TOXIN-DEFICIENT MUTANTS FROM A TOXIN-SENSITIVE TRANSFORMANT OF COCHLIOBOLUS-HETEROSTROPHUS

Tox1 is the only genetic element identified which controls production of T-toxin, a linear polyketide involved in the virulence of Cochliobo lus heterostrophus to its host plant, corn. Previous attempts to induc e toxin-deficient (Tox(-)) mutants, using conventional mutagenesis and screening procedures, have been unsuccessful. As a strategy to enrich for Tox(-) mutants, we constructed a Tox1(+) strain that carried the corn T-urf13 gene (which confers T-toxin sensitivity) fused to a funga l mitochondrial signal sequence; the fusion was under control of the i nducible Aspergillus nidulans pelA promoter which, in both A. nidulans and C. heterostrophus, is repressed by glucose and induced by polygal acturonic acid (PGA). We expected that a transformant carrying this co nstruction would be sensitive to its own toxin when the T-urf13 gene w as expressed. Indeed, the strain grew normally on medium containing gl ucose but was inhibited on medium containing PGA. Conidia of this stra in were treated dth ethylmethanesulfonate and plated on PGA medium. Am ong 362 survivors, 9 were defective in T-toxin production. Authenticit y of each mutant was established by the presence of the transformation vector, proper mating type, and a restiction fragment length polymorp hism tightly linked to the Tox1(+) locus. Progeny of each mutant cross ed to a Tox1(+) tester segregated 1:1 (for wild type toxin production vs. no or reduced toxin production), indicating a single gene mutation in each case. Progeny of each mutant crossed to a Tox1(-) tester segr egated 1 : 1 (for no toxin production vs. no or reduced toxin producti on) indicating that each mutation mapped at the Tox1 locus. Availabili ty of Tox(-) mutants will permit mapping in the Tox1 region without in terference from a known Tox1 linked translocation breakpoint.