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Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinerea sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful. Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant (ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance, where introns are not required for expression, emphasising the variability in importance of these elements.

To detect and ultimately isolate genes of Phytophthora capsici the expression of which is induced during its interaction with pepper, a comparative analysis of gene expression in the wild-type pathogenic fungus with expression in a non-pathogenic (Nop) mutant reduced in cutinase and esterase activities was performed by the differential display of mRNAs. Discrimination of fungal genes induced in planta, from plant genes induced in response to the pathogen, was accomplished by exposure of the mycelium to bare-rooted seedlings of pepper (Capsicum annuum) in sterile water, to allow the initiation of infection, and then physical removal of the induced mycelium. With six sets of primer combinations, eight cDNA fragments (representing fungal genes) were present in planta only for the pathogenic strain. RNA-blot analysis showed that the transcripts detected accumulated to detectable levels only at early stages of the interaction. Sequence analysis and database searches revealed homology of one of the cDNA clones to fungal cutinases. The 218 amino-acid sequence predicted from sequencing a genomic clone of P. capsici suggested a protein of molecular weight of 23 980 Da with similarity to fungal cutinases previously characterized. These results indicated that differential-display analysis is sufficiently sensitive to be applied for the detection and isolation of fungal genes induced during a plant-pathogen interaction.

The Magnaporthe grisea ERG2 gene, encoding delta 8-->delta 7 sterol isomerase, was isolated from a genomic library by heterologous hybridization to a fragment of the Ustilago maydis ERG2 gene. The isolated gene contained a reading frame of 745 bp which encoded a protein of 221 amino acids. The coding region was interrupted by a single putative 79-bp-long intron. The deduced amino-acid sequence exhibited similarity to the ERG2 gene products of U. maydis and of Saccharomyces cerevisiae, particularly in the central region of the proteins. The NH2-terminal of all three proteins contained a long stretch of amino acids that were strongly hydrophobic, suggesting that they may function by anchoring the protein to a membrane surface. The M. grisea ERG2 gene complemented a U. maydis deletion mutant in which the ERG2 gene had been removed using a one-step gene replacement procedure. The delta 8-->delta 7 sterol isomerase produced by the M. grisea ERG2 gene exhibited a level of sensitivity to the sterol biosynthesis inhibitor, tridemorph, similar to that of the enzyme derived from the U. maydis ERG2 gene.

Phytophthora capsici, P. citricola, P. cinnamomi and P. citrophthora were transformed without the removal of cell walls by particle acceleration with plasmids containing the beta-glucuronidase gene and hygromycin B resistance. Transformants were detected by histochemical and fluorometric beta-glucuronidase assays and confirmed by Southern-blot hybridization. It was found that the promoter of a plant virus is functional in Phytophthora. In addition, a method was designed to visually identify homogeneous transformed colonies, derived from zoospores of transformed multinucleated Phytophthora mycelia, based on blue color development on plates containing X-Gluc.