Amplification of Citrus tristeza virus from a cDNA clone and infection of Citrus trees

Isolates of the Ciosterovirus, 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 t o obtain pure cultures of the virus, it is not known what components of the population are primarily responsible for induction of diseases. We previou sly developed an infectious cDNA clone from which in vitro-produced RNA tra nscripts could infect protoplasts (Satyanarayana st al, 1999, Proc. Natl. A cad. Sci. USA 96, 7433-7438). However, neither the RNA transcripts nor viri ons from transcript-infected protoplasts were competent for infection of ci trus trees. Using a green fluorescent protein-marked virus as inoculum, we found that the similar to 20-kb RNA from virions or transcripts of cDNA inf ected only a small percentage of protoplasts (similar to0.01%), 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 successiv e passages in protoplasts using virions in crude sap as inoculum. By the th ird to seventh passages in protoplasts, maximal amounts of recombinant prog eny virus were produced, which were used for inoculation of small citrus tr ees by slashing stems in the presence of virion preparations. A relatively high percentage of plants became infected with the recombinant virus from p rotoplasts, resulting in the first defined pure culture of CTV in pla nts. 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 functi ons of replication, movement, and aphid transmissibility, and had a symptom phenotype indistinguishable from that of the parental population. Addition ally, fulfilling Koch's postulates of the first pure culture of CTV in plan ts suggested that the major genotype of the CTV T36 population is the prima ry determinant of the symptom phenotype. We could distinguish no biological contributions resulting from the minor genotypes and defective RNAs of the parental population. (C) 2001 Academic Press.