Tanaka et al. used a forward genetics approach to create mutants of the endophyte that would be unable to establish or maintain a mutualistic relationship with perennial ryegrass. They inserted foreign DNA randomly into the genome of Epichlo festucae, resulting in a population of fungal strains having disruptions in different genes throughout the fungal genome. From this collection they isolated a mutant that is unable to synchronize its growth with that of the plant host.
Plants infected with the mutant fungus showed stunted growth, premature senescence, and death, whereas those infected with the wild-type fungus exhibited their usual growth pattern. This was accompanied by a dramatic increase in fungal endophyte growth within the plant compared with plants inoculated with wild-type fungus. The fungal hyphae of the wild type fungus showed limited branching and were mostly oriented parallel to the intercellular spaces of the leaf. On the other hand, the hyphae of the mutant fungus showed extensive colonization of the leaf--similar to a pathogenic infection. As a result, the biomass of the mutant fungus increased significantly compared to wild type. Thus a mutualistic interaction became an antagonistic one with the mutation of a single gene.
Tanaka et al. then went on identify and sequence the fungal gene responsible for the mutant phenotype. They determined that the foreign DNA had disrupted a fungal gene, called noxA, which encodes an enzyme that catalyzes the conversion of molecular oxygen to superoxide. The altered symbiotic phenotype is due to a mutation (caused by the insertion of a segment of foreign DNA) in the E. festucae noxA gene.