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

• Published in: Plant and cell physiology 1994, Vol.35 (1), p.93-98
• Main Authors: Hatsuyama, Yoshimichi, Sunaga, Natsuo, Habu, Yoshiki, Ishikawa, Masayuki, Kawamoto, Shinichi, Naito, Satoshi, Ohno, Takeshi
• Format: Article
• Language: English
• Subjects: Arabidopsis; Arabidopsis - genetics; Arabidopsis thaliana; Biological and medical sciences; Biotechnology; Cell fusion; Cloning, Molecular; DNA transfer; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Methods. Procedures. Technologies; Plasmids - genetics; Polyethylene Glycols; Protoplasts; Reporter gene; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Transfection - methods; Transgenic animals and transgenic plants; Transgenic plants
• ISSN: 0032-0781; 1471-9053; 1471-9053
• DOI: 10.1093/oxfordjournals.pcp.a078575

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, β-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2μm-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 × 108 of S. cerevisiae spheroplasts harboring the plasmid and 2 × 106 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 × 108 of S. cerevisiae spheroplasts which did not carry the reporter gene, 2 × 106 of A. thaliana protoplasts and the same amount of the reporter plasmid DNA as that contained in 1 × 108 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.