Exsymbiotic, Chlorella-like eukaryotic green algae act as hosts for a group of large double-stranded DNA-containing viruses. The algae can be readily cultured in the presence of light. The viruses synchronously infect, rapidly replicate, and ultimately lyse their algal host. As a result of the development of a plaque assay, this is the first and, thus far, only example of a plant-virus system which lends itself to investigation using established technology adapted from bacteriophage studies. An additional benefit of this system is that the viral-algal lysate is a rich source of cell wall degrading enzyme activity which can be used to produce protoplasts into which recombinant DNA may be introduced. The ultimate goal of this project is to develop an algal transformation system. Toward this end, it is proposed to: (1) develop a protoplast regeneration system for this alga; (2) search for DNA sequences from either the virus or the host which will confer autonomous replication of plasmids in this alga; (3) determine the intracellular sites of viral DNA replication; (4) study viral DNA for clues as to the regulation of viral gene expression; (5) study vectors containing reporter molecules, to measure transient expression; and (6) construct vectors with selectable markers. Success in these goals will allow the characterization and exploitation of this interesting algal system, and will have significant impact on plant genetics and cell biology.
