William E. Hintz

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 pathogencity 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.

Characterization of the Class I α-mannosidase gene family in the filamentous fungus Aspergillus nidulans

Abstract We describe the cloning and sequence characterization of three Class I α-1,2-mannosidase genes from the filamentous fungus Aspergillus nidulans. We used degenerate PCR primers to amplify a portion of the α-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 α-1,2-mannosidase IB gene and the 1759 nt coding region of the α-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 α-1,2-mannosidases. The deduced protein sequences were aligned with 11 published Class I α-1,2-mannosidases to determine sequence relationships. All three genes exhibited high similarity to other fungal α-1,2-mannosidases. The α-1,2-mannosidase IB exhibited very high similarity to the Aspergillus satoi and Penicillium citrinum α-1,2-mannosidases and likely represents an orthologue of these genes. Phylogenetic analysis suggests that the three A. nidulans Class I α-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.

Endopolygalacturonase is encoded by a multigene family in the basidiomycete Chondrostereum purpureum

The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG). Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C. purpureum. Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD). The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity. Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes. C. purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.

Effects of Transgenic Hybrid Aspen Overexpressing Polyphenol Oxidase on Rhizosphere Diversity

ABSTRACT This study assessed the potential effects of transgenic aspen overexpressing a polyphenol oxidase gene on diversity in rhizosphere communities. Cultivation-independent methods were used to better delineate bacterial and fungal populations associated with transgenic and nontransgenic trees. Gene libraries for the bacterial component of the rhizosphere were established using 16S rRNA and chaperonin-60 (CPN-60) gene sequences, while the fungal community was characterized using 18S rRNA gene sequences. The 16S rRNA gene libraries were dominated by alphaproteobacterial sequences, while the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group. In both the CPN-60 and 16S rRNA libraries, there were differences in only minor components of the bacterial community between transgenic and unmodified trees, and no significant differences in species diversity were observed. Compared to the bacterial gene libraries, greater coverage of the underlying population was achieved with the fungal 18S rRNA libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. The dominant groups of fungi associated with each tree type were very similar, although there were some qualitative differences in the recovery of less-abundant fungi, likely as a result of the underlying heterogeneity of the fungal population. The methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees.

A comparison of the nucleotide sequence of the cerato-ulmin gene and the rDNA ITS between aggressive and non-aggressive isolates ofOphiostoma 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 ceratoulmincu gene product has been associated with disease symptoms. Nucleotide-sequence analysis ofcu 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 forcu in the promoter, coding, and transcription termination regions. Sequence data for the ITS clearly distinguish the subgroups.

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.

Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93–1224)

BackgroundOphiostoma novo-ulmi is the causative agent of Dutch elm disease (DED). It is an ascomycetous filamentous fungus that ranks as the third most devastating fungal pathogen in Canada. The disease front has spread eastward and westward from the epicentre in Ontario and Quebec and is threatening elm populations across the country. Numerous mitigation strategies have been tried to eradicate this pathogen, but success has thus far been limited. An alternative approach might utilize double-stranded RNA (dsRNA) mycoviruses which have been reported to induce hypovirulence in other fungi.MethodsUsing a modified single primer amplification technique (SPAT) in combination with chromosomal walking, we have determined the genome sequence of two RdRp encoding dsRNA viruses from an O. novo-ulmi isolate (93–1224) collected from the disease front in Winnipeg.ResultsWe propose that these viruses, which we have named OnuMV1c and OnuMV7 based on sequence similarity to other Ophiostoma mitoviruses, are two new members of the genus Mitovirus in the family Narnaviridae.ConclusionsThe discovery of such dsRNA elements raises the potential for engineering these viruses to include other genetic elements, such as anti-sense or interfering RNAs, to create novel and highly specific biological controls. Naïve fungal hosts could be infected with both the engineered molecule and a helper mitovirus encoding an RdRp which would provide replication capacity for both molecules.

Suppression of polygalacturonase gene expression in the phytopathogenic fungus Ophiostoma novo-ulmi by RNA interference

The fungal pathogen, Ophiostomo novo-ulmi, has been responsible for the rapid decline of American elm (Ulmus americana) across North America and remains a serious threat to surviving elm populations. The production of pectinolytic polygalacturonase enzymes has been implicated as a virulence factor for many fungal pathogens, including O. novo-ulmi. Previous work has shown that the targeted disruption of the endopolygalacturonase gene locus epg1 of O. novo-ulmi reduced, but did not eliminate pectinase activity. In the present study, we evaluated the use of RNA interference (RNAi) as a method of suppressing expression of the epg1 locus in O. novo-ulmi and compared its efficiency to the gene disruption method. While there was a reduction in epg1-specific mRNA transcripts and in the amount of polygalacturonase enzyme secreted for both methods of gene regulation, neither method completely suppressed the expression of pectinase activity. There was, however, a significantly greater reduction in both transcript levels and secreted enzyme observed for some of the RNAi transformants. As the first demonstration of RNAi in O. novo-ulmi, this method of gene regulation shows promise in future studies of gene expression and pathogenicity.

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.

AbstractCerato-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.

Functional categorization of unique expressed sequence tags obtained from the yeast-like growth phase of the elm pathogen Ophiostoma novo-ulmi

BackgroundThe highly aggressive pathogenic fungus Ophiostoma novo-ulmi continues to be a serious threat to the American elm (Ulmus americana) in North America. Extensive studies have been conducted in North America to understand the mechanisms of virulence of this introduced pathogen and its evolving population structure, with a view to identifying potential strategies for the control of Dutch elm disease. As part of a larger study to examine the genomes of economically important Ophiostoma spp. and the genetic basis of virulence, we have constructed an expressed sequence tag (EST) library using total RNA extracted from the yeast-like growth phase of O. novo-ulmi (isolate H327).ResultsA total of 4,386 readable EST sequences were annotated by determining their closest matches to known or theoretical sequences in public databases by BLASTX analysis. Searches matched 2,093 sequences to entries found in Genbank, including 1,761 matches with known proteins and 332 matches with unknown (hypothetical/predicted) proteins. Known proteins included a collection of 880 unique transcripts which were categorized to obtain a functional profile of the transcriptome and to evaluate physiological function. These assignments yielded 20 primary functional categories (FunCat), the largest including Metabolism (FunCat 01, 20.28% of total), Sub-cellular localization (70, 10.23%), Protein synthesis (12, 10.14%), Transcription (11, 8.27%), Biogenesis of cellular components (42, 8.15%), Cellular transport, facilitation and routes (20, 6.08%), Classification unresolved (98, 5.80%), Cell rescue, defence and virulence (32, 5.31%) and the unclassified category, or known sequences of unknown metabolic function (99, 7.5%). A list of specific transcripts of interest was compiled to initiate an evaluation of their impact upon strain virulence in subsequent studies.ConclusionsThis is the first large-scale study of the O. novo-ulmi transcriptome. The expression profile obtained from the yeast-like growth phase of this species will facilitate a multigenic approach to gene expression studies to assess their role in the determination of pathogenicity for this species. The identification and evaluation of gene targets in such studies will be a prerequisite to the development of biological control strategies for this pathogen.