RESTORATION OF WILD-TYPE VIRULENCE TO TRI5 DISRUPTION MUTANTS OF GIBBERELLA-ZEAE VIA GENE REVERSION AND MUTANT COMPLEMENTATION

Gibberella zeae is a pathogen of small grain crops and produces tricho thecene mycotoxins in infected host tissue. The role of trichothecenes in the virulence of C. zeae was previously investigated using trichot hecene-non-producing mutants that were generated via transformation-me diated disruption of a gene (Tri5) that encodes the first enzyme in th e trichothecene biosynthetic pathway. The mutants were less virulent o n some hosts than the wild-type strain from which they were derived. H ere, we used two approaches to determine whether the reduced virulence of mutants was due specifically to Tri5 disruption or to non-target e ffects caused by the transformation process. First, we generated a rev ertant from a Tri5 disruption mutant by allowing the mutant to pass th rough the sexual phase of its life cycle. In approximately 2% of the r esulting progeny the disrupted Tri5 had reverted to wild-type; however , only one of three revertant progeny also regained the ability to pro duce trichothecenes. In the second approach, we complemented the Tri5 mutation in a disruption mutant by transforming the mutant with a plas mid carrying a functional copy of Tri5. In all transformants examined, the ability to produce trichothecenes was restored. The restoration o f trichothecene production in the revertant progeny and in the complem ented mutant was accompanied by restoration of wild-type or near wild- type levels of virulence on wheat seedlings (cultivar Wheaten). The re sults indicate that the reduced virulence of the mutants was caused by disruption of Tri5 rather than nontarget effects resulting from the t ransformation process. The results also provide further evidence that trichothecenes contribute to the virulence of plant-pathogenic fungi.