Genetic Engineering of an Ectomycorrhizal Fungus Laccaria bicolor for Use as a Biological Control Agent

We are developing genetically engineered ectomycorrhizal fungi for use as biological control agents. Using the gold particle bombardment technique, we transformed Laccaria bicolor, an ectomycorrhizal fungus capable of forming ectomycorrhizae on a wide range of plant hosts. Structural gene sequences of the hygromycin B phosphotransferase (hph) gene (hygromycin resistance for selection of transformants), β-glucuronidase (GUS) gene (reporter gene for tracking transformants) and Bacillus thuringiensis δ-endotoxin (BtCryIIIA) gene (Coleopteran specific insecticidal gene from Bacillus thuringiensis var tenebrionis) were introduced into L. bicolor simultaneously. Southern and western blot analyses showed that the introduced sequences were inserted into the fungal genome and transcribed and translated. One of the transformants produced the insecticidal toxin; however, the levels were not sufficient to perform toxicity assays. The technique developed can be employed to generate superior strains of L. bicolor suitable for use as biological control agents in various soil and plant conditions.