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Replication of Citrus Tristeza Closterovirus in Citrus Protoplasts. M. Price, University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850-2299; J. Schell(2), J. Grosser(3), S. S. Pappu(4), H. R. Pappu(5), V. Febres(6), K. L. Manjunath(7), C. L. Niblett(8), K. S. Derrick(9), and R. F. Lee(10). (2)(3)(7)(9)(10)University of Florida, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred 33850-2299; (4)(5)(6)(8)University of Florida, Plant Pathology Department, P.O. Box 110680, Gainesville 32611-0680. Phytopathology 86:830-833. Accepted for publication 25 April 1996. Copyright 1996 The American Phytopathological Society. DOI: 10.1094/Phyto-86-830.

The study of citrus tristeza closterovirus (CTV) has been hindered by particle fragility, the inherently low virus titers, and its woody citrus hosts. We report the development of an in vitro Citrus sinensis cv. Hamlin protoplast system useful for the study of CTV replication. Full-length CTV genomic RNA from purified virions was inoculated into ‘Hamlin’ protoplasts in the presence of polyethylene glycol. After 24 h, the protoplasts were assayed for virus replication. Positive- and negative-strand CTV genomic RNA were detected by Northern hybridization using digoxigenin-labeled RNA probes. Three viral-encoded proteins of 20, 25 (capsid protein), and 27 kDa were detected by Western blotting, demonstrating that the viral RNA was translated in vivo. CTV virus particles also were isolated and observed in the electron microscope. This was the first demonstration of the infectivity of CTV RNA and the first report of CTV replication in protoplasts. This system will enable a more detailed study of the replication strategy of this economically important and complex virus.