Analysis of Tox5 gene expression in Gibberella pulicaris strains with different trichothecene production phenotypes.

The Tox5 gene encodes trichodiene synthase, the first unique enzyme in the trichothecene biosynthetic pathway. In Gibberella pulicaris R-6380, the level of Tox5 mRNA was found to increase 47-fold in early stationary phase. Sequence analysis of the Tox5 promoter regions from geographically distinct strains of G. pulicaris revealed the existence of two Tox5 alleles (Tox5-1 and Tox5-2). All G. pulicaris strains that produce high levels of trichothecenes in liquid culture carry a 42-nucleotide (nt) tandem repeat sequence (Tox5-1) in the Tox5 promoter region, whereas strains that produce low levels of trichothecenes carry a single copy of this sequence (Tox5-2). A genetic cross between high- and low-level trichothecene producers resulted in the cosegregation of higher-level trichothecene production with the Tox5-1 allele. To determine the importance of the 42-nt repeat sequence in the regulation of Tox5 expression, reporter gene constructs carrying either the Tox5-1 or the Tox5-2 promoter region fused to the beta-galactosidase gene of Escherichia coli were introduced into the high-level-trichothecene-producing strain, R-6380. Expression of reporter gene activity in transformants was found to be regulated in a manner similar to Tox5 expression but appeared to be independent of the 42-nt sequence copy number. These results indicate that transcriptional controls play an important role in the regulation of Tox5 expression and that genes involved in trichothecene biosynthesis in G. pulicaris may be linked to Tox5.