Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus

The biocontrol agent Trichoderma harzianum colonises sclerotia of the plant pathogenic fungus Sclerotinia sclerotiorum. Plating of sclerotia typically has been used to determine the incidence of mycoparasitism, but does not quantify the extent to which individual sclerotia are colonised. We developed a specific PCR primer/probe set for the green fluorescent protein (GFP)-transformant T. harzianum ThzID1-M3, which exhibited high precision and reproducibility. Quantitative real-time PCR was evaluated along with epifluorescence microscopy and image analysis to investigate dynamics of colonisation of sclerotia in non-sterile soil. Amounts of ThzID1-M3 DNA and S. sclerotiorum DNA from entire individual sclerotia were quantified using real-time PCR. Epifluorescence micrographs were captured from sclerotial thin-section samples, and GFP fluorescence from these was quantified using computer image analysis in order to estimate colonisation on a per-sclerotium basis. As determined by either method, ThzID1-M3 colonised sclerotia in soil, and both methods quantified colonisation dynamics over time. In a separate experiment, colonisation of sclerotia on agar plates was observed using confocal laser scanning microscopy to view the GFP-fluorescing hyphae of ThzID1-M3. This method, while highly labour-intensive, provided high spatial resolution of colonisation dynamics. Thus, each method has advantages: microscopy combined with image analysis can provide useful information on the spatial and temporal dynamics of colonisation, while real-time PCR can provide a more precise assessment of the extent of sclerotial colonisation over time and can more easily be used to sample entire sclerotia.     

Sclerotial Formation of Polyporus umbellatus by Low Temperature Treatment under Artificial Conditions

Background

Polyporus umbellatus sclerotia have been used as a diuretic agent in China for over two thousand years. A shortage of the natural P. umbellatus has prompted researchers to induce sclerotial formation in the laboratory.

Methodology/Principal Finding

P. umbellatus cultivation in a sawdust-based substrate was investigated to evaluate the effect of low temperature conditions on sclerotial formation. A phenol-sulfuric acid method was employed to determine the polysaccharide content of wild P. umbellatus sclerotia and mycelia and sclerotia grown in low-temperature treatments. In addition, reactive oxygen species (ROS) content, expressed as the fluorescence intensity of mycelia during sclerotial differentiation was determined. Analysis of ROS generation and sclerotial formation in mycelia after treatment with the antioxidants such as diphenyleneiodonium chloride (DPI), apocynin (Apo), or vitamin C were studied. Furthermore, macroscopic and microscopic characteristics of sclerotial differentiation were observed. Sclerotia were not induced by continuous cultivation at 25°C. The polysaccharide content of the artificial sclerotia is 78% of that of wild sclerotia. In the low-temperature treatment group, the fluorescent intensity of ROS was higher than that of the room temperature (25°C) group which did not induce sclerotial formation all through the cultivation. The antioxidants DPI and Apo reduced ROS levels and did not induce sclerotial formation. Although the concentration-dependent effects of vitamin C (5–15 mg mL⁻¹) also reduced ROS generation and inhibited sclerotial formation, using a low concentration of vitamin C (1 mg mL⁻¹) successfully induced sclerotial differentiation and increased ROS production.

Conclusions/Significance

Exposure to low temperatures induced P. umbellatus sclerotial morphogenesis during cultivation. Low temperature treatment enhanced ROS in mycelia, which may be important in triggering sclerotial differentiation in P. umbellatus. Moreover, the application of antioxidants impaired ROS generation and inhibited sclerotial formation. Our findings may help to provide new insights into the biological mechanisms underlying sclerotial morphogenesis in P. umbellatus.     

Electrophoretic and Immunological Comparisons of Developmentally Regulated Proteins in Members of the Sclerotiniaceae and Other Sclerotial Fungi

The fungal stroma is a distinct developmental stage, a compact mass of hyphal cells enveloped by a melanized layer of rind cells which is produced from vegetative mycelium. Two types of stromata that are characteristic of members of the Sclerotiniaceae but are also produced in a wide range of other fungi, i.e., the determinate tuberlike sclerotium and the indeterminate platelike substratal stroma, were compared in these studies. Developmental proteins found in determinate sclerotial and indeterminate substratal stromata, but not in mycelia, were characterized and compared in 52 isolates of fungi, both ascomycetes (including 18 species in the Sclerotiniaceae and 5 species of Aspergillus) and the basidiomycete Sclerotium rolfsii. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of mycelial, stromatal initial, and stromatal extracts demonstrated that all members of the Sclerotiniaceae produced proteins unique to stromatal extracts within a molecular weight range of 31,000 to 39,500 which composed 13 to 58% of the total protein in stromata. Proteins unique to the sclerotial stage were also produced in Sclerotium rolfsii and the Aspergillus species but within a generally lower-molecular-weight range of 11,000 to 30,000. The proteins were then characterized by two-dimensional electrophoresis to determine the number and isoelectric point of polypeptides composing each protein. Polyclonal antibodies were raised to the major 36-kDa sclerotial protein of Sclerotinia sclerotiorum (Ssp). Immunoblots demonstrated that all sclerotial proteins from species in the Sclerotiniaceae cross-reacted with anti-Ssp antibodies, while no cross-reaction was observed with proteins from substratal stromatal species in the Sclerotiniaceae, sclerotial species of Aspergillus, or Sclerotium rolfsii. Results of discriminant analysis of the data from competitive inhibition enzyme-linked immunosorbent assays were consistent with the results of immunoblotting. Three groupings, sclerotial species in the Sclerotiniaceae, substratal stromatal species in the family, and sclerotial species outside the family, were delimited on the basis of relative decreasing ability to compete for anti-Ssp antibody. These data demonstrate that stromatal proteins differ among different taxonomic groups of fungi and suggest that the Sclerotiniaceae may include two distinct lineages of genera.     

Involvement of Ca2+ Channel Signalling in Sclerotial Formation of Polyporus umbellatus

Growth and morphogenesis transformation in Polyporus umbellatus were examined in the presence of various pharmacological compounds, to investigate signal transduction pathways that influence the development of sclerotia. Both the calcium channel blocker nifedipine and the calcium ionophor A23187 reduced sclerotial production in P. umbellatus; four classes of Ca²⁺ signal agent—including calcium chelators, calcium channel blockers, calcium ionophors and calmodulin inhibitors—were further studied. Among them, EGTA and BAPTA, as calcium chelators, exhibited a complete inhibitory effect on sclerotial formation, among the levels tested. Calcium channel blockers and calcium ionophors at the concentrations used in this study could not eliminate sclerotia formation completely, but did greatly reduce sclerotial production. Notoginsenoside in dosages >250 μg/ml produced a significant negative effect on mycelial growth, and it prevented sclerotial formation entirely at a dosage of 500 μg/ml; no other drug influenced vegetative growth at all. The calcium ionophor A23187 did not decrease sclerotial mean weight at low doses (20 nM); at higher doses (200 nM), however, sclerotial development was significantly reduced, albeit not completely halted. The CaM inhibitors (W-7 and chlorpromazine) could each completely stop sclerotial formation. Using Fluo-3/AM as the indicator of cytosolic free calcium, the Ca²⁺ content in the cytoplasm was found to have decreased significantly when hyphae were treated with different drugs, and there was no active Ca²⁺ signal in the sclerotial mycelium. In general, the results suggest that Ca²⁺ signal transduction may play an important role in sclerotial formation in P. umbellatus.     

Effect of copper-induced oxidative stress on sclerotial differentiation and antioxidant properties of Penicillium thomii PT95 strain

Penicillium thomii PT95 strain was able to form abundant orange, sand-shaped sclerotia in which carotenoids were accumulated. The aim of this work was to determine the effects of copper-induced oxidative stress on the sclerotial differentiation and antioxidant properties of PT95 strain. The results showed that the time of exudates initiation, sclerotial initiation and sclerotial maturation of PT95 strain were advanced in 1–2 days under the copper-induced oxidative stress growth conditions. The analytical results of sclerotial biomass, carotenoids content in sclerotia showed that copper-induced oxidative stress favored the sclerotial differentiation and biosynthesis of carotenoids. Under the copper-induced oxidative stress growth conditions, the total phenolics content and DPPH free radical scavenging activity of sclerotia of this fungus were decreased as compared with the control. However, the oxidative stress induced by a lower amount of CuSO₄ in media could enhance significantly the reducing power of sclerotia.     

A new method to induce sclerotial differentiation in Polyporus umbellatus by split-plate culture

Polyporus umbellatus is one of the most valuable medicinal fungi, and its sclerotium has been used as a diuretic agent and an antidote in traditional Chinese medicine. In nature, Polyporus umbellatus has almost been depleted because of over-exploitation and lack of natural habitats. Thus, artificial sclerotia production has increased. This study aimed at finding an effective method to induce sclerotia, and selected the split-plate culture method. One side contained fructose agar medium (FAM), while the other side contained nutrient-limited medium. It was observed that sclerotia were only formed on the nutrient-limited medium side but scarcely emerged on the FAM side, even when the fructose concentration on both sides were the same. The sclerotial differentiation rate was 100% and the sclerotial yield was 106% higher than in the conventional way. In conclusion, the split-plate culture method is an effective way to induce P. umbellatus sclerotia in the laboratory.     

药用真菌猪苓溶血素基因克隆和序列分析

利用3'-RACE-PCR方法首次从药用真菌猪苓中克隆得到与真菌形态发育相关的溶血素基因。结果表明,猪苓溶血素基因的全长cDNA为744bp,其中编码区占447bp,共编码148个氨基酸,推测其分子量约为15.79kDa,理论等电点为4.89。推定的猪苓溶血素蛋白具有与杨树菇溶血素类蛋白家族相同的结构域和功能位点,两者同源性为60%。系统进化树结果显示猪苓溶血素隶属于担子菌类群。实时荧光定量PCR分析结果表明在菌核形成初期猪苓溶血素基因表达量较高,且显著高于菌丝体中猪苓溶血素基因的转录水平,说明溶血素基因参与了猪苓菌核的形态发育。     

Isolation of a non-sclerotial mutant ofSclerotium rolfsii and its cellulolytic activity

A mutant strain EMS-1 ofSclerotium rolfsii lacking the ability to develop mature sclerotia was isolated following chemical mutagenesis of macerated sclerotia with ethyl methane-sulfonate. The mutant failed to form sclerotia even in the presence of lactose, threonine or iodo-acetic acid which promoted sclerotial development in the wild strain and the UV-8 mutant. EMS-1 exhibited higher (1.5 – 3.0 times) cellulase and hemicellulase activity compared to the wild strain. Possible correlation between sclerotial morphogenesis and cellulase and/or oxalic acid production is discussed.     

Polyamine metabolism during sclerotial development of Sclerotinia sclerotiorum

A study on polyamine metabolism and the consequences of polyamine biosynthesis inhibition on the development of Sclerotinia sclerotiorum sclerotia was conducted. Concentrations of the triamine spermidine and the tetramine spermine, as well as ornithine decarboxylase and S-adenosyl-methionine decarboxylase activities, decreased during sclerotia maturation. In turn, the concentration of the diamine putrescine was reduced at early stages of sclerotial development but it increased later on. This increment was not related to de novo biosynthesis, as demonstrated by the continuous decrease in ornithine decarboxylase activity. Alternatively, it could be explained by the release of putrescine from the conjugated polyamine pool. α-Difluoro-methylornithine and cyclohexylamine, which inhibit putrescine and spermidine biosynthesis, respectively, decreased mycelial growth, but did not reduce the number of sclerotia produced in vitro even though they disrupted polyamine metabolism during sclerotial development. It can be concluded that sclerotial development is less dependent on polyamine biosynthesis than mycelial growth, and that the increase of free putrescine is a typical feature of sclerotial development. The relationship between polyamine metabolism and sclerotial development, as well as the potential of polyamine biosynthesis inhibition as a strategy for the control of plant diseases caused by sclerotial fungi are discussed.     

Major sclerotial polypeptides of psychrophilic pathogenic fungi: Intracellular localization and antigenic relatedness

Summary Major sclerotial polypeptides from the psychrophiles,Myriosclerotinia borealis (W 51),Coprinus psychromorbidus (LRS 131),Typhula idahoensis (W 21), andTyphula incarnata (W 21) were purified by using polyacrylamide gel electrophoresis and electroelution. Polyclonal antibodies were raised against these major sclerotial polypeptides. Immunofluorescence microscopy showed that the major sclerotial polypeptides from all four psychrophilic species were sequestered in discrete protein bodies of cultured and field-grown sclerotia. Western blot analysis indicated that all antisera reacted positively with their respective antigens, the major sclerotial polypeptides. Reciprocal immunological cross-reactions were observed between the major sclerotial polypeptides ofM. borealis (W 51) andT. idahoensis (W 21). Antiserum to the major sclerotial polypeptides of bothM. borealis andT. idahoensis also recognized the major sclerotial polypeptides ofC. psychromorbidus (LRS 131). It is suggested that the major sclerotial polypeptides of these psychrophilic plant pathogens may act as storage proteins.Abbreviations W 51 Myriosclerotinia borealis (W 51) - LRS 131 Coprinus psychromorbidus (LRS 131) - W 21 Typhula idahoensis (W 21) - W 29 Typhula incarnata (W 29) - anti W 51 antiserum to the major sclerotial polypeptide ofM. borealis W 51 - anti LRS 131 antiserum to the major sclerotial polypeptides ofC. psychromorbidus (LRS 131) - anti W 21 antiserum to the major sclerotial polypeptides ofT. idahoensis (W 21) - anti W 29 antiserum to the major sclerotial polypeptides ofT. incarnata (W 29) - SDS sodium dodecylsulfate - kDa kilodalton - PAGE polyacrylamide gel electrophoresis - HRP horseradish peroxidase - PBS phosphate buffered saline - TBS Tris buffered saline - FITC fluorescein isothiocyanate     

Scanning Electron Microscopy of Sclerotia of Sclerotinia trifoliorum and Related Species

The microanatomy of immature 'white', 'slightly pigmented' and mature, 1-month-old 'black' sclerotia of Sclerotinia trifoliorum , S. sclerotiorum , and S. minor were studied by scanning electron microscopy (SEM). A surface mycelial network was present over sclerotia at maturity. Also dried exudate on the superficial, sclerotial cells at maturity was observed. At this stage of morphogenesis an outer layer of the wall of medullary hyphae was synthesized. Two zones (i.e., rind and medulla) of hyphal tissue in sections of mature sclerotia were distinguished. The wall of rind cells was thick and one-layered, whereas the wall of medullary hyphae was thick and bi-layered.
No lacunac (intercellular spaces) in sclerotial rind were found but the sclerotial medulla appeared to be lacunate in all three species. At the SEM level the structural organization of sclerotia of S. trifoliorum was identical to that one of sclerotia of S. sclerotiorum and S. minor. Thus, in the conducted investigation of the sclerotial stromata, a unique, structural characteristic of taxonomic importance to distinguish S. trifoliorum from the other Sclerotinia species was not found. Observations on the sclerotial morphogenesis in S. trifoliorum and the related species agree with and supplement the light and transmission electron microscope studies of other researchers.     

ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA

Rhizoctonia solani AG-1 IA is the causal agent of rice sheath blight (RSB) and causes severe economic losses in rice-growing regions around the world. The sclerotia play an important role in the disease cycle of RSB. In this study, we report the effects of reactive oxygen species (ROS) and trehalose on the sclerotial development of R. solani AG-1 IA. Correlation was found between the level of ROS in R. solani AG-1 IA and sclerotial development. Moreover, we have shown the change of ROS-related enzymatic activities and oxidative burst occurs at the sclerotial initial stage. Six genes related to the ROS scavenging system were quantified in different sclerotial development stages by using quantitative RT-PCR technique, thereby confirming differential gene expression. Fluorescence microscopy analysis of ROS content in mycelia revealed that ROS were predominantly produced at the hyphal branches during the sclerotial initial stage. Furthermore, exogenous trehalose had a significant inhibitory effect on the activities of ROS-related enzymes and oxidative burst and led to a reduction in sclerotial dry weight. Taken together, the findings suggest that ROS has a promoting effect on the development of sclerotia, whereas trehalose serves as an inhibiting factor to sclerotial development in R. solani AG-1 IA.     

The development-specific ssp1 and ssp2 genes of Sclerotinia sclerotiorum encode lectins with distinct yet compensatory regulation

The Ssp1 development-specific protein is the most abundant soluble protein in sclerotia and apothecia of Sclerotinia sclerotiorum. Although closely associated with these developmental stages, the functions of the Ssp1 protein and its paralog, Ssp2, are not known. In this study, protein structure prediction analysis revealed that Ssp1 and Ssp2 are structurally similar to fucose-specific lectins. In an effort to understand the function of these abundant, development-specific proteins, a homokaryotic ssp1 deletion mutant was generated. The resulting mutant (Δssp1) displays a wild-type growth and development phenotype in culture but produces approximately 50% fewer sclerotia in cultures supplemented with hygromycin. Genetic complementation with a wild-type copy of ssp1 restores normal sclerotium formation in the presence of hygromycin. This suggests that Ssp1 might play a role in resistance to glycoside-containing antibiotics encountered in the environment. Although a slight delay in carpogenic germination was observed, no additional effects of ssp1 loss-of-function were found in regards to apothecial morphology or fecundity. When the expression of ssp2 was examined in the Δssp1 mutant, it was found to be expressed earlier in sclerotial development and its encoded protein accumulated to higher levels in both sclerotia and apothecia. These findings suggest regulatory compensation for loss of Ssp1 coupled with potential functional redundancy among lectins accumulating in sclerotia and apothecia.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Increase in Endogenous and Exogenous Cyclic AMP Levels Inhibits Sclerotial Development in Sclerotinia sclerotiorum

Growth and development of a wild-type Sclerotinia sclerotiorum isolate were examined in the presence of various pharmacological compounds to investigate signal transduction pathways that influence the development of sclerotia. Compounds known to increase endogenous cyclic AMP (cAMP) levels in other organisms by inhibiting phosphodiesterase activity (caffeine and 3-isobutyl-1-methyl xanthine) or by activating adenylate cyclase (NaF) reduced or eliminated sclerotial development in S. sclerotiorum. Growth in the presence of 5 mM caffeine correlated with increased levels of endogenous cAMP in mycelia. In addition, incorporation of cAMP into the growth medium decreased or eliminated the production of sclerotia in a concentration-dependent manner and increased the accumulation of oxalic acid. Inhibition of sclerotial development was cAMP specific, as exogenous cyclic GMP, AMP, and ATP did not influence sclerotial development. Transfer of developing cultures to cAMP-containing medium at successive time points demonstrated that cAMP inhibits development prior to or during sclerotial initiation. Together, these results indicate that cAMP plays a role in the early transition between mycelial growth and sclerotial development.     

外源β-胡萝卜素、光照对青霉PT95菌株菌核分化和类胡萝卜素产率的影响

初步研究了外源β-胡萝卜素和光照对青霉PT5菌株菌核分化和类胡萝卜素产率的影响。结果表明,在培养基中加入外源β-胡萝卜素后,PT5菌株渗出液出现的时间、菌核出现的时间延迟了,但菌核成熟的时间没变。培养基中的外源β-胡萝卜素浓度越大,其渗出液、菌核出现的时间越迟。外源β-胡萝卜素亦能降低PT5菌株的脂质过氧化水平和菌核中的类胡萝卜素含量。高氧胁迫的光照培养条件有利于PT95菌株的菌核分化和色素在菌核中的积累;与低氧胁迫的黑暗培养条件相比,其菌核生物量和类胡萝卜素产率分别增加了18．7％和101％。以上实验结果表明,若想获得高的菌核生物量和类胡萝卜素产率,应该尽可能在高氧胁迫、无抗氧化剂存在的条件下培养PT5菌株。