Development of a fungal transformation system based on selection of sequences with promoter activity.

Abstract

A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5-to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.A selectable gene and a promoter sequence that drives its expression are essential to the development of a transformation system. In our work with the plant pathogenic fungus Cochliobolus heterostrophus (anamorph: Helminthosporium maydis = Bipolaris maydis), we are interested in transformation based on drug resistance, because such a system obviates the need for inducing mutations in the recipient strain and might be generally useful in genetically undeveloped organisms, such as most of the economically important fungi.As a selectable marker, we chose the Escherichia coli hygB gene, which encodes hygromycin B phosphotransferase. This enzyme detoxifies the aminocyclitol antibiotic hygromycin B by phosphorylation (12,15). Unlike many drugs, hygromycin B is toxic at a relatively low concentration (25 to 100 pug/ml) to a variety of ascomycetes, including the maize pathogen C. heterostrophus. Resistance to hygromycin B has been used as the basis of transformation systems for a number of eucaryotes, including Saccharomyces cerevisiae (12, 15), higher plants (16,27,28), and mouse L cells (22). In all cases, the procaryotic hygB gene was fused to a eucaryotic promoter.We reasoned that C. heterostrophus sequences capable of promoting hygB expression might be selected directly by inserting a library of small DNA fragments in front of a promoterless hygB gene and then transforming the fungus to hygromycin B resistance with this promoter probe library. Ther...

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