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Virology
Satyanarayana, T., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Bar-Joseph, M., Volcani Institute, Bet-Dagan, 50250, Israel
Mawassi, M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Albiach-Martí, M.R., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Ayllón, M.A., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Gowda, S., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Hilf, M.E., USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, United States
Moreno, P., Instituto Valenciano De Investigaciones Agrarias, 46113 Moncada, Valencia, Spain
Garnsey, S.M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Dawson, W.O., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the ∼20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts (∼0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population. © 2001 Academic Press.
פותח על ידי קלירמאש פתרונות בע"מ -
הספר "אוצר וולקני"
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תנאי שימוש
Amplification of Citrus tristeza virus from a cDNA clone and infection of Citrus trees
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Satyanarayana, T., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Bar-Joseph, M., Volcani Institute, Bet-Dagan, 50250, Israel
Mawassi, M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Albiach-Martí, M.R., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Ayllón, M.A., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Gowda, S., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Hilf, M.E., USDA-ARS, 2001 South Rock Road, Fort Pierce, FL 34945, United States
Moreno, P., Instituto Valenciano De Investigaciones Agrarias, 46113 Moncada, Valencia, Spain
Garnsey, S.M., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Dawson, W.O., Department of Plant Pathology, University of Florida, Citrus Research and Education Center, Lake Alfred, FL 33850, United States
Amplification of Citrus tristeza virus from a cDNA clone and infection of Citrus trees
Isolates of the Closterovirus, Citrus tristeza virus (CTV), are populations of disparate genotypes and defective RNAs developed during long periods of vegetative propagation of citrus trees. Because it has not been possible to obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previously developed an infectious cDNA clone from which in vitro-produced RNA transcripts could infect protoplasts (Satyanarayana et al., 1999, Proc. Natl. Acad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor virions from transcript-infected protoplasts were competent for infection of citrus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the ∼20-kb RNA from virions or transcripts of cDNA infected only a small percentage of protoplasts (∼0.01%), but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone (recombinant virus) by successive passages in protoplasts using virions in crude sap as inoculum. By the third to seventh passages in protoplasts, maximal amounts of recombinant progeny virus were produced, which were used for inoculation of small citrus trees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from protoplasts, resulting in the first defined pure culture of CTV in plants. The comparative biology of the pure culture of recombinant CTV with that of the parental population in planta demonstrated that the recombinant virus retained through all of the recombinant DNA manipulations the normal functions of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Additionally, fulfilling Koch's postulates of the first pure culture of CTV in plants suggested that the major genotype of the CTV T36 population is the primary determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population. © 2001 Academic Press.
Scientific Publication
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