T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tum our-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) g enes, also located on the Ti plasmid, encode proteins involved in the trans port of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechan ism. The host range of A. tumefaciens is not restricted to plant species. A . tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cer evisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either dire ct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. There fore, the outcome of gene targeting experiments in some organisms may depen d strongly upon the DNA substrate used.