Doenças causadas por virus do gênero Begomovirus (família Geminiviridae) são incomuns em espécies de passifloras. Nos últimos dez anos, entretanto, foram encontrados no Brasil begomovirus infectando passifloras em pomares nos municipios de São Fidelis (RJ), Paragominas (PA), Patos de Minas (MG) e Araguari (MG). Estes isolados foram transmitidos mecanicamente para plantas de Nicotiana benthamiana, que apresentaram sintomas de mosaico e deformação foliar. Também foi possível a transmissão para plantas de Sida rhombifolia através de inoculação por biobalística com o DNA amplificado do isolado de São Fidelis. Estas plantas demonstraram sintomas de mosaico amarelo e deformação foliar. O DNA total extraído de plantas infectadas foi amplificado por RCA, sendo que o componente A (DNA-A) dos isolados de Paragominas e Patos de Minas foram sequenciados diretamente por \"primer walking\". O DNA-A dos isolados de São Fidelis e Araguari foram clonados e sequenciados. As sequências de nucleotideos dos isolados de Paragominas e de São Fidelis apresentaram 90% de similaridade ao Sida mottle virus (SiMoV), enquanto a sequência de nucleotídeos do isolado de Araguari apresentou 96% de similaridade ao Sida micrantha mosaic virus (SimMV). Assim esses isolados encontrados em maracujazeiro podem ser considerados estirpes do SiMoV e SimMV, respectivamente. Não foi possivel a transmissão desses isolados através de Bemisia tabaci biótipo B, no entanto, os insetos foram capazes de adquirir o vírus. O isolado de São Fidelis foi detectado separadamente na região onde se encontra a glandula salivar (cabeça e protórax) e na região posterior do inseto, indicando que o vírus transpos a barreira do mesenteron e circulou pela hemolinfa do inseto. Alves (2008) obteve uma forma atenuada do isolado de São Fidelis através de inoculações mecanicas em plantas de N. benthamiana. O DNA-A desta forma atenuada foi sequênciado e apresentou 90% de identidade ao isolado do qual se originou. A forma atenuada do begomovirus foi capaz de proteger plantas de maracujazeiro contra a estirpe severa do vírus. / Diseases caused by begomoviruses (family Geminiviridae) are hardly found in Passiflora species. In the last years, however, begomovirus infected passionflowers were found in orchards in the counties of São Fidelis (state of Rio de Janeiro), Paragominas (Pará), Araguari and Patos de Minas (Minas Gerais). These isolates were mechanically transmitted to Nicotiana benthamiana plants, which showed variable symptoms of mosaic and leaf distortion. Another susceptible host is Sida rhombifolia, which was biolistic inoculated with amplified DNA of São Fidelis isolate, and showed symptoms of yellow mosaic and leaf distortion. Total DNA extracted from field infected passiflora was amplified by RCA, and the DNA-A of Paragominas and Patos de Minas isolates were directly sequenced by primer walking. The A component of São Fidelis and Araguari isolates were cloned and also completely sequenced. The complete nucleotide sequence of DNA-A of Araguari isolate shared 96% identity with that of Sida micrantha mosaic virus (SimMV), whereas the DNA-A of Paragominas and São Fidelis isolates shared 90% identity with that of Sida mottle virus (SiMoV). These viruses may be consider as strains of SiMoV and SimMV, respectively. It was not possible to transmit these isolates by Bemisia tabaci biótipo B, although the insects were able to acquire the virus. São Fidelis isolate could be detected separately at salivary gland region and posterior region of the insect, indicating that the virus could cross the digestive tract and circulate in the hemolymph. Alves (2008) obtained a mild strain of São Fidelis isolate by mechanical inoculation in N. benthamiana plants. The mild isolate was able to protect passionflower against the severe isoalte of this begomovirus.

BACKGROUND:Tomato leaf curl Sudan virus (ToLCSDV) is a single-stranded DNA begomovirus of tomato that causes downward leaf curl, yellowing, and stunting. Leaf curl disease results in significant yield reduction in tomato crops in the Nile Basin. ToLCSDV symptoms resemble those caused by Tomato yellow leaf curl virus, a distinct and widespread begomovirus originating in the Middle East. In this study, tomato samples exhibiting leaf curl symptoms were collected from Gezira, Sudan. The associated viral genome was molecularly characterized, analyzed phylogenetically, and an infectious clone for one isolate was constructed.FINDINGS:The complete genomes for five newly discovered variants of ToLCSDV, ranging in size from 2765 to 2767-bp, were cloned and sequenced, and subjected to pairwise and phylogenetic analyses. Pairwise analysis indicated that the five Gezira isolates shared 97-100% nucleotide identity with each other. Further, these variants of ToLCSDV shared their highest nucleotide identity at 96-98%, 91-95%, 91-92%, and 91-92% with the Shambat, Gezira, Oman and Yemen strains of ToLCSDV, respectively. Based on the high maximum nucleotide identities shared between these ToLCSDV variants from Gezira and other previously recognized members of this taxonomic group, they are considered isolates of the Shambat strain of ToLCSDV. Analysis of the complete genome sequence for these new variants revealed that they were naturally occurring recombinants between two previously reported strains of ToLCSDV. Finally, a dimeric clone constructed from one representative ToLCSV genome from Gezira was shown to be infectious following inoculation to tomato and N. benthamiana plants.CONCLUSION:Five new, naturally occurring recombinant begomovirus variants (>96% shared nt identity) were identified in tomato plants from Gezira in Sudan, and shown to be isolates of the Shambat strain of ToLCSDV. The cloned viral genome was infectious in N. benthamiana and tomato plants, and symptoms in tomato closely resembled those observed in field infected tomato plants, indicating the virus is the causal agent of the leaf curl disease. The symptoms that developed in tomato seedlings closely resembled those observed in field infected tomato plants, indicating that ToLCSDV is the causal agent of the leaf curl disease in Gezira.

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Geminiviruses (family Geminiviridae) are characterized structurally by twinned (geminate) morphology of virions (ca. 18-30 nm) and genetically by a genome comprising one or two small circular single stranded DNA (ssDNA) molecules and they are responsible for major crop losses worldwide. The genus Begomovirus (type member Bean golden yellow mosaic virus) is the largest genus of the family Geminiviridae. The members of this genus have either monopartite or bipartite genomes. They are transmitted by whiteflies and infect only dicotyledonous plants. DNA β molecules are symptom modulating single-stranded sat-DNA molecules which are associated with certain monopartite begomoviruses. These molecules are around half the size (approximately 1350 nt in length) of their helper viruses and rely on the helper begomovirus for movement in plant tissues, replication and plant-to-plant transmission by the whitefly (Bemisia tabaci). They contribute to production of symptoms and enhance helper virus accumulation in certain hosts. DNA β molecules encode a single gene, called βC1, on the complementary strand which is important for pathogenicity and suppression of post transcriptional gene silencing. In this study the regulation of βC1 gene expression, a host factor interacting with βC1 and its role in the pathogenicity of DNA β are described. Transient expression studies using Nicotiana tabacum plants and GUS as a reporter gene, identified the sequences important for transcription of βC1 from DNA β associated with Cotton leaf curl Multan virus (CLCuMV). A 68 nt fragment (between -139 to -207), which contains a G-box motif was sufficient for DNA β promoter activity. Deletion of this region also led to loss of DNA β replication capacity. Mutation of the G-box, located at 143 nucleotides upstream of the βC1 start codon, resulted in a two to three times reduction in the DNA β promoter activity. This motif was shown to bind specifically to the nuclear factors isolated from tobacco leaf tissues. Histochemical staining of transgenic tobacco plants expressing the gus gene driven by full length DNA β promoter showed phloem specific localisation patterns. It was concluded that a G-box motif is required for binding of host nuclear factors and is necessary for efficient expression of this phloem specific βC1 gene. An ubiquitin-conjugating enzyme, called SlUBC, was retrieved from screening of a tomato cDNA library, using βC1 encoded by DNA β associated with CLCuMV as the bait. The SlUBC was shown to complement yeast deficient in the ubiquitin-conjugating enzyme. It is thought that this enzyme is a key factor in the ubiquitin proteasome pathway, which plays a central role in many eukaryotic cellular processes. The authenticity and specificity of this interaction was confirmed both in vivo, using a bimolecular fluorescence complementation assay, and in vitro. Domain mapping of βC1 showed that a myristoylation-like motif is required for the interaction with SlUBC in the yeast system and induction of DNA β specific symptoms in host plants. Western blot analysis showed that expression of βC1 in transgenic tobacco plants decreased the level of poly-ubiquitinated proteins as compared with wild type plants. However, the level of expression of homologous SlUBC remained stable in these transgenic plants. These results indicated that interaction of βC1 with the SlUBC is required for DNA β specific symptom induction possibly through down-regulation of the host ubiquitin proteasome pathway. Using GFP transgenic N. benthamiana plants, the βC1 encoded by DNA β associated with CLCuMV showed suppression of post transcriptional gene silencing. This protein inhibited both local and systemic silencing. However, the low level of GFP fluorescence and also the results of RNA analysis in patch co-infiltration assay indicated that βC1 is a weak suppressor of local RNA silencing as compared with P19 protein from Tomato bushy stunt virus. A three-way grafting assay and separate patch infiltration assays showed that βC1 interferes with the activity of GFP silencing signal. Mutation of Gly103 in βC1 which was shown to be required for interaction with SlUBC and induction of DNA β specific symptoms in host plants, had no effect on the silencing suppression activity of βC1 protein. This work has provided a new insight into the importance of a G-box motif in expression of βC1 gene of DNA β and also for binding to the host nuclear proteins. In addition, interaction with a host factor, SlUBC, has been shown to be required for induction of DNA β specific symptoms in experimental plants using ToLCV as a helper virus. However, this interaction was not required for silencing suppression activity of βC1. The results of this study can be adapted to determine the mode of pathogenesis and regulation of expression of βC1 in cotton leaf curl disease. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1337164 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2016-06-14T11:23:47Z No. of bitstreams: 1 texto completo.pdf: 947659 bytes, checksum: e40a84800499bceebcf0b0b2051d2265 (MD5) / Made available in DSpace on 2016-06-14T11:23:47Z (GMT). No. of bitstreams: 1 texto completo.pdf: 947659 bytes, checksum: e40a84800499bceebcf0b0b2051d2265 (MD5) Previous issue date: 2007-07-17 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Geminivírus constituem um grupo de vírus com genoma composto de DNA de fita simples que se replicam no núcleo de células do hospedeiro através de um intermediário dupla fita, podendo ser constituídos por um genoma mono ou bi-segmentado. O transporte do DNA viral do núcleo para o citoplasma de geminivírus bi-segmentados requer a proteína viral designada NSP, Nuclear Shuttle Protein (BV1). A localização de NSP e seu papel proposto no movimento célula-a-célula do DNA viral predizem que podem ocorrer interações com fatores do hospedeiro tanto no citoplasma quanto no núcleo. Inicialmente foi demonstrado que a proteína NSP de geminivírus que infecta Arabidopsis Cabbage leaf Curl Virus (CaLCuV) interage com receptores cinase (RLK) contendo repetições ricas em leucina (LRR) designadas NIK (NSP-Interacting Kinase). A interação NSP-NIK foi mapeada em NIK1 e ocorre através de uma região de 81 resíduos de aminoácidos do domínio cinase (aa 422-502) que compreende o potencial sítio ativo ser/tre cinase (subdomínio VIbHrDvKssNxLLD) e a alça de ativação (subdomínio VII-DFGAk/rx, mais subdomínio VIII GtxGyiaPEY). A proteína comportou-se como um autêntico receptor cinase, sofrendo autofosforilação in vitro. Sendo, entretanto, a atividade cinase inibida pela proteína NSP. A fim de analisar o papel biológico da interação NSP-NIK, ensaios de infecção foram realizados em plantas contendo alelos nulos para o gene NIK1. Estes resultados demonstraram que a inativação dos alelos NIK1 e NIK3 aumentou a suscetibilidade à infecção causada pelo CaLCuV. Dada a sobreposição do domínio de interação com NSP com domínios regulatórios, foram realizados estudos bioquímicos através de mutagênese sítio dirigida em NIK1. A fim de mapear os sítios envolvidos na ativação da fosforilação de NIK1, o resíduo Thr-474 na alça de ativação foi substituído para resíduos de Ala, Asp ou Glu. Além disso, os resíduos Thr-468, Thr-469 e Ser-465 foram trocados por Ala. Foi demonstrado que a proteína receptora NIK1 exibe um padrão complexo de sítios de fosforilação, que agem independentemente em reações de transfosforilação e fosforilação de substratos. O resíduo de Thr-474 possui um papel central na ativação da proteína cinase, uma vez que os mutantes T474A e T474E apresentaram baixas atividades de auto e fosforilação do substrato. Similarmente, substituição do resíduo Gly-473 por Val juntamente com substituição de Thr-474 por Ala aboliu a autofosforilação e a fosforilação do substrato, sugerindo que, esta mutação acarreta modificações estruturais da alça de ativação que promovem impedimentos estéricos. Em contraste, a substituição dos resíduos Thr-469 e Ser465 por alaninas não causou impacto acentuado na autofosforilação, mas aumentou a atividade de fosforilação do substrato. Provavelmente, fosforilação desses resíduos provocam um efeito inibitório na atividade cinase. Além disso, foram conduzidos experimentos de complementação em A. thaliana nocautes para NIK. Complementação de NIK1 restaurou o fenótipo selvagem com a diminuição da suscetibilidade ao vírus. Em contraste, a expressão de NIK1 mutante com o domínio cinase inativo não reverteu o fenótipo do nocaute nik1, mantendo a elevada taxa de infecção. Estes resultados suportam o argumento de que o domínio cinase de NIK1 medeia uma via de sinalização que culmina em uma resposta de defesa. / Geminiviruses are small, single-stranded DNA viruses that replicate through doublestranded DNA intermediates in the nuclei of their plant hosts and can be mono or bipartite. The transport of bipartite viral DNA from the nucleus to the cytoplasm requires the viral protein NSP, and the nuclear shuttle protein NSP (BV1), both required for systemic infection. The localization of NSP and its proposed role in cell-to-cell movement of the viral DNA predict that interactions with host factors may occur in both the cytoplasm and the nucleus. Initially we have demonstrated that the NSP from an Arabidopsis-infecting geminivirus, Cabbage leaf Curl Virus (CaLCuV) interacts with a leucine rich repeat (LRR) receptor like kinases (RLK) designated NIK (NSP-Interacting Kinase). The NSP-NIK interaction occurs in NIK1 through an 81 amino acid region of the kinase domain (aa 422502) that encompasses the putative active site for ser/thr kinases (sub-domain VIbHrDvKssNxLLD) and the activation loop (sub-domain VII-DFGAk/rx, plus subdomain VIII -GtxGyiaPEY).The protein behaved like authentic receptor kinase undergo autophosphorylation in vitro. However, NSP protein inhibits the kinase activity. To unravel the biological significance of the NSP-NIK interaction we have selected nik null alleles for infection assays. Inactivation of NIK1 and NIK3 alleles enhanced the susceptibility to geminivirus infection. Given the overlap of the NSP-interacting domain with regulatory domains for kinase activity, we focused on site-directed mutagenesis studies on NIK1 To map the phosphorylation sites involved in activation of NIK1, we have replaced Thr-474 in the activation loop by Ala, Asp or Glu residues, whereas Thr-468, Thr-469 and Ser 465 were replaced by Ala. We found that NIK1 exhibits a complex pattern of phosphorylation sites that function independently in auto- and in substrate phosphorylation. The Thr-474 residue seems to play a pivotal role in the activation of the kinase protein, since the mutants T474A and T474E exhibited low auto- and substrate phosphorylation activities. Furthermore, the replacement of Gly-473 by Val in addition to Thr-474 by Ala abolished autophosphorylation and phosphorylation of substrate, sugesting that this mutation promotes a structural rearrangement of the A-loop that compromises activity. In contrast, the replacement of Thr469 and Ser-465 for alanines did not impact autophosphorylation significantly, but increased substrate phosphorylation activity. Possibly the phosphorylation of these residues impairs the kinase activity. In addition, we conducted complementation experiments in the nik1 genetic background. NIK1 expression in nik1 complemented the mutant and restored the wild type phenotype, with decreased virus susceptibility. In contrast, an inactive kinase domaincontaining NIK1 mutant did not reverse the enhanced susceptibility phenotype of the knockout lines and kept a high infection rate. These results are consistent with a model in which the kinase domain of NIK mediates a signaling pathway that culminates in antiviral defense.

Made available in DSpace on 2015-03-26T13:42:30Z (GMT). No. of bitstreams: 1 texto completo.pdf: 2281509 bytes, checksum: 8e44475ccbcd12f91301ea5e4fda8830 (MD5) Previous issue date: 2013-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Plants are engaged in a continuous co-evolutionary struggle for dominance with their pathogens and the outcomes of these interactions are of particular importance to human activities, as they can have dramatic effects on agricultural systems (Dodds & Rathjen, 2010). Recently, the convergence of molecular studies of plant immunity and pathogen infection strategies is revealing an integrated picture of the plant pathogen interaction (Mukhtar et al., 2011) in which the pathogen effectors interaction converge onto highly connected subgroups of proteins, named hubs. A well-defined hub form plant immune system network corresponds to CSN5A protein, a catalytic subunit of the COP9 signalosome acting as a key regulator in several basic cellular processes. Consistent with the prediction that different effectors from different pathogens target similar connections in plant-pathogen interaction network, it has been shown, independently, that the protein C2 from geminivirus, a DNA virus that infects a wide variety of agronomic crops, interacts to CNS5A (Lozano- Duran et al, 2011). Additionally, it was shown that NIG and the immune receptor NIK, both targets of geminivírus NSP, interact to CSN5A (Machado, 2011; Mukhtar et al., 2011). Based on this information, it is expected that the hub CNS5A is a functional element in the geminivirus-host interaction network. Recently, it was reported that NIG, a cellular partner of CSN5A, also interacts with a unknown function protein, encoded by the locus AT2G41020 in yeast (Machado, 2011). As a possible component from geminivirus-host interaction network converging to CSN5A, AT2G41020 may interact directly or indirectly with virulence factors in defense response or compatibility. The objectives of this research involved biochemical characterization of the protein encoded by the locus AT2G4102, designated AtWWP1, and identification of its possible interactions with viral proteins and host factors. In silico analysis of tWWP1 predicted structure revealed the presence of two WW domains, and a C-terminal domain highly conserved between homologous in plant and animals. Furthermore, it has been shown that AtWWP1 is a nuclear protein capable of forming nuclear bodies via the conserved C-terminal domain. Coimmunoprecipitation and BiFC assays demonstrated that AtWWP1 interacts in vivo with the cytoplasmic protein NIG, redirecting it to nuclear bodies. In order to explore the formative activity of nuclear bodies AtWWP1, the interaction between AtWWP1 and a second protein partner AtMBD2 (methyl CG binding domaincontaining protein) was characterized in vivo. The ability to form nuclear bodies as interaction with AtMBD2 were mapped AtWWP1 occuring via its domain and C-terminal conserved, substantiating the argument that this region of AtWWP1 is responsible for the formation of nuclear bodies. Colocalization assays have shown that nuclear bodies contained in AtWWP1 are distinct from those formed by proteins involved in RNA splicing, but colocalized with nuclear bodies containing CDKC2. Furthermore, it was demonstrated that AtWWP1 does not bind to RNA, but exhibits a binding activity to DNA. These characteristics imply that AtWWP1 should be involved with basic nuclear functions. As a component of a functional hub in geminivirus-host interaction network, it is important to assess whether the viral infection would affect the nuclear bodies formed by AtWWP1. / Estudos moleculares envolvendo o sistema imune de plantas e a infecção por patógenos revelaram um panomara integrado de interações plantapatógeno em que as interações dos efetores de virulência convergem para subconjuntos de proteínas do hospedeiro altamente interconectadas e designadas hubs. Um hub funcional e bem definido do sistema imune de plantas corresponde a interconexões convergentes para a proteína CNS5A que constitui a subunidade catalítica do complexo COP9 signalosome, um regulador chave de diversos processos celulares básicos. Consistente com a previsão de que efetores de diferentes patógenos devem alvejar similares conexões na rede de interações planta-patógeno, foi demonstrado, independentemente, que a proteína C2 de geminivírus, um vírus de DNA que infecta uma grande variedade de culturas agronômicas, interage com a proteína CNS5A. Além disso, foi também demonstrado que tanto a proteína NIG, quanto o receptor imune NIK, ambos alvos da proteína NSP de geminivírus, também interagem com CNS5A. Baseado nestas informações, prevê-se que a interconexão (hub) representada por CNS5A seja um elemento funcional na interação geminivírus-hospedeiro. Recentemente, foi identificado que, além de interagir com CSN5A, a proteína NIG também interage com uma proteína de função desconhecida codificada pelo locus AT2G41020, em leveduras . Como possível componente da rede de interações geminivírus-hospedeiro que converge em CNS5A, AT2G41020 pode interagir direta ou indiretamente com fatores de virulência em resposta de defesa ou de compatibilidade. Sendo assim, os objetivos principais dessa investigação envolveram caracterização bioquímica da proteína codificada pelo locus AT2G41020 e identificação de possíveis interações com proteínas virais e fatores do hospedeiro. Análise in silico da estrutura predita da proteína codificada pelo lócus At2G41020, designada AtWWP1, revelou a presença de dois domínios WW e um domínio C-terminal altamente conservado entre proteínas homólogas de espécies vegetais e animais. Além disso, foi demonstrado que a proteína AtWWP1 é uma proteína nuclear capaz de formar corpos subnucleares via o domínio C-terminal conservado. Ensaios de coimunoprecipitação e BiFC demonstraram que AtWWP1 interage in vivo com a proteína citoplasmática NIG promovendo o seu redirecionamento para corpos nucleares. Com a finalidade de explorar a atividade formadora de corpos nucleares de AtWWP1, a interação entre AtWWP1 e uma segunda proteína parceira AtMBD2 (proteína contendo um domínio de interação com CG metilado) foi caracterizada in vivo. Tanto a capacidade de formar corpos nucleares quanto a interação com AtMBD2 foram mapeadas em AtWWP1 e ocorrem via seu domíno C-terminal conservado, substanciando o argumento de que esta região de AtWWP1 é responsável pela formação de corpos subnucleares. Ensaios de co-localização demonstraram que os corpos nucleares contidos em AtWWP1 são distintos daqueles formados por proteínas envolvidas em splicing do RNA; porém co-localizam com corpos nucleares contendo CDKC2. Além disso, foi demonstrado que AtWWP1 não liga a RNA, mas exibe uma atividade de ligação ao DNA. Estas características implicam que AtWWP1 deve estar envolvida com funções nucleares básicas. Como componente de um hub funcional na interação geminivírus-hospedeiro, torna-se relevante avaliar se a infecção viral afetaria os corpos nucleares formados por AtWWP1.

Les virus du genre Begomovirus (famille Geminiviridae) sont fréquemment détectés en association avec des ADN satellites appelées alphasatellite et betasatellite qui font la moitié de la taille du génome viral. L’alphasatellite est autonome pour sa réplication et dépend du virus pour son mouvement et son encapsidation tandis que le betasatellite est dépendant de ces fonctions virales. L’alphasatellite a rarement été montré comme ayant un impact sur le virus assistant, contrairement au betasatellite qui augmente la virulence de son virus assistant. En dehors des bégomovirus tels que le Cotton leaf curl virus (CLCuV) qui ont besoin d’un betasatellite pour initier une infection symptomatique dans leur hôte naturel, la plupart des bégomovirus peuvent causer des symptômes, même sans les satellites avec lesquels ils sont parfois détectés. Le Tomato yellow leaf curl virus (TYLCV), un des virus les plus dommageables dans le monde a rarement été détecté associé à des ADN satellites. Les souches méditerranéennes qui sont aussi les plus invasives, n’ont jamais été détectées avec des ADN satellites, bien qu’elles soient capables en conditions artificielles de les assister avec pour conséquence une considérable augmentation de la virulence en cas de co-inoculation avec un betasatellite. Le risque potentiel d’association de satellites avec le TYLCV-Mld a été évalué en testant divers facteurs potentiellement impliqués dans le maintien de l’association TYLCV-satellite: (i) l'accumulation relative intra-plante du TYLCV et des satellites, (ii) la fréquence de co-infection au niveau cellulaire du TYLCV et des satellites, et (iii) l'efficacité de transmission des satellites par le vecteur Bemisia tabaci. Trois satellites précédemment isolés sur coton au Burkina Faso ont été montrés comme pouvant être assistés par le TYLCV dans des plantes de tomate: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) et Okra leaf curl Burkina Faso alphasatellite (OLCBFA). La quantification par PCR quantitative des ADN du TYLCV et des trois satellites entre 11 et 150 jours après inoculation (dpi) révèle qu’en général, les satellites ont une accumulation supérieure à celle du virus, et que, contrairement aux alphasatellites qui n’ont aucun impact, le betasatellite affecte l’accumulation du TYLCV-Mld. Bien que le rapport des quantités de virus/satellites varie au cours du temps, les satellites sont maintenus avec le TYLCV-Mld au temps tardif de 150 dpi et sont transmis par B. tabaci à 32 et 150 dpi. Le TYLCV-IL interagit différemment avec le CLCuGB car son accumulation n’est pas affectée dans les plantes coinfectées.L’estimation par la technique FISH à 18 et 32 dpi de la fréquence d’association des molécules au niveau cellulaire montre que plus de la moitié des cellules infectées sont coinfectées par le TYLCV et un satellite. Ce résultat est cohérent avec la fréquence observée d’ADN satellite dans les plantes. Cependant, on observe de manière inattendue un nombre important de cellules ne semblant contenir que le betasatellite, ce qui pose des questions sur le fonctionnement des associations virus/satellites. Comme la multiplicité d'infection (MOI) des bégomovirus et des satellites est attendue pour être un facteur déterminant de l’efficacité de la co-infection cellulaire, deux variants équi-competitifs de TYLCV ont été préparés afin de déterminer ce paramètre. Enfin, des amorces PCR permettant la détection générique de betasatellites ont été dessinées pour être utilisées dans le diagnostic par l'Agence française pour l'alimentation, l'environnement et la santé et sécurité au travail (ANSES). Outre les conséquences agronomiques d’un maintien possible des satellites avec le TYLCV, les résultats de cette étude donnent un aperçu novateur sur les interactions entre les bégomovirus et les satellites, au niveau de la plante, au niveau cellulaire et moléculaire. / Begomoviruses (family Geminiviridae) are frequently detected with half genome sized defective virus DNAs, and for some of them with satellite DNAs of similar size, i.e. alphasatellite and betasatellite. Both molecules rely on the virus for maintenance in plant. The alphasatellite was rarely proved to have an impact on the helper virus but the betasatellite was often shown to increase its virulence. Except some begomoviruses, like Cotton leaf curl virus (CLCuV) which rely on a betasatellite for a full symptomatic infection in its natural host plant, most of the begomoviruses which were frequently detected with satellites do not rely on them for infectivity. Tomato yellow leaf curl virus (TYLCV) is one of the most damaging begomovirus worldwide. The Mediterranean IL and Mld strains, the most invasive ones, were never detected in association with satellites, although they were experimentally proved to readily assist them for replication and movement in plant. This was particularly true for betasatellites and resulted in a dramatic increase in the virulence of TYLCV.The potential of a TYLCV-satellite association was assessed by testing various factors involved in the maintenance of both molecules in tomato plants: (i) the relative intra-plant accumulation of TYLCV and satellites, (ii) the frequency of host cells co-infected with TYLCV and satellites, and (iii) the transmission efficiency of satellites by the natural whitefly vector of TYLCV, Bemisia tabaci. Three satellites previously isolated from okra in Burkina Faso, were shown here to be assisted by TYLCV in tomato plants: Cotton leaf curl Gezira betasatellite (CLCuGB), Cotton leaf curl Gezira alphasatellite (CLCuGA) and Okra leaf curl Burkina Faso alphasatellite (OLCBFA). The dynamic of TYLCV and satellite DNAs monitored between 11 and 150 days post-inoculation (dpi) by quantitative PCR revealed that satellites accumulated at a higher level than the virus, and that, in contrast with alphasatellites which have no impact, betasatellites affected TYLCV-Mld accumulation. Although the ratio of virus/satellite amounts varies over time, satellites were maintained in all test plants up to 150 dpi and were readily transmitted at 32 and 150 dpi. TYLCV-IL interacts differentially with CLCuGB as its accumulation was not affected in the coinfected plants.At 32 dpi, the TYLCV/satellite infection status of plant cells was determined by FISH and more than 50% of the monitored infected cells were co-infected with TYLCV and a satellite. The infection status was consistent with the frequency of satellite DNA in plants. Unexpectedly a substantial number of cells were positive only for betasatellite, suggesting that the coinfection with the virus could be dispensable for replication. This observation raises question on the functioning of virus/satellite association or multipartite viruses. As the multiplicity of infection (MOI) of begomoviruses and satellites is expected to be a determinant of the efficiency of virus/satellite cell coinfection, two equi-competitive TYLCV variants were prepared to determine this parameter for TYLCV. Finally, PCR primers designed for the generic detection of betasatellites were designed to be used as a diagnostic tool by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES).Besides the agronomic concern of the possible maintenance of DNA satellites with TYLCV, the results of our study are expected to provide a new insight on the interactions between begomovirus and satellites, at the plant, cellular and molecular levels.

O tomateiro (Lycopersicon esculentum) é cultivado em várias regiões durante todo o ano, propiciando assim condições favoráveis ao surgimento de inúmeras doenças incluindo as causadas por vírus. Dentre as viroses consideradas como limitantes a esta cultura destacam-se as causadas por begomovírus, que pertencem à família Geminiviridae. Sua transmissão se dá pelo aleirodídeo Bemisia tabaci biótipo B. A partir da década de 90 tornaram-se freqüentes os relatos da disseminação desse aleirodídeo e de begomovírus causando perdas que variam de 40% a 100%. No Estado de São Paulo, o Tomato yellow vein streak virus (ToYVSV), até 2005, estava predominando nos campos de tomateiro, onde foram constatadas incidências de plantas com sintomas deste begomovírus variando de 58% a 100%. O presente trabalho teve como objetivos estudar a interação do ToYVSV com o vetor Bemisia tabaci biótipo B e identificar hospedeiras alternativas deste vírus. Na relação do vírus com o vetor constatou-se que os períodos de acesso mínimo de aquisição (PAA) e de inoculação (PAI) foram de 30 min e 10 min, respectivamente. A porcentagem de plantas infectadas chegou até cerca de 75% após um PAA e PAI de 24 h. O período de latência do vírus no vetor foi de 16 horas. O ToYVSV foi retido pela B. tabaci 20 dias após a aquisição deste. Não foi detectada a transmissão do vírus para progênie da B. tabaci biótipo B oriundas de insetos virulíferos. Das 34 espécies de plantas testadas como hospedeiras somente C. annuum, C. amaranticolor, C. quinoa, D. stramonium, G. globosa, N. tabacum cv. TNN e N. clevelandii foram suscetíveis à infecção com o ToYVSV, por meio de inoculação com a B. tabaci. As transmissões do ToYVSV por Cuscuta campestris e mecanicamente foram ineficientes. As espécies susceptíveis ao ToYVSV serviram de fonte de inóculo para a transmissão do vírus para tomateiros por meio de B. tabaci biótipo B. / Tomato (Lycopersicon esculentum) has been cultivated in various parts of Brazil almost during the entire year, which provides favorable conditions for the incidence of innumerous diseases, including those caused by viruses. Among the virus diseases responsible for yield losses on tomato crops are those caused by species in the genus Begomovirus, family Geminiviridae. These viruses are transmitted by the whitefly Bemisia tabaci biotype B. Yield losses associated with begomovirus infection varying from 40% to 100% have been frequently reported since early 90's. Tomato yellow vein streak virus (ToYVSV), a putative species of begomovirus, was prevalent on tomato crops in São Paulo State until 2005, causing yield losses varying from 58% to 100%. The objectives of this work were to study the interaction between ToYVSV am its vector B. tabaci biotype B and to identify alternative host for the virus. The results indicated that the minimum acquisition and inoculation access periods of ToYVSV by B. tabaci were 30 min and 10 min, respectively. Seventy five percent of tomato-test plants were infected when the acquisition and inoculation access periods were 24 h. The latent period of the virus in the insect was 16 h. The ToYVSV was retained by B. tabaci for 20 days after acquisition. First generation of adult whiteflies obtained from viruliferous females did not have the virus as shown by PCR analysis and did not transmit the virus to tomato plants. Out of 34 species of test-plants inoculated with ToYVSV by means of B. tabaci biotype B, only C. annuum, C. amaranticolor, C. quinoa, D. stramonium, G. globosa, N. tabacum cv. TNN and N. clevelandii were susceptible to infection. Attempts to transmit ToYVSV to susceptible hosts mechanically and with Cuscuta campestris failed. B. tabaci biotype B was able to acquire the virus from all susceptible species, transmitting it to tomato test-plants.

Mastreviruses (family Geminiviridae) are plant-infecting viruses with circular single-stranded (ss) DNA genomes (~2.7kb). The genus Mastrevirus is comprised of thirty-two species which are transmitted by leafhoppers belonging to the genus Cicadulina. Mastreviruses are widely distributed and have been found in the Middle East, Europe, Asia, Australia, Africa and surrounding islands. Only one species, dragonfly-associated mastrevirus has so far been identified in the Americas, isolated from a dragonfly in Puerto Rico. Species can be group based on the host(s) they infect, those which infect monocotyledonous (monocot) plants and those which infect dicotyledonous (dicot) plants. In recent years many new mastrevirus species have been discovered. Several of these new discoveries can largely been attributed to the development of new molecular tools. The current state of sequencing platforms has made it affordable and easier to characterise mastreviruses at a genome level thus allowing scientists to delve deeper into understanding the dynamics of mastreviruses. A few mastrevirus species have been identified as important agricultural pathogens and as a result have been the focus of much of the mastrevirus research. Maize streak virus, strain A (MSV-A) has been the most extensively studied due to the devastating impact it has on maize production in Africa. Studies have shown that MSV-A likely emerged as a pathogen of maize less than 250 years following introduction of maize in Africa by early European settlers. There is compelling evidence to suggest that MSV-A is likely the result of recombination events between wild grass adapted MSV strains. It therefore is equally important to monitor viruses infecting non-cultivated plants in order to gain a greater understanding of the epidemiological dynamics of mastreviruses, which in turn is essential for implementing disease management strategies. The objective of the research undertaken as part of this PhD thesis was to investigate global mastrevirus dynamics focusing on diversity, host and geographic ranges, mechanisms of evolution, phylogeography and possible origins of these viruses. In addition to this a viral metagenomic approach was used in order to identify novel mastreviruses or mastrevirus-like present in New Zealand. The dynamics of the monocot-infecting mastreviruses are investigated in Chapter Two and Three. The work described in these two chapters focus mainly on mastreviruses which infect non-cultivated grasses in Africa and Australia, a total of 161 full mastrevirus genomes were recovered collectively in the two studies. Chapter Two reveals a high level of mastrevirus diversity present in Australia with the discovery of four new species and several new strains of previously characterised species. An extensive sampling effort in Africa undertaken in Chapter Three reveals a broader host range and geographic distribution of the African monocot-infecting mastreviruses than previously documented. Mosaic patterns of recombination are evident among both the Australian and African monocot-infecting mastreviruses. In Chapters Four, Five and Six a comprehensive investigation was undertaken focusing on the dicot-infecting mastreviruses. The study undertaken in Chapter Four entailed the recovery of 49 full mastrevirus genomes from Australia, the Middle East, Africa, Turkey and the Indian Subcontinent to investigate the diversity of dicot-infecting mastreviruses from a global context. Analyses revealed a high degree of CpCDV strain diversity and extended the known geographic range of CpCDV. For the first time phylogeographic analysis was able to investigate the origins of the dicot-infecting mastreviruses. Results revealed the likely origin of the most recent common ancestor (MRCA) of these viruses is likely closer to Australia than anywhere else that dicot-infecting mastreviruses have been sampled and illuminated a supported series of historical movements following the emergence of the MRCA. In Chapter Five two novel mastreviruses Australian-like mastreviruses were isolated from chickpea material from Pakistan. A comprehensive analysis of CpCDV isolates in the major pulse growing regions of Sudan in Chapter Six reveals that this region harbours a high degree of strain diversity. Complex patterns of intra-species recombination indicate these strains are evidently circulating in these regions and infecting the same hosts, driving the emergence of new CpCDV strains. Collectively the results discussed in Chapters Two through Six extended the current knowledge of mastrevirus diversity. The natural host range of many mastreviruses has proven to be more extensive than previously documented, with many species having overlapping host ranges and hence these hosts could be acting as ‘mixing vessels’ enabling inter-species recombination. Patterns of recombination and selection were observed in both the monocot-infecting and the dicot-infecting mastreviruses further elucidating the mechanisms these viruses employ to evolve rapidly. Extensive sampling in a wide range of geographic regions provides insights into the true geographic range of species such as MSV and CpCDV. Given that mastreviruses have been able to move globally and Australia has been identified as a major mastrevirus diversity hotspot it is conceivable that mastreviruses are also present in New Zealand. In Chapter Seven and Eight this is explored by using a viral metagenomic approach to investigate the ssDNA viral populations associated with wild grasses and sewage material in New Zealand. Although no mastreviruses were recovered, this endeavour resulted in the discovery of more than 50 novel circular Rep-encoding ssDNA (CRESS DNA) viruses associated with non-cultivated grasses and treated sewage material, many of which are similar to mastreviruses and other geminiviruses. These discoveries expand current knowledge on the diversity of ssDNA viruses present in New Zealand and further highlight this viral metagenomic approach as an effective method for ssDNA virus discovery. Overall the results discussed in this thesis provide insights into mastrevirus diversity and dynamics as well as revealing a wealth of novel CRESS DNA viruses, some of which share similarities to geminiviruses.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 4 uppsatser.