MybA, a transcription factor involved in conidiation and conidial viability of the human pathogen Aspergillus fumigatus.

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.

A novel dehydrin-like protein from Aspergillus fumigatus regulates freezing tolerance.

We recently reported on DprA and DprB, dehydrin-like proteins involved in the protection against oxidative, osmotic and pH stress in Aspergillus fumigatus. A third dehydrin-like gene, DprC, was detected in the genome of the filamentous fungus and repressed during conidial germination. The deletion mutant was impaired in freezing tolerance. The DprC-mediated response was dependent on the SakA MAP kinase pathway, and accordingly, SakA phosphorylation was induced by cold shock. When fused to eGFP, the protein was associated with the vacuoles.

Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity.

Ste12 and Ste12-like proteins are transcription factors found exclusively in the fungal kingdom. In the yeast model Saccharomyces cerevisiae, where the first member was identified, Ste12p was shown to regulate mating and invasive/pseudohyphal growth. In recent literature, there have been several reports of Ste12-like factors in multiple fungal systems, yeasts or filamentous fungi, with saprophytic or parasitic life-styles. In all these models, Ste12 and Ste12-like factors are involved in the regulation of fungal development and pathogenicity. In this review, we discuss the features, the regulation, and the role of Ste12 and Ste12-like factors by highlighting the similarities and dissimilarities that occur within this group.

Host and nonhost resistance in Medicago-Colletotrichum interactions.

Medicago truncatula lines resistant (A17) or susceptible (F83005.5) to the alfalfa pathogen Colletotrichum trifolii were used to compare defense reactions induced upon inoculation with C. trifolii or with the nonadapted pathogens C. lindemuthianum and C. higginsianum. Nonadapted Colletotrichum spp. induced a hypersensitive response (HR)-like reaction similar to the one induced during the host-incompatible interaction. Molecular analyses indicated an induction of PR10 and chalcone synthase genes in host and nonhost interactions but delayed responses were observed in the F83005.5 line. The clste12 penetration-deficient C. lindemuthianum mutant induced an HR and defense gene expression, showing that perception of nonadapted strains occurs before penetration of epidermal cells. Cytological and transcriptomic analyses performed upon inoculation of near-isogenic M. truncatula lines, differing only at the C. trifolii resistance locus, Ct1, with the nonadapted Colletotrichum strain, showed that nonhost responses are similar in the two lines. These included a localized oxidative burst, accumulation of fluorescent compounds, and transient expression of a small number of genes. Host interactions were characterized by a group of defense and signaling-related genes induced at 3 days postinoculation, associated with an accumulation of salicylic acid. Together, these results show that M. truncatula displays a rapid and transient response to nonadapted Colletotrichum strains and that this response is not linked to the C. trifolii resistance locus.

Galactofuranose attenuates cellular adhesion of Aspergillus fumigatus.

Galactofuranose (Galf) is a major molecule found in cell wall polysaccharides, secreted glycoproteins, membrane lipophosphoglycans and sphingolipids of Aspergillus fumigatus. The initial step in the Galf synthetic pathway is the re-arrangement of UDP-galactopyranose to UDP-Galf through the action of UDP-galactopyranose mutase. A mutant lacking the AfUGM1 gene encoding the UDP-galactopyranose mutase has been constructed. In the mutant, though there is a moderate reduction in the mycelial growth associated with an increased branching, it remains as pathogenic and as resistant to cell wall inhibitors and phagocytes as the wild-type parental strain. The major phenotype seen is a modification of the cell wall surface that results in an increase in adhesion of the mutants to different inert surfaces (glass and plastic) and epithelial respiratory cells. The adhesive phenotype is due to the unmasking of the mannan consecutive to the removal of galactofuran by the ugm1 mutation. Removal of the mannan layer from the mutant surface by a mannosidase treatment abolishes mycelial adhesion to surfaces.

An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen.

Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Delta mutant and a Colletotrichum lindemuthianum clste12Delta mutant. * Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

A novel family of dehydrin-like proteins is involved in stress response in the human fungal pathogen Aspergillus fumigatus.

During a search for genes controlling conidial dormancy in Aspergillus fumigatus, two dehydrin-like genes, DprA and DprB, were identified. The deduced proteins had repeated stretches of 23 amino acids that contained a conserved dehydrin-like protein (DPR) motif. Disrupted DprAΔ mutants were hypersensitive to oxidative stress and to phagocytic killing, whereas DprBΔ mutants were impaired in osmotic and pH stress responses. However, no effect was observed on their pathogenicity in our experimental models of invasive aspergillosis. Molecular dissection of the signaling pathways acting upstream showed that expression of DprA was dependent on the stress-activated kinase SakA and the cyclic AMP-protein kinase A (cAMP-PKA) pathways, which activate the bZIP transcription factor AtfA, while expression of DprB was dependent on the SakA mitogen-activated protein kinase (MAPK) pathway, and the zinc finger transcription factor PacC. Fluorescent protein fusions showed that both proteins were associated with peroxisomes and the cytosol. Accordingly, DprA and DprB were important for peroxisome function. Our findings reveal a novel family of stress-protective proteins in A. fumigatus and, potentially, in filamentous ascomycetes.

Regulation and role of a STE12-like transcription factor from the plant pathogen Colletotrichum lindemuthianum.

In phytopathogenic fungi, STE12-like genes encode transcription factors essential for appressorium-mediated host penetration. However, their regulation and downstream targets are still unknown. In the present study, a STE12-like gene (CLSTE12) from Colletotrichum lindemuthianum was isolated. We identified a spliced variant whose expression was negatively regulated during early stages of pathogenesis, whereas the correctly spliced mRNA remained expressed up to the penetration step, suggesting distinct roles for these two transcripts. Indeed, the full-length sequence was able to complement a yeast STE12 mutant, whereas overexpression of the transcript variant had a dominant-negative effect on yeast invasive growth and C. lindemuthianum pathogenicity. To further investigate the downstream genes that could be regulated by CLSTE12, disruption mutants were generated. Phenotypic analyses of the mutants revealed reduced pectinase activity and conidial adhesion to polystyrene. Analysis of cell surface proteins allowed the identification of a major protein, Clsp1p, which was absent from the mutants. Clsp1p belongs to a new family of wall-associated proteins only found in euascomycetous fungi. Overall, these results suggest that the activity of CLSTE12 can be modulated by a regulated alternative splicing mechanism and that this factor is involved in the production of cell surface proteins and host cell wall degrading enzymes.