The glyceraldehyde-3-phosphate dehydrogenase gene of Moniliophthoraperniciosa, the causal agent of witches' broom disease of Theobroma cacao

This report describes the cloning, sequence and expression analysis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene of Moniliophthora perniciosa, the most important pathogen of cocoa in Brazil. Southern blot analysis revealed the presence of a single copy of the GAPDH gene in the M. perniciosa genome (MpGAPDH). The complete MpGAPDH coding sequence contained 1,461 bp with eight introns that were conserved in the GAPDH genes of other basidiomycete species. The cis-elements in the promoter region of the MpGAPDH gene were similar to those of other basidiomycetes. Likewise, the MpGAPDH gene encoded a putative 339 amino acid protein that shared significant sequence similarity with other GAPDH proteins in fungi, plants, and metazoans. Phylogenetic analyses clustered the MPGAPDH protein with other homobasidiomycete fungi of the family Tricholomataceae. Expression analysis of the MpGAPDH gene by real-time PCR showed that this gene was more expressed (~1.3X) in the saprotrophic stage of this hemibiotrophic plant pathogen than in the biotrophic stage when grown in cacao extracts

FRUITING GENES OF SCHIZOPHYLLUM-COMMUNE ARE TRANSCRIPTIONALLY REGULATED

Fruiting genes in Schizophyllum commune are controlled by the mating-type genes and other regulatory genes. To examine whether differential accumulation of mRNAs for these fruiting genes is caused by transcriptional regulation, run-on transcription assaYs were performed with nuclei isolated from cultures differing in expression of these genes. It could be shown that accumulation of mRNAs followed enhanced transcription of most of the fruiting genes. Transcription continued during subsequent decrease of these mRNAs. In young not-yet-fruiting dikaryons and non-fruiting monokaryons the fruiting genes appeared to be transcribed at a low basal level. The GPD gene was used a control in these experiments and was transcribed at all stages

A homologous gene-reporter system for the basidiomycete Schizophyllum commune based on internally deleted homologous genes

Problems encountered in our attempts to achieve expression of heterologous genes, driven by ascomycetous regulatory sequences, in homobasidiomycetes led us to develop a gene-reporter system based on the expression of homologous genes in Schizophyllum commune. Internal deletions were made in the coding sequences of the regulated Sc4 gene and the constitutively expressed GPD gene. After introduction of these constructs into S. commune it was found that the expected truncated transcripts were produced. The internally deleted Sc4 gene, containing 1140 bp of upstream and 200 bp of downstream sequences, was only expressed in dikaryons at the time of fruiting (as was the resident Sc4 gene) but not at all in monokaryons, indicating that the construct contained all regulatory sequences necessary and sufficient to confer control by the mating-type genes and expression during fruiting. The internally deleted GPD gene, containing 1300 bp of upstream and 150 bp of downstream sequences, was expressed both in monokaryons and dikaryons at levels similar to those of the resident GPD gene, indicating that all sequences necessary for proper expression were present. This reporter-gene system may be applicable to the analysis of cis-regulatory sequences of these genes. Furthermore, heterologous genes may be inserted into the well-expressed GPD deletion construct to obtain expression of such genes in S. commune and possibly in other homobasidiomycetes

Targeted mutation of the SC3 hydrophobin gene of Schizophyllum commune affects formation of aerial hyphae

The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic

The Sc7/Sc14 Gene Family of Schizophyllum Commune Codes for Extracellular Proteins Specifically Expressed During Fruit-Body Formation

The Sc7 and Sc14 genes are specifically expressed in the dikaryon of the basidiomycete fungus Schizophyllum commune during fruiting. These genes are closely linked (within 6 kb) and highly similar in gene structure and nucleotide sequence (70% identical nucleotides in their coding regions). The encoded proteins (204 and 214 amino acids, respectively) have 87% similarity in amino acids (56% of the amino acids are identical). They contain putative signal sequences for secretion, are rich in aromatic amino acids which are generally located at similar positions, and they are generally hydrophilic. Inspection of databanks showed similarities with pathogenesis-related proteins (PR1) from plants, testis-specific proteins from mammals and venom allergen proteins from insects. An antibody raised against a Sc7 fusion protein showed the presence of the Sc7 protein in the culture medium and in the fruit bodies where it is apparently loosely associated with hyphal walls