Nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in Neurospora crassa

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions.     

Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae

ABSTRACT

In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae.     

Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system

Vegetative incompatibility is a widespread phenomenon in filamentous ascomycetes, which limits formation of viable heterokaryons. Whether this phenomenon plays a role in maintaining the homokaryotic state of the hyphae during the vegetative growth of Tuber spp. Gene expression, polymorphism analysis as well as targeted in vitro experiments allowed us to test whether a heterokaryon incompatibility (HI) system operates in Tuber melanosporum. HI is controlled by different genetic systems, often involving HET domain genes and their partners whose interaction can trigger a cell death reaction. Putative homologues to HI-related genes previously characterized in Neurospora crassa and Podospora anserina were identified in the T. melanosporum genome. However, only two HET domain genes were found. In many other ascomycetes HET domains have been found within different genes including some members of the NWD (NACHT and WD-repeat associated domains) gene family of P. anserina. More than 50 NWD homologues were found in T. melanosporum but none of these contain a HET domain. All these T. melanosporum paralogs showed a conserved gene organization similar to the microexon genes only recently characterized in Schistosoma mansoni. Expression data of the annotated HI-like genes along with low allelic polymorphism suggest that they have cellular functions unrelated to HI. Moreover, morphological analyses did not provide evidence for HI reactions between pairs of genetically different T. melanosporum strains. Thus, the maintenance of the genetic integrity during the vegetative growth of this species likely depends on mechanisms that act before hyphal fusion.     

A novel gene, phcA from Pseudomonas syringae induces programmed cell death in the filamentous fungus Neurospora crassa

The phytopathogen Pseudomonas syringae competes with other epiphytic organisms, such as filamentous fungi, for resources. Here we characterize a gene in P. syringae pv. syringae B728a and P. syringae pv. tomato DC3000, termed phcA , that has homology to a filamentous fungal gene called het-c . phcA is conserved in many P. syringae strains, but is absent in one of the major clades, which includes the P. syringae pathovar phaseolicola. In the filamentous fungus Neurospora crassa , HET-C regulates a conserved programmed cell death pathway called heterokaryon incompatibility (HI). Ectopic expression of phcA in N. crassa induced HI and cell death that was dependent on the presence of a functional het-c pin-c haplotype. Further, by co-immunoprecipitation experiments, a heterocomplex between N. crassa HET-C1 and PhcA was associated with phcA- induced HI . P. syringae was able to attach and extensively colonize N. crassa hyphae, while an Escherichia coli control showed no association with the fungus. We further show that the P. syringae is able to use N. crassa as a sole nutrient source. Our results suggest that P. syringae has the potential to utilize phcA to acquire nutrients from fungi in nutrient-limited environments like the phyllosphere by the novel mechanism of HI induction.     

Nonself recognition is mediated by HET-C heterocomplex formation during vegetative incompatibility

Nonself recognition during vegetative growth in filamentous fungi is mediated by heterokaryon incompatibility (het) loci. In Neurospora crassa, het-c is one of 11 het loci. Three allelic specificity groups, termed het-c(OR), het-c(PA) and het-c(GR), exist in natural populations. Heterokaryons or partial diploids that contain het-c alleles of alternative specificity show severe growth inhibition, repression of conidiation and hyphal compartmentation and death (HCD). Using epitope-tagged HET-C, we show that nonself recognition is mediated by the presence of a heterocomplex composed of polypeptides encoded by het-c alleles of alternative specificity. The HET-C heterocomplex localized to the plasma membrane (PM); PM-bound HET-C heterocomplexes occurred in all three het-c incompatible allelic interactions. Strains containing het-c constructs deleted for a predicted signal peptide sequence formed HET-C heterocomplexes in the cytoplasm and showed a growth arrest phenotype. Our finding is a step towards understanding nonself recognition mechanisms that operate during vegetative growth in filamentous fungi, and provides a model for investigating relationships between recognition mechanisms and cell death.     

The fungus-specific HET domain mediates programmed cell death in Podospora anserina

Vegetative incompatibility is a programmed cell death reaction that occurs when fungal cells of unlike genotypes fuse. Genes defining vegetative incompatibility (het genes) are highly polymorphic, and most if not all incompatibility systems include a protein partner bearing the fungus-specific domain termed the HET domain. The nonallelic het-C/het-E incompatibility system is the best-characterized incompatibility system in Podospora anserina. Cell death is triggered by interaction of specific alleles of het-C, encoding a glycolipid transfer protein, and het-E, encoding a HET domain and a WD repeat domain involved in recognition. We show here that overexpression of the isolated HET domain from het-E results in cell death. This cell death is characterized by induction of autophagy, increased vacuolization, septation, and production of lipid droplets, which are hallmarks of cell death by incompatibility. In addition, the HET domain lethality is suppressed by the same mutations as vegetative incompatibility, but not by the inactivation of het-C. These results establish the HET domain as the mediator of cell death by incompatibility and lead to a modular conception of incompatibility systems whereby recognition is ensured by the variable regions of incompatibility proteins and cell death is triggered by the HET domain.     

Approaches for refining heterologous protein production in filamentous fungi

Fungi combine the advantages of a microbial system such as a simple fermentability with the capability of secreting proteins that are modified according to a general eukaryotic scheme. Filamentous fungi such as Aspergillus niger efficiently secrete genuine proteins but the secretion of recombinant proteins turned out be a difficult task. Aspergillus niger is an attractive organism because of its high secretion capacity and is frequently used as a model organism. Whereas high production yields can be obtained when homologous proteins are expressed, much lower amounts are obtained with the production of heterologous proteins. To fully exploit the potential of filamentous fungi, understanding of the molecular genetics, their physiology, and the glycosylation metabolism has to be investigated and clarified in more detail. This review summarizes recent developments in heterologous protein production by filamentous fungi and also generalizes the possibilities of improving the protein production by various genetic and bioprocessing approaches, thereby easing recognition of filamentous fungi as a relevant and reliable expression platform.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

丝状真菌的细胞凋亡

凋亡是一种程序性细胞死亡类型,为多细胞生物发育和维持生命所必需的,也普遍存在于细菌等原核生物和酵母、丝状真菌等真核生物中。丝状真菌既具有酵母和哺乳动物共有的凋亡同源蛋白,也具有酵母所不具备的哺乳动物凋亡同源蛋白,所以其凋亡机制较酵母更为复杂,而又较哺乳动物简单。凋亡在丝状真菌的发育、繁殖、衰老等过程中具有重要的作用。近年,丝状真菌作为新的凋亡研究的模式生物被广泛研究,而且进展迅速。综述丝状真菌的凋亡现象和检测方法,丝状真菌中凋亡的生物学功能,丝状真菌凋亡的诱导条件,以及丝状真菌凋亡相关基因的功能研究进展。     

全基因组水平扫描鉴定粗糙脉孢菌糖转运蛋白及其在酿酒酵母己糖发酵中的评价

木质纤维素降解真菌粗糙脉孢菌天然具有吸收利用多种单糖和寡糖的能力,但是目前基因组中注释的预测糖转运蛋白仍然有过半功能未知。本研究从全基因组水平系统分析了粗糙脉孢菌预测糖转运蛋白的转运底物。研究发现两个转运蛋白(NCU01868和NCU08152)具有转运多种己糖底物的功能,因此分别命名为NcHXT-1和NcHXT-2。利用荧光共振能量转移技术(FRET)确认了NcHXT-1/-2具有葡萄糖转运功能。在己糖转运蛋白全缺酿酒酵母EBY.VW4000中分别过表达NcHXT-1/-2,能恢复其在葡萄糖、半乳糖或甘露糖的液体培养基中生长并生成乙醇的能力。NcHXT-1/-2在很多纤维素降解真菌中均具有保守的同源蛋白。本研究通过全基因组扫描鉴定,发现了两个保守的丝状真菌己糖转运蛋白,为真菌降解利用木质纤维素及酵母利用单糖发酵提供了新的改造靶点。     

Inmunopatología de las micosis invasivas por hongos filamentosos

Invasive fungal infections caused by filamentous fungi are devastating diseases that occur in patients with a variety of immunosuppressive conditions. This review focuses on the pathogenesis of the most important invasive mycosis in the human being caused by the filamentous fungi Aspergillus, Fusarium, Scedosporium and mucorales. The first contact between the mould and the patient, the host defense to different fungi, including the role of mucosa in the innate immune system, the whole innate immune recognition receptors, and the pathways connecting innate and adaptive immunity, as well as the virulence factors of fungi, are discussed in this paper.     

Sex in fungi: lessons of gene regulation

Fungi have been very useful for gene regulation studies. Mating implicates in a series of events influenced by many types of environmental input that are interpreted into regulatory pathways, including signal transduction. Although various aspects of mating and signal transduction in the yeast Saccharomyces cerevisiae have long been characterized, recent findings in filamentous fungi indicate that pheromones and pheromone receptors may be essential for mating partner recognition and also for nucleus recognition in sorting before meiosis. A brief overview on mating-type genes of ascomycete fungi and recent contributions to the understanding of their role in the regulation of multicellularity and sexual dimorphism is presented in this review.     

Programmed cell death correlates with virus transmission in a filamentous fungus

Programmed cell death (PCD) is an essential part of the defence response in plants and animals against pathogens. Here, we report that PCD is also involved in defence against pathogens of fungi. Vegetative incompatibility is a self/non-self recognition system in fungi that results in PCD when cells of incompatible strains fuse. We quantified the frequency of cell death associated with six vegetative incompatibility (vic) genes in the filamentous ascomycete fungus Cryphonectria parasitica. Cell death frequencies were compared with the effects of vic genes on transmission of viruses between the same strains. We found a significant negative correlation between cell death and virus transmission. We also show that asymmetry in cell death correlates with asymmetry in virus transmission; greater transmission occurs into vic genotypes that exhibit delayed or infrequent PCD after fusion with an incompatible strain. Furthermore, we found that virus infection can have a significant, strain-specific, positive or negative effect on PCD. Specific interactions between vic gene function and viruses, along with correlations between cell death and transmission, strongly implicate PCD as a host-mediated pathogen defence strategy in fungi.     

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.     

Fungal apoptosis: function, genes and gene function

Cells of all living organisms are programmed to self-destruct under certain conditions. The most well known form of programmed cell death is apoptosis, which is essential for proper development in higher eukaryotes. In fungi, apoptotic-like cell death occurs naturally during aging and reproduction, and can be induced by environmental stresses and exposure to toxic metabolites. The core apoptotic machinery in fungi is similar to that in mammals, but the apoptotic network is less complex and of more ancient origin. Only some of the mammalian apoptosis-regulating proteins have fungal homologs, and the number of protein families is drastically reduced. Expression in fungi of animal proteins that do not have fungal homologs often affects apoptosis, suggesting functional conservation of these components despite the absence of protein-sequence similarity. Functional analysis of Saccharomyces cerevisiae apoptotic genes, and more recently of those in some filamentous species, has revealed partial conservation, along with substantial differences in function and mode of action between fungal and human proteins. It has been suggested that apoptotic proteins might be suitable targets for novel antifungal treatments. However, implementation of this approach requires a better understanding of fungal apoptotic networks and identification of the key proteins regulating apoptotic-like cell death in fungi.