Self-eating and self-killing: crosstalk between autophagy and apoptosis

The functional relationship between apoptosis ('self-killing') and autophagy ('self-eating') is complex in the sense that, under certain circumstances, autophagy constitutes a stress adaptation that avoids cell death (and suppresses apoptosis), whereas in other cellular settings, it constitutes an alternative cell-death pathway. Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis; in other instances, the cell switches between the two responses in a mutually exclusive manner. On a molecular level, this means that the apoptotic and autophagic response machineries share common pathways that either link or polarize the cellular responses.     

Regulation of secondary metabolite production in filamentous ascomycetes

Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.     

Molecular genetics of heterokaryon incompatibility in filamentous ascomycetes.

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

构巢曲霉与30种丝状子囊菌的基因组比较

为探讨丝状子囊菌的序列同源性,文章利用公开发表的真菌基因组序列构建本地基因组数据库,设置E值统计阈值为0.1,将构巢曲霉(Aspergillus nidulans)基因组的10560个注释基因分别与30种丝状子囊菌基因组比较。结果表明,同源匹配基因数量的多少可反映子囊菌之间的进化关系。构巢曲霉基因组的924个基因与这30种子囊菌基因组同时存在匹配序列,其中E值在10-5～0.1、10-30～10-5、10-100～10-30、0～10-100范围内都存在匹配序列的基因分别为6个、3个、6个和6个。ClustalX多序列比对分析显示,E值10-5～0.1的6组序列和E值10-30～10-5的3组序列均显示变异性过大而E值0～10-100的6组序列过于保守,E值介于10-100～10-30之间的6组同源序列可用于本研究的31种子囊菌系统学分析。     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

Curbing autophagy and histone deacetylases to kill cancer cells

Cells respond to cytotoxicity by activating a variety of signal transduction pathways. One pathway frequently upregulated during cytotoxic response is macroautophagy (hereafter referred to as autophagy). Previously, we demonstrated that pan-histone deacetylase (HDAC) inhibitors, such as the anticancer agent suberoylanilide hydroxamic acid (SAHA, Vorinostat), can induce autophagy. In this study, we show that HDAC inhibition triggers autophagy by suppressing MTOR and activating the autophagic kinase ULK1. Furthermore, autophagy inhibition can sensitize cells to both apoptotic and nonapoptotic cell death induced by SAHA, suggesting the therapeutic potential of autophagy targeting in combination with SAHA therapy. This study also raised a series of questions: What is the role of HDACs in regulating autophagy? Do individual HDACs have distinct functions in autophagy? How do HDACs regulate the nutrient-sensing kinase MTOR? Since SAHA-induced nonapoptotic cell death is not driven by autophagy, what then is the mechanism underlying the apoptosis-independent death? Tackling these questions should lead to a better understanding of autophagy and HDAC biology and contribute to the development of novel therapeutic strategies.     

A complete inventory of fungal kinesins in representative filamentous ascomycetes

Complete inventories of kinesins from three pathogenic filamentous ascomycetes, Botryotinia fuckeliana, Cochliobolus heterostrophus, and Gibberella moniliformis, are described. These protein sequences were compared with those of the filamentous saprophyte, Neurospora crassa and the two yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Data mining and phylogenetic analysis of the motor domain yielded a constant set of 10 kinesins in the filamentous fungal species, compared with a smaller set in S. cerevisiae and S. pombe. The filamentous fungal kinesins fell into nine subfamilies when compared with well-characterized kinesins from other eukaryotes. A few putative kinesins (one in B. fuckeliana and two in C. heterostrophus) could not be defined as functional, due to unorthodox organization and lack of experimental data. The broad representation of filamentous fungal kinesins across most of the known subfamilies and the ease of gene manipulation make fungi ideal models for functional and evolutionary investigation of these proteins.     

基于比较基因组学方法的丝状子囊菌同源序列分析

为探讨丝状子囊菌基因组的同源保守序列作为标记基因,利用Standalone BLASTN方法将构巢曲霉全基因组基因分别与30种丝状子囊菌基因组比较.构巢曲霉与每个丝状子囊菌基因组之间的同源匹配基因数量似乎可反映子囊菌之间的进化关系,构巢曲霉（10,560个基因）与15种散囊菌纲其他真菌间的匹配基因数量为5,179-7,747个,其中与另外7个同属的种匹配的基因数量为7,434-7,747个,而与亲缘关系较远的2种锤舌菌纲真菌灰葡萄孢和核盘菌的匹配基因数量分别仅有4,318个和4,242个.构巢曲霉的10,560个基因与20余种子囊菌基因组同时匹配的基因数为3,509个,占33.2％,构巢曲霉基因与30种子囊菌共同匹配的基因仅924个.此外,E值大小在10-30_0.1范围的同源序列变异性大,而在0-10-100范围的同源序列高度保守.随着基因组序列数据的增加,比较基因组方法将会在真菌系统学研究领域发挥更大的作用.     

Biofungicide utilizations of antifungal proteins of filamentous ascomycetes: current and foreseeable future developments

Today’s dearth of effective antimicrobial agents can be overcome by the use of antimicrobial proteins, which are produced naturally by a wide range of organisms including microorganisms, plants and mammals. These small basic proteins are highly stable, easy to manufacture on a large scale, and any resistance against them develops only rarely. These proteins are therefore good candidates for the treatment and prevention of various fungal infections. Importantly, these protein-based antimycotics can even be expressed heterologously in suitable organisms and can be used for various agricultural purposes in the future including biocontrol applications. In this review, we summarize today’s knowledge on the sources, structures, large-scale productions, direct surface applications as well as on the heterologous expressions in host plants of the small molecular mass antifungal proteins produced by filamentous fungi. Future developments foreseeable in this promising area of antifungal protein research are also presented and discussed in this review.     

Origin and distribution of epipolythiodioxopiperazine (ETP) gene clusters in filamentous ascomycetes

Background  

Genes responsible for biosynthesis of fungal secondary metabolites are usually tightly clustered in the genome and co-regulated with metabolite production. Epipolythiodioxopiperazines (ETPs) are a class of secondary metabolite toxins produced by disparate ascomycete fungi and implicated in several animal and plant diseases. Gene clusters responsible for their production have previously been defined in only two fungi. Fungal genome sequence data have been surveyed for the presence of putative ETP clusters and cluster data have been generated from several fungal taxa where genome sequences are not available. Phylogenetic analysis of cluster genes has been used to investigate the assembly and heredity of these gene clusters.     

Cell growth: how to grow and where to grow

Root hairs provide a model system for studying tip growth in plants. The recent cloning of genes required for tip growth has shed new light on the link between ionic regulation, cell wall assembly and the cytoskeleton in cell growth.     

Development of ToxA and ToxB promoter-driven fluorescent protein expression vectors for use in filamentous ascomycetes

The green fluorescent protein (GFP) has been established as the premier in vivo reporter for investigations of gene expression, protein localization, and cell and organism dynamics. The fungal transformation vector pCT74, with sGFP under the control of the ToxA promoter from Pyrenophora tritici-repentis, effectively expresses GFP in a diverse group of filamentous ascomycetes. Due to the versatility of ToxA promoter-driven expression of GFP, we constructed an additional set of fluorescent protein expression vectors to expand the color palette of fluorescent markers for use in filamentous fungi. EYFP, ECFP and mRFP1 were successfully expressed from the ToxA promoter in its fungus of origin, P. tritici-repentis, and a distant relative, Verticillium dahliae. Additionally the ToxB promoter from P. tritici-repentis drove expression of sGFP in V. dahliae, suggesting a similar potential to the ToxA promoter for heterologous expression in ascomycetes. The suite of fungal transformation vectors presented here promise to be useful for a variety of fungal research applications.     

An interrelationship between autophagy and filamentous growth in budding yeast

Over the last 15 years, yeast pseudohyphal growth (PHG) has been the focus of intense research interest as a model of fungal pathogenicity. Specifically, PHG is a stress response wherein yeast cells deprived of nitrogen form filaments of elongated cells. Nitrogen limitation also induces autophagy, a ubiquitous eukaryotic stress response in which proteins are trafficked to the vacuole/lysosome for degradation and recycling. Although autophagy and filamentous growth are both responsive to nitrogen stress, a link between these processes has not been investigated to date. Here, we present several studies describing an interrelationship between autophagy and filamentous growth. By microarray-based expression profiling, we detect extensive upregulation of the pathway governing autophagy during early PHG and find both processes active under conditions of nitrogen stress in a filamentous strain of budding yeast. Inhibition of autophagy results in increased PHG, and autophagy-deficient yeast induce PHG at higher concentrations of available nitrogen. Our results suggest a model in which autophagy mitigates nutrient stress, delaying the onset of PHG; conversely, inhibition of autophagy exacerbates nitrogen stress, resulting in precocious and overactive PHG. This physiological connection highlights the central role of autophagy in regulating the cell's nutritional state and the responsiveness of PHG to that state.     

Inhibition of cellular autophagy in kidney tubular cells stimulated to grow by unilateral nephrectomy

Cytoplasmic growth (hypertrophy) presupposes a positive metabolic balance brought about by increased anabolic and/or decreased catabolic processes. Degradation of cytoplasmic components takes place in autophagic vacuoles (AVs) whose volume fraction may be taken as a measure of the relative rate of degradation of cytoplasmic components. Male adult Sprague-Dawley rats (n = 80) were unilaterally nephrectomized (n = 40) or sham-operated (n = 40) and were killed 3.5-57.5 h p.o. The volume density of AVs in parenchymal cells of renal cortical convoluted tubules was determined morphometrically by systematic evaluation of large test fields in the electron microscope. During compensatory renal growth, the volume densities of autophagic vacuoles were reduced at day 0 (3.5-8 h p.o.), day 1 (20.5-33.5 h p.o.) and day 2 (44.5-57.5 h p.o.) by 49% (p less than 0.01), 43% (p less than 0.05), and 19% (n.s.), respectively, when compared with sham-operated controls. No decrease, and even an increase, in the AV-volume fraction was found in liver parenchymal cells of the unilaterally nephrectomized animals. This indicates that inhibition of autophagy is not a general response after unilateral nephrectomy, but is confined to the growing kidney, where it may represent a significant factor in the increase of cytoplasmic mass.     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

Use of pH auxostats to grow filamentous fungi in continuous flow culture at maximum specific growth rate

Abstract The pH auxostat employs a pH-derived feedback control of biomass concentration and allows continuous flow cultures of organisms to be grown at their maximum specific growth rate. We examine three modifications of the pH auxostat system for the growth of filamentous fungi and discuss the suitability of each method according to the biomass concentration desired, the medium used and the equipment available. Fusarium graminearum and Geotrichum candidum were used for this work and it was possible to maintain steady state pH auxostats of these filamentous fungi for up to 20 days. pH auxostats have not previously been described for filamentous fungi.