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- Olive mild mosaic virus coat protein and p6 are suppressors of RNA silencing and their silencing confers resistance against OMMVPublication . Varanda, Carla M. R.; Materatski, Patrick; Campos, Maria Doroteia; Clara, Maria Ivone E.; Nolasco, Gustavo; Felix, Maria do RosarioRNA silencing is an important defense mechanism in plants, yet several plant viruses encode proteins that suppress this mechanism. In this study, the genome of the Olive mild mosaic virus (OMMV) was screened for silencing suppressors. The full OMMV cDNA and 5 OMMV open reading frames (ORFs) were cloned into the Gateway binary vector pK7WG2, transformed into Agrobacterium tumefaciens, and agroinfiltrated into N. benthamiana 16C plants. CP and p6 showed suppressor activity, with CP showing significantly higher activity than p6, yet activity that was lower than the full OMMV, suggesting a complementary action of CP and p6. These viral suppressors were then used to induce OMMV resistance in plants based on RNA silencing. Two hairpin constructs targeting each suppressor were agroinfiltrated in N. benthamiana plants, which were then inoculated with OMMV RNA. When silencing of both suppressors was achieved, a significant reduction in viral accumulation and symptom attenuation was observed as compared to those of the controls, as well as to when each construct was used alone, proving them to be effective against OMMV infection. This is the first time that a silencing suppressor was found in a necrovirus, and that two independent proteins act as silencing suppressors in a virus member of the Tombusviridae family.
- Genetic diversity of the coat protein of Olive Mild Mosaic Virus (OMMV) and Tobacco Necrosis Virus D (TNV-D) isolates and its structural implicationsPublication . Varanda, C. M. R.; Machado, M.; Martel, P.; Nolasco, Gustavo; Clara, M. I. E.; Felix, M. R.The genetic variability among 13 isolates of Olive mild mosaic virus (OMMV) and of 11 isolates of Tobacco necrosis virus D (TNV-D) recovered from Olea europaea L. samples from various sites in Portugal, was assessed through the analysis of the coat protein (CP) gene sequences. This gene was amplified through reverse transcriptase polymerase chain reaction (RTPCR), cloned, and 5 clone sequences of each virus isolate, were analysed and compared, including sequences from OMMV and TNV-D isolates originally recovered from different hosts and countries and available in the GenBank, totalling 131 sequences. The encoded CP sequences consisted of 269 amino acids (aa) in OMMV and 268 in TNV-D. Comparison of the CP genomic and amino acid sequences of the isolates showed a very low variability among OMMV isolates, 0.005 and 0.007, respectively, as well as among TNV-D isolates, 0.006 and 0.008. The maximum nucleotide distances of OMMV and TNV-D sequences within isolates were also low, 0.013 and 0.031, respectively, and close to that found between isolates, 0.018 and 0.034, respectively. In some cases, less variability was found in clone sequences between isolates than in clone sequences within isolates, as also shown through phylogenetic analysis. CP aa sequence identities among OMMV and TNV-D isolates ranged from 84.3% to 85.8%. Comparison between the CP genomic sequences of the two viruses, showed a relatively low variability, 0.199, and a maximum nucleotide distance between isolates of 0.411. Analysis of comparative models of OMMV and TNV-D CPs, showed that naturally occurring substitutions in their respective sequences do not seem to cause significant alterations in the virion structure. This is consistent with a high selective pressure to preserve the structure of viral capsid proteins.
- Occurrence of genetic bottlenecks during citrus tristeza virus acquisition by Toxoptera citricida under field conditionsPublication . Nolasco, Gustavo; Fonseca, Filomena; Silva, GoncaloIn this study, we address the involvement of T. citricida in strain segregation and genetic bottleneck events by comparing the nucleotide diversity of CTV coat protein (CP) gene variants present in field-grown trees with that of variants retrieved from single apterous aphids. Plant material and aphids were collected in orange orchards in the northern part of Portugal. Shoots from two trees that were found to be positive using ELISA and twenty-four apterous aphids from these same trees were selected for individual molecular assays. CTV was detected in 60% of the aphids by amplification of a 417-bp fragment of the CP gene. Analysis of molecular variance (AMOVA) of this fragment revealed that most of the variation of the virus was found among individual aphids (FSC: 0.766) within each location. Nucleotide diversity comparison between the pool of sequences obtained from a given shoot and sequences obtained from individual aphids present on that shoot showed a reduction of more than one order of magnitude in most cases. Computer simulations of random virus acquisition by single aphids showed that in 54% of the cases only a single CP gene phylogenetic group was acquired. However, a small number of aphids (e.g. 6) was enough to acquire the full complement of phylogenetic groups present.
- Presence of Citrus tristeza virus in Angola and São Tomé e Princípe: characterization of isolates based on coat protein gene analysisPublication . Silva, Goncalo; Fonseca, Filomena; Santos, C.; Nolasco, GustavoA first report is given of the presence of Citrus tristeza virus (CTV) in Angola and Sao Tome e Principe. Two out of twenty citrus samples from Sao Tome e Principe and all of the seven samples from Angola were shown by ELISA to contain CTV. The capsid protein gene variants obtained by IC/RT-PCR were characterized by SSCP analysis to test for the presence of different haplotypes in each isolate and nucleotide sequence analysis of each variant leading to a different SSCP pattern, followed by in silico comparison with previously characterized CTV groups. The results confirmed that each isolate could contain different variants, which clustered with the different groups. Variants obtained from each country fitted into the same three clusters.
- Rupestris stem pitting associated virus isolates are composed by mixtures of genomic variants which share a highly conserved coat proteinPublication . Nolasco, Gustavo; Santos, C.; Santos, M. T.; Cortez, I.; Fonseca, Filomena; Petrovic, N.; Boben, J.; Pereira, A. M. N.; Sequeira, O. A.Broad spectrum primers were used to amplify a fragment comprising the CP gene and putative ORF6 by RT-PCR from ds-RNA templates originating from 46 Portuguese varieties, totalling 190 samples, including some wild Vitis ssp sylvestris vines, and 2 vines from Slovenia. SSCP analysis was used as a preliminary screen to avoid cloning and sequencing very similar variants. Four groups of variants were recognized. In pair wise comparisons between nucleotide sequences the minimal homology found was 81%. In case of the cultivated varieties, no relationship could be seen between the phylogenetic groups and geographic origin or grape variety. Several isolates were found harbouring mixed infections with genomic variants from different groups, but the mixing did not lead to an extensive recombination between them. The deduced amino-acid sequences revealed a conserved CP subjected to strong purifying selection pressure. Analysis of the selection pressure operating on the putative ORF6 suggests that this ORF does not exist. Previously produced polyclonal antiserum raised against the recombinant CP of RSPaV expressed in Escherichia coli was shown to be able to detect all four groups of variants of RSPaV included in this study, which might enable the diagnosis of the virus on a serological basis.
- Typing of Egyptian Citrus tristeza virus (CTV) isolates based on the capsid protein genePublication . Amin, H. A.; Fonseca, Filomena; Santos, C.; Nolasco, GustavoThe capsid protein gene of three Egyptian CTV isolates from two locations was amplified by immunocapture RT-PCR and analysed by single stranded conformation polymorphism and sequencing. The CTV isolates studied did not differ significantly in sequence composition and each isolate consisted of very similar haplotypes. Comparison with reference sequences from isolates elsewhere in the world showed that these haplotypes clustered very close to the severe strain T3 from Florida causing quick decline and stem pitting. Analysis of the deduced amino acid sequence showed the epitope characteristic of reactivity with the MCA13 antibody. Sequence comparison with the sequence of an Egyptian isolate (Qaha) available in the Genbank showed a distance of about 8%, suggesting that it had a different origin.
- The p19.7 RNA silencing suppressor from Grapevine leafroll-associated virus 3 shows different levels of activity across phylogenetic groupsPublication . Gouveia, P.; Nolasco, GustavoAt least five phylogenetic groups have been reported for Grapevine leafroll-associated virus 3 (GLRaV-3). The p19.7 protein encoded by the GLRaV-3 was previously identified as an RNA silencing suppressor. In this study, five constructs of p19.7 belonging to different groups were compared for their suppressing activity. For each p19.7 variant, the accumulation level of green fluorescent protein mRNA and specific siRNAs were determined using co-infiltration assays in transgenic 16C Nicotiana benthamiana. Differences in the suppressing activity were found among the variants assayed. Some constructs originated viral-like mosaic symptoms that evolved to necrosis. The intensity of these symptoms appeared to be related to the strength of the suppressor activity. A comparison of the protein sequences revealed a few amino acid substitutions that may be associated with the observed differences in the suppressing activity.
- Identification of an RNA silencing suppressor encoded by Grapevine leafroll-associated virus 3Publication . Gouveia, P.; Dandlen, S. A.; Costa, A.; Marques, N T.; Nolasco, GustavoGLRaV-3, a member of the Closteroviridae family and type member of the genus Ampelovirus, is involved in the grapevine leafroll disease. Until now no RNA silencing suppressor has been found among viruses of this genus, contrary to what happens with a large number of other viral genera. In the sister genus Closterovirus, RNA silencing suppressors are present in the 3' end of the genome and have molecular weights close to 20 KDa. To test for RNA suppressing activity screening of p21, p19.6 and p19.7 proteins, coded for in an analogous genomic location of the GLRaV-3 was undertaken. Only p19.7 revealed suppressor activity demonstrated in diverse silencing inducing systems. This suppressor is able to overcome strong silencing inducers and shares several properties with the BYV p21-like family of suppressors of the closteroviruses. This is the first report of an RNA silencing suppressor in the genus Ampelovirus.
- Técnicas biomoleculares no diagnòstico e tipificação em virologia vegetalPublication . Nolasco, Gustavo; Sequeira, Oscar Amaro deO diagnóstico é um dos principais meios de protecção de plantas em virologia vegetal. Muito frequentemente seria vantajoso poder ainda complementar o diagnóstico com informação sobre as estirpes que estão presentes num dado contexto epidemiológico. Na maior parte dos casos isto não é possível devido à inexistência de métodos apropriados de tipificação viral. Esta limitação tem também dificultado estudos de âmbito mais teórico que envolvem a identificação de estirpes presentes em populações naturais e que permitiriam melhor delinear as estratégias protecção. Num pequeno número de casos tem sido possível obter esta informação pela análise dos perfis electroforéticos obtidos pelo fracionamento de RNA bicatenário de origem viral ou, mais recentemente, a partir da análise dos genomas virais amplificados por PCR. Neste trabalho estudam-se métodos capazes de serem aplicados em condições de rotina ao diagnóstico viral e à identificação de isolamentos de vírus, desenvolvendo-se em três partes. No primeiro capítulo estuda-se a aplicabilidade da análise de RNA bicatenário ao diagnóstico viral, empregando para tal diversos vírus e hospedeiros. Verifica-se que este método é muito dependente do hospedeiro e desenvolvem-se protocolos de extracção de ácidos nucleicos que podem ser empregues com hospedeiros considerados difíceis, tais como a videira. Não são contudo protocolos que possam ser empregues em condições de rotina. Outras limitações provêm ainda da existência de RNA bicatenários endógenos que foram encontrados em vários hospedeiros. Para além destes problemas, a possibilidade de identificar estirpes depende ainda da estratégia de expressão genómica do vírus e do hospedeiro estar a vegetar em condições apropriadas para a manifestação do perfil completo de bandas. Em comparação com outros métodos que vieram a ser desenvolvidos neste trabalho, a análise de RNA bicatenário apresenta um interesse limitado quer no diagnóstico quer na tipificação viral. Poderá ter interesse como método exploratório no estudo de doenças de etiologia presumivelmente viral ainda não esclarecida, como por exemplo o mosaico da figueira, tratado neste trabalho. Esta doença aparece relacionada com um perfil electroforético composto de várias bandas de que sobressaem duas correspondentes a fragmentos de RNA bicatenário com cerca de 10-12 Kbp e 2,2 Kbp. Estas moléculas poderão estar relacionadas com a replicação de vírus de RNA possívelmente envolvido(s) na patogénese. Na segunda parte estudam-se os métodos de diagnóstico baseados na amplificação in vitro de determinadas partes do genoma viral. Deste estudo resultou um método de diagnóstico (IC/RT-PCR) de elevada sensitividade que evita a extracção de ácidos nucleicos, o que não acontecia na metodologia até então disponível. Os viriões são capturados directamente do extracto da planta mediante anticorpos adsorvidos sobre a superfície de uma fase sólida, provavelmente sofrendo uma distorção que possibilita a iii extracção e transcrição do genoma pela RTase, seguindo-se a sua amplificação por PCR e quantificação dos resultados por fluorimetria. Todo o processo pode ser efectuado numa placa de microtitulação, sendo concebível um grau de automatização equivalente ao da técnica ELISA. Este método foi objecto de um pedido de patente (Nolasco et al., 1992). A sua validade foi verificada com vírus de diversos grupos (Potyvirus, Nepovirus, Cucumovirus, Closterovirus, Luteovirus, Tobamovirus, Tospovirus) e com RNA satélites de CMV e GFLV. Num pequeno rastreio de CTV e GFLV, comprovou-se a incapacidade da técnica ELISA em detectar o vírus em várias amostras positivas por IC/RT-PCR. Por outro lado, no caso do GFLV não foi possível amplificar alguns isolamentos positivos por ELISA. Este facto foi atribuído a uma inesperada variabilidade genómica e reforça a necessidade de se efectuarem estudos de variabilidade genómica de vírus. Alternativamente à captura por anticorpos específicos, é possível capturar os genomas virais por meio de anticorpos para RNA bicatenário, o que permite alargar o âmbito desta metodologia a patogéneos desprovidos de proteína estrutural. Na terceira parte estudou-se a conjugação da amplificação genómica por IC/RTPCR com métodos de análise mutacional com vista ao desenvolvimento de métodos de tipificação genómica de vírus. Dois destes métodos, a análise de polimorfismos conformacionais monocatenários (SSCP) e de polimorfismos de locais de restrição (RSP) empregam como técnica preparativa a amplificação específica por IC/RT-PCR e foram objecto de um pedido de patente (Nolasco et al., 1993a). Um terceiro método, a Síntese Aleatória de cDNA (SAcDNA), baseia a tipificação no aproveitamento das condições pouco restritivas da transcrição reversa para iniciar, por jusante, mediante um iniciador inespecífico, um conjunto de moléculas característico do isolamento viral, que é em seguida amplificado em condições normais de restritividade. Este método foi também objecto de um pedido de patente (Nolasco et al., 1994b). São características gerais dos métodos apresentados poderem ser aplicados directamente ao extracto da planta a analisar, serem independentes do hospedeiro e, comparativamente com outros métodos, serem muito pouco laboriosos. Como modelo de aplicação empregou-se uma série de isolamentos de GFLV e CTV, grande parte mantidos em condições naturais. Obteve-se o melhor poder discriminante com SAcDNA. Para além da discriminação, a análise por RSP permitiu ainda o estudo de relações entre isolamentos e a avaliação quantitativa da diversidade genética de populações de GFLV e CTV, o que não havia sido feito prèviamente. Verificou-se que o gene da proteína do capsídeo do GFLV é altamente variável e que o distanciamento genético entre isolamentos não se relaciona com o distanciamento espacial entre as plantas, mesmo quando lado a lado. A diversificação do CTV segue um modelo distinto, relacionando-se a proximidade geográfica com a proximidade genética entre os isolamentos e não se atingindo diversidades tão elevadas como para o GFLV.
- Stem pitting and seedling yellows symptoms of Citrus tristeza virus infection may be determined by minor sequence variantsPublication . Cerni, S.; Ruscic, J.; Nolasco, Gustavo; Gatin, Z.; Krajacic, M.; Skoric, D.The isolates of Citrus tristeza virus (CTV), the most destructive viral pathogen of citrus, display a high level of variability. As a result of genetic bottleneck induced by the bud-inoculation of CTV-infected material, inoculated seedlings of Citrus wilsonii Tanaka displayed different symptoms. All successfully grafted plants showed severe symptoms of stem pitting and seedling yellows, while plants in which inoculated buds died displayed mild symptoms. Since complex CTV population structure was detected in the parental host, the aim of this work was to investigate how it changed after the virus transmission, and to correlate it with observed symptoms. The coat protein gene sequence of the predominant genotype was identical in parental and grafted plants and clustered to the phylogenetic group 5 encompassing severe reference isolates. In seedlings displaying severe symptoms, the low-frequency variants clustering to other phylogenetic groups were detected, as well. Indicator plants were inoculated with buds taken from unsuccessfully grafted C. wilsonii seedlings. Surprisingly, they displayed no severe symptoms despite the presence of phylogenetic group 5 genomic variants. The results suggest that the appearance of severe symptoms in this case is probably induced by a complex CTV population structure found in seedlings displaying severe symptoms, and not directly by the predominant genomic variant.