Submitted to: Virology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 1/10/2001
Publication Date: 10/10/2001
Citation: Satyanaryana, T., Bar-Joseph, M., Mawassi, M., Albiach-Marti, M. R., Ayllon, M. A., Gowda, S., Hilf, M. E., Moreno, P., Garnsey, S. M., Dawson, W. O. 2001. Amplification of Citrus Tristeza Virus from a CDNA Clone and Infection of Citrus Trees. Virology, 280, 87-96. Interpretive Summary: This paper describes the development of a process to infect citrus plants with citrus tristeza virus (CTV) that has been synthesized in the laboratory. This technique overcomes what had been a major barrier to use genetic analysis to study the function of CTV genes involved in disease development in citrus plants. Since this laboratory-produced virus can now be put back into citrus plants, genetic analysis of CTV genes can be done.
Technical Abstract: 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. 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, but virions could infect more than 80% of the protoplasts. Based on this information, we amplified the virus from the cDNA clone 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.