Real time RT-PCR quantification and Northern analysis of cerato-ulmin ( CU) gene transcription in different strains of the phytopathogens Ophiostoma ulmi and O. novo-ulmi.

Cerato-ulmin is a surface protein that belongs to the class of fungal proteins known as hydrophobins. This class II hydrophobin is produced throughout the life cycle and in all developmental stages of Ophiostoma novo-ulmi and O. ulmi; the aggressive and non-aggressive pathogens responsible for Dutch elm disease. Since yeast/mycelial transitions are often important to pathogenesis in dimorphic fungi such as Ophiostoma, we have examined the levels and abundance of cu mRNA in the yeast and mycelial stages of this fungus. The fungus contains one copy of the cu gene per haploid genome, located on chromosome IV. Our studies have been done using phosphoimager-based Northern analysis and real-time quantitative RT-PCR (qRT-PCR) to measure levels of cu mRNA. These measurements were made in both yeast-like and mycelial stages of the pathogen. Two wild-type, aggressive, strains of O. novo-ulmi (VA30 and H327) and one wild type non-aggressive strain of O. ulmi (H5) were analysed. As controls, we have utilized two types of mutants that we had previously generated, the null cu mutants THEK5-8 and THEK5-8-1, and a cu over-expression mutant, H5-tf16. Data generated by both Northern hybridization and real-time qRT-PCR analyses demonstrate that there is no cu mRNA transcription in the null mutants. The Northern analysis clearly showed that the over-expressing mutant H5-tf16 produces much more cu mRNA than the non-aggressive or aggressive strains. The quantitative data generated using qRT-PCR demonstrated that mycelium generally had 20-60% more cu mRNA than the yeast form. The non-aggressive strain of O. ulmi (H5) produces one-tenth as much cu mRNA as the aggressive strains (VA30 and H327). When transformed with additional copies of the cu gene, this same non-aggressive strain (H5-tf16) expressed about 20 times more cu mRNA than the wild type H5 strain. These data were consistently generated in multiple real-time qRT-PCR experiments with different RNA preparations, clearly demonstrating that the quantitative abundance values obtained were reproducible. Our study represents the first report on the use of real-time qRT-PCR to compare and quantify gene transcription in different growth phases of a fungal pathogen.

Characterization of the class I alpha-mannosidase gene family in the filamentous fungus Aspergillus nidulans.

We describe the cloning and sequence characterization of three Class I alpha-1,2-mannosidase genes from the filamentous fungus Aspergillus nidulans. We used degenerate PCR primers to amplify a portion of the alpha-1,2-mannosidase IA gene and used the PCR fragment to isolate the 2495 nt genomic gene plus several hundred bases of flanking region. Putative introns were confirmed by RT-PCR. Coding regions of the genomic sequence were used to identify two additional members of the gene family by BLAST search of the A. nidulans EST sequencing database. Specific PCR primers were designed to amplify portions of these genes which were used to isolate the genomic sequences. The 1619 nt coding region of the alpha-1,2-mannosidase IB gene and the 1759 nt coding region of the alpha-1,2-mannosidase IC gene, plus flanking regions, were fully sequenced. All three genes appeared to encode type-II transmembrane proteins that are typical of Class I alpha-1,2-mannosidases. The deduced protein sequences were aligned with 11 published Class I alpha-1, 2-mannosidases to determine sequence relationships. All three genes exhibited high similarity to other fungal alpha-1,2-mannosidases. The alpha-1,2-mannosidase IB exhibited very high similarity to the Aspergillus satoi and Penicillium citrinum alpha-1,2-mannosidases and likely represents an orthologue of these genes. Phylogenetic analysis suggests that the three A. nidulans Class I alpha-1, 2-mannosidases arose from duplication events that occurred after the divergence of fungi from animals and insects. This is the first report of the existence of multiple Class I mannosidases in a single fungal species.

A gene associated with filamentous growth in Ophiostoma novo-ulmi has RNA-binding motifs and is similar to a yeast gene involved in mRNA splicing.

The COL1 gene was isolated from Ophiostoma novo-ulmi using the techniques of insertional mutagenesis and plasmid rescue. Sequence analyses suggest that the COL1 gene encodes a unique protein of 826 amino acids with consensus-type RNA-binding domains, most similar to a putative protein from Schizosaccharomyces pombe which resembles the C-terminus of the Saccharomyces cerevisiae U4/U6 splicing factor PRP24. Disruption of the COL1 gene produced the yeast-like col1 mutant. The inability of the mutant to synthesize the COL1 gene product was confirmed by transcript analysis. Transformation of the col1 mutant with the COL1 gene restored the wild phenotype and production of the 4.0-kb mRNA. The results from this study demonstrate that the COL1 RNA-binding protein is associated with filamentous growth in O. novo-ulmi.

Sequence analysis of the chitin synthase A gene of the Dutch elm pathogen Ophiostoma novo-ulmi indicates a close association with the human pathogen Sporothrix schenckii.

Degenerate oligonucleotide primers were designed according to conserved regions of the chitin synthase gene family and used to amplify a 621 basepair (bp) fragment from genomic DNA of Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The amplification product was used as a hybridization probe to screen a library of genomic DNA sequences and to retrieve a full-length chitin synthase gene (chsA). The putative coding region of the gene was 2619 bp long, lacked introns, and encoded a polypeptide of 873 amino acids. Based on the similarity of the predicted amino acid sequence to the full-length chsC gene of Aspergillus nidulans and chsA gene of Ampelomyces quisqualis, the O. novo-ulmi chsA was classified as a Class I chitin synthase. The phylogenies constructed, according to a subregion of all available chitin synthases, showed that O. novo-ulmi consistently clustered most closely with the human pathogen Sporothrix schenckii, recently classified as a member of the mitosporic Ophiostomataceae. Disruption of the chsA gene locus had no obvious effects on the growth or morphology of the fungus.

Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans.

A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain. Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, and compose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.

Cerato-ulmin, a hydrophobin secreted by the causal agents of Dutch elm disease, is a parasitic fitness factor.

Dutch elm disease is caused by the aggressive Ophiostoma novo-ulmi and the nonaggressive O. ulmi. Both secrete the protein cerato-ulmin (CU). To determine what role CU plays in the pathology of Dutch elm disease, we constructed a CU overexpression mutant of the nonaggressive O. ulmi H5. Stable integration of a single copy of the cu gene from the aggressive O. novo-ulmi into the genome of the nonaggressive isolate resulted in increased secretion of CU protein. Trials with American elm, Ulmus americana, suggested no alteration of virulence of this overexpressing transformant. Using aggressive and nonaggressive wild types, the cu overexpressing mutant, and our cu- mutant (Bowden et al., 1996), we have demonstrated that CU production is correlated with an altered phenotype and more hydrophobic and adherent yeast-like cells. Our results also demonstrate that CU has a role in protecting infectious propagules from desiccation. These biological roles for CU would affect transmission of Dutch elm disease, and we therefore propose that this hydrophobin acts as a parasitic fitness factor.

A comparison of the nucleotide sequence of the cerato-ulmin gene and the rDNA ITS between aggressive and non-aggressive isolates of Ophiostoma ulmi sensu lato, the causal agent of Dutch elm disease.

Little genetic information exists comparing aggressive and non-aggressive isolates of the causal agent of Dutch elm disease, Ophiostoma ulmi. Two genetic elements were compared between the subgroups. The ceratoulmin cu gene product has been associated with disease symptoms. Nucleotide-sequence analysis of cu and the internal transcribed spacer (ITS) region of the rDNA were made from three aggressive and three non-aggressive isolates of the pathogen. Our results suggested uniformity within, and unique differences between, subgroups. Differences were detected for cu in the promoter, coding, and transcription termination regions. Sequence data for the ITS clearly distinguish the subgroups.

Isolation and characterization of the cerato-ulmin toxin gene of the Dutch elm disease pathogen, Ophiostoma ulmi.

The hydrophobic protein cerato-ulmin (CU), produced by Ophiostoma ulmi, has been implicated in the pathogenicity of this fungus on elm. Primers were designed based on the nucleotide sequence deduced from the published CU amino-acid sequence, and a DNA fragment of the cu gene was amplified using the polymerase chain reaction. The amplified cu fragment was used as a hybridization probe to identify and isolate the cu gene from a genomic DNA library of an aggressive isolate of O. ulmi ( = O. novo-ulmi). The cu coding region is interrupted by two introns and encodes a 100 amino-acid prepro-CU polypeptide that is processed to a 75 amino-acid mature protein upon secretion. CU shows significant sequence similarity to hydrophobins secreted by certain other fungi.

A glucose-derepressed promoter for expression of heterologous products in the filamentous fungus Aspergillus nidulans.

We describe a putative binding sequence (GCGGGGC) for the glucose-responsive repressor protein CreA at two positions upstream of the transcription start site of the alcohol dehydrogenase I (alcA) gene of Aspergillus nidulans. To positively identify the putative binding sites as CreA-specific, the GCGGGGC blocks were mutated at five internal nucleotide positions to GTACTAC and reintroduced into the wild type alcA promoter driving expression of the endogenous alcohol dehydrogenase I gene. This CreA-binding site variant was then transformed into an AlcR constitutive A. nidulans host strain (T2625) and growth was monitored in the presence of the non-metabolized glucose analogue, 2-deoxyglucose. Positive transformants were selected by their ability to grow using ethanol as a carbon source in the presence of 2-deoxyglucose. Similar CreA binding site variant alcA promoters should permit the alcA-driven expression of heterologous genes in A. nidulans in the presence of glucose, the preferred carbon source for biomass accumulation and provides a model for controlling carbon-catabolite regulated expression in other expression systems.

A genetic survey of the pathogenic fungus Ophiostoma ulmi across a Dutch elm disease front in western Canada.

The natural population structure of the Dutch elm pathogen Ophiostoma ulmi was determined from isolated collected from across a Western Canadian disease front through an analysis of restriction-site polymorphisms in the ribosomal DNA repeat, length mutations in the mitochondrial genomes, and through DNA fingerprinting of the nuclear genomes using a minisatellite DNA probe. The 8.8-kbp rDNA repeat was selected from a genomic library, and restriction-site and genic maps were constructed for the nonaggressive and aggressive subgroups of O. ulmi. There were only three restriction-site differences that distinguished these two subgroups and no intrasubgroup variation was detected. All of the isolates collected from the disease front were of the aggressive subgroup and were represented by two distinct nuclear and four mitochondrial genotypes. The minority of the isolates were of a single genotype (type A nuclear DNA; type I mtDNA), indicating the presence of a single very large clone extending across much of Manitoba and into Saskatchewan.