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.

Quantification of microcystin-producing cyanobacteria and E. coli in water by 5'-nuclease PCR.

AIMS: 5'-Nuclease (real-time, quantitative) PCR methodologies were developed and applied as diagnostic tools for the detection of microcystin-producing cyanobacteria and Escherichia coli in water. METHODS AND RESULTS: PCR was used to detect regions of the lacZ gene in E. coli, and the microcystin synthetase gene in microcystin-producing cyanobacteria. In environmental water samples, natural inhibitors to PCR were effectively removed with a prefiltration step and an EDTA wash. A lower detection limit of 10 cells ml(-1) was obtained with endpoint PCR detection. 5'-Nuclease PCR was used for microbial quantification of 1 ml inoculated water samples. We were able to detect down to three copies of our target genes per sample within about 2 h (post-DNA isolation) for both E. coli and microcystin-producing cyanobacteria. CONCLUSIONS: 5'-Nuclease PCR offers a rapid and sensitive method of bacterial quantification in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: 5'-Nuclease PCR can be adopted as an effective diagnostic tool for monitoring microbiological water quality, through coliform quantification, and detection of other waterborne microbial pathogens.

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.

Widespread distribution of low-copy-number variants of mitochondrial plasmid pEM in the genus Agaricus.

The incidence of the linear mitochondrial plasmid pEM in Agaricus spp. was believed to be rare, based on visualization by gel electrophoresis and Southern hybridization. However, we report in this study PCR amplification of pEM-like sequences from all but one species of Agaricus examined. Regions amplified included (1) the pEM RNA polymerase gene and (2) adjoining carboxy-termini of the DNA and RNA polymerase genes. Sequence data from the RNA polymerase-like products support a plasmid, rather than mitochondrial, origin for these sequences. Sequence variation was low, and most differences were silent or conservative at the amino acid level. Stop codons were found in two of seven sequence types suggesting that functional constraints are low. A parsimony-derived phylogeny for these sequences did not match expected phylogenies for the host species. Recent acquisition of the plasmid is presented as the most likely hypothesis explaining these observations.

Isolation of adenylate cyclase gene-specific sequences from Ophiostoma novo-ulmi, Candida albicans, and Agaricus bisporus by PCR.

Degenerate primers corresponding to consensus sequences in the catalytic domains of known fungal adenylate cyclases were used to isolate gene-specific homologs from the Dutch elm disease pathogen Ophiostoma novo-ulmi, the dimorphic human pathogen Candida albicans, and the commercial mushroom Agaricus bisporus. All three fungi gave the expected PCR product of about 390 bp. Computer searches of the databases revealed that the products generated from O. novo-ulmi and C. albicans were highly similar to the adenylate cyclase gene of Magnaporthe grisea, the rice blast fungus (91% and 79%, respectively). The PCR product from the homobasidiomycete A. bisporus, on the other hand, showed 78% similarity to the uac1 gene of the heterobasidiomycete smut fungus, Ustilago maydis. Southern hybridization indicated that all three fungi contain a single adenylate cyclase gene. Our data suggest that PCR will be highly successful for the isolation of adenylate cyclase sequences from other fungi.

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.

Mitochondrial Haplotype Influences Mycelial Growth of Agaricus bisporus Heterokaryons.

We evaluated the influence of mitochondrial haplotype on growth of the common button mushroom Agaricus bisporus. Ten pairs of heterokaryon strains, each pair having the same nuclear genome but different mitochondrial genomes, were produced by controlled crosses among a group of homokaryons of both wild and commercial origins. Seven genetically distinct mitochondrial DNA (mtDNA) haplotypes were evaluated in different nuclear backgrounds. The growth of heterokaryon pairs differing only in their mtDNA haplotypes was compared by measuring mycelial radial growth rate on solid complete yeast medium (CYM) and compost extract medium and by measuring mycelial dry weight accumulation in liquid CYM. All A. bisporus strains were incubated at temperatures similar to those utilized in commercial production facilities (18, 22, and 26(deg)C). Statistically significant differences were detected in 8 of the 10 heterokaryon pairs evaluated for one or two of the three growth parameters measured. Some heterokaryon pairs showed differences in a single growth parameter at all three temperatures of incubation, suggesting a temperature-independent difference. Others showed differences at only a single temperature, suggesting a temperature-dependent difference. The influence of some mtDNA haplotypes on growth was dependent on the nuclear genetic background. Our results show that mtDNA haplotype can influence growth of A. bisporus heterokaryons in some nuclear backgrounds. These observations demonstrate the importance of including a number of mitochondrial genotypes and evaluating different nuclear-mitochondrial combinations of A. bisporus in strain improvement programs.

Lack of association between cerato-ulmin production and virulence in Ophiostoma novo-ulmi.

Cerato-ulmin (CU), a hydrophobin produced by Ophiostoma novo-ulmi, has been implicated in the pathogenicity of this fungus on elm. We have generated a CU- mutant by transformation-mediated gene disruption of a highly virulent (aggressive) strain of O. novo-ulmi. The inability of the mutant to synthesize CU was confirmed by transcript analysis as well as turbidity and immunological measurements. Bioassay of the CU- strain in highly susceptible elm trees indicated no difference in the virulence parameters, percent vascular discoloration, and percent foliar wilting, when compared with the wild type. Our results indicate that the inability to produce CU had no measurable effect on the ability of O. novo-ulmi to produce symptoms of Dutch elm disease on inoculated elms.

Plasmid RNA polymerase-like mitochondrial sequences in Agaricus bitorquis.

A linear mitochondrial plasmid, pEM, found in certain isolates of the basidiomycete Agaricus bitorquis, potentially encodes virus-like DNA and RNA polymerases. Mitochondrial DNA from Agaricus bisporus that hybridizes to an internal region of pEM contains a fragmented and potentially non-functional version of the carboxy terminal end of the plasmid RNA polymerase. In this study, we present the sequence of the corresponding region of mitochondrial DNA from A. bitorquis. This sequence contained the same region of the plasmid RNA polymerase gene as was reported for the mitochondrial DNA of A. bisporus, and the level of similarity between the A. bisporus and A. bitorquis mitochondrial sequences was much higher than the level of similarity between either mitochondrial sequence and the plasmid. We propose that this plasmid RNA polymerase-like sequence was present in the Agaricus mitochondrial genome before the divergence of A. bisporus and A. bitorquis, and thus is unlikely to be a recent derivative of the plasmid pEM.

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.

Uniparental Mitochondrial Transmission in the Cultivated Button Mushroom, Agaricus bisporus.

A uniparental mitochondrial (mt) transmission pattern has been previously observed in laboratory matings of the cultivated mushroom Agaricus bisporus on petri dishes. In this study, four sets of specific matings were further examined by taking mycelial plugs from the confluent zone of mated homokaryons and inoculating these plugs into rye grain for laboratory fruiting and for fruiting under industrial conditions. Examination of the mt genotype of each individual fruit body for mt-specific restriction fragment length polymorphisms further confirmed that the mt genome was inherited uniparentally. The vegetative radial growth and the fruiting activity of two pairs of intraspecific heterokaryons, each pair carrying the same combination of nuclear genomes but different mt genotypes, were compared. Our results suggested that the mt genotype did not appreciably affect radial growth or fruiting activity. The failure to recover both heterokaryons, each carrying either parental mt genotype in any given cross, therefore clearly indicated that in matings of A. bisporus, the mt genome from one of the parental homokaryons is either selectively excluded in the newly formed heterokaryon or selectively eliminated in the immediate heterokaryotic mitotic progeny of the newly formed heterokaryon.

Inheritance of Strain Instability (Sectoring) in the Commercial Button Mushroom, Agaricus bisporus.

The button mushroom, Agaricus bisporus, is a commercially important cultivated filamentous fungus. During the last decade, the button mushroom industry has depended mainly on two strains (or derivatives of these two strains). Using one of these highly successful strains (strain U1) we examined the phenomenon of strain instability, specifically, the production of irreversible sectors. Three "stromatal" and three "fluffy" sectors were compared with a healthy type U1 strain and with a wild-collected isolate. Compost colonization and fruit body morphology were examined. The main objective of this study, however, was to examine the meiotic stability of the sectored phenotype. Single basidiospores were isolated and subjected to a grain bioassay in which the ability to produce sectors was measured. Our results were as follows: (i) basidiospore cultures obtained from a wild-collected isolate showed no tendency to produce sectors; (ii) approximately 5% of the basidiospore cultures obtained from healthy type U1 strains produced irreversible sectors in the grain bioassay; (iii) the five primary sectors examined produced basidiospore cultures, half of which produced normal-looking growth in the grain bioassay and half of which produced some degree of sectoring; and (iv) the one sectored isolate that represented the F2 generation gave ratios similar to the 1:1 ratio observed for the F1 cultures.

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.

Localization of the Mating Type Gene in Agaricus bisporus.

The cultivated mushroom Agaricus bisporus is secondarily homothallic. Most basidia produce two basidiospores, each of which receives two of the four postmeiotic nuclei. Usually, the two packaged nuclei carry compatible mating types. Previous studies suggested that there may be only a single mating type locus in A. bisporus. In this study, we determined whether the mating type segregated as a single Mendelian determinant in a cross marked with 64 segregating molecular markers. To score mating types, each of the 52 homokaryotic offspring from this cross was paired with each of the two progenitor homokaryons. Compatible matings were identified by the formation of genetically stable heterokaryons which were verified by assay of restriction fragment length polymorphisms (RFLPs). Data for screening mycelial interactions on petri plates as well as fruit body formation were compared with the RFLP results. Mating types of 43 of the 52 homokaryotic offspring were determined on the basis of RFLP analysis. Our results indicate (i) there is a segregating mating type gene in A. bisporus, (ii) this mating type gene is on the largest linkage group (chromosome I), (iii) mycelial interactions on petri plates were associated with heterokaryon formation under selected conditions, (iv) fruit body formation was dependent upon the mating type gene, and (v) compatible mating types may not always be sufficient for fruiting.

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.

Further characterization of a large inverted repeat in the mitochondrial genomes of Agaricus bisporus (= A. brunnescens) and related species.

The mitochondrial (mt) genome of Agaricus bisporus Ag50 (a heterokaryon) is a 136-kilobase (kb) circular molecule which contains a pair of large inverted repeats (IRs). Two large BAMHI fragments (B1 and B2) which contain the IR regions were further mapped. The repeated regions were determined to be approximately 7.7 kb in length. The mt small ribosomal RNA (S rRNA) gene is located adjacent to one of the repeated regions. Orientational isomers, generated by homologous recombination between the repeated regions, were not observed in mtDNA extractions from Ag50 mycelium (liquid culture) or from Ag50 fruit bodies. We also did not observe any orientational isomers in Ag50HA or Ag50HB, two homokaryons somatically isolated from Ag50. DNA homologous to the Ag50 mt repeated regions was observed in ten other isolates of Agaricus including four isolates of A. bisporus, two isolates of A. subperonatus, two isolates of A. subfloccosus, one isolate of A. bitorquis, and one isolate of A. pattersonae. The repeated regions and the small unique regions in two other heterokaryotic strains of A. bisporus, Ag2 and Ag85, were physically mapped. The repeated regions in these two strains are also in the inverted forms. Restriction endonuclease mapping indicated that the two copies of the IR in Ag85 were not identical.

Meiotic behavior and linkage relationships in the secondarily homothallic fungus Agaricus bisporus.

This study followed the transmission of 64 segregating genetic markers to 52 haploid offspring, obtained from both homokaryotic and heterokaryotic meiospores, of a cross (AG 93b) of Agaricus bisporus, the commonly cultivated "button mushroom." The electrophoretic karyotypes of the AG 93b component nuclei were determined concurrently (n = 13). Eleven distinct linkage groups were identified by two-point analysis. DNA-DNA hybridization showed that nine of these corresponded to unique chromosome-sized DNAs. Two other chromosomal DNAs were marked with nonsegregating markers, including the rDNA repeat. Two remaining chromosomes remained unmarked but hybridized to repeated-sequence probes. Cross 93b had an essentially conventional meiosis in which both independent assortment and joint segregation of markers occurred, but in which crossing over was infrequent over much of the mapped genome. The 48 homokaryotic spore-offspring had overall crossover frequencies that were similar to, but possibly slightly less than, those of three homokaryon constituents of heterokaryotic spore-offspring. These daa provide support for our earlier cytogenetic model of sporogenesis in A. bisporus, that explains why heterokaryotic spore-offspring usually appear to exhibit no recombination. No evidence favoring an alternative, mitotic model of sporogenesis was found. The resulting genetic map appears to survey the genome extensively and for the first time permits localization of loci determining economically important traits in this fungal crop species. Large differences in the vigor of homokaryotic offspring were correlated with the inheritance of certain chromosome segments and were also often associated with significant departures from Mendelian segregation ratios.

Investigation of Mitochondrial Transmission in Selected Matings between Homokaryons from Commercial and Wild-Collected Isolates of Agaricus bisporus (= Agaricus brunnescens).

Ten heterokaryons of Agaricus bisporus (= Agaricus brunnescens) were shown to carry four different mitochondrial (mt) genotypes by analysis of mt restriction fragment length polymorphisms (RFLPs). Fifteen homokaryons derived from these strains were used to investigate mt inheritance in A. bisporus. One hundred eighty-nine pairings were performed in 25 different combinations. Pairings in 15 different combinations produced heterokaryons on the basis of nuclear RFLP analyses and/or fruiting trials. The mt genotype of each new intraspecies hybrid was examined by using mt RFLPs as genetic markers. Our results suggest the following. (i) Recombination between the mt genomes was not a common event. (ii) From most individual pairings, all heterokaryons carried the same mt genotype. (iii) Heterokaryons carrying either of the two possible mt genotypes were observed in certain crosses after modification of the pairing procedure. A biparental transmission pattern was demonstrated for some crosses, but there appears to be a preference for one of the mt genotypes to predominate in any specific pairing.

Homology between mitochondrial DNA of Agaricus bisporus and an internal portion of a linear mitochondrial plasmid of Agaricus bitorquis.

Agaricus bisporus, the cultivated mushroom, contains a mitochondrial fragment (50H) which was previously demonstrated by Southern hybridization to have sequence similarity to an internal region of pEM, a linear mitochondrial plasmid of Agaricus bitorquis. The nucleotide sequence of 50H was determined and compared to the sequence of the corresponding pEM fragment. The region of sequence homology on pEM is contained within an open reading frame (ORF) that may encode an RNA polymerase, but 50H is neither an intact nor a complete copy of the ORF. pEM also contains an ORF with characteristics of genes for virus-encoded DNA polymerases. pEM appears to be very similar to other linear mitochondrial plasmids (in fungi and higher plants) reported to contain ORFs that may encode the same types of polymerases. The potential functionality of the pEM sequence suggests that it has diverged less than the mitochondrial fragment from a common ancestor.

Analysis of a high frequency transformation system for Ophiostoma ulmi, the causal agent of Dutch elm disease.

A transformation system for Ophiostoma ulmi (Buism.) Nannf. was developed and analyzed. Protoplasts were generated from actively budding yeastlike cells by digestion with NovoZym 234 in MgSO4 after pretreatment with 2-mercaptoethanol. Protoplast regeneration was most efficient when 0.6 M sucrose was used as the osmoticum. Several plasmids containing fusions between fungal promoters and a bacterial gene for hygromycin phosphotransferase successfully transformed O. ulmi to hygromycin resistance. One of these vectors, pPS57, which contains a promoter for isopenicillin N synthetase from Penicillium chrysogenum, consistently conferred the greatest resistance to hygromycin. Linearization of the vector and inclusion of 2-mercaptoethanol in the transformation reaction resulted in enhanced transformation efficiency. Approximately 4 x 10(3) transformants/micrograms DNA per 10(7) protoplasts were obtained using the optimized procedure. Southern hybridization after alternating field and standard electrophoresis suggested random insertion of tandem repeats (some greater than 250 kb) into the fungal chromosomes. Antibiotic resistance was stable through mitosis. However, expression of the transforming DNA after meiosis was highly variable.