Variabilidade genómica de Citrus tristeza virus e transformação genética de Citrus aurantium L. com vista à obtenção de resistência

Tese de dout., Biologia, Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2003

Estudo preliminar do efeito supressor de genes do Citrus tristeza virus (CTV) no silenciamento pós-transcripcional em Nicotiana benthamiana

Dissertação de mest., Engenharia Biológica, Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2011

Molecular characterization of Citrus tristeza virus isolates from Cyprus on the basis of the coat protein gene

Within the context of a program in Cyprus for the control of Citrus tristeza virus (CTV), the coat protein (CP) genes of 12 local isolates of the virus that induced different symptoms on host trees, were compared to those of known isolates. The CP genes were reverse-transcribed (RT) and amplified by polymerase chain reaction (PCR) and the resulting amplicons were cloned and sequenced. Nucleotide sequence analysis revealed no signs of geographic speciation. All the sequences obtained clustered close to those of previously known isolates of worldwide origin that are in five distinct groups. The nucleotide diversity was high compared to that found using a worldwide database of CP gene sequences. These data support the existence of different CTV introductions into Cyprus or an introduction from a location in which CTV is relatively diverse. Some of the isolates induced stem pitting on branches of grapefruit and sweet orange. Such isolates have not been noted often in the Mediterranean basin. They were close in CP sequence to isolate B249 from Venezuela, which induces stem pitting, and are of particular concern for the whole region.

First report of Citrus tristeza virus in the State Union of Serbia and Montenegro.

Citrus production in the State Union of Serbia and Montenegro has a strategic importance to the agricultural sector. Approximately 400,000 trees are now grown in the major citrus producing region, which is the Montenegrin Coastal Region. Satsuma mandarins and lemons grafted on Poncirus trifoliata are the most cultivated varieties. In December 2003, eight samples taken from the coastal region close to the towns of Bar and Ulcinj were analyzed using enzyme- linked immunosorbent assay (ELISA) with SP7 antibodies produced at Universidade do Algarve, Portugal (3). Further analysis was done using immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) targeting the entire coat protein (CP) gene (forward primer CTV1: 5(prime)- ATGGACGACGAAACAAAGAA-3(prime) and reverse primer CTV10: 5 (prime)-ATCAACGTGTGTTGAATTTCC-3(prime)). Using both techniques, seven of eight samples analyzed were found to be infected by Citrus tristeza virus (CTV), including samples from five trees that exhibited chlorosis, gummosis, and fruit deformation, and two trees that were symptomless.

Occurrence of stem-pitting strains of Citrus tristeza virus in Croatia

Citrus is grown in Croatia (approximately 1,500 ha of citrus groves) on the Dalmatian Coast and Islands between 42 and 43°30'N. The major species, Citrus unshiu Marc. (Satsuma mandarin), is grafted on trifoliate rootstock. The presence of Citrus tristeza virus (CTV) in Satsumas in the Neretva Valley Region was previously reported (3). During the course of a biomolecular characterization of isolates from Croatia, 15 budsticks were collected from field- infected, enzyme-linked immunosorbent assay (ELISA)-positive sources during the autumn of 2003 near Kaštela, Split, Metković (Neretva Valley), and on the island of Vis. Isolates were propagated by graft transmission to Madam Vinous sweet orange (SwO) and maintained in an insect-proof greenhouse at 21 to 33° C.

Caraterização dos supressores p20 e p23 do Citrus tristeza virus e avaliação da possibilidade da sua inibição

No presente trabalho pretendeu-se caracterizar a capacidade supressora das proteínas p20 e p23 de diferentes grupos filogenéticos do CTV e o possível silenciamento da p23 de forma a ser incluída numa estratégia de proteção. A atividade supressora local da proteína p23 de todos os grupos filogenéticos foi caracterizada em Nicotiana benthamiana da linha 16C. Todas as proteínas testadas foram capaz de suprimir o silenciamento local, mas não o silenciamento a curta distância. A supressão local mais eficiente verificou-se para a p23 do Gp 5 e a menos eficiente para os isolados dos Gps 2 e M. Surpreendentemente, a p23 do Gp 5 aboliu completamente o silenciamento sistémico, sugerindo que existe uma relação entre a intensidade do silenciamento local e sistémico. A capacidade supressora local conjunta das proteínas p20 e p23 foi avaliada. A coexpressão de ambas as proteínas revelou atividade supressora mais forte comparada com a capacidade de cada proteína individual, mesmo quando inoculada com metade da densidade ótica, sugerindo a existência de sinergismo entre as proteínas p20 e p23. Para analisar as propriedades supressoras a longo prazo, as proteínas p20 e p23 foram inseridas no vetor viral TRV que assegurou a sua disseminação pela planta e expressão por um período mais alargado. Foram observados sintomas em N. benthamiana para todas as modalidades testadas, tais como, nanismo da planta, lesões necróticas severas nas folhas inoculadas e nas folhas novas ligeiros sintomas de mosaico e enrolamento. Contudo, sistemicamente não foram registadas diferenças na capacidade supressora das proteínas p20 e p23. A possibilidade para silenciar sistemicamente a proteína p23 quando incluída num genoma viral foi avaliada através do uso de plantas e enxertos transgénicos para a p23. A estratégia que envolve o uso de enxertos transgénicos parece indicar resultados promissores que conduzem ao silenciamento da p23, contudo, são resultados que devem ser encarados como preliminares.

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

Filodinâmica do vírus da tristeza dos citrinos: epidemiologia molecular e biologia evolutiva

O Citrus tristeza virus (CTV), responsável por várias doenças em citrinos, é um dos maiores condicionantes da citricultura a nível mundial. Existem diversos isolados de CTV com diferentes características biológicas e moleculares, sendo que os sintomas causados pelo vírus dependem essencialmente do isolado viral e da combinação variedade/porta-enxerto. A implementação de medidas de controlo da doença depende, em grande parte, do tipo de isolados presentes numa dada região. No Capítulo 2, efetuou-se uma análise comparativa entre dois métodos de tipificação de isolados de CTV e verificou-se que a caracterização por PCR assimétrico-ELISA, que considera a existência de sete grupos, é mais adequada à descrição da estrutura genética de CTV. Estes resultados foram complementados com o estudo da dinâmica de colonização de cada grupo filogenético através de um imuno-ensaio in situ (Capítulo 3). Os resultados obtidos sugerem que os isolados de CTV diferem na quantidade de células infetadas e que essa diferença parece estar relacionada com a severidade do isolado. No Capítulo 4, o estudo da variabilidade genómica da região 3’ terminal permitiu verificar que a estrutura de grupos obtida para o gene da proteína da cápside (CP) é extensível a toda a região 3’ terminal que contém os genes mais fortemente implicados na interação com o hospedeiro. A estabilidade da estrutura genética nesta região foi também inferida a partir da pesquisa de eventos de recombinação. Os resultados sugerem uma baixa frequência de recombinação entre isolados de CTV, mesmo em isolados contendo mistura de haplótipos e mantidos há mais de 12 anos no mesmo hospedeiro. Adicionalmente, foi estimada a taxa de evolução de CTV através de um método estatístico Bayesiano (Capítulo 5). Para tal, foram usadas sequências do gene da CP de isolados de diversas regiões do mundo, pertencentes a diferentes grupos filogenéticos e obtidas entre 1990 e 2010. A taxa média de evolução estimada foi de 1,58 X 10-4 substituições nucleotídicas / ano. No geral, os resultados destes dois capítulos mostram que os isolados de CTV mantêm uma elevada estabilidade genética ao longo do tempo. Finalmente, no Capítulo 6, foi estudada a situação epidemiológica de CTV em Portugal continental a partir de isolados de CTV recolhidos no campo, onde se verificou que a maioria das árvores infetadas era composta por isolados de CTV pertencentes ao grupo M, ou seja isolados considerados suaves e que não provocam sintomas severos.

Nuclear magnetic resonance characterization of metabolite disorder in orange trees caused by citrus sudden death disease

Citrus sudden death (CSD) is a new disease of sweet orange and mandarin trees grafted on Rangpur lime and Citrus volkameriana rootstocks. It was first seen in Brazil in 1999, and has since been detected in more than four million trees. The CSD causal agent is unknown and the current hypothesis involves a virus similar to Citrus tristeza virus or a new virus named Citrus sudden death-associated virus. CSD symptoms include generalized foliar discoloration, defoliation and root death, and, in most cases, it can cause tree death. One of the unique characteristics of CSD disease is the presence of a yellow stain in the rootstock bark near the bud union. This region also undergoes profound anatomical changes. In this study, we analyse the metabolic disorder caused by CSD in the bark of sweet orange grafted on Rangpur lime by nuclear magnetic resonance (NMR) spectroscopy and imaging. The imaging results show the presence of a large amount of non-functional phloem in the rootstock bark of affected plants. The spectroscopic analysis shows a high content of triacylglyceride and sucrose, which may be related to phloem blockage close to the bud union. We also propose that, without knowing the causal CSD agent, the determination of oil content in rootstock bark by low-resolution NMR can be used as a complementary method for CSD diagnosis, screening about 300 samples per hour.

Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to Alternative Host Plants Found in Citrus Orchards

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ontogênese de caneluras em pedúnculo de flores de laranjeira doce infectados pelo vírus da tristeza dos citros estirpe Capão Bonito

Um estudo da ontogênese das caneluras induzidas em ramos de laranjeiras doces suscetíveis por isolados severos do vírus da tristeza dos citros (Citrus tristeza vírus - CTV) foi feito usando-se como modelo pedúnculos florais e de frutos. O menor calibre destes órgãos permite um melhor acompanhamento do processo. As observações foram feitas em laranjeira cv. Pêra infetada pelo isolado severo Capão Bonito do CTV. Cinco fases do processo de formação de caneluras puderam ser deduzidas pelas análises anatômicas. As primeiras alterações são representadas pelo aparecimento de células adensadas, hipertrofia e hiperplasia no parênquima e câmbio do floema e uma desorganização generalizada desta área. Segue-se uma atividade intensa do câmbio do floema adjacente e sua expansão em direção ao xilema. Esta invasão do xilema resulta na ruptura do anel do xilema pela massa celular do floema constituída de células recém formadas de parede celular delgada. Esta invasão do floema em direção ao xilema inicia um processo de degeneração dos vasos e parênquima do xilema. Finalmente há um colapso completo da região do xilema invadida, que é substituída pela massa do floema, resultando na canelura, notada ao se remover a casca.

Complete nucleotide sequence, genomic organization and phylogenetic analysis of Citrus leprosis virus cytoplasmic type

The complete nucleotide sequence of the genomic RNA 1 (8745 nt) and RNA 2 (4986 nt) of Citrus leprosis virus cytoplasmic type (CiLV-C) was determined using cloned cDNA. RNA 1 contains two open reading frames (ORFs), which correspond to 286 and 29 kDa proteins. The 286 kDa protein is a polyprotein putatively involved in virus replication, which contains four conserved domains: methyltransferase, protease, helicase and polymerase. RNA 2 contains four ORFs corresponding to 15, 61, 32 and 24 kDa proteins, respectively. The 32 kDa protein is apparently involved in cell-to-cell movement of the virus, but none of the other putative proteins exhibit any conserved domain. The 5' regions of the two genomic RNAs contain a 'cap' structure and poly(A) tails were identified in the 3'-terminals. Sequence analyses and searches for structural and non-structural protein similarities revealed conserved domains with members of the genera Furovirus, Bromovirus, Tobravirus and Tobamovirus, although phylogenetic analyses strongly suggest that CiLV-C is a member of a distinct, novel virus genus and family, and definitely demonstrate that it does not belong to the family Rhabdoviridae, as previously proposed. Based on these results it was proposed that Citrus leprosis virus be considered as the type member of a new genus of viruses, Cilevirus.

Separação de virus de importância fitopatológica em citros: CTV e CSDaV através de citometria de fluxo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.