Fungal Genome Sequences Research Articles

Comparative genomic analysis of phytopathogenic fungi using expressed sequence tag (EST) collections

SUMMARY We describe the analysis of 57 727 unique expressed sequence tags (ESTs) from 15 species of phytopathogenic and three species of saprophytic fungi. This resource is held within the COGEME phytopathogen EST database (http://cogeme.ex.ac.uk/). Comparative analysis was performed to investigate the differences between pathogenic and free-living fungi based on a substantial collection of expressed gene sequences and available, completed fungal genome sequences. We report that the expressed gene inventories of pathogenic fungi were not significantly more similar to each other than to those of free-living filamentous fungi. As expected, however, filamentous fungi as a group share more sequences in common than with the free-living yeast species Saccharomyces cerevisiae. Interestingly, ESTs of the obligate biotrophic fungus Blumeria graminis f. sp. hordei were more dissimilar to those of all other fungal species assessed, having a lower number of sequences in common with filamentous ascomycetes studied to date and also possessing a larger proportion of unisequences of unknown function. Our analysis of ESTs in the COGEME database enabled identification of a set of functional groups of genes that are more highly represented in the genomes of pathogenic fungi than non-pathogenic species.

Genomics of the fungal kingdom: Insights into eukaryotic biology

The last decade has witnessed a revolution in the genomics of the fungal kingdom. Since the sequencing of the first fungus in 1996, the number of available fungal genome sequences has increased by an order of magnitude. Over 40 complete fungal genomes have been publicly released with an equal number currently being sequenced--representing the widest sampling of genomes from any eukaryotic kingdom. Moreover, many of these sequenced species form clusters of related organisms designed to enable comparative studies. These data provide an unparalleled opportunity to study the biology and evolution of this medically, industrially, and environmentally important kingdom. In addition, fungi also serve as model organisms for all eukaryotes. The available fungal genomic resource, coupled with the experimental tractability of the fungi, is accelerating research into the fundamental aspects of eukaryotic biology. We provide here an overview of available fungal genomes and highlight some of the biological insights that have been derived through their analysis. We also discuss insights into the fundamental cellular biology shared between fungi and other eukaryotic organisms.

Fungal Pathogens Research: Novel and Improved Molecular Approaches for the Discovery of Antifungal Drug Targets

With the rise of fungal infection incidence amongst the patient population, the importance of developing new antifungal drug targets is higher than ever. This review mainly focuses on the three most prevalent fungal pathogens, Candida, Aspergillus and Cryptococcus, and on the most recent progresses in molecular research that contribute to a better understanding of the pathogen itself, but also its host and the interaction with its host. We consider the progress made in comparative genomics following the huge effort of fungal genome sequence projects undertaken in the last few years. We focus not only on currently used mammalian animal models such as mice, but also on novel non-mammalian models, such as the nematode worm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, which offer useful tools in the area of the innate immune response to fungal infections. In addition we relate to the recent genomic and proteomic studies and focus on the use of these approaches in in vivo experiments in the pathogen itself as well as in the host. Finally, we describe the latest targeted mutagenesis strategy available in C. albicans and the use of RNA interference in both Cryptococcus neoformans and A. fumigatus. Our aim is not to give an exhaustive list of all new strategies but rather to give an overview of what will contribute most to the identification of new antifungal drug targets and the establishment of novel antifungal strategies.

A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum

Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysporum. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase ( HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2′-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2′-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F. oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology.

The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis

Epipolythiodioxopiperazines (ETPs) are toxic secondary metabolites made only by fungi. The best-known ETP is gliotoxin, which appears to be a virulence factor associated with invasive aspergillosis of immunocompromised patients. The toxicity of ETPs is due to the presence of a disulphide bridge, which can inactivate proteins via reaction with thiol groups, and to the generation of reactive oxygen species by redox cycling. With the availability of complete fungal genome sequences and efficient gene-disruption techniques for fungi, approaches are now feasible to delineate biosynthetic pathways for ETPs and to gain insights into the evolution of such gene clusters.

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.

Contig assembly and microsynteny analysis using a bacterial artificial chromosome library for Epichloëfestucae, a mutualistic fungal endophyte of grasses

We constructed and characterized a bacterial artificial chromosome (BAC) library for Epichlo ë festucae, a genetically tractable fungal plant mutualist. The 6144 clone library with an average insert size of 87 kb represents at least 18-fold coverage of the 29 Mb genome. We used the library to assemble a 110 kb contig spanning the putative ornithine decarboxylase ( odc) ortholog and subsequently expanded it to 228 kb with a single walking step in each direction. Furthermore, we evaluated conservation of microsynteny between E. festucae and some model filamentous fungi by comparing sequence available from a 43 kb region at the end of one BAC to publicly available fungal genome sequences. Orthologs to the 13 contiguous open reading frames (ORFs) identified in E. festucae are syntenic in Neurospora crassa and Magnaporthe grisea occurring in small sets of two, three or four colinear ORFs. This library is a valuable resource for research into traits important for the development and maintenance of a plant-fungus mutualistic symbiosis.

A relational database for the discovery of genes encoding amino acid biosynthetic enzymes in pathogenic fungi

Fungal phytopathogens continue to cause major economic impact, either directly, through crop losses, or due to the costs of fungicide application. Attempts to understand these organisms are hampered by a lack of fungal genome sequence data. A need exists, however, to develop specific bioinformatics tools to collate and analyse the sequence data that currently is available. A web-accessible gene discovery database (http://cogeme.ex.ac.uk/biosynthesis.html) was developed as a demonstration tool for the analysis of metabolic and signal transduction pathways in pathogenic fungi using incomplete gene inventories. Using Bayesian probability to analyse the currently available gene information from pathogenic fungi, we provide evidence that the obligate pathogen Blumeria graminis possesses all amino acid biosynthetic pathways found in free-living fungi, such as Saccharomyces cerevisiae. Phylogenetic analysis was also used to deduce a gene history of succinate-semialdehyde dehydrogenase, an enzyme in the glutamate and lysine biosynthesis pathways. The database provides a tool and methodology to researchers to direct experimentation towards predicting pathway conservation in pathogenic microorganisms.