Transformation of filamentous fungi by electroporation.

Transformation systems have been reported for a number of filamentous fungi (1) based on the procedure perfected for Neurospora crassa by Case et al. employing sphaeroplasts (2). While efficient transformation is achieved with sphaeroplasts, their preparation demands a careful monitoring of the individual steps, along with optimization of conditions for each batch of cell wall-degrading enzymes. Therefore, a new efficient transformation system was developed involving direct electroporation of germinating conidia of N. crassa. Initially we employed a recombinant plasmid harboring the qa-2+ gene, encoding the catabolic dehydroquinase (2) in conjunction with a double mutant (qa-2 arom-9) recipient strain, devoid of both the biosynthetic and catabolic dehydroquinase activities. Fifteen-day-old conidia were grown in 0.5 xFries' medium (3) with aromatic amino acid supplements, for 4 h at 30°C, while shaking. Germinated conidia were washed and suspended in 1 mM HEPES buffer (pH 7.0)-5O mM mannitol and 100-/il (6x 10 6) samples were mixed with 2 to 7 /ig of plasmid DNA and subjected to electroporation using the Bio-Rad Gene Pulser apparatus. After electroporation, 1 ml Vogel's minimal medium, Vm (3) was added and incubation resumed at 30°C for 3 h. Transformants were selected by plating on Vm-1.0% sorbose-0.1% glucose-0.1% fructose-1.5% agar. Virtually no transformants were recovered when field strength ranging from 3.0 to 9 KV/cm was employed, with capacitance values of 1 and 3 /iF. On increasing the field strength to 12.5 KV/cm, at 25 /tF capacitance and 5 msec pulse length, 2 to 3 stable transformants per ng DNA were obtained. Raising the pulse length to 25 msec resulted in suppression of transformation efficiency. Assuming that the cell wall impedes the uptake of DNA, we added 1 mg/ml /3-glucuronidase (Sigma: from Helix pomatia) to the growth medium for the final 2 h of germination. With this treatment a dramatic improvement in the efficiency of transformation was achieved: yields averaging 21 stable transformants per ^g DNA, comparable to those reported for other filamentous fungi (1) were observed routinely. Southern blot analysis of restriction endonuclease-digested DNA from a random sample of transformants demonstrated the integration of the plasmid in the genome. Hybridization with 32 P-labelled probes showed only the resident qa-2 genes in the untransformed recipient strain (Figure 1A, B: lane 1). In contrast, the DNA of transformants revealed ectopically integrated copies of qa-2+ gene (Figure 1A: lanes 2—5) as well as vector sequences (Figure IB: lanes 2-5), a majority of the integration …