Identification of the First Fungal Annexin: Analysis of Annexin Gene Duplications and Implications for Eukaryotic Evolution

Annexin homologues have been found in animals, plants, and distinct protist lineages. We report the identification of the first fungal annexin, encoded by the anx14 gene of the filamentous ascomycete Neurospora crassa. Annexins have a complex evolutionary history and exhibit a large number of gene duplications and gene losses in various taxa, including the complete loss of annexin sequences from another ascomycete, the budding yeast Saccharomyces cerevisiae. Surprisingly, the N. crassa annexin homologue is most closely related to the annexin homologue of the slime mold Dictyostelium discoideum, suggesting a phylogenetic link between cellular slime molds and true fungi. Both of these annexin homologues are closely related to the family of annexin homologues present in animals, an observation consistent with the existence of the animal–fungal clade. These data further suggest that the gene duplications that generated the family of annexin sequences present in animals, fungi, and slime molds began prior to the divergence of these taxa. Received: 10 December 1997 / Accepted: 17 April 1998     

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α‐aminoadipate, a precursor for penicillin and lysine via the α‐aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.     

The major cellulases CBH‐1 and CBH‐2 of Neurospora crassa rely on distinct ER cargo adaptors for efficient ER‐exit

Filamentous fungi are native secretors of lignocellulolytic enzymes and are used as protein‐producing factories in the industrial biotechnology sector. Despite the importance of these organisms in industry, relatively little is known about the filamentous fungal secretory pathway or how it might be manipulated for improved protein production. Here, we use Neurospora crassa as a model filamentous fungus to interrogate the requirements for trafficking of cellulase enzymes from the endoplasmic reticulum to the Golgi. We characterized the localization and interaction properties of the p24 and ERV‐29 cargo adaptors, as well as their role in cellulase enzyme trafficking. We find that the two most abundantly secreted cellulases, CBH‐1 and CBH‐2, depend on distinct ER cargo adaptors for efficient exit from the ER. CBH‐1 depends on the p24 proteins, whereas CBH‐2 depends on the N. crassa homolog of yeast Erv29p. This study provides a first step in characterizing distinct trafficking pathways of lignocellulolytic enzymes in filamentous fungi.     

The rust transferred proteins—a new family of effector proteins exhibiting protease inhibitor function

Only few fungal effectors have been described to be delivered into the host cell during obligate biotrophic interactions. RTP1p, from the rust fungi Uromyces fabae and U. striatus, was the first fungal protein for which localization within the host cytoplasm could be demonstrated directly. We investigated the occurrence of RTP1 homologues in rust fungi and examined the structural and biochemical characteristics of the corresponding gene products. The analysis of 28 homologues showed that members of the RTP family are most likely to occur ubiquitously in rust fungi and to be specific to the order Pucciniales. Sequence analyses indicated that the structure of the RTPp effectors is bipartite, consisting of a variable N‐terminus and a conserved and structured C‐terminus. The characterization of Uf‐RTP1p mutants showed that four conserved cysteine residues sustain structural stability. Furthermore, the C‐terminal domain exhibits similarities to that of cysteine protease inhibitors, and it was shown that Uf‐RTP1p and Us‐RTP1p are able to inhibit proteolytic activity in Pichia pastoris culture supernatants. We conclude that the RTP1p homologues constitute a rust fungi‐specific family of modular effector proteins comprising an unstructured N‐terminal domain and a structured C‐terminal domain, which exhibit protease inhibitory activity possibly associated with effector function during biotrophic interactions.     

Fungal community associated with marine macroalgae from Antarctica

Filamentous fungi and yeasts associated with the marine algae Adenocystis utricularis, Desmarestia anceps, and Palmaria decipiens from Antarctica were studied. A total of 75 fungal isolates, represented by 27 filamentous fungi and 48 yeasts, were isolated from the three algal species and identified by morphological, physiological, and sequence analyses of the internal transcribed spacer region and D1/D2 variable domains of the large-subunit rRNA gene. The filamentous fungi and yeasts obtained were identified as belonging to the genera Geomyces, Antarctomyces, Oidiodendron, Penicillium, Phaeosphaeria, Aureobasidium, Cryptococcus, Leucosporidium, Metschnikowia, and Rhodotorula. The prevalent species were the filamentous fungus Geomyces pannorum and the yeast Metschnikowia australis. Two fungal species isolated in our study, Antarctomyces psychrotrophicus and M. australis, are endemic to Antarctica. This work is the first study of fungi associated with Antarctic marine macroalgae, and contributes to the taxonomy and ecology of the marine fungi living in polar environments. These fungal species may have an important role in the ecosystem and in organic matter recycling.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

The different morphologies of yeast and filamentous fungi trigger distinct killing and feeding mechanisms in a fungivorous amoeba

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.     

Presence of weed fungus in a non‐social beetle–fungus cultivation mutualism

1. Farming by non‐human organisms has arisen independently in several animal lineages, allowing them to survive on food sources that are otherwise difficult to access. However, agricultural gardens are prone to invasion by parasites that overgrow cultivars in the absence of host animals. The presence of garden parasites and associated host adaptations are well studied in advanced fungal agriculture practised by social insects (ants, termites, and ambrosia beetles), but the impact of garden parasites in more primitive forms of agriculture is poorly known. The aim of the present study was to report the presence of weed fungi in fungal gardens of the non‐social lizard beetle Doubledaya bucculenta Lewis (Coleoptera: Erotylidae: Languriinae), which cultivates the yeast Wickerhamomyces anomalus (E.C. Hansen) Kurtzman, Robnett & Bas.‐Powers in the internodes of recently dead bamboo culms. 2. The filamentous fungi Arthrinium spp., Fusarium spp., and an unidentified species were isolated from the inner wall and insect‐made wall debris of bamboo internodes inhabited by the beetle. 3. When cultured together with the yeast in the absence of host larvae, Arthrinium sp.1 outcompeted the yeast, suggesting that this filamentous fungus can invade yeast gardens but is normally suppressed under natural conditions. 4. Rearing experiments showed that beetle larvae grew faster when grown on yeast cultures than when fed only Arthrinium sp.1. 5. These results suggest that Arthrinium sp.1 acts as a weed in the fungal gardens of D. bucculenta, inhibiting both growth of the beetle larvae and proliferation of the beneficial yeast.     

Biodiversity in sulfur metabolism in hemiascomycetous yeasts

The evolution of the metabolism of sulfur compounds among yeast species was investigated. Differences between species were observed in the cysteine biosynthesis pathway. Most yeast species possess two pathways leading to cysteine production, the transsulfuration pathway and the O-acetyl-serine (OAS) pathway, with the exception of Saccharomyces cerevisiae and Candida glabrata, which only display the transsulfuration pathway, and Schizosaccharomyces pombe, which only have the OAS pathway. An examination of the components of the regulatory network in the different species shows that it is conserved in all the species analyzed, as its central component Met4p was shown to keep its functional domains and its partners were present. The analysis of the presence of genes involved in the catabolic pathway shows that it is evolutionarily conserved in the sulfur metabolism and leads us to propose a role for two gene families which appeared to be highly conserved. This survey has provided ways to understand the diversity of sulfur metabolism products among yeast species through the reconstruction of these pathways. This diversity could account for the difference in metabolic potentialities of the species with a biotechnological interest.