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.

The Canadian Ophiostoma Genome Project, which was initiated in 2001, is a collaborative effort between research teams in four different universities. Its general objective is to conduct a large-scale identification and analysis of genes controlling important aspects of the life cycle of Ophiostomatoid fungi. To this end, several expressed sequence tag (EST) libraries were obtained for the Dutch elm disease pathogen Ophiostoma novo-ulmi and the sapstainer O. piceae, following partial, single-pass automated sequencing of complementary DNA clones. The largest EST library, prepared from yeast like cells of O. novo-ulmi grown at 24 °C, contains over 3,400 readable sequences and serves as a general reference library for Ophiostomatoid fungi. Smaller, specific EST libraries were constructed from mycelia of O. novo-ulmi grown at suboptimal temperatures, from perithecia formed in laboratory crosses, as well as from O. piceae grown on different carbon sources. Ongoing bioinformatic searches in public databases have so far identified over 750 Ophiostoma unique ESTs which show significant homology with other fungal genes of known function, although a high proportion of Ophiostoma ESTs are either orphans (no match to any known gene) or show homology to genes of unknown function. In addition to EST analysis, differential expression of selected genes and structural genomics are also being studied.