A mutation in the eln2 gene encoding a cytochrome P450 of Coprinus cinereus affects mushroom morphogenesis

A dominant mutation of the elongationless2 (eln2) gene of the mushroom Coprinus cinereus (eln2-1) affects pattern formation in the development of fruit body primordia, causing dumpy primordia which culminate in mature fruit bodies with short stipes. Phenotypic analyses revealed disturbance of tissue organization and cell morphogenesis in the primordial shaft and suggested that the defects in the primordial shaft interfere with differentiation of the stipe tissue in the mutant primordia. The eln2 locus was assigned to chromosome XII and the eln2-1 mutant gene was cloned from a chromosome XII-specific cosmid library as a genomic fragment that brings about the dominant mutant phenotype. The eln2 gene encodes a novel type of microsomal cytochrome P450 enzyme (CYP502) and is constitutively expressed. The eln2-1 mutation is a 4-bp deletion in the 3'-terminal region of the gene, leading to truncation of C-terminal 18 amino acids. We suggest that the truncation affects the activity of the CYP502 enzyme.     

Gravitropism of the basidiomycete Flammulina velutipes: morphological and physiological aspects of the graviresponse

The fruiting body of the basidiomycete Flammulina velutipes shows a distinct negative gravitropic response. Maturing fruiting bodies in the rapid elongation phase become graviresponsive with basidiospore differentiation. Lateral gravistimulation by horizontal arrangement of the fruiting body results in unilateral growth regulation. Elongation in the upper Stipe side decreases to 40% during gravitropic reorientation of the fruiting body. Overshooting of the gravitropic response during reorientation is precisely regulated. The graviresponsiveness is concentrated to the apical area of the stipe, the transition zone between pileus and stipe, which features a prominent elongation capability. The small size and low vacuolization of the transition zone hyphae compared with differentiated basal stipe hyphae correspond with this physiological function on the light and electron microscopical levels. Curvature experiments using intact and explanted fruiting bodies demonstrated the graviperceptive role of the transition zone. The excision of various amounts of pilear tissue, even the disruption of the whole pileus, had no severe effect on gravitropic curvature, until the transition zone was damaged. Removal of the transition zone resulted in a dramatic loss of graviresponse, whereas the decrease of elongation was less drastic.     

Elongation growth and gravitropic curvature in the Flammulina velutipes (Agaricales) fruiting body

Differential elongation of stipe hyphae drives the gravitropic reorientation of Flammulina velutipes (Agaricales) fruiting bodies. The gravitropic curvature is strictly dependent on the presence of the transition zone between pileus and stipe. Elongation growth, providing the driving force for curvature, is also promoted by the pileus. Gravitropic curvature is successfully suppressed by clinostatic rotation, but the elongation rate is not affected. Explantation of fruiting body stipes lowers curvature and elongation rates corresponding to explant size reduction. In Flammulina, 25 mm length of transition zone explants is an efficient size for reproducible curvature and elongation during 48- to 72-h curvature tests. Submersion of specimens in aqueous medium causes cessation of the gravitropic curvature, but does not affect elongation. Thus the involvement of a diffusible factor in transmission of the curvature signal is probable. Splitting the fruiting body stipe in segments of 1/8 diameter does not suppress the gravitropic response, and the segments are individually reoriented to the vertical. It is concluded that the graviresponse of the Flammulina fruiting body is based on cellular perception of the gravistimulus and that a differential growth signal is transmitted in the stipe by a soluble factor that regulates hyphal elongation.     

金针菇外切-β-1,3-葡聚糖酶家族基因鉴定及在不同发育时期的差异表达

金针菇具有很高的营养与保健价值,菌柄长短决定金针菇的产量与品质,而菌柄伸长的相关作用酶及分子机理尚不清楚。前期草菇中发现外切-β-1,3-葡聚糖酶基因（exg2）可能与菌柄伸长相关,但在金针菇中尚没有exg基因的相关报道。本研究首先在金针菇全基因组中鉴定到3个外切-β-1,3-葡聚糖酶家族基因（分别命名为：Ffexg1、Ffexg2、Ffexg3）,并进行了克隆验证。进一步采用定量PCR对3个基因在金针菇不同发育时期及组织部位的差异性表达进行了分析。结果显示：Ffexg1只在菌柄中高表达,Ffexg2与Ffexg3在菌柄中表达量先上升后下降,在菌盖中呈逐渐上升趋势。3个Ffexg基因均在菌柄发生伸长的部位表达量较高,且在菇体水平放置后菌柄弯曲程度较大的部位表达量较高。结果显示金针菇exg家族3个基因存在时空差异性表达,在菌柄中伸长较快的时期及部位伴随着Ffexg基因的高表达。结合其功能预测,Ffexg家族基因可能作用于细胞壁成分β-1,3-葡聚糖链,从而在金针菇菌柄及菌盖发育中起作用。     

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。     

MADS-box转录因子编码基因Vvrin1在草菇不同发育时期的表达分析

草菇是我国土特产出口的主要种类之一,而采后易开伞问题限制了草菇的贮藏及运输。MADS-box转录因子对植物的成熟衰老和真菌的子实体发育起到重要的调控作用。目前尚未见草菇MADS-box转录因子的相关报道。本研究通过生物信息方法及分子生物学的手段对草菇的基因组、转录组数据进行分析,获得了草菇MADS-box转录因子基因Vvrin1。该基因全长1 392bp,含2个内含子,编码419个氨基酸残基。该转录因子含有一个MADS-box结构域,序列与双孢蘑菇Agaricus bisporus、斑玉蕈Hypsizygus marmoreus和灰盖鬼伞Coprinopsis cinerea的MADS-box转录因子相似性分别为71%、67%和61%。通过表达谱数据及荧光定量PCR分析表明Vvrin1基因在草菇子实体伸长期菌柄的表达量出现高峰,具有极显著性差异（P<0.01）,推测该转录因子参与调控草菇菌柄的伸长,菌盖的开伞。这些结果为草菇成熟衰老（特别是开伞）的调控研究提供数据支持。     

Identification and expression analysis of a new glycoside hydrolase family 55 exo-β-1,3-glucanase-encoding gene in Volvariella volvacea suggests a role in fruiting body development

The edible straw mushroom Volvariella volvacea is an important crop in South East Asia and is predominantly harvested in the egg stage. Rapid stipe elongation and cap expansion result in a swift transition from the egg to elongation and maturation stage, which are subjected to fast senescence and deterioration. In other mushrooms, β-1,3-glucanases have been associated with degradation (softening) of the cell wall during stipe elongation and senescence. We present a new glycoside hydrolase family 55 (GH55) exo-β-1,3-glucanase gene, exg2, and highly conserved deduced EXG2 protein. The 3D model and presumed catalytic residues of V. volvacea EXG2 are identical to Lentinula edodes EXG2 and Phanerochaete chrysosporium Lam55A, supporting similar enzymatic functions. In addition to previous association to stipe elongation and senescence, our data clearly indicates a role for cap (pileus) expansion. Digital gene expression, quantitative PCR and isobaric tags for relative and absolute quantification analysis showed low exg2 and EXG2 levels in primordia, button, egg and elongation stages and significantly increased levels in the maturation stage. Subsequent relative quantitative PCR analysis designated expression of exg2 to the stipe in the elongation stage and to the pileus and stipe in the maturation stage. EXG2 cell wall softening activity, close correlation of exg2 expression with the principal expanding mushroom tissues and a strong conservation of expression patterns and protein sequences in other mushrooms, make V. volvacea exg2 an important candidate for future studies on mechanisms of fruiting body expansion and senescence causing commodity value loss.     

A mutation in a methionine tRNA gene suppresses the prp2-1 Ts mutation and causes a pre-mRNA splicing defect in Saccharomyces cerevisiae.

The PRP2 gene in Saccharomyces cerevisiae encodes an RNA-dependent ATPase that activates spliceosomes for the first transesterification reaction in pre-mRNA splicing. We have identified a mutation in the elongation methionine tRNA gene EMT1 as a dominant, allele-specific suppressor of the temperature-sensitive prp2-1 mutation. The EMT1-201 mutant suppressed prp2-1 by relieving the splicing block at high temperature. Furthermore, EMT1-201 single mutant cells displayed pre-mRNA splicing and cold-sensitive growth defects at 18 degrees. The mutation in EMT1-201 is located in the anticodon, changing CAT to CAG, which presumably allowed EMT1-201 suppressor tRNA to recognize CUG leucine codons instead of AUG methionine codons. Interestingly, the prp2-1 allele contains a point mutation that changes glycine to aspartate, indicating that EMT1-201 does not act by classical missense suppression. Extra copies of the tRNA(Leu)(UAG) gene rescued the cold sensitivity and in vitro splicing defect of EMT1-201. This study provides the first example in which a mutation in a tRNA gene confers a pre-mRNA processing (prp) phenotype.     

The molecular mechanism of stipe cell wall extension for mushroom stipe elongation growth

Stipe elongation growth is one of the remarkable characteristics of the growth and development of basidiomycete fruiting bodies. Stipe elongation is resulting from the lateral extension of stipe cells. The stipe cell is enclosed within a thin cell wall which must be loosened to expand the wall surface area for accommodation of the enlarged protoplast as the stipe cell elongates. In fungal cell walls, chitin molecules associate with each other by interchain hydrogen bonds to form chitin microfibrils which are cross-linked covalently to matrix polysaccharides. Early, some scientists proposed that stipe elongation was the result of enzymatic degradation of wall polysaccharides, whereas other researchers suggested that stipe elongation resulted from nonhydrolytic disruption of the hydrogen bonds by turgor pressure between wall polysaccharides. Recently, an extensometer was used to determine stipe wall extension for elucidation of the molecular mechanism of stipe elongation. In Coprinopsis cinerea, the native stipe cell wall is induced to extend by acidic buffers and the acid-induced native wall extension activity is located in the growing apical stipe region. A series of current experiments indicate that chitinases play a key role in the stipe wall extension, and β-glucanases mainly function in the wall remodeling for regulation of stipe wall expansibility to cooperate with chitinase to induce stipe wall extension. In addition, fungal expansin-like proteins can bind to chitin to enhance chitin hydrolysis, and their expression pattern is consistent with the stipe elongation growth, which is suggested to play an auxiliary role in the stipe wall extension.     

Growth of fruit-bodies inFavolus arcularius

The fruiting ofFavolus arcularius in culture is described. When the cultures, which have been pre-incubated in darkness to allow the inoculum mycelia to become thick and white wooly in texture, are exposed to light, fruit-body primordia, 1 mm in height, are formed about 4 days after the start of illumination. The primordium develops into a cylindrical stipe, the growth of which mainly occurs in the final 1 mm of the terminal region. Hyphal elongation in the region within 3 mm of the apex is predominant in the growth of the pileate stipe. With maturation of the stipe, changes in hyphal orientation occur on the periphery of the subapical region, and then the pileus-primordium is formed. The differentiation into the inner layer and the outer layer (pre-hymenial layer) in the pileus tissue is completed at this stage. The early growth of the pileus may be due to rapid elongation of the hyphae on the margin in addition to gradual expansion of the hyphae in the preformed pseudo-tissue. When the pileus has grown to about 3 mm in diameter, the subsequent three to four fold increase in size may be due to parallel expansion of the hyphae constituting the young pileus tissue.     

Stipe elongation during basidiocarp maturation in Coprinus macrorhizus: Mechanical properties of stipe cell wall

Stipe elongation during basidiocarp maturation in the wild-type,#5026+5132, and the elongationless mutant, NG0398, of Coprinusmacrorhizus was studied, and the following results were obtained.

1. In the wild-type the middle zone of the stipe elongated 8.4times in 15 hr during maturation, while in the mutant it elongatedoaiy 2.2 times.
2. Component cells of the stipe elongated inparallel with thestipe elongation in both the wild-type andthe mutant. The widthof stipe cells was almost constant duringelongation in thewild-type, while it increased 2 times in themutant. Cell volumeincreased ca. 8 times in both stocks.
3. Theosmotic value of stipe cells was almost constant (0.45–0.50M) throughout elongation of both the wild-type and the elongationlessstipes.
4. Mechanical properties of the cell wall were examinedby measuringshrinkage, extensibility and minimum stress-relaxationtime(To) of the stipe during maturation. These parameters weredirectlyproportional to the elongation rate to follow.
5. Whenthe wild-type stipes were incubated in various concentrationsof mannitol solution and then in plain buffer solution, theextensibility of the stipe after the incubation in mannitolsolutions changed proportionally with the stipe length afterthe mannitol treatment, and To with the elongation capacityin plain buffer solution.

(Received March 3, 1977; )     

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.     

Stigmatella aurantiaca fruiting body formation is dependent on the fbfA gene encoding a polypeptide homologous to chitin synthases.

Stigmatella aurantiaca is a prokaryotic organism that undergoes a multicellular cycle of development resulting in the formation of a fruiting body. For analyzing this process, mutants defective in fruiting body formation have been induced by transposon mutagenesis using a Tn5-derived transposon. About 800 bp upstream of the transposon insertion of mutant AP182 which inactivates a gene (fbfB) involved in fruiting, a further gene (fbfA) needed for fruiting body formation was detected. Inactivation of fbfA leads to mutants which form only non-structured clumps instead of the wild-type fruiting body. The mutant phenotype of fbfA mutants can be partially suppressed by mixing the mutant cells with cells of some independent mutants defective in fruiting body formation. The fbfA gene is transcribed after 8 h of development as determined by measuring the induction of beta-galactosidase activity of a fbfA-delta(trp)-lacZ fusion gene and by Northern (RNA) analysis using an insertion encoding a stable mRNA. The predicted polypeptide FbfA shows a homology of about 30% to NodC of rhizobia, an N-acetylglucosamine-transferase which is involved in the synthesis of the sugar backbone of lipo-oligosaccharides. These induce the formation of the root nodules in the Papilionaceae. Besides the predicted molecular mass of 45.5 kDa, the hydropathy profile reveals a structural relationship to the NodC polypeptide.     

Temperature-sensitive inhibition of development in Dictyostelium due to a point mutation in the piaA gene

The Dictyostelium mutant HSB1 is temperature-sensitive for development, undergoing aggregation and fruiting body formation at temperatures below 18 degrees C but not above. In vivo G protein-linked adenylyl cyclase activation is defective in HSB1, and the enzyme is not stimulated in vitro by GTPgammaS; stimulation is restored upon addition of wild-type cytosol. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant. We excluded the possibility that HSB1 cells fail to express PIA and show that the HSB1 piaA gene harbors a point mutation, resulting in the amino acid exchange G(917)D. Both wild-type and HSB1 cells were also transfected with the HSB1 piaA gene. The piaA(HSB1) gene product displayed a partial inhibitory effect on wild-type cell development. We hypothesize that PIA couples the heterotrimeric G protein to adenylyl cyclase via two binding sites, one of which is altered in a temperature-sensitive way by the HSB1 mutation. When overexpressed in the wild-type background, PIA(HSB1) competes with wild-type PIA via the nonmutated binding site, resulting in dominant-negative inhibition of development. Expression of GFP-fused PIA shows that PIA is homogeneously distributed in the cytoplasm of chemotactically moving cells.     

Sporulation timing in Myxococcus xanthus is controlled by the espAB locus

The fruiting body development of Myxococcus xanthus consists of two separate but interacting pathways: one for aggregation of many cells to form raised mounds and the other for sporulation of individual cells into myxospores. Sporulation of individual cells normally occurs after mound formation, and is delayed at least 30 h after starvation under our laboratory conditions. This suggests that M. xanthus has a mechanism that monitors progress towards aggregation prior to triggering sporulation. A null mutation in a newly identified gene, espA (early sporulation), causes sporulation to occur much earlier compared with the wild type (16 h earlier). In contrast, a null mutation in an adjacent gene, espB, delays sporulation by about 16 h compared with the wild type. Interestingly, it appears that the espA mutant does not require raised mounds for sporulation. Many mutant cells sporulate outside the fruiting bodies. In addition, the mutant can sporulate, without aggregation into raised mounds, under some conditions in which cells normally do not form fruiting bodies. Based on these observations, it is hypothesized that EspA functions as an inhibitor of sporulation during early fruiting body development while cells are aggregating into raised mounds. The aggregation-independent sporulation of the espA mutant still requires starvation and high cell density. The espA and espB genes are expressed as an operon and their translations appear to be coupled. Expression occurs only under developmental conditions and does not occur during vegetative growth or during glycerol-induced sporulation. Sequence analysis of EspA indicates that it is a histidine protein kinase with a fork head-associated (FHA) domain at the N-terminus and a receiver domain at the C-terminus. This suggests that EspA is part of a two-component signal transduction system that regulates the timing of sporulation initiation.     

Growth regulation inCoprinus radiatus

Some of the morphological and physiological parameters of stipe growth or elongation inCoprinus radiatus were investigated. During the development of the fruit body the number of cells in a row in the growing portion of the stipe doubled during the development of the button, and again during the phase of rapid stipe elongation. Also during the stage of rapid elongation the cells in the upper 2/3 of the stipe increased 6–8 fold in length. The existence of a growth regulator synthesized in the cap and exerting control over the stipe was demonstrated through decapitation experiments. The cap appears to be required for normal stipe development until the stipe reaches about 1/4 of its final length. Through decapitation and cap-stipe exchanges it was found that the cap produced growth regulator up to the time of autodigestion; however, the stipe responded to the regulator only during a brief period at the onset of elongation.     

Effects of deletion of the gene for the development-specific protein S on differentiation in Myxococcus xanthus

A deletion mutation of the gene for protein S (tps), a development-specific protein of Myxococcus xanthus, was constructed. No significant differences in the process of fruiting body formation or the yield of myxospores were observed between mutant and wild-type cells. On the other hand, when the tps gene was deleted together with a 2.0-kilobase sequence including the ops gene immediately upstream of the tps gene, fruiting body formation was substantially delayed, and the yield of myxospores was reduced. These results indicate that protein S is not essential for differentiation of M. xanthus, whereas a gene product(s) coded from the sequence upstream of the tps gene appears to be required for normal fruiting body formation.