A homologous and self-replicating system for efficient transformation of Fusarium oxysporum.

Abstract

A highly efficient transformation system has been developed for Fusarium oxysporum f. sp. lycopersici based on the complementation of a nitrate-reductase mutant with the homologous nit1 gene and on the presence of ARS and telomeric sequences in the vector. Preliminary transformation experiments with the niaD gene from Aspergillus niger generated self-replicating plasmids within the transformed entity that contained extra-fungal DNA. A fragment of the extra DNA was inserted into pUC19 together with the F. oxysporum nit1 gene, resulting in plasmid pFNit-Lam. This allowed the isolation of a new linear plasmid within self-replicative F. oxysporum transformants (pFNit-Lam-TLam, linear). The circular form of this vector yielded 5600 fungal transformants per microgram of DNA. All of the transformants contained autonomous linear plasmids harboring direct repeats of fungal DNA at both ends. The sequence of the 1.2-kb fragment from F. oxysporum responsible for autonomous replication, and maintenance as linear plasmid molecules, has been determined. Comparison analysis with the ARS from different organisms has shown that this fragment contained the commonly identified ARS consensus sequence, 5'A/TTTTATA/GTTTA/T3' and, in addition to this core, ten copies of the ARS-box, 5'TNTA/GAA3'. Adjacent to this presumed ARS, the telomeric hexanucleotide sequence (TTAGGG)n was present in six tandem copies followed by 18 copies of its complementary sequence.