Abstract
This study focuses on the cloning and characterization of the major nitrogen regulator element from the ectomycorrhizal fungus Tuber borchii, TbNre1. Sequence analysis of the predicted protein and complementation experiments in Neurospora crassa demonstrated that the cloned gene is orthologous to areA/nit-2 gene. Transcriptional expression investigations by real-time RT-PCR showed TbNre1 up-regulation in the presence of nitrate or in the absence of nitrogen during free-living mycelium growth. On the contrary, TbNre1 mRNA levels remained at basal values in the presence of preferred nitrogen sources like ammonium and glutamine. Furthermore, TbNre1 mRNA was found to be up-regulated during T. borchii and T. platyphyllos interaction. All these data suggest that the regulatory protein TBNRE1 could play a major role in regulating N metabolism genes of T. borchii in the free living mycelium and in T. borchii–T. platyphyllos interaction. Finally, the possible role of the transcription factor TBNRE1 in the induction of proteases and virulence-like genes, necessary in ectomycorrhizal establishment, was also discussed.
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Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Attwood TK, Beck ME (1994) PRINTS-a protein motif fingerprint database. Prot Eng 7:841–848
Bailly J, Debaud JC, Verner MC, Plassard C, Chalot M, Marmeisse R, Fraissinet-Tachet L (2007) How does a symbiotic fungus modulate expression of its host-plant nitrite reductase? New Phytol 175:155–165
Ballario P, Vittorioso P, Magrelli A, Talora C, Cabibbo A, Macino G (1996) White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. EMBO J 15:1650–1657
Bücking H (2004) Phosphate absorption and efflux of three ectomycorrhizal fungi as affected by external phosphate, cation and carbohydrate concentrations. Mycol Res 108:599–609
Buscot F, Munch JC, Charcosset JY, Gardes M, Nehls U, Hampp R (2000) Recent advances in exploring physiology and biodiversity of ectomycorrhizas highlight the functioning of these symbioses in ecosystems. FEMS Microbiol Rev 24:601–614
Caddick MX, Peters D, Platt A (1994) Nitrogen regulation in fungi. Anton Leeuw 65:169–177
Chalot M, Blaudez D, Brun A (2006) Ammonia: a candidate for nitrogen transfer at the mycorrhizal interface. Trends Plant Sci 11:263–266
Crawford NM, Arst HN Jr (1993) The molecular genetics of nitrate assimilation in fungi and plants. Ann Rev Genet 27:115–146
Dabas N, Morschhäuser J (2008) A transcription factor regulatory cascade controls secreted aspartic protease expression in Candida albicans. Mol Microbiol 69:586–602
De Bellis R, Agostini D, Piccoli G, Vallorani L, Potenza L, Polidori E, Sisti D, Amoresano A, Pucci P, Arpaia G, Macino G, Balestrini R, Bonfante P, Stocchi V (1998) The tbf-1 gene from the white truffle Tuber borchii codes for a structural cell wall protein specifically expressed in fruitbody. Fungal Genet Biol 25:87–99
Ducic T, Parlade J, Polle A (2008) The influence of the ectomycorrhizal fungus Rhizopogon subareolatus on growth and nutrient element localisation in two varieties of Douglas fir (Pseudotsuga menziesii var. menziesii and var. glauca) in response to manganese stress. Mycorrhiza 18:227–239
Froeliger EH, Carpenter BE (1996) NUT1, a major nitrogen regulatory gene in Magnaporthe grisea, is dispensable for pathogenicity. Mol Gen Genet 251:647–656
Fu YH, Marzluf GA (1990) Nit-2, the major nitrogen regulatory gene of Neurospora crassa, encodes a protein with a putative zinc finger DNA-binding domain. Mol Cell Biol 10:1056–1065
Gente S, Poussereau N, Fevre M (1999) Isolation and expression of a nitrogen regulatory gene, nmc, of Penicillium roqueforti. FEMS Microbiol Lett 175:291–297
Guescini M, Pierleoni R, Palma F, Zeppa S, Vallorani L, Potenza L, Sacconi C, Giomaro G, Stocchi V (2003) Characterization of the Tuber borchii nitrate reductase gene and its role in ectomycorrhizae. Mol Genet Genom 269:807–816
Guescini M, Zeppa S, Pierleoni R, Sisti D, Stocchi L, Stocchi V (2007) The expression profile of the Tuber borchii nitrite reductase suggests its positive contribution to host plant nitrogen nutrition. Curr Genet 51:31–41
Henikoff S, Henikoff JG (1994) Protein family classification based on searching a database of blocks. Genomics 19:97–107
Jargeat P, Gay G, Debaud JC, Marmeisse R (2000) Transcription of a nitrate reductase gene isolated from the symbiotic basidiomycete fungus Hebeloma cylindrosporum does not require induction by nitrate. Mol Gen Genet 263:948–956
Jargeat P, Rekangalt D, Verner MC, Gay G, Debaud JC, Marmeisse R, Fraissinet-Tachet L (2003) Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum. Curr Genet 43:199–205
Kudla B, Caddick MX, Langdon T, Martinez-Rossi NM, Bennett CF, Sibley S, Davies RW, Arst HN Jr (1990) The regulatory gene areA 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
Lau G, Hamer JE (1996) Regulatory Genes Controlling MPG1 Expression and Pathogenicity in the Rice Blast Fungus Magnaporthe grisea. Plant Cell 8:771–781
Lilleskov EA, Fahey TJ, Horton TR, Lovett GM (2002) Belowground ectomycorrhizal fungal community change over a nitrogen deposition gradient in Alaska. Ecology 83:104–115
Lindahl BD, Ihrmark K, Boberg J, Trumbore SE, Hogberg P, Stenlid J, Finlay RD (2007) Spatial separation of litter decomposition and mycorrhizal nitrogen uptake in a boreal forest. New Phytol 173:611–620
Martin F, Cliquet J, Stewart G (2001) Nitrogen acquisition and assimilation in mycorrhizal symbioses. In: Lea P, Morot-Gaudry JF (eds) The assimilation of nitrogen in plants. Springer, Berlin, pp 147–166
Martin F, Aerts A, Ahren D, Brun A, Danchin EG, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buee M, Brokstein P, Canback B, Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbe J, Lin YC, Legue V, Le Tacon F, Marmeisse R, Melayah D, Montanini B, Muratet M, Nehls U, Niculita-Hirzel H, Oudot-Le Secq MP, Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, Kues U, Lucas S, Van de Peer Y, Podila GK, Polle A, Pukkila PJ, Richardson PM, Rouze P, Sanders IR, Stajich JE, Tunlid A, Tuskan G, Grigoriev IV (2008) The genome of Laccaria bicolor provides insights into mycorrhizal symbiosis. Nature 452:88–92
Marzluf GA (1997) Genetic regulation of nitrogen metabolism in the fungi. Microbiol Mol Biol Rev 61:17–32
Merika M, Orkin SH (1993) DNA-binding specificity of GATA family transcription factors. Mol Cell Biol 13:3999–4010
Miozzi L, Balestrini R, Bolchi A, Novero M, Ottonello S, Bonfante P (2005) Phospholipase A2 up-regulation during mycorrhiza formation in Tuber borchii. New Phytol 167:229–238
Molina R (1979) Ectomycorrhizal inoculation of containerized Douglas-fir and lodgepole pine seedlings with six isolates of Pisolithus tinctorius. Forest Sci 25:585–590
Montanini B, Moretto N, Soragni E, Percudani R, Ottonello S (2002) A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii. Fungal Genet Biol 36:22–34
Montanini B, Betti M, Marquez AJ, Balestrini R, Bonfante P, Ottonello S (2003) Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus. Biochem J 373:357–368
Montanini B, Gabella S, Abba S, Peter M, Kohler A, Bonfante P, Chalot M, Martin F, Ottonello S (2006a) Gene expression profiling of the nitrogen starvation stress response in the mycorrhizal ascomycete Tuber borchii. Fungal Genet Biol 43:630–641
Montanini B, Viscomi AR, Bolchi A, Martin Y, Siverio JM, Balestrini R, Bonfante P, Ottonello S (2006b) Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete. Biochem J 394:125–134
Morozov IY, Galbis-Martinez M, Jones MG, Caddick MX (2001) Characterization of nitrogen metabolite signalling in Aspergillus via the regulated degradation of areA mRNA. Mol Microbiol 42:269–277
Pellier AL, Lauge R, Veneault-Fourrey C, Langin T (2003) CLNR1, the AREA/NIT2-like global nitrogen regulator of the plant fungal pathogen Colletotrichum lindemuthianum is required for the infection cycle. Mol Microbiol 48:639–655
Perez-Garcia A, Snoeijers SS, Joosten MH, Goosen T, De Wit PJ (2001) Expression of the Avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by the global nitrogen response factor NRF1. Mol Plant Microbe In 14:316–325
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45
Pierleoni R, Vallorani L, Sacconi C, Sisti D, Giomaro G, Stocchi V (2001) Evaluation of the enzymes involved in primary nitrogen metabolism in Tilia platyphyllos–Tuber borchii ectomycorrhizae. Plant Physiol Biochem 39:1111–1114
Platt A, Langdon T, Arst HN Jr, Kirk D, Tollervey D, Sanchez JM, Caddick MX (1996) Nitrogen metabolite signalling involves the C-terminus and the GATA domain of the Aspergillus transcription factor AREA and the 3′ untranslated region of its mRNA. EMBO J 15:2791–2801
Read D (1999) Mycorrhiza—the state of the art. In: Varma A, Hock B (eds) Mycorrhiza: structure, function, molecular biology and biotecnology. Springer, Berlin, pp 3–34
Saltarelli R, Ceccaroli P, Vallorani L, Zambonelli A, Citterio B, Malatesta M, Stocchi V (1998) Biochemical and morphological modifications during the growth of Tuber borchii mycelium. Mycol Res 102:403–409
Sambrook J, Russel DW (2001) Molecular cloning. A laboratory manual, 3rd edn, vol 1. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 50–58
Scazzocchio C (2000) The fungal GATA factors. Curr Opin Microbiol 3:126–131
Selle A, Willmann M, Grunze N, Gessler A, Weiss M, Nehls U (2005) The high-affinity poplar ammonium importer PttAMT1.2 and its role in ectomycorrhizal symbiosis. New Phytol 168:697–706
Snoeijers SS, Vossen P, Goosen T, Van den Broek HW, De Wit PJ (1999) Transcription of the avirulence gene Avr9 of the fungal tomato pathogen Cladosporium fulvum is regulated by a GATA-type transcription factor in Aspergillus nidulans. Mol Gen Genet 261:653–659
Snoeijers SS, Pérez-Garcia A, Joosten MH, De Wit PJ (2000) The effect of nitrogen on disease development and gene expression in bacterial and fungal pathogens. Eur J Plant Pathol 106:493–506
Soragni E, Bolchi A, Balestrini R, Gambaretto C, Percudani R, Bonfante P, Ottonello S (2001) A nutrient-regulated, dual localization phospholipase A(2) in the symbiotic fungus Tuber borchii. EMBO J 20:5079–5090
Tao Y, Marzluf GA (1999) The NIT2 nitrogen regulatory protein of Neurospora: expression and stability of nit-2 mRNA and protein. Curr Genet 36:153–158
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Tudzynski B, Homann V, Feng B, Marzluf GA (1999) Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi. Mol Gen Genet 261:106–114
Vallorani L, Polidori E, Sacconi C, Agostini D, Pierleoni R, Piccoli G, Zeppa S, Stocchi V (2002) Biochemical and molecular characterization of NADP-glutamate dehydrogenase from the ectomycorrhizal fungus Tuber borchii Vittad. New Phytol 154:779–790
Zeppa S, Potenza L, Polidori E, Guescini M, Agostini D, Giomaro G, Stocchi V (2001) Cloning and characterisation of a polyubiquitin gene from the ectomycorrhizal fungus Tuber borchii Vittad. Curr Genet 40:49–53
Acknowledgments
We are very grateful to Prof. Viotti A. for kindly providing the mycelium genomic DNA library, Marzluf G.A. for the gift of nit-2 RIP-350 mutant strain of N. crassa, Ballario P. and Grimaldi B. for kindly providing the pMYX2 vector.
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Communicated by U. Kües.
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Guescini, M., Stocchi, L., Sisti, D. et al. Characterization and mRNA expression profile of the TbNre1 gene of the ectomycorrhizal fungus Tuber borchii . Curr Genet 55, 59–68 (2009). https://doi.org/10.1007/s00294-008-0222-x
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DOI: https://doi.org/10.1007/s00294-008-0222-x