Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

Abstract

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, beta-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 microns-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae spheroplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplasts was treated with PEG and high pH-high Ca2+ solution (0.4 M mannitol, 50 mM CaCl2, 50 mM glycine-NaOH pH 10.5), GUS activity was detected in the extract of the plant cells after a three-day culture. The GUS activity was higher than that of a reconstitution experiment in which the mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 x 10(8) of S. cerevisiae spheroplasts, was treated with PEG and high pH-high Ca2+ solution. Moreover, the GUS gene expression was resistant to micrococcal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transferred from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.