The Gibberella fujikuroi niaD gene, encoding nitrate reductase, has been isolated and used to develop an efficient homologous transformation system. A cosmid vector designated pGFniaD was generated based on niaD selection and shown to give comparable transformation efficiencies. Using pGFniaD, a genomic library was prepared and used for genetic transformations, giving frequencies of up to 200 transformants per μg DNA. Of 15 transformants analysed by Southern blots, six showed homologous integration whilst the remaining nine integrated at heterologous sites, indicating that the vector may be used reliably for both types of integration. The system therefore may be used both for self-cloning of gibberellin biosynthetic genes on the basis of complementation of defective mutants, and also for gene disruption experiments. Electrophoretic karyotype determination suggested at least 11 chromosomes ranging from 2 to 6 Mb, the total genome size being at least 37 Mb. The niaD gene was assigned to chromosome V by Southern blot analysis. The niaD gene is interrupted by one intron, and remarkably the promoter sequence, but not the 3′ untranslated sequence, is highly homologous to that of the corresponding Fusarium oxysporum gene. This situation appears to be unique with respect to the promoter regions of corresponding genes in related species of filamentous fungi.
AvalosJ.,
CasadesusJ.,
Cerda-OlmedoE.1994; Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N-nitro-N-nitrosoguanidine. Appi Environ Microbiol 49:187–191
BanksG. R.,
SheltonP. A.,
KanugaN.,
HoldenD. W.,
SpanosA.1993; The Ustilago maydis nari gene encoding nitrate reductase activity: structural and transcriptional regulation. Gene 131:69–78
BearderJ. R.1983; In vivo diterpenoid biosynthesis in Gibberella fujikuroi: the pathway after ent-kaurene. The Biochemistry and Physiology of Gibberellins251–387CrozierA.
New York: Praeger Publications;
BrücknerB.,
UnklesS. E.,
WeltringK.-M.,
KinghornJ. R.1992; Transformation of Gibberella fujikuroi: effect of the Aspergillus nidulans AMAI sequence on frequency and integration. Curr Genet 22:314–316
Bu’LockJ. D.,
DetroyR. W.,
HostalekZ.,
Munim-al-ShakarchiA.1974; Regulation of secondary metabolite biosynthesis in Gibberella fujikuroi
. Trans Br Mycol Soc 62:377–389
BurgerG.,
StraussJ.,
ScazzocchioC.,
LangB. F.1991; nirA the pathway-specific regulatory gene of nitrate assimilation in Aspergillus nidulans encodes a putative GAL4-type zinc finger protein and contains four introns in highly conserved regions. Mol Cell Biol 11:5746–5755
CandauR.,
AvalosJ.,
Cerda-OlmedoE.1991; Gibberellins and carotenoids in the wild type and mutants of Gibberella fujikuroi
. Appl Environ Microbiol 57:3378–3382
DiolezA.,
LanginT.,
Gerlinger C.,
BrygooY.,
DaboussiM.-J.1993; The nia gene of Fusarium oxysporum : isolation, sequence and development of a homologous transformation system. Gene 131:61–67
FuY.-H.,
KneessiJ. Y.,
MarzlufG. A.1989; Isolation of nit-4, the minor regulatory gene which mediates nitrate induction in Neurospora crassa
. J Bacteriol 171:4067–4070
JohnstoneI. L.,
MacCabeP. C.,
GreavesP.,
ColeG. E.,
BrowM. A. D.,
GurrS. J.,
UnklesS. E.,
ClutterbuckA. J.,
KinghornJ. R.,
InnisM. A.1990; Isolation and characterisation of the crnA–niiA–niaD gene cluster for nitrate assimilation in Aspergillus nidulans
. Gene 90:181–192
KudlaB.,
CaddickM. X.,
LangdonT.,
Martinez-RossiN. M.,
BennettC. F.,
SibleyS.,
DaviesR. W.,
ArstH. N.1990; The regulatory gene are A mediating nitrogen metabolite repression in Aspergillus nidulans Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–1364
OkamotoP. M.,
FuY.-H.,
MarzlufG. A.1991; nit-3, the structural gene of nitrate reductase in Neurospora crassa : nucleotide sequence and regulation of mRNA synthesis and turnover. Mol & Gen Genet 227:213–223
PitelD. W.,
ViningL. C.,
ArsenaultG. P.1971a; Improved methods for preparing pure gibberellins from cultures of Gibberella fujikuroi Isolation by adsorption or partition chromatography on silicic acid and by partition chromatography on Sephadex columns. Can J Biochem 49:185–193
PitelD. W.,
ViningL. C.,
ArsenaultG. P.1971b; Biosynthesis of gibberellins in Gibberella fujikuroi The sequence after gibberellin GA4. Can J Biochem 49:194–200
Sanchez-FernandezR.,
UnklesS. E.,
CampbellE. I.,
MacroJ. A.,
Cerda-OlmedoE.,
KinghornJ. R.1991; Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus nidulans niaD gene encoding nitrate reductase. Mol & Gen Genet 225:231–233
SmithC. L.,
MatsumotoT.,
NiwaO.,
KlcoS.,
FanJ.-B.,
YanagidaM.,
CantorC. R.1987; An electrophoretic karyotype for Schizosaccharomyes pombe by pulsed field gel electrophoresis. Nucleic Acids Res 15:4481–4489
UnklesS. E.,
CampbellE. I.,
PuntP. J.,
HawkerK. L,
ContrerasR.,
HawkinsA. R.,
van den HondelC. A. M. J. J.,
KinghornJ. R.1992; The Aspergillus niger niaD gene encoding nitrate reductase : upstream nucleotide and amino acid comparisons. Gene 111:149–155
WilliamsR. S. B.,
DavisM. A.,
HowlettB. J.1994; Nitrate reductase of the ascomycetous fungus, Leptospbaeria maculans : gene sequence and chromosomal location. Mol & Gen Genet 244:1–8