NUT1, a major nitrogen regulatory gene inMagnaporthe grisea,is dispensable for pathogenicity

NUT1, a gene homologous to the major nitrogen regulatory genesnit-2 ofNeurospora crassa andareA ofAspergillus nidulans, was isolated from the rice blast fungus,Magnaporthe grisea. NUT1 encodes a protein of 956 amino acid residues and, likenit-2 andareA, has a single putative zinc finger DNA-binding domain. Functional equivalence ofNUT1 toareA was demonstrated by introducing theNUT1 gene by DNA-mediated transformation into anareA loss-of-function mutant ofA. nidulans. The introducedNUT1 gene fully complemented theareA null mutation, restoring to the mutant the ability to utilize a variety of nitrogen sources. In addition, the sensitivity ofAspergillus NUT1 transformants to ammonium repression of extracellular protease activity was comparable to that of wild-typeA. nidulans. Thus,NUT1 andareA encode functionally equivalent gene products that activate expression of nitrogen-regulated genes. A one-step gene disruption strategy was used to generatenutl ^? mutants ofM. grisea by transforming a rice-infecting strain with a disruption vector in which a gene for hygromycin B phosphotransferase (Hyg) replaced the zinc-finger DNA-binding motif ofNUT1. Of 31 hygromycin B (hyg B)-resistant transformants shown by Southern hybridization to contain a disruptedNUT1 gene (nut1::Hyg), 26 resulted from single-copy replacement events at theNUT1 locus. Althoughnut1 ^? transformants ofM. grisea failed to grown on a variety of nitrogen sources, glutamate, proline and alanine could still be utilized. This contrasts withA. nidulans where disruption of the zinc-finger region ofareA prevents utilization of nitrogen sources other than ammonium and glutamine. The role ofNUT1 and regulation of nitrogen metabolism in the disease process was evaluated by pathogenicity assays. The infection efficiency ofnut1 ^? transformants on susceptible rice plants was similar to that of the parental strain, although lesions were reduced in size. These studies demonstrate that theM. grisea NUT1 gene activates expression of nitrogen-regulated genes but is dispensable for pathogenicity.     

Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi

The gene areA-GF, a homologue of the major nitrogen regulatory genes nit-2, areA, nre and NUT1 of Neurospora crassa, Aspergillus nidulans, Penicillium chrysogenum and Magnaporthe grisea, respectively, was cloned from the gibberellin (GA)-producing rice pathogen Gibberella fujikuroi.areA-GF encodes a protein of 972 amino acid residues which contains a single putative zinc finger DNA-binding domain that is at least 98% identical to the zinc finger domains of the homologous fungal proteins. The areA-GF gene has been shown to be functional in N. crassa by heterologous complementation of a RIP induced nit-2 mutant. The transformation rate was nearly as high as in a homologous complementation control. Transformants were able to utilize nitrate and expressed a normally regulated nitrate reductase activity. To generate areA-GF  ⁻ mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc finger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave significantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production. Received: 23 April 1998 / Accepted: 16 October 1998     

Nitrogen regulation in fungi

Nitrogen regulation has been extensively studied in fungi revealing a complex array of interacting regulatory genes. The general characterisation of the systems inAspergillus nidulans andNeurospora crassa shall be briefly described, but much of this paper will concentrate specifically on the recent molecular characterisation ofareA, the principle regulatory gene fromA. nidulans which mediates nitrogen metabolite repression. Three areas shall be explored in detail, firstly the DNA binding domain, which has been characterised extensively by both molecular and genetic analysis. Secondly we shall report recent analysis which has revealed the presence of related DNA binding activities inA. nidulans. Finally we shall discuss the mechanism by which the nitrogen state of the cell is monitored by theareA product, in particular localisation of the domain within theareA product which mediates the regulatory response within the protein.     

Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants

Summary A plasmid construct carrying the hygromycin phosphotransferase (hph) gene fused to the expression elements of the trpC gene of Aspergillus nidulans was used to obtain hygromycin B (Hyg)-resistant transformants of Neurospora crassa. The plasmid does not have any homology with the N. crassa genome. Here we demonstrate that most of the transformants arise from integration of the transforming DNA into only one of the nuclei present in the protoplasts. Furthermore, in most of the transformants the integrated transforming DNA is physically stable after growth of the transformants for about 25 nuclear divisions without Hyg selection, in spite of being present in multiple copies. In transformants carrying only a single insertion, phenotypic expression of the hph gene remains unaltered in conidial isolates obtained withoug Hyg selection. On the other hand, about 40% of transformants harbouring plasmid DNA integrated at more than one location yield conidial isolates showing reversible inactivation of the hph genes. Interestingly, the presence of methylated cytosine residues in the integrated DNA is strongly correlated with the number of plasmid copies. The hph genes are heavily methylated in transformants harbouring multiple copies but not in those harbouring only one copy of the plasmid. Phenotypic expression of the inactive hph genes can be restored by growing the transformants either under Hyg selection pressure or in the presence of 5-azacytidine. In the first case the hph genes are again inactivated when Hyg selection pressure is removed, while the activation of the hph gene by 5-azacytidine gives stable Hyg^r strains.Dedicated to Dr. T.A. Trautner on the occasion of his 60^th birthday     

Transformation of the cultivated mushroom,Agaricus bisporus, to hygromycin B resistance

Application of biotechnology to the cultivated mushroom,Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain ofA. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into theA. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomyceteAspergillus nidulans and fromA. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from theA. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.     

TheAspergillus nidulans geneschsA andchsD encode chitin synthases which have redundant functions in conidia formation

We previously isolated three chitin synthase genes (chsA, chsB, andchsC) fromAspergillus nidulans. In the present work, we describe the isolation and characterization of another chitin synthase gene, namedchsD, fromA. nidulans. Its deduced amino acid sequence shows 56.7% and 55.9% amino acid identity, respectively, with Cal1 ofSaccharomyces cerevisiae and Chs3 ofCandida albicans. Disruption ofchsD caused no defect in cell growth or morphology during the asexual cycle and caused no decrease in chitin content in hyphae. However, double disruption ofchsA andchsD caused a remarkable decrease in the efficiency of conidia formation, while double disruption ofchsC andchsD caused no defect. Thus it appears thatchsA andchsD serve redundant functions in conidia formation.     

Disruption of a Magnaporthe grisea cutinase gene.

Summary Using a one-step strategy to disrupt CUT1, a gene for cutinase, cut1 ^– mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3–12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB ⁺ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1 : : ArgB ⁺). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1:. Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1 ^– disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Fungal catabolic gene regulation: Molecular genetic analysis of theamdS gene ofAspergillus nidulans

Aspergillus nidulans is an excellent experimental organism for the study of gene regulation. Genetic and molecular analyses oftrans-acting andcis-acting mutations have revealed a complex pattern of regulation involving multiple independent controls. Expression of theamdS gene is regulated by thefacB andamdA genes which encode positively acting regulatory proteins mediating a major and a minor form of acetate induction respectively. The product of theamdR gene mediates omega amino acid induction ofamdS. The binding sites for each of these proteins have been localised throughamdS cis-acting mutations which specifically affect the interaction with the regulatory protein. The global controls of nitrogen metabolite repression and carbon catabolite repression regulate the expression of many catabolic genes, includingamdS. Nitrogen control is exerted through the positively actingareA gene product and carbon control is dependent on thecreA gene product. Each of the characterized regulatory genes encodes a DNA-binding protein which recognises particular sequences in theamdS promoter to activate or repress gene expression. In addition, there is evidence for other genetically uncharacterised proteins, including a CCAAT-binding complex, which interact with the 5 region of theamdS gene.     

Mutational analysis of the C-terminal region of AREA,the transcription factor mediating nitrogen metabolite repression inAspergillus nidulans

InAspergillus nidulans the positive-acting, wide domain regulatory geneareA mediates nitrogen metabolite repression. Previous analysis demonstrated that the C-terminal 153 residues of theareA product (AREA) are inessential for at least partial expression of most genes subject to regulation byareA. Paradoxically,areA ^r 2, a ?1 frameshift replacing the wild-type 122 C-terminal residues with a mutant peptide of 117 amino acids, leads to general loss of function. To determine the basis for theareA ^r 2 mutant phenotype, and as a means of delineating functional domains within the C-terminal region of AREA, we have selected and characterisedareA ^r 2 revertants. Deletion analysis, utilising direct gene replacement, extended this analysis. A mutantareA product truncated immediately after the last residue of the highly conserved GATA (DNA-binding) domain retains partial function. TheareA ^r 2 product retains some function with respect to the expression ofuaZ (encoding urate oxidase) and the mutant allele is partially dominant with respect to nitrate reductase levels. Consistent with theareA ^r 2 product having a debilitating biological activity, we have demonstrated that a polypeptide containing both the wild-type DNA-binding domain and the mutant C-terminus of AREA2 is able to bind DNA in vitro but no longer shows specificity for GATA sequences.     

Mutations affecting extracellular protease production in the filamentous fungusAspergillus nidulans

The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA.     

Genetic analysis of amdS transformants of Aspergillus niger and their use in chromosome mapping

Summary TheAspergillus nidulans gene coding for acetamidase (amdS) was introduced intoA. niger by transformation. Twelve Amd⁺ transformants were analysed genetically. TheamdS inserts were located in seven different linkage groups. In each transformant the plasmid was integrated in only a single chromosome. Our (non-transformed)A. niger strains do not grow on acetamide and are more resistant to fluoroacetamide than the transformants. Diploids hemizygous for theamdS insert have the Amd⁺ phenotype. We exploited the opportunity for two-way selection inA. niger: transformants can be isolated based on the Amd⁺ phenotype, whereas counter-selection can be performed using resistance to fluoroacetamide. On this basis we studied the phenotypic stability of the heterologousamdS gene inA. niger transformants as well as in diploids. Furthermore, we mapped the plasmid insert of transformant AT1 to the right arm of chromosome VI betweenpabA1 andcnxA1, providing evidence for a single transformational insert. The results also show that theamdS transformants ofA. niger can be used to localize non-selectable recessive markers and that the method meets the prerequisites for efficient mitotic mapping. We suggest the use ofamdS transformants for mitotic gene mapping in other fungi.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

ThehapC gene ofAspergillus nidulans is involved in the expression of CCAAT-containing promoters

The 5 regulatory region of theamdS gene ofAspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence which is required for setting the basal level ofamdS expression. Mobility shift studies have identified a factor inA. nidulans nuclear extracts which binds to this CCAAT sequence. InSaccharomyces cerevisiae theHAP3 gene encodes one component of a multisubunit complex that binds CCAAT sequences. A search of the EMBL and SwissProt databases has revealed anA. nidulans sequence with significant homology to theHAP3 gene adjacent to the previously cloned regulatory geneamdR. Sequencing of the remainder of this region has confirmed the presence of a gene, designatedhapC, with extensive homology toHAP3. The predicted amino acid sequence of HapC shows extensive identity to HAP3 in the central conserved domain, but shows little conservation in the flanking sequences. A haploid carrying ahapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially slowly on acetamide as a sole carbon and nitrogen source, indicating thathapC plays a role inamdS expression. In agreement with this notion, it has been shown that thehapC deletion results in reduced levels of expression of anamdS::lacZ reporter gene and this effect is particularly evident under conditions of carbon limitation. Nuclear extracts prepared from thehapC deletion mutant show no CCAAT binding activity to theamdS orgatA promoters, indicating thathapC may encode a component of the complex binding at this sequence.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

Regulation of acid phosphatases in anAspergillus niger pacC disruption strain

AnAspergillus niger strain has been constructed in which the pH-dependent regulatory gene,pacC, was disrupted. ThepacC gene ofA. niger, like that ofA. nidulans, is involved in the regulation of acid phosphatase expression. Disruptants were identified by a reduction in acid phosphatase staining of colonies. Southern analysis demonstrated integration of the disruption plasmid at thepacC locus and Northern analysis showed that the disruption strain produced a truncatedpacC mRNA of 2.2 kb (as compared to 2.8 kb in the wild type). The strain carrying thepacC disruption was used to assign thepacC gene to linkage group IV; this was confirmed by CHEF electrophoresis and Southern analysis. This strain further allowed us to determine which extracellular enzyme and transport systems are under the control ofpacC inA. niger. Expression of theA. niger pacC wild-type gene and the truncatedpacC gene showed that, in contrast to the auto-regulated wild-type expression, which was elevated only at alkaline pH, the truncatedpacC gene was deregulated, as high-level expression occurred regardless of the pH of the culture medium. Analysis of the phosphatase spectrum by isoelectric focussing and enzyme activity staining both in the wild-type and thepacC disruptant showed that at least three acid phosphatases are regulated by thepacC. For the single alkaline phosphatase no pH regulation was observed.     

The glucose repressor genecre1 ofTrichoderma: Isolation and expression of a full-length and a truncated mutant form

Thecre1 genes of the filamentous fungiTrichoderma reesei andT. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor genecreA ofAspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C₂H₂ type. Thecre1 promoters contain several sequence elements that are identical to the previously identified binding sites forA. nidulans CREA. Steady-state mRNA levels forcre1 of theT. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest thatcre1 expression may be autoregulated. TheT. reesei strain Rut-C30, a hyperproducer of cellulolytic enzymes, was found to express a truncated form of thecre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-lengthcre1 gene into this strain caused glucose repression ofcbh1 expression, demonstrating thatcre1 regulates cellulase expression.     

Transformation of the cultivated mushroom,Agaricus bisporus,to hygromycin B resistance

Application of biotechnology to the cultivated mushroom,Agaricus bisporus, has been hampered thus far by the lack of a transformation system. Here, transformation of both a homo- and a heterokaryotic strain ofA. bisporus to hygromycin B resistance is described. Transforming DNA was integrated into theA. bisporus genome and stably maintained throughout vegetative growth. Transformants of the heterokaryotic strain formed transgenic fruiting bodies. Promoters derived from the unrelated ascomyceteAspergillus nidulans and fromA. bisporus itself, were able to drive expression of the hygromycin B resistance gene. Expression controlled by a fragment of 265 bp from theA. bisporus GPD promoter was sufficient to generate transformants. However, transformation efficiency was not enhanced by using this homologous promoter.     

Studies of partially repressed mutants at the tamA and areA loci in Aspergillus nidulans

Summary Mutants, designated tamA ^r, have been isolated on the basis of simultaneous resistance to toxic analogues thiourea, aspartate hydroxamate and chlorate with L-alanine as the sole nitrogen source. tamA ^r mutants are also resistant to methylammonium. This resistance of tamA ^r mutants is correlated with partially repressed activity of a number of enzyme and transport systems regulated by ammonium. Furthermore, tamA ^r mutants have low NADP-glutamate dehydrogenase (NADP-GDH) activity and also efflux ammonium under certain growth conditions.Mutants at the areA locus (areA ^r) have also been isolated on the basis of resistance to these analogues, with nitrate or L-aspartate as the nitrogen source. These, similar to tamA ^r lesions, result in resistance to methylammonium and are partially repressed for ammonium repressible systems, but in contrast to tamA ^r, areA ^r alleles have wild-type NADP-GDH activity and normal ammonium efflux. tamA ^r and areA ^r mutants grow as wild type on all nitrogen or carbon sources tested, are recessive, and appear to be epistatic to all other mutations (gdhA1, meaA8 and meaB6) which result in derepressed levels of ammonium regulated system. Whereas tamA ^r and areA ^r phenotypes are additive, tamA ^r is epistatic to areA ^d phenotype.