Repellents have functionally replaced hydrophobins in mediating attachment to a hydrophobic surface and in formation of hydrophobic aerial hyphae in Ustilago maydis.

Ustilago maydis contains one repellent and two class I hydrophobin genes in its genome. The repellent gene rep1 has been described previously. It encodes 11 secreted repellent peptides that result from the cleavage of a precursor protein at KEX2 recognition sites. The hydrophobin gene hum2 encodes a typical class I hydrophobin of 117 aa, while hum3 encodes a hydrophobin that is preceded by 17 repeat sequences. These repeats are separated, like the repellent peptides, by KEX2 recognition sites. Gene hum2, but not hum3, was shown to be expressed in a cross of two compatible wild-type strains, suggesting a role of the former hydrophobin gene in aerial hyphae formation. Indeed, aerial hyphae formation was reduced in a Delta hum2 cross. However, the reduction in aerial hyphae formation was much more dramatic in the Delta rep1 cross. Moreover, colonies of the Delta rep1 cross were completely wettable, while surface hydrophobicity was unaffected and only slightly reduced in the Delta hum2 and the Delta hum2 Delta hum3 cross, respectively. It was also shown that the repellents and not the hydrophobins are involved in attachment of hyphae to hydrophobic Teflon. Deleting either or both hydrophobin genes in the Delta rep1 strains did not further affect aerial hyphae formation, surface hydrophobicity and attachment. From these data it is concluded that hydrophobins of U. maydis have been functionally replaced, at least partially, by repellents.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis.

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune.

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete Schizophyllum commune. For instance, the SC3 and SC15 genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The SC15 gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of S. commune after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.

The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins.

Streptomycetes form hydrophobic aerial hyphae that eventually septate into hydrophobic spores. Both aerial hyphae and spores possess a typical surface layer called the rodlet layer. We present here evidence that rodlet formation is conserved in the streptomycetes. The formation of the rodlet layer is the result of the interplay between rodlins and chaplins. A strain of Streptomyces coelicolor in which the rodlin genes rdlA and/or rdlB were deleted no longer formed the rodlet layer. Instead, these surfaces were decorated with fine fibrils. Deletion of all eight chaplin genes (strain DeltachpABCDEFGH) resulted in the absence of the rodlet layer as well as the fibrils at surfaces of aerial hyphae and spores. Apart from coating these surfaces, chaplins are involved in the escape of hyphae into the air, as was shown by the strong reduction in the number of aerial hyphae in the DeltachpABCDEFGH strain. The decrease in the number of aerial hyphae correlated with a lower expression of the rdl genes in the colony. Yet, expression per aerial hypha was similar to that in the wild-type strain, indicating that expression of the rdl genes is initiated after the hypha has sensed that it has grown into the air.