NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog

Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.     

金针菇信息素信号通路基因在子实体发育过程中的差异表达

【背景】子实体是食用菌的主要商品部位,也是真菌生殖生长的重要结构,其发育受到多种信号途径的调控。【目的】以金针菇(Flammulina filiformis)为材料,对转录组和基因组数据的信息素信号通路基因进行分析获得差异表达的基因,并对其在菌丝生长和子实体发育过程中的表达情况进行分析,以期为研究食用菌子实体发育提供参考。【方法】基于已有的金针菇基因组数据,注释了金针菇信息素信号通路。进一步通过转录组测序鉴定了该通路中参与金针菇子实体发育的关键基因,并对关键基因进行荧光定量PCR验证。【结果】cdc24和ste12基因在子实体发育不同时期的5个样品(原基、伸长期菌柄、伸长期菌盖、成熟期菌柄和成熟期菌盖)中的表达具有显著差异,使用荧光定量PCR技术进行验证与上述结果一致。【结论】cdc24和ste12这2个关键基因可能参与了金针菇子实体发育过程中的组织分化调控机制。     

Protein expression during Flammulina velutipes fruiting body formation

Formation of the Flammulina velutipes fruiting body can be induced by lowering the ambient temperature (first treatment) in complete darkness. Fruiting bodies formed under these conditions elongate without pileus formation (pinhead fruiting body), suggesting that they cannot mature in complete darkness. However, after light treatment of the pinhead fruiting body (second treatment), a pileus develops immediately, and the stipe also thickens and becomes increasingly pigmented. The apical region swells as a result of cell division starting 2 days after light treatment, the pileus–stipe junction fracture and hymenium primordia form on day 4, and gills appear at day 6. Pf1 and Pf3 are specifically expressed after exposure to low temperature without light. The cell wall-associated protein [pileus-specific hydrophobin-like protein (PSH)] is specifically induced in the pileus, but not in the stipe, following the second light treatment to the pinhead fruiting body. These results suggest that Pf1 and Pf3 would be involved in fruiting body induction and that PSH would be involved in pileus formation. These phenomena will aid further histological and molecular biological investigations into the mechanisms behind fruiting body development in F. velutipes.     

Cmku70基因敲除对蛹虫草生长发育的影响

ku70和ku80是非同源末端连接修复通路的关键基因,在一些丝状真菌中其基因敲除株可作为底盘菌株,提高同源重组效率和基因敲除效率.本研究从蛹虫草基因组中鉴定得到Cmku70 及 Cmku80基因,分别编码分子量为71.50kDa和80.96kDa的蛋白,均含有Ku core结构域,预测均定位于细胞核.系统进化分析表明K...     

糙皮侧耳生长发育过程中漆酶基因家族的表达研究

漆酶参与木质素的降解和食用菌的生长发育。为了明晰漆酶基因在糙皮侧耳生长发育过程中的作用,本文采用real-time PCR检测了11条漆酶基因及Lacc2的小亚基sspoxa3在糙皮侧耳生长发育不同阶段的表达。其中lacc6和sspoxa3在整个生长发育阶段表达量均较高;lacc12随着原基的分化和子实体的形成大量表达,与成菇过程有关。lacc4,lacc7和lacc11在原基分化期高表达,与原基的分化有关。lacc2,lacc3和lacc8在成熟子实体阶段表达量显著上升,与子实体的分化和成熟有关。     

Autophagy genes Smatg8 and Smatg4 are required for fruiting-body development,vegetative growth and ascospore germination in the filamentous ascomycete Sordaria macrospora

Autophagy is a tightly controlled degradation process involved in various developmental aspects of eukaryotes. However, its involvement in developmental processes of multicellular filamentous ascomycetes is largely unknown. Here, we analyzed the impact of the autophagic proteins SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. A Saccharomyces cerevisiae complementation assay demonstrated that the S. macrospora Smatg8 and Smatg4 genes can functionally replace the yeast homologs. By generating homokaryotic deletion mutants, we showed that the S. macrospora SmATG8 and SmATG4 orthologs were associated with autophagy-dependent processes. Smatg8 and Smatg4 deletions abolished fruiting-body formation and impaired vegetative growth and ascospore germination, but not hyphal fusion. We demonstrated that SmATG4 was capable of processing the SmATG8 precursor. SmATG8 was localized to autophagosomes, whereas SmATG4 was distributed throughout the cytoplasm of S. macrospora. Furthermore, we could show that Smatg8 and Smatg4 are not only required for nonselective macroautophagy, but for selective macropexophagy as well. Taken together, our results suggest that in S. macrospora, autophagy seems to be an essential and constitutively active process to sustain high energy levels for filamentous growth and multicellular development even under nonstarvation conditions.     

低温刺激对食用菌子实体形成的研究进展

探究低温刺激子实体形成过程的组学数据能为食用菌的工厂化低碳高效栽培及广温型品种的选育提供参考。本文首先通过主成分分析对食用菌低温刺激子实体形成的类型进行低温型(11−19 ℃)和冷压型的划分(≤10 ℃)。在此基础上,综述了食用菌两种类型的低温刺激子实体形成过程的研究进展。食用菌低温刺激子实体形成过程中都涉及信号转导、胁迫响应、基础代谢、细胞分化、细胞结构变化等代谢过程。随着低温型到冷压型的刺激温度下降,糖代谢可能转向脂代谢为子实体形成提供能量。     

Isolation and analysis of genes specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes by fluorescence differential display

Using fluorescence differential display, cDNAs specifically expressed at the primordial stage of fruiting body development were isolated from the basidiomycete, Flammulina velutipes. Seventy-five cDNAs were sequenced and compared with the amino-acid sequences of proteins in the database by BLASTX search. Significant similarity was found for 29 cDNAs coding for proteins with known function, GTP-binding protein, growth factor, ubiquitin-proteasome, cytochrome P450 and hydrophobin, all of which would be associated with fruiting body development. Seventeen cDNAs were not similar to proteins in the database and may represent unique genes that play specific roles in the process of fruiting in F. velutipes.     

兰茂牛肝菌子实体发育的关联物质及其主要通路分析

【背景】兰茂牛肝菌(Lanmaoaasiatica)等外生菌根真菌的子实体形成和发育机制仍然未知。【目的】揭示调控子实体发育的关联物质。【方法】同时运用核磁共振、气相质谱和液相质谱3种代谢组学技术,分析兰茂牛肝菌纯培养8d原基(Y8)与野生子实体(Z0)的小分子物质。【结果】Y8及Z0分别共指认出451、473种化合物;Y8vs.Z0,有362种显著或极显著上调(206种)及下调(156种)差异物质,其涉及47条调控通路。【结论】推测通过9条主要通路完成物质的深度转化及调控,极显著上调及下调差异物质如牛肝菌素可能对子实体的发育起着一定的调控作用,3种方法互相补充扩大了检测的泛度及灵敏度,这为探究兰茂牛肝菌子实体发育机理及人工培养提供了一定的理论参考。     

The development of gene expression systems for filamentous fungi

Filamentous fungi have been used for decades in the commercial production of enzymes, antibiotics, and specialty chemicals. Traditionally, improving the yields of these products has involved either mutagenesis and screening or modification of fermentation conditions. Generally, selective breeding of strains has not been successful, because most of the commercially important fungal species lack a sexual cycle. For a few species, strain improvements have been made possible by employing the parasexual cycle for genetic crosses (30). The recent development of DNA-mediated transformation systems for several industrially important fungal species has spawned a flurry of research activity directed toward the development of gene expression systems for these microorganisms. This technology is now a viable means for novel and more directed approaches to improving existing fungal strains which produce enzymes or antibiotics. In addition, fungal expression systems are now being tested for the production of heterologous gene products such as mammalian pharmaceutical proteins. The goal of this review is to present a summary of the gene expression systems which have recently been developed for some filamentous fungi of commercial importance. To insure that the most recent developments are presented we have included data from not only scientific papers, but also from personal communications, abstracts, symposia, and our own laboratory.     

巴氏蘑菇子实体不同发育阶段的转录组分析

为探讨巴氏蘑菇子实体不同发育阶段基因的表达情况,本研究对巴氏蘑菇子实体不同发育时期（原基、采收期和开伞期）进行转录组测序,以本实验室已获得的巴氏蘑菇JA菌株的不育单孢菌株JA-15036基因组为参考基因组研究原基与采收期及开伞期样本间差异表达基因,并对差异表达基因进行了GO功能和Pathway富集分析。GO功能分析结果显示,差异表达基因主要富集在跨膜转运、碳水化合物代谢途径和膜组分,它们协同调控为子实体生长发育提供稳定的内环境。KEGG富集分析结果表明,原基期上调的差异表达基因主要富集在核糖体蛋白和DNA复制,表明原基期细胞代谢旺盛,其中核糖体蛋白基因上调为后期蛋白质合成提供重要场所;采收期和开伞期子实体时期差异表达基因主要富集在碳水化合物代谢、脂肪酸降解和氨基酸代谢等途径,为巴氏蘑菇子实体的生长发育与成熟提供营养与能量。     

A <Emphasis Type="Italic">Sordaria macrospora</Emphasis> mutant lacking the <Emphasis Type="Italic">leu1</Emphasis> gene shows a developmental arrest during fruiting body formation

Developmental mutants with defects in fruiting body formation are excellent resources for the identification of genetic components that control cellular differentiation processes in filamentous fungi. The mutant pro4 of the ascomycete Sordaria macrospora is characterized by a developmental arrest during the sexual life cycle. This mutant generates only pre-fruiting bodies (protoperithecia), and is unable to form ascospores. Besides being sterile, pro4 is auxotrophic for leucine. Ascospore analysis revealed that the two phenotypes are genetically linked. After isolation of the wild-type leu1 gene from S. macrospora, complementation experiments demonstrated that the gene was able to restore both prototrophy and fertility in pro4. To investigate the control of leu1 expression, other genes involved in leucine biosynthesis specifically and in the general control of amino acid biosynthesis (“cross-pathway control”) have been analysed using Northern hybridization and quantitative RT-PCR. These analyses demonstrated that genes of leucine biosynthesis are transcribed at higher levels under conditions of amino acid starvation. In addition, the expression data for the cpc1 and cpc2 genes indicate that cross-pathway control is superimposed on leucine-specific regulation of fruiting body development in the leu1 mutant. This was further substantiated by growth experiments in which the wild-type strain was found to show a sterile phenotype when grown on a medium containing the amino acid analogue 5-methyl-tryptophan. Taken together, these data show that pro4 represents a novel mutant type in S. macrospora, in which amino acid starvation acts as a signal that interrupts the development of the fruiting body. Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00438-005-0021-8     

曲酸对斑玉蕈子实体形成过程中木质纤维素酶的影响研究

为了探究曲酸增加子实体产量的机制,首先考察了搔菌后外源添加曲酸对不同菌丝培养时间出菇的影响。研究发现当菌丝培养时间过短或者过长添加曲酸都得不到很好的增产效果,菌丝培养时间在60-80d之间增产效率最高,并且后熟期60d的增产效率大于80d的增产效率。进一步研究发现添加曲酸可以提高菌丝利用基质中木质纤维素的利用率。更深入地研究发现,基质中的漆酶和纤维素酶活性在斑玉蕈的不同发育时期受到曲酸调控。漆酶活性在最初的菌丝恢复期和转色期酶活性低于对照组,但是在原基期、钉头期和子实体期酶活性显著地高于对照组;纤维素酶活性在整个发育周期中曲酸组都高于对照组,在子实体发育后期酶活性被提高3.16倍。最后,从分子水平上分析了漆酶基因和纤维素酶基因的表达量,研究显示添加曲酸后漆酶基因和纤维素酶基因在不同程度上被上调,这个结果与酶活的结果相一致。这些结果说明外源添加曲酸通过提高生殖生长阶段的菌丝利用培养基质中的漆酶和纤维素酶活性,进而提高菌丝利用木质纤维素,为斑玉蕈子实体生长发育提供更多的能源,实现增加子实体产量的目的。     

Influences of environmental factors on fruiting body induction,development and maturation in mushroom-forming fungi

Mushroom-forming fungi (restricted to basidiomycetous fungi in this review) differentiate by sensing several environmental factors for fruiting body formation. For fruiting body induction, nutrient, temperature and light conditions are critical environmental factors. Higher nitrogen and carbon sources in the media will suppress fruiting body induction in many mushroom-forming fungi, with induction being triggered by lower nitrogen and carbon concentrations. Low temperature or temperature downshift is another critical influencing factor for fruiting body induction in many cultivated mushrooms, such as Flammulina velutipes, Lentinula edodes, and Volvariella volvacea. Fungal response toward starvation and cold involves the production of sexual spores as the next generation. Species like F. velutipes and Coprinopsis cinerea can form fruiting bodies in the dark; however, light accelerates fruiting body induction in some mushroom-forming fungi. Remarkably, fruiting bodies formed in the dark have tiny or no pileus on heads (called dark stipe, pinhead fruiting body, or etiolated stipe). Light is essential for pileus differentiation in many, but not all mushroom species; one exception is Agaricus bisporus. Mushrooms have positive phototropism and negative gravitropism for effective dispersal of spores. Carbon dioxide concentrations also affect fruiting body development; pileus differentiation is suppressed at a high concentration of carbon dioxide. Thus, the pileus differentiation system of mushrooms may allow the most effective diffusion of spores. Full expansion of the pileus is followed by pileus autolysis or senescence. In C. cinerea, pileus autolysis occurs during spore diffusion. Fruiting body senescence, browning of gill, and softening occur after harvesting in several mushroom species. Fruiting body induction, development, and maturation in mushroom-forming fungi are discussed in this review.     

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth

The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.     

两个赤芝子实体多糖的理化特性分析及结构鉴定

赤芝子实体多糖P32A分子量为506,322,P32B分子量为287,389,两种多糖均以己糖为主构成。P32A由鼠李糖、木糖、甘露糖和葡萄糖组成,四种单糖的摩尔比为：4．3：2．6：6．3：11．4;P32B亦由鼠李糖、木糖、甘露糖和葡萄糖组成,摩尔比为：1．2：1．0：2．1：9．2。该结果显示,P32A与P32B具有较类似的糖组成,且都以葡萄糖和甘露糖为主要单糖组分。根据^13C NMR和甲基化的结果分析知P32A可能含有l→4连接的葡萄糖构成的主链,非还原末端均由葡萄糖构成,此外P32A中还有l,4-连接的甘露糖和l,3—连接的鼠李糖。P32B可能以l→3连接形成主链,部分l,3-连接葡萄糖的6位有分支,该多糖也含有由葡萄糖构成的非还原末端,与P32A类似,P32B中还含有l,4-连接的甘露糖,不同的是不含l,3—连接的鼠李糖。     

大型真菌子实体发生的形态学过程及调控机制

大型真菌中很多种类具有较高的营养价值和药用价值,而食药用部位多为大型真菌的子实体,所以子实体的形成对大型真菌的开发应用就显得尤为重要。在大型真菌生活史中,子实体的发生揭示着真菌完成了从营养生长向生殖生长的转变。从理论上来说,菌丝体只需完成营养生长,即可进入生殖生长;而实际上,子实体的发生受到各种环境因素、遗传因素等的影响。因此,本文从子实体发生的形态学过程、环境影响因素、分子调控机制等方面综述了近年国内外关于大型真菌子实体发生的研究概况,为大型真菌的系统研究和重要食药用菌的栽培驯化提供理论借鉴和参考依据。