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

BACKGROUND: The 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). RESULTS: A 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. CONCLUSIONS: This 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.

Identification of transcripts up-regulated in asexual and sexual fruiting bodies of the Dutch elm disease pathogen Ophiostoma novo-ulmi.

Suppression subtractive hybridization cDNA libraries were prepared from asexual synnemata (S-lib) and sexual perithecia (P-lib) fruiting bodies of the Dutch elm disease pathogen Ophiostoma novo-ulmi subsp. novo-ulmi isolate H327 (mating-type MAT1-1) consisting of 630 and 401 cDNA clones, respectively. Both libraries were differentially screened in duplicate with forward and reverse subtracted probes. Up-regulated S-lib transcripts included those with homologies to phosphoenolpyruvate carboxykinase and aquaporin. Up-regulated P-lib transcripts included those with homologies to aspartyl proteinase, DNA lyase 2, and part of a mating-type (MAT) protein containing a DNA-binding domain of the high-mobility group (HMG) type. Phylogenetic analyses of HMG domains present within the putative O. novo-ulmi MAT protein and within MAT1-1-3 and MAT1-2-1 proteins of other ascomycete fungi identified the O. novo-ulmi protein as a homologue of the MAT1-1-3 protein, which represents part of the so far uncharacterized O. novo-ulmi MAT1-1 idiomorph. Reverse transcription - quantitative real-time PCR indicated up-regulation of the MAT1-1-3 homologue in O. novo-ulmi perithecia and synnemata. The present work identifies, for the first time, proteins involved in the formation of asexual and sexual fruiting bodies in O. novo-ulmi and should be of interest to researchers concerned with reproduction, mating type, and sexuality of filamentous ascomycete fungi.

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.

Functional annotation of the Ophiostoma novo-ulmi genome: insights into the phytopathogenicity of the fungal agent of Dutch elm disease.

The ascomycete fungus Ophiostoma novo-ulmi is responsible for the pandemic of Dutch elm disease that has been ravaging Europe and North America for 50 years. We proceeded to annotate the genome of the O. novo-ulmi strain H327 that was sequenced in 2012. The 31.784-Mb nuclear genome (50.1% GC) is organized into 8 chromosomes containing a total of 8,640 protein-coding genes that we validated with RNA sequencing analysis. Approximately 53% of these genes have their closest match to Grosmannia clavigera kw1407, followed by 36% in other close Sordariomycetes, 5% in other Pezizomycotina, and surprisingly few (5%) orphans. A relatively small portion (∼3.4%) of the genome is occupied by repeat sequences; however, the mechanism of repeat-induced point mutation appears active in this genome. Approximately 76% of the proteins could be assigned functions using Gene Ontology analysis; we identified 311 carbohydrate-active enzymes, 48 cytochrome P450s, and 1,731 proteins potentially involved in pathogen-host interaction, along with 7 clusters of fungal secondary metabolites. Complementary mating-type locus sequencing, mating tests, and culturing in the presence of elm terpenes were conducted. Our analysis identified a specific genetic arsenal impacting the sexual and vegetative growth, phytopathogenicity, and signaling/plant-defense-degradation relationship between O. novo-ulmi and its elm host and insect vectors.

Sequencing of the Dutch elm disease fungus genome using the Roche/454 GS-FLX Titanium System in a comparison of multiple genomics core facilities.

As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30-50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs.

Use of insertional mutagenesis to tag putative parasitic fitness genes in the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi.

We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 microg of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.

Characterization of three DNA transposons in the Dutch elm disease fungi and evidence of repeat-induced point (RIP) mutations.

Transposable elements (TEs) are fundamental components of eukaryotic genomes and can contribute in various ways to genome plasticity and evolution. We describe here the first three DNA transposons in the Dutch elm disease (DED) pathogens Ophiostoma ulmi and O. novo-ulmi, named OPHIO1, OPHIO2 and OPHIO3. We demonstrate that OPHIO transposons, which show high homology to Fot1/pogo TEs within the Tc1/mariner superfamily, have different distribution patterns and specificity in the DED fungi and that interspecific hybrids could act as genetic bridges for transmission of TEs between closely related fungal species. OPHIO3 was found to have undergone repeat-induced point mutations (RIP). We have also developed a complementary method to Margolin's ratios based on the computation of cumulative transition scores (CTS) in order to visualize rapidly RIP signatures on individual DNA strands of OPHIO transposons and TEs found in other ascomycete fungi.