INTEGRATION OF VECTORS BY HOMOLOGOUS RECOMBINATION IN THE PLANT PATHOGEN GLOMERELLA-CINGULATA

An homologous transformation system has been developed for the plant p athogenic fungus Glomerella cingulata (Colletotrichum gloeosporioides) . A transformation vector containing the G. cingulata gpdA promoter fu sed to the hygromycin phosphotransferase gene was constructed. Souther n analyses indicated that this vector integrated at single sites in mo st transformants. A novel method of PCR amplification across the recom bination junction point indicated that the integration event occurred by homologous recombination in more than 95% of the transformants. Del etion studies demonstrated that 505 bp (the minimum length of homologo us promoter DNA analysed which was still capable of promoter function) was sufficient to target integration events. Homologous integration o f the vector resulted in duplication of the gdpA promoter region. When transformants were grown without selective pressure, a high incidence of vector excision by recombination between the duplicated regions wa s evident. The significance of these recombination characteristics is discussed with reference to the feasibility of performing gene disrupt ion experiments.