Methodological improvements in the expression of foreign genes and in gene replacement in the phytopathogenic fungus Botrytis cinerea.

SUMMARY Genetic transformation is generally carried out in Botrytis cinerea by random integration of the foreign DNA into the genome, resulting in transformants that show differences among them in, for example, the expression of a reporter gene. Here we report a system for site-directed integration in which a novel recipient strain containing a 5'-truncated copy of the hygromycin resistance gene, hph, is transformed with a vector containing another truncated copy, now in the opposite end, of the same selection marker. Homologous recombination in the region shared by these two truncated copies of hph is the only way by which antibiotic-resistant transformants can be generated. The transformation frequency obtained for the site-directed strategy was only three-fold lower than that of the standard transformation, and all the transformants had at least one copy of the plasmid integrated at the expected locus. This system was tested by the expression of the green fluorescent protein and we found that the levels of this protein were more homogeneous among the transformants, when compared with those obtained by random integration. On the other hand, in this paper, we also tried to optimize gene replacements in B. cinerea, which are generally carried out by transformation with an antibiotic resistance marker flanked by regions homologous to the target gene. We studied the influence of the length of these regions on the frequency of replacement of the B. cinerea gene cel5A. Lengths between 500 and 2000 bp gave similar frequencies (about 60%), while lengths of 100 bp decreased the frequency to 6%, showing that 500 bp is a convenient size that would give optimal gene replacement frequencies in this fungus.