Characterisation of Aspergillus nidulans polarisome component BemA.

BemA, the orthologue of Saccharomyces cerevisiae Bem1p, was identified through genome sequence comparison. We have shown that it plays a similar role to Bem1p in yeast, acting as a cell growth protein. Deletion of the gene produced a moderately abnormal hyphal tip morphology, and had an extremely detrimental effect on conidiospore production, with development stalling after conidiophore vesicle production. It was also shown that BemA is required for vacuole fusion, similar to Bem1p. This role is dependent on the first SH3 domain of the protein, whose deletion has no detectable effect on cell growth. Localisation studies showed that BemA formed a clear cap at hyphal tips, analogous to the S. cerevisiae polarisome. The relationship between BemA and SepA, a spitzenk?rper protein, was investigated. It was found that localisation of the proteins were interdependent, and a conditional double mutant was inviable.     

Maximal polar growth potential depends on the polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii

We used actin staining and videomicroscopy to analyze the development from a spore to a young mycelium in the filamentous ascomycete Ashbya gossypii. The development starts with an initial isotropic growth phase followed by the emergence of germ tubes. The initial tip growth speed of 6-10 microm/h increases during early stages of development. This increase is transiently interrupted in response to the establishment of lateral branches or septa. The hyphal tip growth speed finally reaches a maximum of up to 200 micro/h, and the tips of these mature hyphae have the ability to split into two equally fast-growing hyphae. A search for A. gossypii homologs of polarisome components of the yeast Saccharomyces cerevisiae revealed a remarkable size difference between Spa2p of both organisms, with AgSpa2p being double as long as ScSpa2p due to an extended internal domain. AgSpa2 colocalizes with sites of polarized actin. Using time-lapse videomicroscopy, we show that AgSpa2p-GFP polarization is established at sites of branch initiation and then permanently maintained at hyphal tips. Polarization at sites of septation is transient. During apical branching the existing AgSpa2p-GFP polarization is symmetrically divided. To investigate the function of AgSpa2p, we generated two AgSPA2 mutants, a partial deletion of the internal domain alone, and a complete deletion. The mutations had an impact on the maximal hyphal tip growth speed, on the hyphal diameter, and on the branching pattern. We suggest that AgSpa2p is required for the determination of the area of growth at the hyphal tip and that the extended internal domain plays an important role in this process.     

Induction of contour sensing in Aspergillus niger by stress and its relevance to fungal growth mechanics and hyphal tip structure

Thigmotropism (contour sensing) has been assigned an important role in both plant and human fungal pathogens. However, outside these systems, our knowledge of the function of thigmotropism in fungal growth control is relatively poor. Furthermore, the effects of environmental stress on thigmotropic responses have received scant attention. To try to elucidate some of the mechanisms behind hyphal contour sensing in response to nutrient-poor environments, we have used micro-engineered substrates and several imaging techniques to investigate the thigmotropic reactions of the ubiquitous fungus Aspergillus niger. This organism not appear to demonstrate thigmotropic growth under normal conditions. Our results show that A. niger undergoes significant morphological changes during growth on solid substrates and demonstrate that the intensity of contour sensing varies depending on the area of the hyphal tip which initiates contact with the substrate. We propose that growth under nutrient-limited conditions triggers several factors that combine to increase thigmotropic sensitivity while conversely creating a 60 degrees arc at the hyphal tip which is blind to topographical variations. This has important consequences for our general understanding of the hyphal mode of growth in fungi as well as more specific aspects of hyphal tip development under stress.     

Distribution and conformation of crystalline nigeran in hyphal walls of Aspergillus niger and Aspergillus awamori.

Hyphal walls of Aspergillus awamori containing increased amount of the alpha-glucan, nigeran, became correspondingly more opaque when viewed in the electron microscope as shadowed preparations. However, increased polymer deposition was not accompanied by any significant change in wall thickness. The nigeran of both A. awamori and Aspergillus niger occurred in situ in a crystalline conformation identical to that of single crystals prepared with pure polysaccharide. Furthermore, this polymer was the dominant crystalline material in the hyphae whether or not they were enriched in nigeran. Enzymic digestion of nigeran in A. niger and A. awamori revealed that the bulk of the polymer was exposed to the cell's exterior. However, a certain fraction was accessible to enzymic attack only after the wall was treated with boiling water. A third portion, detectable only by x-ray diffraction, was associated with other components and could not be extracted, even with prolonged boiling. It was removed by hot, dilute alkali and was associated in the wall with another glucan fraction. Dry heating of A. niger walls altered their susceptibility to enzymic digestion of nigeran in situ. It is proposed that this treatment introduces interstices in the crystal surface that facilitate attack.     

Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger

Five endo-polygalacturonases (poly(1,4-alpha-D-galacturonide) glycanohydrolase, EC 3.2.1.15) and one exo-polygalacturonase (poly(1,4-alpha-D-galacturonide) galacturonohydrolase, EC 3.2.1.67) were isolated from a commercial pectinase preparation derived from Aspergillus niger. All five endo-enzymes could be purified to homogeneity by affinity chromatography on cross-linked alginate, ion-exchange chromatography, chromatofocusing, and gel permeation chromatography. The exo-polygalacturonase was only partially purified but free from endo-polygalacturonase activity. The two most abundant endo-polygalacturonases (endo-I and endo-II), with molecular masses of 55 and 38 kDa, respectively, are quite different with respect to their isoelectric point, specific activity, mode of action on oligomeric substrates, and amino acid composition. The physicochemical properties of the other three endo-polygalacturonases (endo-IIIA, endo-IIIB, and endo-IV), present in low amounts, are quite similar to those of the endo-I type. The pH optima of all these endo-polygalacturonases are in the range of 4.3-4.9.     

Effect of nutrient availability on hyphal maturation and topographical sensing in Aspergillus niger

Topographical sensing (thigmotropism) is an essential component of efficient fungal growth. It is an important element in the complex pathway of sensory and mechanical elements that drive and control the growing hyphal tip, a fuller understanding of which will bring the mycological community a step closer to complete comprehension of the hyphal growth mode. Previous work has led us to hypothesize that the stress induced by nutrient deficiency causes structural changes in the hyphal tip that induces a thigmotropic response in Aspergillus niger, a soil fungus that does not display thigmotropism under normal conditions. In this study, we have sought to identify some of the factors that influence this induction of thigmotropism using a novel combination of microengineered substrates and imaging and analysis techniques to quantify thigmotropic behavior in complex hyphal systems. We have shown that the sensitivity of fungal contour sensing appears to be directly linked to nutrient availability and hypothesize that this may be caused by a stress-induced flattening of the tip and increased immaturity of the hyphal apex. Parts of this paper were presented at the Mycological Society of Japan (MSJ)/British Mycological Society (BMS) Joint Symposium, “The new generation of mycologists in Japan and the UK” held in Chiba, Japan, on June 3, 2006.     

The AMT1 arginine methyltransferase gene is important for plant infection and normal hyphal growth in Fusarium graminearum

Arginine methylation of non-histone proteins by protein arginine methyltransferase (PRMT) has been shown to be important for various biological processes from yeast to human. Although PRMT genes are well conserved in fungi, none of them have been functionally characterized in plant pathogenic ascomycetes. In this study, we identified and characterized all of the four predicted PRMT genes in Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. Whereas deletion of the other three PRMT genes had no obvious phenotypes, the Δamt1 mutant had pleiotropic defects. AMT1 is a predicted type I PRMT gene that is orthologous to HMT1 in Saccharomyces cerevisiae. The Δamt1 mutant was slightly reduced in vegetative growth but normal in asexual and sexual reproduction. It had increased sensitivities to oxidative and membrane stresses. DON mycotoxin production and virulence on flowering wheat heads also were reduced in the Δamt1 mutant. The introduction of the wild-type AMT1 allele fully complemented the defects of the Δamt1 mutant and Amt1-GFP fusion proteins mainly localized to the nucleus. Hrp1 and Nab2 are two hnRNPs in yeast that are methylated by Hmt1 for nuclear export. In F. graminearum, AMT1 is required for the nuclear export of FgHrp1 but not FgNab2, indicating that yeast and F. graminearum differ in the methylation and nucleo-cytoplasmic transport of hnRNP components. Because AMT2 also is a predicted type I PRMT with limited homology to yeast HMT1, we generated the Δamt1 Δamt2 double mutants. The Δamt1 single and Δamt1 Δamt2 double mutants had similar defects in all the phenotypes assayed, including reduced vegetative growth and virulence. Overall, data from this systematic analysis of PRMT genes suggest that AMT1, like its ortholog in yeast, is the predominant PRMT gene in F. graminearum and plays a role in hyphal growth, stress responses, and plant infection.     

The role of microtubules in rapid hyphal tip growth of Aspergillus nidulans

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-alpha-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew approximately 5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10x reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.     

A fungus-specific ras homolog contributes to the hyphal growth and virulence of Aspergillus fumigatus

The Ras family of GTPase proteins has been shown to control morphogenesis in many organisms, including several species of pathogenic fungi. In a previous study, we identified a gene encoding a fungus-specific Ras subfamily homolog, rasB, in Aspergillus fumigatus. Here we report that deletion of A. fumigatus rasB caused decreased germination and growth rates on solid media but had no effect on total biomass accumulation after 24 h of growth in liquid culture. The DeltarasB mutant had an irregular hyphal morphology characterized by increased branching. Expression of rasBDelta113-135, a mutant transgene lacking the conserved rasB internal amino acid insertion, did not complement the deletion phenotype of delayed growth and germination rates and abnormal hyphal morphology. Virulence of the rasB deletion strain was diminished; mice infected with this strain exhibited approximately 65% survival compared to approximately 10% with wild-type and reconstituted strains. These data support the hypothesis that rasB homologs, which are highly conserved among fungi that undergo hyphal growth, control signaling modules important to the directional growth of fungal hyphae.     

黑曲霉对黄曲霉生长、产毒及黄曲霉毒素B1的影响

目的研究黑曲霉对黄曲霉生长、产毒的抑制作用及对AFB1的降解作用。方法将黑曲霉分别与黄曲霉、AFB1共同培养,定期测定培养液pH、菌丝体干重、黄曲霉孢子数、AFB1含量。结果黑曲霉与黄曲霉混合培养时,黄曲霉孢子数、AFB1含量均比单独培养的低,2组之间差异有统计学意义(P<0.05),抑制率达到68.06%~91.52%;加入黑曲霉后,AFB1含量降低,实验组与对照组之间差异有统计学意义(P<0.05),降解率为46.19%。结论黑曲霉既能抑制黄曲霉生长、产毒,又能降解AFB1。     

Amylose-like polysaccharide accumulation and hyphal cell-surface structure in relation to citric acid production by Aspergillus niger in shake culture

When 120 mg glucose/ml was used as a carbon source, in shake culture Aspergillus niger Yang no. 2 maximally produced only 15.4 mg citric acid/ml but accumulated 3.0 mg extracellular polysaccharide/ml. The polysaccharide secreted by mycelia of Yang no. 2 in shake culture was confirmed to be an amylose-like alpha-1,4-glucan by hydrolysis analysis with acid, amylase and glucoamylase. However, in static cultures, such as semisolid and surface cultures free from physical stresses caused by shaking damage, Yang no. 2 produced more citric acid but did not accumulate the polysaccharide. With cultivation time in shake culture, the amount of extracellular polysaccharide and the viscosity of the culture broth increased. The increase of shaking speed caused a remarkable increase in the accumulation of extracellular polysaccharide, e.g. 11.2 mg extracellular polysaccharide/ml was accumulated in the medium at a shaking speed of 200 rpm. The addition of 2.0 mg carboxymethylcellulose (CMC)/ml as a viscous additive to the medium reduced drastically the amount of extracellular polysaccharide accumulated to 1.5 mg/ml, but increased the citric acid produced to 52.0 mg/ml. However, intracellular polysaccharide accumulation kept up a steady rate of 0.26 microgram/mg dried mycelium through the entire period of cultivation. The addition of 3.0 mg polysaccharide/ml purified from the culture broth to the medium at the start of a culture resulted in a decrease of extracellular polysaccharide accumulation but an increase of citric acid accumulation. From electronmicroscopic observation, cell surfaces of hyphae cultivated with CMC were smooth, while hyphae cultivated without CMC had fibrous and granular polysaccharide on the cell surface. These results suggested that Yang no. 2 secreted the polysaccharide on the cell surface as a viscous substance and/or a shock absorber to protect itself from physical stresses caused by shaking damage in shake culture.     

Localization of growth and secretion of proteins in Aspergillus niger

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.     

The Aspergillus niger annexin, anxC3.1 is constitutively expressed and is not essential for protein secretion

An annexin, anxC3.1, was isolated and characterised from the industrially important filamentous fungus Aspergillus niger. anxC3.1 is a single copy gene encoding a 506 amino acid predicted protein which contains four annexin repeats. Disruption of the anxC3.1 gene did not lead to any visible changes in phenotype, nor in the levels of secreted protein, nor specifically in glucoamylase production, suggesting no major role in secretion. anxC3.1 expression was found to be unaltered under a variety of conditions such as increased secretion, altered nitrogen source, heat shock, and decreased Ca2+ levels, indicating that anxC3.1 is constitutively expressed. This is the first reported functional characterisation of a fungal annexin.     

Arabidopsis thaliana Rop GTPases are localized to tips of root hairs and control polar growth

Plants contain a novel unique subfamily of Rho GTPases, vital components of cellular signalling networks. Here we report a general role for some members of this family in polarized plant growth processes. We show that Arabidopsis AtRop4 and AtRop6 encode functional GTPases with similar intrinsic GTP hydrolysis rates. We localized AtRop proteins in root meristem cells to the cross-wall and cell plate membranes. Polar localization of AtRops in trichoblasts specifies the growth sites for emerging root hairs. These sites were visible before budding and elongation of the Arabidopsis root hair when AtRops accumulated at their tips. Expression of constitutively active AtRop4 and AtRop6 mutant proteins in root hairs of transgenic Arabidopsis plants abolished polarized growth and delocalized the tip-focused Ca2+ gradient. Polar localization of AtRops was inhibited by brefeldin A, but not by other drugs such as latrunculin B, cytochalasin D or caffeine. Our results demonstrate a general function of AtRop GTPases in tip growth and in polar diffuse growth.     

Aspergillus nidulans RhoA is involved in polar growth, branching, and cell wall synthesis

Growth of the filamentous fungus Aspergillus nidulans begins when the conidium breaks dormancy and grows isotropically. Eventually a germ tube emerges and the axis of growth remains fixed in the primary hypha while new growth axes are established basally to form secondary germ tubes and lateral branches. Rho1 is a Rho family GTPase that has been shown to be involved in polarity establishment and cell wall deposition in Saccharomyces cerevisiae. A gene predicted to encode a Rho1 homolog was cloned from A. nidulans and named rhoA. Strains carrying ectopic copies of the constitutively active rhoA(G14V) allele or the dominant rhoA(E40I) allele were created and characterized. The constitutively active rhoA(G14V) strain grew slowly relative to wild type and showed an abnormal clustered pattern of branch emergence. The rhoA(G14V) strain also labeled intensely with calcofluor, showed elevated levels of cell wall N-acetylglucosamine and had unusually thick cell walls. The dominant rhoA(E40I)strain was accelerated in the emergence of secondary and tertiary germ tubes, and lateral branches relative to wild type and showed lysis with prolonged incubation. The rhoA(E40I) strain also was hypersensitive to the cell wall disrupting agents calcofluor and caspofungin acetate and showed an increase in cell wall N-acetylglucosamine levels. Our results suggest that rhoA plays a role in polarity, proper branching pattern, and cell wall deposition.     

Fungal metabolites from Aspergillus niger AN27 related to plant growth promotion

Two metabolites have been isolated from Aspergillus niger AN27, a biocontrol agent, and identified as 2-carboxymethyl 3-n-hexyl maleic acid (compound 1) and 2-methylene-3-hexylbutanedioic acid (compound 2). Their biological activities related to crop growth promotion have been assayed. Both the compounds increased germination and improved crop vigour. Compound 1 was more effective for increase in germination and shoot length, whereas compound 2 had relatively greater role in increasing the root length and biomass of cauliflower seedlings.     

The protein kinase kin1, the fission yeast orthologue of mammalian MARK/PAR-1, localises to new cell ends after mitosis and is important for bipolar growth

The kin1 protein kinase of the fission yeast Schizosaccharomyces pombe is a member of the PAR-1/MARK (partitioning-defective 1/microtubule-associated protein/microtubule affinity-regulating kinase) family important in eukaryotic cell polarity and cytoskeletal dynamics. We show here that kin1 plays a role in establishing the characteristic rod-shaped morphology of fission yeast. Cells in which kin1 was deleted are viable but are impaired in growth, and are rounded at one end or both ends. They are monopolar because after mitosis they fail to activate bipolar growth, and are delayed in cytokinesis, resulting in a high proportion of septated cells often with multiple septa. This phenotype can be partially rescued by heterologous expression of human MARKs, which restore bipolar growth in most cells, but do not correct the delay in cytokinesis. Using chromosomal epitope tagging, we show that kin1p localises to the cell ends, except during mitosis when it disappears from cell ends. After mitosis, kin1p first reappears at the new cell end. Overexpression of kin1 results in a loss of polarity, with partially or fully rounded cells. From these results we suggest that kin1 is required to direct the growth machinery to the cell ends.     

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.