Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer

Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an ef ficient tool for insertional mutagenesis. In this paper, we report the cons truction of four novel binary vectors for fungal transformation and the opt imization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fasrium oxysporum and other phytopathoge nic fungi to be achieved. Employing the binary vectors, carrying the bacter ial hygromycin B phosphotransferase gene (hph) under the control of the Asp ergillus nidulans trpC promoter as a selectable marker, led to the producti on of 300 to 500 hygromycin B resistant transformants per 1 x 10(6) conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaci ens cells and significantly with the number of Agrobacterium tumefaciens ce lls present during the cocultivation period (r = 0.996; n = 3; P < 0.01). A ll transformants tested remained mitotically stable, maintaining their hygr omycin B resistance. Growing Agrobacterium tumefaciens cells in the presenc e of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased t o over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DN A per genome and the colony diameter of the transformants, the period of co cultivation or the quantity of Agrobacterium tumefaciens cells present duri ng cocultivation. To isolate the host sequences flanking the inserted T-DNA , we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) techn ique. Utilizing just one arbitrary primer resulted in the successful amplif ication of desired products in 90% of those transformants analyzed. The ins ertion event appeared to be a random process with truncation of the inserte d T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and l eft border sequences. Considering the size and design of the vectors descri bed here, coupled with the efficiency and flexibility of this ATMT protocol , it is suggested that ATMT should be regarded as a highly efficient altern ative to other DNA transfer procedures in characterizing those genes import ant for the pathogenicity of F. oxysporum and potentially those of other fu ngal pathogens.