biótipo B de B. tabaci no início da década de 1990, a incidência de begomovírus em tomateiro tornou-se freqüente, com vários relatos de novas espécies virais. Algumas dessas espécies tornaram-se prevalentes em condições de campo no Brasil, incluindo o Tomato severe rugose virus (ToSRV) e o Tomato yellow vein streak virus (ToYVSV). O objetivo deste trabalho foi caracterizar dois isolados dessas espécies, por meio da clonagem de seus genomas completos seguida de análise molecular, e da obtenção de clones infecciosos para determinação de suas gamas de hospedeiros. Extratos de DNA total de plantas infectadas foram utilizados para a amplificação do genoma viral completo utilizando-se a DNA polimerase do fago φ29. Os produtos da amplificação foram clonados em plasmídeos e completamente seqüenciados. A análise das seqüências do DNA-A e DNA-B dos isolados ToSRV- [BR:Pir1:05] e ToYVSV- [BR:Pda30:05] indicou valores de identidade acima de 90% com outros isolados de ToSRV e ToYVSV, respectivamente. A análise molecular indica que o DNA-A do isolado BR:Pir1:05 pode ser resultado de um evento de recombinação, no qual o vírus adquiriu a ORF rep do Tomato rugose mosaic virus (ToRMV) e a ORF cp de um vírus não identificado. A análise do DNA-B do mesmo isolado indica a existência de relacionamento com vírus que infectam plantas daninhas/silvestres no Brasil, reforçando a hipótese de que vírus presentes em hospedeiros silvestres deram origem aos vírus atualmente encontrados em tomateiro. Adicionalmente, devido à alta identidade observada entre os DNAs-B dos isolados BR:Pir1:05 e do ToRMV, é possível que este componente genômico seja compartilhado pelos dois vírus. Nos testes de gama de hospedeiros, plantas apresentando infecções latentes foram observadas para os dois isolados. Tais plantas podem atuar como reservatórios naturais e servir de fonte de inóculo primário para plantas hospedeiras de importância econômica, como o tomateiro. Os clones infecciosos do isolado BR:Pda30:05 apresentaram baixa infectividade nos testes de gama de hospedeiros. É possível que o método de inoculação não tenha sido eficiente na transmissão deste isolado, ou que os clones apresentem mutações que reduzam a eficiência de sua replicação.

Made available in DSpace on 2015-03-26T13:37:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 668759 bytes, checksum: 554c480543a7e27e41087d744a247db0 (MD5) Previous issue date: 2013-03-15 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Begomoviruses (genus Begomovirus, family Geminiviridae) comprise a group of plant viruses of great economic importance causing serious economic losses in tropical and subtropical crops. It is believed that the emergence of begomoviruses in Brazil occurred through horizontal transfer of viruses previously restricted to non-cultivated plants by the B biotype of Bemisia tabaci. Little is known about the genetic variability of weedinfecting begomoviruses. The study of this variability is important to understand how viruses evolve in order to adopt strategies for the development of crop cultivars with durable resistance. In this study we investigated the genetic variability of two weedinfecting begomoviruses, Euphorbia yellow mosaic virus (EuYMV) and Macroptilium yellow vein virus (MaYVV), which infect Euphorbia heterophylla and Macroptilium lathyroides, respectively. Our results, based on 19 DNA-A sequences of EuYMV and 18 of MaYVV obtained from samples collected in 2011 and 2012, support the hypothesis that the genetic structure of begomoviruses can be modulated by their hosts by common processes of selection, mutation and recombination. We observed distinct degrees of genetic variability between the two viruses. EuYMV presented a higher variability, similar to other weed-infecting begomoviruses, while MaYVV presented a lower variability. The nucleotide diversity of EuYMV (0.00819) was four-fold higher than that of MaYVV (0.00197). The mutation frequency of EuYMV (2.5×10-3) was higher than that of MaYVV (4.2×10-4). This difference was supported by the higher nucleotide diversity of all genes of EuYMV compared to MaYVV: CP (~three-fold), Rep (~seven-fold), Trap (~32-fold), Ren (~four-fold), AC4 (~eight-fold). Therefore the higher variability of EuYMV can be explained mostly by the Trap gene. The lower diversity observed for MaYVV could be due to its recent emergence compared to EuYMV, reported since the 1950's in Brazil. / Os begomovírus (gênero Begomovirus, familia Geminiviridae) são um grupo vírus de plantas de grande importância econômica causando sérias perdas em diversas culturas tropicais e subtropicais. Acredita-se que a emergência dos begomovírus presentes no Brasil se deu por meio da transferência horizontal de vírus anteriormente restritos a plantas silvestres e invasoras para plantas cultivadas mediada pelo biótipo B de Bemisia tabaci. A maioria dos trabalhos realizados até o presente tiveram enfoque na caracterização molecular dos begomovírus e pouco se sabe sobre a variabilidade genética das populações destes no campo. O estudo desta variabilidade é importante para entender como os vírus evoluem no sentido de serem adotadas estratégias para o desenvolvimento de cultivares com resistência durável. Neste trabalho foi investigada a variabilidade genética de dois begomovírus encontrados em plantas invasoras, Euphorbia yellow mosaic virus (EuYMV) e Macroptilium yellow vein virus (MaYVV), que infectam Euphorbia heterophylla e Macroptilium lathyroides, respectivamente. Os resultados, baseados em 19 sequências de EuYMV e 18 de MaYVV obtidas de amostras coletadas em 2011 e 2012, reforçam a hipótese de que a estrutura genética de begomovírus pode ser modulada pelo hospedeiro por processos comuns de seleção, mutação e recombinação. Foram observados diferentes graus de variabilidade genética entre os dois vírus. O EuYMV apresentou maior variabilidade, semelhante a outros begomovírus que infectam plantas não cultivadas, enquanto o MaYVV apresentou baixa variabilidade. A diversidade nucleotídica do EuYMV (0,00819) foi aproximadamente quatro vezes mais elevada do que a do MaYVV (0,00197). A frequência de mutação do EuYMV (2,5×10-3) foi superior à do MaYVV (4,2×10-4). Esta diferença foi suportada pela maior diversidade nucleotídica de todos os genes do EuYMV em comparação ao MaYVV: CP (aprox. três vezes maior), Rep (sete vezes), Trap (32 vezes), Ren (quatro vezes), AC4 (oito vezes). Esses resultados indicam que a maior variabilidade genética de EuYMV pode ser explicada, principalmente, pelo gene Trap. A menor variabilidade observada para o MaYVV pode ser devido à sua provável emergência recente em comparação ao EuYMV, relatado desde a década de 1950 no Brasil.

Made available in DSpace on 2015-03-26T13:42:06Z (GMT). No. of bitstreams: 1 texto completo.pdf: 737365 bytes, checksum: 3b9e993a4f8a367d7f1b82a2dded22f8 (MD5) Previous issue date: 2009-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Begomoviruses belong to the Geminiviridae family which includes viruses with genomes containing one or two single stranded DNA molecules within icosahedral geminate particles. Begomoviruses are transmitted by the whitefly Bemisia tabaci and may cause diseases of economical importance in several crops mainly in tropical and subtropical regions. In Brazil there are several reports of begomovirus causing serious losses to the common bean and tomato crops, and sporadic reports of infection of soybean. Virus-induced gene silencing (VIGS) is an attractive and fast alternative for studying the expression and function of genes because it does not require plant transformation. Viruses with RNA or DNA genomes have been successfully used as vectors to induce gene silencing in plants. However, there are few examples of efficient vectors for inoculation of tomato and soybean. The main goal of this work was to build a viral vector based on the DNA-A of the begomovirus Tomato rugose mosaic virus (ToRMV) to induce gene silencing in plants of soybean, tomato and Nicotiana benthamiana. The viral vector, designated pToR-A1.4ΔCP, was built from an infectious ToRMV-A clone from which the gene encoding the capsid protein was removed and replaced by the multiple cloning site of the plasmid vector pBluescript KS+ (pKS+). Like other begomoviruses, ToRMV does not require the capsid protein to cause a systemic infection. The construction of the viral vector was confirmed by PCR, enzymatic cleavage and DNA sequencing. To be used on gene silencing experiments, fragments from the following genes were PCR amplified from cDNA clones: phytoene desaturase (PDS) from soybean and tomato, myo-inositol-1-phosphate synthase (MIPS) from soybean and stachyose synthase (STS) from soybean. All fragments were amplified with primers contained restriction sites for cloning. After amplification, the fragments were cloned in pGEM-T Easy vector, sequenced and aligned to the corresponding cDNAs to confirm their identity. To clone these fragments in the viral vector they were cleaved out of the pGEM-T Easy vector, purified and cloned into the pToR-A1.4ΔCP vector previously cleaved at the multiple cloning site with the same enzymes used to cleave the plasmid vector. The ligation reaction used T4 DNA ligase and ultracompetent Escherichia coli cells were transformed by heat shock. Transformants colonies were analyzed by PCR and enzymatic cleaved. Systemic infection of soybean, tomato and Nicotiana benthamiana plants with the empty pToR-A1.4ΔCP vector co-inoculated with ToRMV DNA-B was checked by PCR 21 days after inoculation. In the case of N. benthamiana, the viral vector presented 82% infectivity, indicating the high potential of this vector for VIGS studies in this plant species. In the case of soybean and tomato, the viral vector presented low replication efficiency. New infectivity tests need to be done to confirm these results. It is expected that this viral vector will represent an alternative for functional genomic studies and for the analysis of genes involved in several biological processes. / Os begomovírus pertencem à família Geminiviridae, que inclui vírus com genoma composto por uma ou duas moléculas de DNA circular de fita simples, encapsidado em partículas icosaédricas geminadas. Os begomovírus são transmitidos pela mosca branca Bemisia tabaci e causam doenças de importância econômica em diversas culturas, principalmente em regiões tropicais e subtropicais. No Brasil, há diversos relatos de begomovírus causando sérias perdas nas culturas do feijoeiro e tomateiro e relatos esporádicos de incidência na cultura da soja. O silenciamento gênico induzido por vírus (VIGS) é uma alternativa atraente e rápida para o estudo da expressão e função de genes, pois não há necessidade de transformação genética da planta. Vírus com genoma de RNA e de DNA têm sido utilizados com sucesso como vetores para indução de silenciamento gênico em plantas. Entretanto, existem poucos vetores eficientes para a inoculação em plantas de tomate e de soja. O objetivo deste trabalho foi a construção de um vetor viral, baseado no DNA-A do begomovírus Tomato rugose mosaic virus (ToRMV), para a indução do silenciamento de genes em plantas de soja, tomate e Nicotiana benthamiana. O vetor viral, denominado pToR-A1.4ΔCP, foi construído a partir de um clone infeccioso do ToRMV-A do qual foi removido o gene da proteína capsidial, substituindo pelo sítio múltiplo de clonagem do vetor pBluescript KS+ (pKS+). Assim como outros begomovírus, o ToRMV não requer a proteína capsidial para causar infecção sistêmica. A construção do vetor viral foi confirmada por PCR, clivagem enzimática e sequenciamento. Para serem utilizados em experimentos de silenciamento gênico, fragmentos referentes aos genes fitoeno dessaturase (PDS) de soja e tomate, myo-inositol- 1-fosfato sintase (MIPS) de soja e estaquiose sintase (STS) de soja foram isolados a partir de cDNA dessas plantas, utilizando primers específicos, contendo sítios de restrição adequados para as clonagens. Após a amplificação, todos os fragmentos obtidos foram clonados em pGEM-T Easy, sequenciados e sua identificação foi confirmada por meio de alinhamento utilizando o algoritmo BLASTn. Para a clonagem no vetor viral, os fragmentos foram liberados do vetor pGEM-T Easy com as enzimas de restrição adequadas, purificados e inseridos no vetor pToR-A1.4ΔCP, previamente clivado em seu sítio múltiplo de clonagem com as mesmas enzimas. A reação de ligação foi realizada utilizando T4 DNA ligase e células de Escherichia coli ultracompetentes foram transformadas por meio de choque térmico. Os transformantes foram analisados por PCR e reação de clivagem enzimática. A infecção sistêmica de plantas de soja, tomate e Nicotiana benthamiana pelo vetor pToR-A1.4ΔCP vazio, inoculado conjuntamente com o DNA-B do ToRMV, foi diagnosticada via PCR aos 21 dias após a inoculação. Em N. benthamiana, o vetor viral apresentou 82% de infectividade, indicando um grande potencial de uso em estudos de VIGS nessa planta. Já em plantas de soja e tomate, o vetor viral apresentou baixa taxa de replicação. Portanto, novo teste de infectividade deve ser realizado para confirmar os resultados obtidos. Dessa forma, espera-se que este vetor viral sirva como um complemento e uma alternativa em estudos de genômica funcional e na análise de genes envolvidos em vários processos biológicos.

Viruses are submicroscopic obligate parasites and depend on the host cell for their growth and reproduction. Plants are infected by diverse group of viruses that mostly possess RNA as their genome. As exception, viruses belonging to the family Geminiviridae are DNA viruses and infect both mono and dicotyledonous plants causing a large economic loss. These viruses are smaller in size encoding fewer proteins and employ the host cell machinery for successful infection and spread. Geminiviruses undergo frequent recombinations due to mixed infection resulting in vast diversity across the family and account for driving evolution in these viruses. Movement in these viruses is complex since they have to cross two important barriers, nuclear and cell wall barrier to establish systemic spread. All these factors play very important role while designing control measures against these viruses. Thus a detailed understanding of these processes at molecular level is essential. Cotton is the major cash crop in Indian subcontinent with huge export values. India has become the second largest producer of cotton in the world. However, the major constraint in cotton cultivation has been crop loss due to diseases caused by viruses, particularly the cotton leaf curl disease (CLCuD) caused by begomoviruses. Present thesis deals with the analysis of genetic variability of CLCuD in India and functional characterization of proteins involved in the movement of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab). CLCuKV-Dab belongs to family Geminiviridae and subgroup Begomovirus. A review of the literature on Geminiviridae classification, genome organization, virus entry, replication, transcription, translation, assembly and movement is presented in Chapter 1. This chapter also includes the review of host factors involved in replication, geminiviral proteins involved in gene silencing and a detailed report on CLCuD complexes and sub viral DNAs that are associated with CLCuD. The materials used in this study and the experimental protocols followed such as construction of recombinant clones, their overexpression in both bacterial and baculovirus expression systems, Protein purification techniques, site directed mutagenesis and all other biochemical, molecular biology and cell biology methods are described in detail in Chapter 2. Previous study has reported the complete genomic sequences of CLCuKV-Dab and Tomato leaf curl Bangalore virus-cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequence of CLCuKV-Gang and the Cotton leaf curl Rajasthan virus (CLCuRV-Ban). Phylogenetic analysis of DNA-A sequences of these viruses with other CLCuD causing viruses is discussed in detail in Chapter 3. Chapter 4 deals with overexpression, purification and functional characterization of CLCuKV-Dab CP in terms of its interaction with DNA, the kinetics and its role in cell to cell movement. The proposed partners to CP in the cell to cell movement of monopartite begomoviruses are AV2 and AC4. Thus the Chapter 5 describes the functional characterization of recombinant AV2 of CLCuKV-Dab. Chapter 6 deals with expression of CP and AV2 as GFP fusion proteins in insect cells using baculovirus expression system to study the localization patterns of these proteins. Chapter 7 describes functional characterization of CLCuKV-Dab AC4. Bioinformatic analysis of AC4 showed that it belongs to the rare group of natively unfolded proteins that are functionally active In conclusion, there is a large genetic variability that exists among the begomoviruses and in particular, among the CLCuD causing begomoviruses in India. Functional characterization of the proteins involved in the cell to cell movement in CLCuKV-Dab led to a possible model for its movement; the CP translated in the cytoplasm is targeted the nucleus via its NLS and there binds to progeny ssDNA and exports the ssDNA out of nucleus through its export signals. AC4 or some other host proteins yet to be identified transports the ssDNA-CP complex from the nuclear periphery to AV2 present at the cell periphery. The complex is then transported from one cell to the neighboring cell via plasmodesmata. AC4 being an ATPase/NTPase could provide energy for the process.

1) Geminiviruses are a group of plant viruses which contain circular single stranded DNA molecules as their genomes and the capsid consists of two icosahedra fused together to form twinned or geminate particles. The largest genus in the family Geminiviridae is that of begomoviruses which are of two kinds; the monopartite begomoviruses which contain only one circular single stranded DNA molecule as their genome and the bipartite begomoviruses which contain two circular single stranded DNA molecules (designated DNA-A and DNA-B) as their genomes. In bipartite viruses, the two DNA molecules are enclosed in separate geminate capsids. 2) In bipartite begomoviruses, the DNA-A encodes the proteins essential for replication and encapsidation of the viral genome while the DNA-B encodes the proteins involved in movement. The DNA-B encodes two proteins: the BV1 or the nuclear shuttle protein (NSP) and BC1 or the cell-to-cell movement protein. Geminiviruses have DNA genomes which replicate inside the host cell nucleus. The NSP, which contains nuclear localization signal, brings the viral DNA from nucleus to the cytoplasm while the BC1 serves to take the viral genome to the cell periphery for movement to the neighbouring cell through the plasmodesmata. 3) The monopartite begomoviruses do not contain DNA-B (which, in bipartite begomoviruses, encodes the proteins involved in movement) and it has been suggested that some of the proteins encoded by DNA-A take up the movement function. Based on studies on TYLCV and CLCuV, a model has been proposed for the movement of monopartite begomoviruses according to which the coat protein (CP) of monopartite begomoviruses serves as the functional equivalent of the NSP of bipartite begomoviruses 4) The present thesis deals with the biochemical characterization of the C4 protein of the monopartite begomovirus CLCuKV-Dab. As stated in statement (3) above, the V2 and C4 proteins of monopartite begomoviruses have been implicated to be involved in cell-to¬cell movement of the viral genome. In TYLCV, both the proteins were shown to be localized to the cell periphery and could move from one cell to another through the plasmodesmata. Further, the V2 protein of CLCuKV-Dab was shown to interact with the coat protein and bind to single stranded DNA. The biochemical properties of the C4 protein needed to be elucidated in order to strengthen the proposal of its probable involvement in movement. 5) The objectives of the present study were: i) Bioinformatic analysis of the C4 protein of CLCuKV-Dab ii) Biochemical characterization of ATPase and pyrophosphatase activities of the C4 protein. iii) Studies on the effect of V2-C4 interaction on the enzymatic properties of C4. iv) Functional characterization of C4 in planta. 6) The FoldIndex© and PONDR analyses predicted the C4 protein of CLCuKV-Dab to be natively unfolded. Similarly, in PSIpred analysis, most of the C4 protein was predicted to be a random coil without any well-defined secondary structure. Further, the protein sequence was analyzed using the motifscan server. However, no motif for any specific function was predicted in the C4 Protein. 7) The C4 gene was initially cloned into pRSET-C vector and overexpressed as histidine tagged protein and the solubility of the protein was tested in various conditions including low temperature (18° C) after inducing the expression of the protein, buffers of various pH and different salt concentrations but the protein remained insoluble. Subsequently, the protein was purified under denaturing conditions and attempts were made to refold the protein but the protein precipitated during refolding. In order to get the C4 protein in soluble form, the C4 gene was subcloned into pGEX-5X2 vector and overexpressed as a GST-tagged fusion protein (GST-C4). Some of the GST-C4 protein was soluble which was purified by using GST-bind resin. The purified fusion protein was observed as a 37 kDa band on SDS-PAGE gel. The purified protein was accompanied by a degraded product of approximately 30 kDa size. Both the intact GST-C4 protein and the degraded product were detected in western blot analysis using anti-GST antibody. 8) Because C4 has been implicated to be involved in movement of monopartite begomoviruses and movement is an energy requiring process, it was of interest to determine if GST-C4 possesses ATPase activity. The purified GST-C4 protein was incubated with γ-[32P]-ATP, the product of the reaction was separated by thin layer chromatography and the chromatography plate was analyzed by phosphorimager. The hydrolysis of ATP by GST-C4 and the release of inorganic phosphate was clearly observed, suggesting that GST-C4 might possess ATPase activity. 9) The reaction conditions for the ATPase activity of GST-C4 were standardized. The activity increased linearly upto 2.60μM of the protein. The optimum temperature and pH for the ATPase activity were found to be 30 C and 6.0 respectively. The activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was stimulated by Mg+2, Mn+2 and Zn+2 but inhibited by Ca+2ions. Further, in the time course experiment, it was observed that the ATPase activity increased linearly upto one hour. 10) The Km, Vmax and kcat for the ATPase activity of GST-C4 were found to be 51.72 ± 2.5 µM, 7.2 ± 0.54 nmoles/min/mg of the protein and 0.27 min-1 respectively. Some of the other virally encoded ATPases have been found to exhibit kcat similar to that found for GST-C4 but it is much lower than those of most of the prokaryotic and eukaryotic ATPases (as mentioned in Table 3.3, page 100, chapter 3). Further, the presence of the degraded product did not affect the kinetic constants as described in chapter 3, pages 95¬-98. It is possible that the enzymatic activity might increase upon interaction with some ligand. 11) In the absence of any putative ATP binding motifs, systematic deletions from N-and C-termini were made to delineate the regions of C4 important for the ATPase activity. GST-N∆15-C4 and GST-N∆30-C4 exhibited approximately 70 % reduction in the ATPase activity while all the C-terminal deletion mutants (GST-C∆10-C4, GST-C∆20¬C4 and GST-C∆30-C4) retained the activity similar to the full length GST-C4 protein. This suggested that the N-terminal region of C4 may contain the residues important for the ATPase activity of GST-C4. 12) In the N-terminal region of C4, there is a sequence CSSSSR which closely resembles the sequence present at the active site of phosphotyrosine phosphatases (CXXXXXR). However, GST-C4 did not catalyze the hydrolysis of p-Nitrophenyl phosphate, a substrate analogue commonly used to assay phosphotyrosine phosphatase activity. It was of interest to determine if the cysteine and arginine in this sequence are important for the ATPase activity of GST-C4. GST-R13A-C4 exhibited an approximately two fold reduction in Vmax suggesting that R13 in C4 may be catalytically important for the ATPase activity of GST-C4. On the other hand, the C8A mutation did not affect the ATPase activity of GST-C4. 13) The GST-C4 protein was tested for its ability to hydrolyze several other phosphate containing compounds as mentioned chapter 2, pages 53-55. Among these compounds, GST-C4 catalyzed the hydrolysis of sodium pyrophosphate, that is, GST-C4 exhibited an inorganic pyrophosphatase activity. 14) The reaction conditions for the inorganic pyrophosphatase activity of GST-C4 were initially standardized. The pyrophosphatase activity of GST-C4 increased linearly upto 3.38 µM of the protein. The optimum temperature and pH for the pyrophosphatase activity were found to be 37° C and 7.0 respectively. The pyrophosphatase activity was inhibited by EDTA, suggesting that it is dependent on divalent metal ions. The activity was most efficiently stimulated by Mg+2, although it was also stimulated by Mn+2and Zn+2but inhibited by Ca+2ions. Thus, the pyrophosphatase activity of GST-C4 resembles the family I inorganic pyrophosphatases in metal ion requirements. Further, the pyrophosphatase activity increased linearly upto 1 hour 30 minutes. 15) The Km, Vmax and Kcat for the pyrophosphatase activity of GST-C4 were found to be 0.76 ± 0.04 mM, 141.16 ± 20 nmoles/min/mg of the protein and 5.2 minrespectively. The kcat for the pyrophosphatase activity was approximately 20 fold higher than that for the ATPase activity (0.27 min-1). 16) GST-N∆15-C4 and GST-N∆30-C4 exhibited >70 % reduction in the pyrophosphatase activity, a finding similar to that for the ATPase activity. On the other hand, while GST-C∆10-C4 retained the activity similar to the full length GST-C4 protein, GST-C∆20-C4 and GST-C∆30-C4 exhibited 20 % and 60 % reduction in the pyrophosphatase activity, respectively, as compared to the full length GST-C4 protein. This suggested that the C-terminal region of C4 may also contain the residues important for the pyrophosphatase activity of GST-C4. However, the C-terminal deletion mutants retained the ATPase activity similar to the full length protein. 17) The pyrophosphatase activity of GST-C4 was stimulated more than three fold by several reducing agents. The C4 protein contains only one cysteine (at position 8 in the C4 sequence). This was the first clue that the cysteine may be important for the pyrophosphatase activity of the GST-C4 protein. Further, the pyrophosphatase activity of GST-C4 did not exhibit preference for a particular kind of reducing agent like that of the pyrophosphatase activity in Streptococcus faecalis. 18) GST-C8A-C4 exhibited more than two fold reduction in Vmax, suggesting that the C8 may be catalytically important for the pyrophosphatase activity of GST-C4. On the other hand, the R13A mutation did not affect the pyrophosphatase activity of the GST-C4 protein. Thus, it is possible that during catalysis, the cysteine thiolate of C4 makes a 19) The pyrophosphatase activity of GST-C4 was inhibited by vanadate and fluoride. Vanadate was found to be a competitive inhibitor with Ki 0.33 mM while fluoride was a non-competitive inhibitor with Ki 2.82 mM. A comparative account of the two enzymatic activities of GST-C4 is presented in table 6.1