Pyrone derivatives from the soil fungus Fusarium solani PSU-RSPG37

Fusarpyrones A (1) and B (2), two new pyrone derivatives, were isolated from the soil fungus Fusarium solani PSU-RSPG37 together with eight known compounds, anhydrofusarubin, fusarubin, 5-hydroxy-8-methoxy-2,4-dimethylnaphtho[1,2,b]furan-6,9-dione, 2,3-dihydro-5-hydroxy-8-methoxy-2,4-dimethylnaphtho[1,2,b]furan-6,9-dione, javanicin, fusalanipyrone, p-hydroxyacetophenone, and tyrosol. The cytotoxic, antimalaria, and antimycobacterial activities of isolated compounds were examined.     

Replication of double-stranded RNA in mycovirus from the plant pathogenic fungus, Fusarium solani

Abstract A mycovirus (named FusoV) from the plant pathogenic fungus Fusarium solani possessed two types of double-stranded (ds) RNA genome, designated Ml and M2. RNA-dependent RNA polymerase activity was detected in FusoV particle fractions. An in vitro RNA polymerase reaction using purified FusoV particles that was supplemented with NTPs revealed the synthesis of single-stranded (ss) RNA species and a subsequent formation of dsRNAs having the same size as Ml and M2. The ssRNA species synthesized in the first stage were proved to be of positive polarity (coding strand) for both M1 and M2 by dot blot hybridization analysis. These results suggest that FusoV genomic dsRNA replicates in a conservative manner.     

Light-driven diurnal zonation in the filamentous fungus Fusarium solani

Zonation in growing mycelia of Fusarium solani was induced by diurnal light/dark cycles. Only those parts of the hyphae that grew in darkness for less than 20 hours developed a zone of conidia after illumination. In continuous darkness, in continuous illumination, or after a transition from light to darkness, a conidiation zone failed to appear. Only light periods exceeding a few seconds but lasting less than 21 hours during a 24 hour light/dark cycle induced zonation. This zonation was not caused by periodic staling of the growth medium.     

Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase,in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, one of the main structural components of the cell wall is 1,3-beta-glucan produced by 1,3-beta-glucan synthase (GS). Yeast GS is composed of a putative catalytic subunit encoded by FKS1 and FKS2 and a regulatory subunit encoded by RHO1. A combination of amino acid alterations in the putative catalytic domain of Fks1p was found to result in a loss of the catalytic activity. To identify upstream regulators of 1,3-beta-glucan synthesis, we isolated multicopy suppressors of the GS mutation. We demonstrate that all of the multicopy suppressors obtained (WSC1, WSC3, MTL1, ROM2, LRE1, ZDS1, and MSB1) and the constitutively active RHO1 mutations tested restore 1,3-beta-glucan synthesis in the GS mutant. A deletion of either ROM2 or WSC1 leads to a significant defect of 1,3-beta-glucan synthesis. Analyses of the degree of Mpk1p phosphorylation revealed that among the multicopy suppressors, WSC1, ROM2, LRE1, MSB1, and MTL1 act positively on the Pkc1p-MAPK pathway, another signaling pathway regulated by Rho1p, while WSC3 and ZDS1 do not. We have also found that MID2 acts positively on Pkc1p without affecting 1,3-beta-glucan synthesis. These results suggest that distinct networks regulate the two effector proteins of Rho1p, Fks1p and Pkc1p.     

A double-stranded RNA mycovirus from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

Abstract Two kinds of double-stranded RNA (dsRNA), estimated to be 1.9 and 1.7 kb in size, were detected in the plant pathogenic fungus, Fusarium solani f. sp. robiniae . Isometric virus-like particles (VLPs), 30 nm in diameter, were recovered from cell extracts as a discrete band when centrifuged through a CsCl density gradient. The dsRNA molecules extracted from VLP preparations were identical in electrophoretic mobility to the dsRNAs obtained directly from cells. SDS-PAGE analysis of the VLPs revealed a single polypeptide of 38 kDa. The dsRNAs obtained directly from cells. SDS-PAGE analysis of the asexual cycle).     

Cytotoxic naphthoquinone and a new succinate ester from the soil fungus Fusarium solani PSU-RSPG227

A new naphthoquinone, solaninaphthoquione (1), and a new succinate ester derivative, 4-(4-hydroxyphenethoxy)-4-oxobutanoic acid (2), were isolated from the soil fungus Fusarium solani PSU-RSPG227 together with five previously reported compounds; javanicin (3), monaspilosin (4), aspergillol B (5), tyrosol (6) and 4-hydroxyphenylacetic acid (7). Their structures were elucidated primarily by NMR spectroscopic data. Due to paucity of materials, compounds 2, 4 and 5 as well as analogues of 5 were prepared for biological activity evaluation. Compound 1 showed significant cytotoxic activity against breast cancer (MCF-7) cells and mild cytotoxic activity against oral human carcinoma (KB) cells (IC₅₀ values of 21.3 and 22.6 μM, respectively) compared to standard compounds. Compound 1 also displayed weak antimalarial activity (IC₅₀ of 26.1 μM).     

Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.     

Purification and characterization of a lectin from endophytic fungus Fusarium solani having complex sugar specificity

A lectin from the mycelial extract of an endophytic strain of Fusarium solani was purified. Its hemagglutinating activity was inhibited by glycoproteins possessing N-linked as well as O-linked glycans. The thermodynamics and kinetics of binding of glycans and glycoproteins to F. solani lectin was studied using surface plasmon resonance. The lectin showed high affinity for asialofetuin, asialomucin, asialofibrinogen, and thyroglobulin; and comparatively low affinity for mucin, fetuin, fibrinogen, and holotransferrin. Glycoproteins showed several fold higher affinity than their corresponding glycans with significant contribution from enthalpy and positive entropy, suggesting the involvement of non-polar protein-protein interaction. Moreover, the higher affinity of the glycoproteins was due to their faster association rates and low activation energy.     

Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe.

Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin-based contractile ring. A division septum is formed centripetally, concomitant with ring constriction. Although several genes essential for cytokinesis have been described previously, enzymes that participate in the assembly of the division septum have not been identified. Here we describe a temperature-sensitive mutation, drc1-191, that prevents division septum assembly and causes mutant cells to arrest with a stable actomyosin ring. Unlike the previously characterized cytokinesis mutants, which undergo multiple mitotic cycles, drc1-191 is the first cytokinesis mutant that arrests with two interphase nuclei. Interestingly, unlike drc1-191, drc1-null mutants proceed through multiple mitotic cycles, leading to the formation of large cells with many nuclei. drc1 is allelic to cps1, which encodes a 1,3-beta-glucan synthase subunit. We conclude that Drc1p/Cps1p is not required for cell elongation and cell growth, but plays an essential role in assembly of the division septum. Furthermore, it appears that constriction of the actomyosin ring might depend on assembly of the division septum. We discuss possible mechanisms that account for the differences in the phenotypes of the drc1-191 and the drc1-null mutants and also reflect the potential links between Drc1p and other cytokinesis regulators.     

Adenylate cyclase from the phytopathogenic fungus Alternaria solani

Abstract Adenylate cyclase activity of Alternaria solani bound to the particulate cell fraction was solubilized by 2.5% Ficoll. The apparent K _m of the solubilized enzyme for adenosine 5'-triphosphate was 2.5 mM, and it required Mn²⁺ for maximum activity. M _r as determined by gel filtration was approximately 500. Fluoride ions at millimolar concentrations, GTP, adenosine, and N⁶-phenylisopropyl adenosine at micromolar concentrations did not stimulate adenylate cyclase activity. The enzyme was inhibited 25–55% by millimolar concentrations of 2'-deoxyadenosine, 2'-0 methyl adenosine, 9-β- d -arabinofuranosyl adenosine and 2'-3'-isopropylidene adenosine. Partially purified enzyme obtained after DEAE-BioGel chromatography was very unstable. The amount of extractable enzyme activity varied during the fungal growth cycle.     

Observations on the pathogenesis of the imperfect fungus,Fusarium solani,in the California brown shrimp,Penaeus californiensis

The pathogenesis of Fusarium solani infections was studied using a highly susceptible species, Penaeus californiensis. In two experiments, F. solani infections were established in artificially wounded and infected juvenile and adults of P. californiensis, while similar control groups were wounded but not artificially infected. In the first experiment, the progress of F. solani infections in 15-g cultured juveniles of P. californiensis was followed by gross inspection and by light and electron microscopical study of the wound area. Developing F. solani infections were produced with a success rate of 100% within 14 days postinfection, and deaths due to disease of greater than 50% of the artificially infected group had occurred by 24 days postinfection. Infections were histologically characterized by (1) expansion of lesions into the tissue surrounding the point of entry, (2) destruction of invaded tissues by host granuloma formation and possibly by fungal enzymes and toxins, and (3) hemocyte encapsulation of hyphae with eventual melanization of the encapsulating hemocytes. In the second study, wild adult P. californiensis were artificially infected with F. solani, and changes in several of their hemolymph constituents [alkaline phosphatase, serum glutamic oxaloacetic transaminase (SGOT), glucose, total protein, hemocyte count, and hematopoietic tissue mitotic index] were determined as the disease developed. Significant differences occurred in these hemolymph parameters of shrimp with advanced F. solani infections compared to those of uninfected, unwounded control shrimp or those with early F. solani infections. Hemolymph from severely infected shrimp was hypoproteinemic, contained lower numbers of circulating hemocytes, and frequently failed to coagulate.     

Homologous subunits of 1,3-beta-glucan synthase are important for spore wall assembly in Saccharomyces cerevisiae

During sporulation in Saccharomyces cerevisiae, the four haploid nuclei are encapsulated within multilayered spore walls. Glucan, the major constituent of the spore wall, is synthesized by 1,3-beta-glucan synthase, which is composed of a putative catalytic subunit encoded by FKS1 and FKS2. Although another homolog, encoded by FKS3, was identified by homology searching, its function is unknown. In this report, we show that FKS2 and FKS3 are required for spore wall assembly. The ascospores of fks2 and fks3 mutants were enveloped by an abnormal spore wall with reduced resistance to diethyl ether, elevated temperatures, and ethanol. However, deletion of the FKS1 gene did not result in a defective spore wall. The construction of fusion genes that expressed Fks1p and Fks2p under the control of the FKS2 promoter revealed that asci transformed with FKS2p-driven Fks1p and Fks2p were resistant to elevated temperatures, which suggests that the expression of FKS2 plays an important role in spore wall assembly. The expression of FKS1p-driven Fks3p during vegetative growth did not affect 1,3-beta-glucan synthase activity in vitro but effectively suppressed the growth defect of the temperature-sensitive fks1 mutant by stabilizing Rho1p, which is a regulatory subunit of glucan synthase. Based on these results, we propose that FKS2 encodes the primary 1,3-beta-glucan synthase in sporulation and that FKS3 is required for normal spore wall formation because it affects the upstream regulation of 1,3-beta-glucan synthase.     

Purification to homogeneity and characterization of a 1,3-beta-glucan (callose) synthase from germinating Arachis hypogaea cotyledons.

A 1,3-beta-D-glucan (callose) synthase (CS) from a plasma membrane fraction of germinating peanut (Arachis hypogaea L.) cotyledons has been purified to apparent homogeneity as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), amino-terminal analysis, and the Western blots pattern. The purification protocol involved preparation of a high specific activity plasma membrane fraction, selective solubilization of the enzyme from the membrane with 0.5% digitonin at a protein-to-detergent ratio of 1:6, sucrose gradient centrifugation, and chromatography on hydroxylapatite and DEAE-Sephadex A-50. The purified CS shows a molecular mass of approximately 48,000 by SDS-PAGE, pH optimum of 7.4, leucine as the amino-terminal residue, Km for UDP-glucose of 0.67 mM, and Vmax of 6.25 mumol/min/mg protein. The enzyme is specific for UDP-glucose as the glucosyl donor and required Ca2+, at an optimum concentration of 2-5 mM, for activity. The enzyme activity was inhibited by nucleotides (ATP, GTP, CTP, UTP, UDP, and UMP). The enzyme activity was also inhibited by the addition of EDTA or EGTA to the enzyme, but this inhibition was fully reversible by the addition of Ca2+. The reaction product formed during incubation of UDP-[14C]glucose and cellobiose with purified enzymes was susceptible to digestion by exo-(1,3)-beta-glucanase, but was resistant to alpha- and beta-amylases and to periodate oxidation, indicating that the polymer formed was 1,3-beta-glucan, and beta-1,4 and beta-1,6 linkages were absent.     

Bgs3p,a putative 1,3-beta-glucan synthase subunit,is required for cell wall assembly in Schizosaccharomyces pombe

β-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of β-glucan synthase-related genes. We describe here the cloning and characterization of bgs3⁺, a new member of this family. bgs3⁺ was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the β-glucan, and raises the amount of α-glucan polymer. These cells finally died. bgs3⁺ is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.     

Bgs2p, a 1,3-beta-glucan synthase subunit, is essential for maturation of ascospore wall in Schizosaccharomyces pombe

Previously we have reported that Drc1p/Cps1p, a 1,3-beta-glucan synthase subunit, is essential for division septum assembly in Schizosaccharomyces pombe. In this report, we present evidence that S. pombe Bgs2p, a 1,3-beta-glucan synthase that shows 56% identity to Drc1p/Cps1p, is essential for maturation of ascospore wall in S. pombe, but is not required for vegetative growth. Diploid cells homozygous for the bgs2-null mutation, as well as homothallic bgs2-null mutant haploids undergo meiosis normally. However, a 1, 3-beta-glucan containing spore wall is not assembled in these cells. The spores resulting from meiosis of a bgs2-null mutant lyse upon release from the ascus and are therefore inviable. Using a green fluorescent protein-tagged Bgs2p, we demonstrate that Bgs2p is localized at the periphery of the ascospores during meiosis and sporulation. However, Bgs2p is not detected in vegetative cells. We conclude that Bgs2p is required for 1,3-beta-glucan synthesis during ascospore wall maturation.     

Prenylation of Rho1p is required for activation of yeast 1, 3-beta-glucan synthase

One of the essential protein substrates of geranylgeranyl transferase type I in the budding yeast Saccharomyces cerevisiae is a rho-type GTPase, Rho1p, which is a regulatory subunit of 1, 3-beta-glucan synthase. Previous studies have indicated that modification of Rho1p is significantly reduced in a mutant of the beta subunit of geranylgeranyl transferase type I called cal1-1. Here we present genetic and biochemical evidence showing that modification of Rho1p is required for activity of 1,3-beta-glucan synthase. The 1,3-beta-glucan synthase activity of the cal1-1 membrane was significantly reduced compared with that of the wild-type membrane. The impaired activity was partly due to the reduced amount of Fks1p, a putative catalytic subunit of 1, 3-beta-glucan synthase, but also partly due to reduced affinity between unmodified Rho1p and Fks1p. Glutathione S-transferase (GST)-Rho1 proteins with or without the C-terminal motif required for the modification were purified and used to analyze the interaction. The modified form of GST-Rho1p was specifically able to restore the 1,3-beta-glucan synthase of the rho1-3 membrane. Gel overlay analysis indicated that an unmodified form of GST-Rho1p fails to interact with Fks1p. These results indicated that the geranylgeranylation of Rho1p is a prerequisite to the assembly and activation of 1,3-beta-glucan synthase in vitro. Increased cytoplasmic levels of divalent cations such as Ca(2+) restored both Rho1p modification and the 1,3-beta-glucan synthase activity of cal1-1, suggesting that cytoplasmic levels of the divalent cations affect geranylgeranyl transferase type I activity in vivo.