Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis

Small non-protein-coding RNAs (ncRNAs) have systematically been studied in various model organisms from Escherichia coli to Homo sapiens. Here, we analyse the small ncRNA transcriptome from the pathogenic filamentous fungus Aspergillus fumigatus. To that aim, we experimentally screened for ncRNAs, expressed under various growth conditions or during specific developmental stages, by generating a specialized cDNA library from size-selected small RNA species. Our screen revealed 30 novel ncRNA candidates from known ncRNA classes such as small nuclear RNAs (snRNAs) and C/D box-type small nucleolar RNAs (C/D box snoRNAs). Additionally, several candidates for H/ACA box snoRNAs could be predicted by a bioinformatical screen. We also identified 15 candidates for ncRNAs, which could not be assigned to any known ncRNA class. Some of these ncRNA species are developmentally regulated implying a possible novel function in A. fumigatus development. Surprisingly, in addition to full-length tRNAs, we also identified 5′- or 3′-halves of tRNAs, only, which are likely generated by tRNA cleavage within the anti-codon loop. We show that conidiation induces tRNA cleavage resulting in tRNA depletion within conidia. Since conidia represent the resting state of A. fumigatus we propose that conidial tRNA depletion might be a novel mechanism to down-regulate protein synthesis in a filamentous fungus

HapX-Mediated Iron Homeostasis Is Essential for Rhizosphere Competence and Virulence of the Soilborne Pathogen Fusarium oxysporum

Soilborne fungal pathogens cause devastating yield losses and are highly persistent and difficult to control. During the infection process, these organisms must cope with limited availability of iron. Here we show that the bZIP protein HapX functions as a key regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. Deletion of hapX does not affect iron uptake but causes derepression of genes involved in iron-consuming pathways, leading to impaired growth under iron-depleted conditions. F. oxysporum strains lacking HapX are reduced in their capacity to invade and kill tomato (Solanum lycopersicum) plants and immunodepressed mice. The virulence defect of ΔhapX on tomato plants is exacerbated by coinoculation of roots with a biocontrol strain of Pseudomonas putida, but not with a siderophore-deficient mutant, indicating that HapX contributes to iron competition of F. oxysporum in the tomato rhizosphere. These results establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals

The mechanistic basis of pH-dependent 5-flucytosine resistance in Aspergillus fumigatus

ABSTRACT The antifungal drug 5-flucytosine (5FC), a derivative of the nucleobase cytosine, is licensed for the treatment of fungal diseases; however, it is rarely used as a monotherapeutic to treat Aspergillus infection. Despite being potent against other fungal pathogens, 5FC has limited activity against Aspergillus fumigatus when standard in vitro assays are used to determine susceptibility. However, in modified in vitro assays where the pH is set to pH 5, the activity of 5FC increases significantly. Here we provide evidence that fcyB , a gene that encodes a purine-cytosine permease orthologous to known 5FC importers, is downregulated at pH 7 and is the primary factor responsible for the low efficacy of 5FC at pH 7. We also uncover two transcriptional regulators that are responsible for the repression of fcyB and, consequently, mediators of 5FC resistance, the CCAAT binding complex (CBC) and the pH regulatory protein PacC. We propose that the activity of 5FC might be enhanced by the perturbation of factors that repress fcyB expression, such as PacC or other components of the pH-sensing machinery. </jats:p

Iron competition in fungus-plant interactions the battle takes place in the rhizosphere

Soilborne fungal pathogens are highly persistent and provoke important crop losses. During saprophytic and infectious stages in the soil, these organisms face situations of nutrient limitation and lack of essential elements, such as iron. We investigated the role of the bZIP transcription factor HapX as a central regulator of iron homeostasis and virulence in the vascular wilt fungus Fusarium oxysporum. This root-infecting plant pathogen attacks more than hundred different crops and is an emerging human opportunistic invader. Although iron uptake remains unaffected in a strain lacking HapX, de-repression of genes implicated in iron-consuming processes such as respiration, amino acid metabolism, TCA cycle and heme biosynthesis lead to severely impaired growth under iron-limiting conditions. HapX is required for full virulence of F. oxysporum in tomato plants and essential for infection in immunodepressed mice. Virulence attenuation of the ΔhapX strain on tomato plants is more pronounced by co-inoculation of roots with the biocontrol strain Pseudomonas putida KT2440, but not with a mutant deficient in siderophores production. These results demonstrate that HapX is required for iron competition of F. oxysporum in the tomato rhizosphere and establish a conserved role for HapX-mediated iron homeostasis in fungal infection of plants and mammals