RESUMO
Tobacco plants were transformed with the open reading frame 3 gene from Potato virus X (PVX) coding for the p12 protein. Although the transgenic plants exhibited a normal morphological aspect, microscopic examination revealed extensive alterations in leaf tissue structure. After being challenged with PVX, the transgenic plants showed resistance to PVX infection and formation of specific leaf symptoms consisting of concentric rings encircled by necrotic borders. These novel symptoms were accompanied by biochemical changes normally associated with the hypersensitive response (HR) and were absent in noninfected transgenic plants or in PVX-infected nontransgenic plants. No equivalent virus resistance was observed after inoculation with Tobacco mosaic virus or Potato virus Y, suggesting the presence of a specific resistance mechanism. Despite development of HR-like symptoms, systemic acquired resistance was not induced in PVX-infected p12 transgenic plants. No evidence of an RNA-mediated resistance mechanism was found.
Assuntos
Proteínas do Capsídeo , Nicotiana/genética , Nicotiana/virologia , Potexvirus/genética , Potexvirus/patogenicidade , Capsídeo/genética , Capsídeo/metabolismo , Genes Virais , Fases de Leitura Aberta , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/metabolismo , Folhas de Planta/virologia , Plantas Geneticamente Modificadas , Nicotiana/metabolismoRESUMO
The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.
Assuntos
Potexvirus/genética , Potexvirus/fisiologia , Vírus do Mosaico do Tabaco/patogenicidade , Tobamovirus/patogenicidade , Proteínas Virais/genética , Proteínas Virais/fisiologia , Animais , Anticorpos Antivirais , Sequência de Bases , Primers do DNA/genética , Mutação da Fase de Leitura , Expressão Gênica , Teste de Complementação Genética , Movimento , Fases de Leitura Aberta , Fenótipo , Plantas Geneticamente Modificadas , Plantas Tóxicas , Coelhos , NicotianaRESUMO
In vitro culture was used to study morphogenetic aspects of the tuberization process under controlled conditions in potato (Solanum tuberosum L.) plants. This paper accurately defines four stages of tuber development and their correlation to external morphological characteristics and histological structures. Protein kinase activity, assayed in each stage using Historic HAS as substrate, was differentially expressed during the tuberization process. Phosphorylation was maximum in the first stages of tuber formation. The incorporation of [32PO4 -1] to endogenous peptides containing serine/threonine amino acidic residues followed the same pattern that the protein kinase activity did.
RESUMO
Partial genomic sequences from an unknown garlic potyvirus and from an onion isolate of the onion yellow dwarf potyvirus (OYDV) were obtained. Comparison of the deduced amino acid sequences showed a similarity of 88% between the respective viral coat proteins. The garlic potyvirus coat protein was expressed in E. coli cells, purified, and subjected to Western blot analysis using antibodies raised against different garlic-infecting viruses. The expression protein was consistently recognised by anti-OYDV antibodies and did not react with antibodies specific for leek yellow stripe potyvirus (LYSV), garlic common latent carlavirus (GCLV) and shallot latent carlavirus (SLV). Besides, the garlic potyvirus coat protein was obtained as a fusion protein and used as antigen to produce polyclonal antibodies. These antibodies reacted with purified OYDV virions, but failed to recognise LYSV particles. In the light of this evidence the garlic potyvirus was identified as the garlic strain of OYDV.
Assuntos
Capsídeo/genética , Potyvirus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Capsídeo/imunologia , Capsídeo/metabolismo , Clonagem Molecular , Escherichia coli/genética , Alho/virologia , Expressão Gênica , Dados de Sequência Molecular , Cebolas/virologia , Plantas Medicinais , Potyvirus/imunologia , Potyvirus/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Recombinação Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The complete cDNA sequence corresponding to the genomic RNA of a South American strain of potato virus X (PVXc) is reported. The sequence (6432 nucleotides) contains five open reading frames coding for polypeptides with molecular weights of 165.3, 24.3, 12.3, 7.6 and 25.0 and displays an overall homology of 77.4% with those previously reported for two European isolates. Comparison of amino acid sequences shows an average homology of 87%. Two major domains of variability, located between amino acids 476-615 of ORF 1 and 64-100 of ORF 5, are identified. Sequence similarities between RNA stretches lying upstream of ORFs 2, 4 and 5, and at the 3'-non coding regions of PVX and other plus-strand RNA viruses are described.
Assuntos
Genes Virais , Vírus de Plantas/genética , RNA Viral , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Viral , Europa (Continente) , Dados de Sequência Molecular , RNA Viral/isolamento & purificação , Homologia de Sequência do Ácido Nucleico , Solanum tuberosum/microbiologia , América do SulRESUMO
cDNA libraries, representative of potato viruses X (PVXc strain) and Y (PVY degrees strain) genomes were obtained. A PVX cDNA cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. Dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral RNA. The level of detection in infected plant extracts was as effective as that obtained with the ELISA. The presence of biotinylated PVY cDNA in the hybridization mixture did not affect sensitivity of the PVX detection assay, suggesting that a single diagnostic assay for several potato viruses and virus-related pathogens could be developed.