DIRECT TRANSFER OF PLASMID DNA FROM INTACT YEAST SPHEROPLASTS INTO PLANT-PROTOPLASTS

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidop sis thaliana protoplasts. To monitor the DNA transfer from yeast to pl ant cells, beta-glucuronidase (GUS) reporter gene in which a plant int ron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 mum-based plasmid vector was not expressed in yeast transforma nts, while it expressed GUS activity when the plasmid DNA was introduc ed into plant cells. When a mixture of 1 x 10(8) of S. cerevisiae sphe roplasts harboring the plasmid and 2 x 10(6) of A. thaliana protoplast s 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 i n the extract of the plant cells after a three-day culture. The GUS ac tivity was higher than that of a reconstitution experiment in which th e mixture of 1 x 10(8) of S. cerevisiae spheroplasts which did not car ry the reporter gene, 2 x 10(6) of A. thaliana protoplasts and the sam e 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 microco ccal nuclease treatment before and during PEG treatment. From these re sults, 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.