Roles of Aspergillus nidulans Cdc42/Rho GTPase regulators in hyphal morphogenesis and development.

The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A nidulans.

Production of a Chaetomium globosum enolase monoclonal antibody.

Chaetomium globosum is a hydrophilic fungal species and a contaminant of water-damaged building materials in North America. Methods to detect Chaetomium species include subjective identification of ascospores, viable culture, or molecular-based detection methods. In this study, we describe the production and initial characterization of a monoclonal antibody (MAb) for C. globosum enolase. MAb 1C7, a murine IgG1 isotype MAb, was produced and reacted with recombinant C. globosum enolase (rCgEno) in an enzyme-linked immunosorbent assay and with a putative C. globosum enolase in a Western blot. Epitope mapping showed MAb 1C7 specific reactivity to an enolase decapeptide, LTYEELANLY, that is highly conserved within the fungal class Sordariomycetes. Cross-reactivity studies showed MAb 1C7 reactivity to C. atrobrunneum but not C. indicum. MAb 1C7 did not react with enolase from Aspergillus fumigatus, which is divergent in only two amino acids within this epitope. The results of this study suggest potential utility of MAb 1C7 in Western blot applications for the detection of Chaetomium and other Sordariomycetes species.

Internal transcribed spacer rRNA gene sequencing analysis of fungal diversity in Kansas City indoor environments.

Compared to traditional methods of fungal exposure assessment, molecular methods have provided new insight into the richness of fungal communities present in both indoor and outdoor environments. In this study, we describe the diversity of fungi in the homes of asthmatic children located in Kansas City. Fungal diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA derived from fungi collected in air and dust samples from 31 homes participating in the Kansas City Safe and Healthy Homes Program (KCSHHP). Sequencing results were then compared to data obtained using viable and non-viable fungal exposure assessment methods. ITS clone libraries were predominantly derived from the phylum Ascomycota in both air (68%) and dust (92%) samples and followed by the Basidiomycota and Zygomycota. The majority of Ascomycota clones belonged to four orders including the Pleosporales, Eurotiales, Capnodiales, and Dothideales. ITS sequencing revealed the presence of a number of rarely documented fungal species placed in the Pleosporales. Several species placed in the Basidiomycota were detected in ITS clone libraries but not by viable or non-viable methods. The prevalence of organizational taxonomic units (OTUs) was significantly higher in air than in dust samples (p < 0.0001); however, no differences between OTUs in air samples collected in the subjects' room and basement were observed. These sequencing results demonstrate a much broader diversity of Ascomycota and Basidiomycota communities in KCSHHP indoor environments than previously estimated using traditional methods of assessment.

Glycosylphosphatidylinositol-anchored proteins in Fusarium graminearum: inventory, variability, and virulence.

The contribution of cell surface proteins to plant pathogenicity of fungi is not well understood. As such, the objective of this study was to investigate the functions and importance of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the wheat pathogen F. graminearum. GPI-APs are surface proteins that are attached to either the membrane or cell wall. In order to simultaneously disrupt several GPI-APs, a phosphoethanolamine transferase-encoding gene gpi7 was deleted and the resultant mutant characterized in terms of growth, development, and virulence. The Δgpi7 mutants exhibited slower radial growth rates and aberrantly shaped macroconidia. Furthermore, virulence tests and microscopic analyses indicated that Gpi7 is required for ramification of the fungus throughout the rachis of wheat heads. In parallel, bioinformatics tools were utilized to predict and inventory GPI-APs within the proteome of F. graminearum. Two of the genes identified in this screen (FGSG_01588 and FGSG_08844) displayed isolate-specific length variability as observed for other fungal cell wall adhesion genes. Nevertheless, deletion of these genes failed to reveal obvious defects in growth, development, or virulence. This research demonstrates the global importance of GPI-APs to in planta proliferation in F. graminearum, and also highlights the potential of individual GPI-APs as diagnostic markers.

Morphogenetic and developmental functions of the Aspergillus nidulans homologues of the yeast bud site selection proteins Bud4 and Axl2.

The yeast bud site selection system represents a paradigm for understanding how fungal cells regulate the formation of a polarity axis. In Saccharomyces cerevisiae, Bud4 and Axl2 are components of the axial bud site marker. To address the possibility that these proteins regulate cellular morphogenesis in filamentous fungi, we have characterized homologues of Bud4 and Axl2 in Aspergillus nidulans. Our results show that Bud4 is involved in septum formation in both hyphae and developing conidiophores. Whereas Axl2 appears to have no obvious role in hyphal growth, it is required for the regulation of phialide morphogenesis during conidiation. In particular, Axl2 localizes to the phialide-spore junction, where it appears to promote the recruitment of septins. Furthermore, the developmental regulators BrlA and AbaA control the expression of Axl2. Additional studies indicate that Axl2 is also involved in the regulation of sexual development, not only in A. nidulans, but also in the phylogenetically unrelated fungus Fusarium graminearum. Our results suggest that Axl2 plays a key role in phialide morphogenesis and/or function during conidiation in the aspergilli.

Immunologic, spectrophotometric and nucleic acid based methods for the detection and quantification of airborne pollen.

Microscopic identification of pollen morphological phenotypes has been the traditional method used to identify and quantify pollen collected by air monitoring stations worldwide. Although this method has enabled a semi-standardized approach for the assessment of pollen exposure, limitations including labor intensiveness, required expertise, examiner bias, and the inability to differentiate species, genera, and in some cases families have limited data derived from the these stations. Recent advances in chemical, biochemical and molecular detection methods have provided standardized alternatives to this microscopic approach. In this review, we examine the applicability of alternative methodologies, in particular nucleic acid based assays involving the quantitative polymerase chain reaction, for the standardized detection of airborne pollen.

Comparison of DNA extraction methodologies used for assessing fungal diversity via ITS sequencing.

Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected.

Control of glucosylceramide production and morphogenesis by the Bar1 ceramide synthase in Fusarium graminearum.

The contribution of plasma membrane proteins to the virulence of plant pathogenic fungi is poorly understood. Accordingly, the objective of this study was to characterize the acyl-CoA dependent ceramide synthase Bar1 (previously implicated in plasma membrane organization) in the wheat pathogen Fusarium graminearum. The role of Bar1 in mediating cell membrane organization was confirmed as ΔBAR1 mutants failed to display a distinct sterol-rich domain at the hyphal tip. The ΔBAR1 mutants were non-pathogenic when inoculated onto wheat heads, and their in vitro growth also was severely perturbed. ΔBAR1 mutants were incapable of producing perithecia (sexual fruiting structures) and only produced macroconidia (asexual spores) in the presence of NaCl. Sphingolipid analyses indicated that Bar1 is specifically necessary for the production of glucosylceramides in both F. graminearum and Aspergillus nidulans. Interestingly, glucosylceramides appear to mediate sensitivity to heat stable antifungal factor (HSAF), as, in addition to ΔBAR1 mutants, a glucosylceramide synthase deficient mutant of Yarrowia lipolytica is also resistant to HSAF.

An in vitro method for the analysis of infection-related morphogenesis in Fusarium graminearum.

Fusarium graminearum is a significant pathogen of many cereal crops. With its genetic tractability, ease of culture, genome sequence availability and economic significance, F. graminearum has become the subject of intensive molecular research. Although molecular tools have been developed to enhance research into virulence determinants of F. graminearum, simple assays for infection-related development are lacking. As such, the objective of this study was to develop an in vitro protocol for the analysis of infection-related morphogenesis in F. graminearum. We demonstrate that two morphologically distinct hyphal structures are produced by F. graminearum during the invasion of detached wheat glumes: subcuticular hyphae and bulbous infection hyphae. Specialized wheat epidermal cells (papillae) appear to act as sites of invasion by F. graminearum on the adaxial side of detached wheat glumes. In addition, the development of bulbous infection hyphae is dependent on the pathogenicity mitogen-activated protein kinase Gpmk1, further supporting the infection-related nature of these structures. This relatively simple assay will contribute to the tractability of the F. graminearum system and help to uncover molecular requirements for infection-related development.

Characterization of Fusarium graminearum Mes1 reveals roles in cell-surface organization and virulence.

The surfaces of fungal hyphae are mosaics of carbohydrates and cell-surface proteins. Presently, very little is known about the role of these proteins and their organization at the cell surface. Here, we characterize two Fusarium graminearum genes implicated in cell-surface organization, mes1 (FGSG_06680) and pls1 (FGSG_08695). Mes1 is a homologue of mesA, which is required for the formation of stable polarity axes in Aspergillus nidulans. Pls1 encodes a tetraspanin, which belongs to a class of proteins that have been shown to aggregate in membrane rafts along with integrins and other signaling proteins. Our results indicate that Pls1 is dispensable for saprophytic growth and wheat head infection by F. graminearum (a pathogen that does not form appressoria). However, deletion of mes1 reduces sexual and asexual reproduction, severely perturbs the shape of macroconidia and hyphae, alters the pattern of cell wall deposition and the organization of sterol-rich rafts, and attenuates virulence on wheat heads. Our results provide a basis for identifying determinants of fungal virulence that may localize to specialized domains at the cell surface.

Association of ergot alkaloids with conidiation in Aspergillus fumigatus.

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festucla-vine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (brlA), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation.