Analysis of flocculins in Ashbya gossypii reveals FIG2 regulation by TEC1.

For 95% of the Ashbya gossypii protein-encoding genes there is a Saccharomyces cerevisiae homolog. Out of these 90% are arranged in a conserved, syntenic, gene order. Interestingly, A. gossypii adhesins, encoded by homologs of S. cerevisiae FLO-genes, are found in non-syntenic positions. A. gossypii contains only a small set of adhesins: two FLO5, a FLO11 and a FIG2 homolog, but no FLO1, FLO9, or FLO10 homolog. Here we present the functional analysis of the A. gossypii adhesins and their potential transcriptional regulators SFL1, FLO8, and TEC1. Deletion of individual classes of FLO-genes did not reveal any phenotype. Lack of SFL1 or FLO8 showed reduced growth. The expression of adhesins in different strain backgrounds was tested using promoter-lacZ-fusions. We found that SFL1 acts as a suppressor of one of the FLO5 genes and FLO8 but particularly of FIG2. Interestingly, FIG2 expression was abolished in a tec1 mutant. We identified three potential Tec1-binding sites in the FIG2-promoter by similarity to S. cerevisiae Tec1-binding sites. The AgCHT2 promoter, which regulates a sporulation specific chitinase, also harbours potential Tec1-binding sites. Consequently, expression of CHT2 was not detected in a tec1 strain. This suggests that Tec1- binding sites are conserved between A. gossypii and S. cerevisiae even though there are different Tec1 target genes in each of these organisms.

Comparative genomics of MAP kinase and calcium-calcineurin signalling components in plant and human pathogenic fungi.

Mitogen-activated protein kinase (MAPK) cascades and the calcium-calcineurin pathway control fundamental aspects of fungal growth, development and reproduction. Core elements of these signalling pathways are required for virulence in a wide array of fungal pathogens of plants and mammals. In this review, we have used the available genome databases to explore the structural conservation of three MAPK cascades and the calcium-calcineurin pathway in ten different fungal species, including model organisms, plant pathogens and human pathogens. While most known pathway components from the model yeast Saccharomyces cerevisiae appear to be widely conserved among taxonomically and biologically diverse fungi, some of them were found to be restricted to the Saccharomycotina. The presence of multiple paralogues in certain species such as the zygomycete Rhizopus oryzae and the incorporation of new functional domains that are lacking in S. cerevisiae signalling proteins, most likely reflect functional diversification or adaptation as filamentous fungi have evolved to occupy distinct ecological niches.

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea.

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in beta-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of beta1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAc beta 1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.

Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs.

The ink cap Coprinopsis cinerea is a model organism for studying fruiting body (mushroom) formation in homobasidiomycetes. Mutant screens and expression studies have implicated a number of genes in this developmental process. Functional analysis of these genes, however, is hampered by the lack of reliable reverse genetics tools for C. cinerea. Here, we report the applicability of gene targeting by RNA silencing for this organism. Efficient silencing of both an introduced GFP expression cassette and the endogenous cgl1 and cgl2 isogenes was achieved by expression of homologous hairpin RNAs. In latter case, silencing was the result of a hairpin construct containing solely cgl2 sequences, demonstrating the possibility of simultaneous silencing of whole gene families by a single construct. Expression of the hairpin RNAs reduced the mRNA levels of the target genes by at least 90%, as determined by quantitative real-time PCR. The reduced mRNA levels were accompanied by cytosine methylation of transcribed and nontranscribed DNA at both silencing and target loci in the case of constitutive high-level expression of the hairpin RNA but not in the case of transient expression. These results suggest the presence of both posttranscriptional and transcriptional gene silencing mechanisms in C. cinerea and demonstrate the applicability of targeted gene silencing as a powerful reverse genetics approach in this organism.