Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Arst HN, Scazzocchio C (1985) Formal genetics and molecular biology of the control of gene expression inAspergillus nidulans. In: Bennett JW, Lasure LL (eds) Gene manipulations in fungi. Academic Press, New York, pp 309–343
Caddick MX (1992) Characterization of a majorAspergillus regulatory gene,areA. In: Stahl U, Tudzynski P (eds) Molecular biology of filamentous fungi. VCH, Weinheim, pp 141–152
Cohen BL (1972) Ammonium repression of extracellular protease inAspergillus nidulans. J Gen Microbiol 71:293–299
Crawford MS, Arst HN (1993) The molecular genetics of nitrate assimilation in fungi and plants. Annu Rev Genet 27:115–146
Crawford MS, Chumley FG, Weaver CG, Valent B (1986) Characterization of the heterokaryotic and vegetative diploid phases ofMagnaporthe grisea. Genetics 114:1111–1129
Davis MA, Hynes MJ (1987) Complementation ofareA − regulatory gene mutations ofAspergillus nidulans by the heterologous regulatory genenit-2 ofNeurospora crassa. Proc Natl Acad Sci USA 84:3753–3757
Davis MA, Hynes MJ (1991) Regulatory circuits inAspergillus nidulans. In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, New York, pp 151–189
Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395
Dobinson KF, Harris RE, Hamer JE (1993)Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungusMagnaporthe grisea. Mol Plant-Microbe Interact 6:114–126
Dunn-Coleman NS, Tomsett AB, Garrett RH (1979) Nitrogen metabolite repression of nitrate reductase inNeurospora crassa: effect of thegln-1a locus. J Bacteriol 139:697–700
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Feng B, Xiao X, Marzluf GA (1993) Recognition of specific nucleotide bases and cooperative DNA binding by the trans-acting nitrogen regulatory protein NIT2 ofNeurospora crassa. Nucleic Acids Res 21:3989–3996
Fu YH, Marzluf GA (1990a)nit-2, the major nitrogen regulatory gene ofNeurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol 10:1056–1065
Fu YH, Marzluf GA (1990b) Site-directed mutagenesis of the ‘zic finger’ DNA-binding domain of the nitrogen-regulatory protein NIT2 ofNeurospora. Mol Microbiol 4:1847–1852
Froeliger EH, Munoz-Rivas AM, Specht CA, Ullrich RC, Novotny CP (1989) The isolation of specific genes from the basidiomyceteSchizophyllum commune. Curr Genet 12:547–554
Kafer E (1977) Meiotic and mitotic recombination inAspergillus and its chromosomal aberrations. Adv Genet 19:A1-A28
Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF, Sibley S, Davies RW, Arst HN (1990) The regulatory geneareA mediating nitrogen metabolite repression inAspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. EMBO J 9:1355–1364
Leung H, Borromeo ES, Bernardo MA, Notteghem JL (1988) Genetic analysis of virulence in the rice blast fungusMagnaporthe grisea. Phytopathol 78:1227–1233
Magasanik B (1992) Regulation of nitrogen utilization. In: Jones EW, Pringle JR, Broach JR (eds) The molecular and cellular biology of the yeastSaccharomyces: Gene expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp 283–317
Marzluf GA (1981) Regulation of nitrogen metabolism and gene expression in fungi. Microbiol Rev 45:437–461
Marzluf GA (1993) Regulation of sulfur and nitrogen metabolism in filamentous fungi. Annu Rev Microbiol 47:31–55
Minehart PL, Magasanik B (1991) Sequence and expression ofGLN3, a positive nitrogen regulatory gene ofSaccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol 11:6216–6228
Müller B, Russo VEA (1989) Nitrogen starvation or glucose limitation induces conidiation in constantly shaken liquid cultures ofNeurospora crassa. Fungal Genet Newsl 36:58–60
Orr-Weaver TL, Szostak JW (1985) Fungal recombination. Microbiol Rev 49:33–58
Peters GP, Caddick MX (1994) Direct analysis of native and chimeric GATA specific DNA binding proteins fromAspergillus nidulans. Nucleic Acids Res 22:5164–5172
Rothstein RJ (1983) One-step gene disruption in yeast. Methods Enzymol 101:202–211
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schowalter DB, Sommer SS (1989) The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Anal Biochem 177:90–94
Talbot NJ, Ebbole DJ, Hamer JE (1993) Identification and characterization ofMPG1, a gene involved in pathogenicity from the rice blast fungusMagnaporthe grisea. Plant Cell 5:1575–1590
Timberlake WE (1990) Molecular genetics ofAspergillus development. Annu Rev Genet 24:5–36
Timberlake WE (1991) Cloning and analysis of fungal genes. In: Bennett JW, Lasure LL (eds) More gene manipulations in fungi. Academic Press, New York, pp 51–85
Valent B, Chumley FG (1991) Molecular genetic analysis of the rice blast fungus,Magnaporthe grisea. Annu Rev Phytopathol 29:443–467
Valent B, Farrall L, Chumley FG (1991)Magnaporthe grisea genes for pathogenicity and virulence identified through a series of backcrosses. Genetics 127:87–101
Van den Ackerveken GFJM, Dunn RM, Cozijnsen AJ, Vossen JPMJ, Van den Broek HWJ, DeWit PJGM (1994) Nitrogen limitation induces expression of the avirulence geneavr9 in the tomato pathogenCladosporium fulvum. Mol Gen Genet 243:277–285
Wei Z-M, Sneath BJ, Beer SV (1992) Expression ofErwinia amylovora hrp genes in response to environmental stimuli. J Bacteriol 174:1875–1882
Author information
Authors and Affiliations
Additional information
Communicated by E. Cerdá-Olmedo
Rights and permissions
About this article
Cite this article
Froeliger, E.H., Carpenter, B.E. & Froeliger, E. NUT1, a major nitrogen regulatory gene inMagnaporthe grisea, is dispensable for pathogenicity. Molec. Gen. Genet. 251, 647–656 (1996). https://doi.org/10.1007/BF02174113
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02174113