A copper-transporting ATPase BcCCC2 is necessary for pathogenicity of Botrytis cinerea

Copper is an essential trace element that serves as a cofactor for numerous enzymes. In eukaryotes, copper-transporting ATPases deliver copper to various copper-containing proteins in the trans-golgi network. This study identified a copper-transporting ATPase gene BcCcc2 in a fungus pathogenic to plants, Botrytis cinerea. We investigated the biological roles of BcCCC2 by generating null mutants for BcCcc2. Melanization, conidiation and the formation of sclerotia were severely affected in ∆BcCcc2 mutants. Moreover, a pathogenicity assay using tomato leaves and carnation petals revealed the mutants to be nonpathogenic. Further analysis indicated that they formed fewer appressoria and infection cushions than the wild-type. These structures were aberrant in morphology and in many cases had a significantly reduced ability to penetrate the plant epidermis. An assay also indicated that ∆BcCcc2 mutants were defective in infection through wounds. BcCCC2 is necessary not only for penetrating a host but also for fungal growth within plant tissues. Our results also imply that B. cinerea requires copper-containing proteins for infection that are inactive in the absence of the copper-transporting ATPase BcCCC2.     

Phylogenetic analysis of heavy-metal ATPases in fungi and characterization of the copper-transporting ATPase of Cochliobolus heterostrophus

We performed a phylogenetic analysis of heavy-metal ATPases (HMAs) in fungi and found that HMAs can be divided into three groups, A, B, and C. Group A is predicted to deliver copper ions to copper-containing proteins, while Groups B and C are thought to function as cell-membrane copper-efflux pumps. Furthermore, Groups B and C consist of fungal-specific HMAs, while Group A consists of fungal orthologues that have been well conserved in eukaryotes. We also cloned and characterized a Group A-type HMA gene (i.e., ChCcc2) of the filamentous plant pathogen, Cochliobolus heterostrophus. Mutation of ChCcc2 severely affected growth, pigmentation, conidiation, and colonial morphology. Activity of the copper-containing protein, laccase, was also lost in ChCcc2 mutants, suggesting that ChCCC2 plays an important role in growth and morphology by activating various copper-containing proteins in C. heterostrophus.     

Host Physiology and Pathogenic Variation of Cochliobolus heterostrophus Strains with Mutations in the G Protein Alpha Subunit, CGA1

Conserved eukaryotic signaling proteins participate in development and disease in plant-pathogenic fungi. Strains with mutations in CGA1, a heterotrimeric G protein G alpha subunit gene of the maize pathogen Cochliobolus heterostrophus, are defective in several developmental pathways. Conidia from CGA1 mutants germinate as abnormal, straight-growing germ tubes that form few appressoria, and the mutants are female sterile. Nevertheless, these mutants can cause normal lesions on plants, unlike other filamentous fungal plant pathogens in which functional homologues of CGA1 are required for full virulence. Δcga1 mutants of C. heterostrophus were less infective of several maize varieties under most conditions, but not all, as virulence was nearly normal on detached leaves. This difference could be related to the rapid senescence of detached leaves, since delaying senescence with cytokinin also had differential effects on the virulence of the wild type and the Δcga1 mutant. In particular, detached leaves may provide a more readily available nutrient source than attached leaves. Decreased fitness of Δcga1 as a pathogen may reflect conditions under which full virulence requires signal transduction through CGA1-mediated pathways. The virulence of these signal transduction mutants is thus affected differentially by the physiological state of the host.     

Effect of nutritional parameters on laccase production by the culinary and medicinal mushroom, Grifola frondosa

Summary Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 (M Cu over the basal level (1.6 mM Cu) and peak levels observed at 300 mM Cu were ∼ ∼7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r ∼ ∼70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct second laccase protein band (M _r␣∼ ∼45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. The optimal temperature and pH values for laccase activity were 65 °C and pH 2.2 (using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) {ABTS} as substrate), respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Effect of Nutritional Parameters on Laccase Production by the Culinary and Medicinal Mushroom, <Emphasis Type="Italic">Grifola frondosa</Emphasis>

Extracellular laccase in cultures of Grifola frondosa grown in liquid culture on a defined medium was first detectable in the early/middle stages of primary growth, and enzyme activity continued to increase even after fungal biomass production had peaked. Laccase production was significantly increased by supplementing cultures with 100–500 μM Cu over the basal level (1.6 μM Cu) and peak levels observed at 300 μM Cu were 7-fold higher than in unsupplemented controls. Decreased laccase activity similar to levels detected in unsupplemented controls, as well as an adverse effect on fungal growth, occurred with further supplementation up to and including 0.9 mM Cu, but higher enzyme titres (2- to 16-fold compared with controls) were induced in cultures supplemented with 1–2 mM Cu²⁺. SDS-PAGE combined with activity staining revealed the presence of a single protein band (M _r 70 kDa) exhibiting laccase activity in control culture fluids, whereas an additional distinct laccase protein band (M _r 45 kDa) was observed in cultures supplemented with 1–2 mM Cu. Increased levels of extracellular laccase activity, and both laccase isozymes, were also detected in cultures of G. frondosa supplemented with ferulic, vanillic, veratric and 4-hydroxybenzoic acids, and 4-hydroxybenzaldehyde. Using 2,2′-azino-bis(ethylbenzothiazoline-6-sulfonate) (ABTS) as substrate, the optimal temperature and pH values for laccase activity were 65°C and pH 2.2, respectively, and the enzyme was relatively heat stable. In solid-state cultures of G. frondosa grown under conditions adopted for industrial-scale mushroom production, extracellular laccase levels increased during the substrate colonization phase, peaked when the substrate was fully colonized, and then decreased sharply during fruit body development.     

Two flagellar stators and their roles in motility and virulence in <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> 6605

The motor proteins around the flagellar basal body consist of two cytoplasmic membrane proteins, MotA and MotB, and function as a complex that acts as the stator to generate the torque that drives rotation. Genome analysis of several Pseudomonas syringae pathovars revealed that there are two sets of genes encoding motor proteins: motAB and motCD. Deduced amino acid sequences for MotA/B and MotC/D showed homologies to the H⁺-driven stator from Escherichia coli and Na⁺-driven stator from Vibrio alginolyticus, respectively. However, the swimming motility of P. syringae pv. tabaci (Pta) 6605 was inhibited by the protonophore carbonyl cyanide m-chlorophenylhydrazone but not by the sodium stator-specific inhibitor phenamil. To identify a gene encoding the stator protein required for motility, ∆motAB, ∆motCD, and ∆motABCD mutants were generated. The ∆motCD mutant had remarkably reduced and the ∆motABCD mutant completely abolished swimming motilities, whereas the ∆motAB mutant retained some degree of these abilities. The ∆motCD and ∆motABCD mutants did not produce N-acyl-homoserine lactones (AHLs), quorum-sensing molecules in this pathogen, and remarkably reduced the ability to cause disease in host tobacco leaves, as we previously observed in the ∆fliC mutant strain. These results strongly indicate that both stator pairs in Pta 6605 are proton-dependent and that MotCD is important for not only flagellar motility but also for production of AHLs and the ability to cause disease in host plants.     

Gene inactivation mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> in the filamentous fungi <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.     

Activation of diacylglycerol acyltransferase expressed in Saccharomyces cerevisiae: overexpression of Dga1p lacking the N-terminal region in the ∆snf2 disruptant produces a significant increase in its enzyme activity

We previously found that overexpression of DGA1 encoding diacylglycerol acyltransferase (DGAT) in the ∆snf2 disruptant of Saccharomyces cerevisiae caused a significant increase in lipid accumulation and DGAT activity. The present study was conducted to investigate how Dga1p is activated in the ∆snf2 disruptant. To analyze the expression of Dga1p in wild type and the ∆snf2 disruptant, we overexpressed Dga1p with a 6x His tag at the N-terminus and a FLAG tag at the C-terminus. Immunoblotting using anti-6x His and anti-FLAG antibodies revealed that, in addition to full-length protein, Dga1p lacking the N-terminus was produced only in the ∆snf2 disruptant. Full-length Dga1p and N-terminally truncated Dga1p were separated and purified from the lipid body fraction by using anti-FLAG M2 agarose and TALON metal affinity resin. Major DGAT activity was recovered in the purified fraction of N-terminally truncated Dga1p, indicating that proteolytic cleavage at the N-terminal region is involved in DGAT activation in the ∆snf2 disruptant. Analysis of the cleavage site of N-terminally truncated Dga1p revealed a major site between Lys-29 and Ser-30. We then overexpressed truncated Dga1p variants that lacked different N-terminal amino acids and had a FLAG tag at the C-terminus. The homogenate and lipid body fraction of the ∆snf2 disruptant overexpressing Dga1p lacking the N-terminal 29 amino acids (Dga1∆N2p) had higher DGAT activity than that overexpressing Dga1p, indicating that Dga1∆N2p is activated Dga1p. Dga1∆N2p-FLAG(C-terminus) was purified to near homogeneity by anti-FLAG M2 agarose chromatography and maintained significant DGAT activity. These results provide a new strategy to engineer expression of DGAT.     

Single mating type-specific genes and their 3′ UTRs control mating and fertility in Cochliobolus heterostrophus

To determine the number of proteins required for mating type (MAT) locus-regulated control of mating in Cochliobolus heterostrophus, MAT fragments of various sizes were expressed in MAT deletion strains. As little as 1.5 kb of MAT sequence, encoding a single unique protein in each mating type (MAT-1 and MAT-2), conferred mating ability, although an additional 160 bp of 3^′ UTR was needed for production of ascospores. No other mating type-specific genes involved in mating identity or fertility were found. Thus, although homologs of the C. heterostrophus MAT-1 and MAT-2 genes exist in the filamentous ascomycetes Neurospora crassa and Podospora anserina, C. heterostrophus does not appear to have mating type-specific homologs of two additional genes required by both N. crassa and P.␣anserina for successful sexual reproduction. Three genes were identified in the common DNA flanking the MAT locus: a gene encoding a GTPase-activating protein and an ORF of unknown function lie 5^′ while a β-glucosidase encoding gene lies found 3^′. None of these genes appears to be involving in the mating process. Received: 21 November 1997 / Accepted: 28 April 1998     

A new method for screening and isolation of hypersecretion mutants in Aspergillus niger

Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA₅₁₂). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA₅₁₂–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch. Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous protein that is secreted as a fusion with the glucoamylase protein.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.     

Proton Motive Force in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> Under Anaerobic Conditions in the Dark

In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H₂ production by Rhodobacter sphaeroides, ∆p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, ∆φ was of −94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of −64 mV. The addition of N,N′-dicyclohexylcarbodiimide (DCCD), the F₀F₁–ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ increased from −57.1 mV at pH 6.0 to −103.8 mV at pH 8.0; it was compensated at high external pH by a reversed ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.     

A H<Subscript>2</Subscript>O<Subscript>2</Subscript>-producing glyoxal oxidase is required for filamentous growth and pathogenicity in<Emphasis Type="Italic"> Ustilago maydis</Emphasis>

In the phytopathogenic fungus Ustilago maydis the mating-type loci control the transition from yeast-like to filamentous growth required for pathogenic development. In a large REMI (restriction enzyme mediated integration) screen, non-pathogenic mutants were isolated in a haploid strain that had been engineered to be pathogenic. In one of these mutants, which showed a specific morphological phenotype, the tagged gene, glo1 , was found to encode a product that is highly homologous to a glyoxal oxidase gene from the wood-rot fungus Phanerochaete chrysosporium. Glyoxal oxidase homologues are found in human, plant pathogenic fungi and in plants, but not in other mammals or yeasts. To confirm the function of the glo1 gene, null mutations were generated in compatible haploid U. maydis strains. In crosses null mutants were unable to generate filamentous dikaryons, and were completely non-pathogenic. Using a Glo1-overproducing strain we demonstrated that Glo1 is membrane bound, oxidizes a series of small aldehydes (<C4) and produces H₂O₂. The enzyme needs to be activated, presumably by auto-oxidation, to show full activity. A potential role for Glo1 during filamentous growth and pathogenic development of U. maydis is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by P. J. PuntThe first two authors contributed equally to this workWe dedicate this work to the memory of Jeff Schell, a charismatic and outstanding person who loved science and respected people     

Molecular cloning of the cDNA encoding laccase from Trametes versicolor and heterologous expression in Pichia methanolica

A cDNA encoding for laccase was isolated from the ligninolytic fungus Trametes versicolor by RNA-PCR. The cDNA corresponds to the gene Lcc1, which encodes a laccase isoenzyme of 498 amino acid residues preceded by a 22-residue signal peptide. The Lcc1 cDNA was cloned into the vectors pMETA and pMETαA and expressed in Pichia methanolica. The laccase activity obtained with the Saccharomyces cerevisiae α-factor signal peptide was found to be twofold higher than that obtained with the native secretion signal peptide. The extracellular laccase activity in recombinants with the α-factor signal peptide was 9.79 U ml⁻¹. The presence of 0.2 mM copper was necessary for optimal activity of laccase. The expression level was favoured by lower cultivation temperature. The identity of the recombinant protein was further confirmed by immunodetection using Western blot analysis. As expected, the molecular mass of the mature laccase was 64.0 kDa, similar to that of the native form.     

Absence of repellents in <Emphasis Type="Italic">Ustilago maydis</Emphasis> induces genes encoding small secreted proteins

The rep1 gene of the maize pathogen Ustilago maydis encodes a pre-pro-protein that is processed in the secretory pathway into 11 peptides. These so-called repellents form amphipathic amyloid fibrils at the surface of aerial hyphae. A SG200 strain in which the rep1 gene is inactivated (∆rep1 strain) is affected in aerial hyphae formation. We here assessed changes in global gene expression as a consequence of the inactivation of the rep1 gene. Microarray analysis revealed that only 31 genes in the ∆rep1 SG200 strain had a fold change in expression of ≥2. Twenty-two of these genes were up-regulated and half of them encode small secreted proteins (SSPs) with unknown functions. Seven of the SSP genes and two other genes that are over-expressed in the ∆rep1 SG200 strain encode proteins that can be classified as secreted cysteine-rich proteins (SCRPs). Interestingly, most of the SCRPs are predicted to form amyloids. The SCRP gene um00792 showed the highest up-regulation in the ∆rep1 strain. Using GFP as a reporter, it was shown that this gene is over-expressed in the layer of hyphae at the medium-air interface. Taken together, it is concluded that inactivation of rep1 hardly affects the expression profile of U. maydis, despite the fact that the mutant strain has a strong reduced ability to form aerial hyphae.     

Redundant roles of Srs2 helicase and replication checkpoint in survival and rDNA maintenance in Schizosaccharomyces pombe

Srs2 helicase is believed to function as an anti-recombinase by resolving inappropriate Rad51-DNA filament. We found synthetic lethality or poor growth of srs2 with rad3 or mrc1 in Schizossacharomyces pombe. Lethality may result from a defect in non-checkpoint function of Rad3 or Mrc1 in the absence of Srs2, because srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A (non-phosphorylatable mrc1 allele) did not show significant growth impairment. Notably, the inactivation of rhp51/RAD51 or rad22/RAD52 failed to rescue the growth, suggesting that events that impose lethality are independent of homologous recombination. Incubation of the conditional srs2∆ rad3 ^ts cells at restrictive temperature led not only to a viability decrease but also to a remarkable shortening of rDNA clusters (~100 copies). As opposed to the growth defect, shortening of rDNA clusters was also observed in srs2∆ rad9∆, srs2∆ chk1∆ cds1∆ or srs2∆ mrc1-14A, indicating that proper replication checkpoint signaling is critical for rDNA maintenance. Activation of Chk1 in the unchallenged mrc1-14A srs2∆ cells implies a certain level of spontaneous fork damage that might be the cause for rDNA instability. The data suggest that redundant functions of Srs2 and checkpoint proteins are essential for two independent aspects of genome maintenance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Purification and biochemical characterization of a laccase from the aquatic fungus <Emphasis Type="Italic">Myrioconium</Emphasis> sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO₄ and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k _cat/K _m) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s⁻¹ mM⁻¹, respectively, with K _m values of 4.2, 67.8, and 104.9 μM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15°C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.