SummaryAREA (NIT2) is a general transcription factor involved in derepression of numerous genes responsible for nitrogen utilization in Gibberella fujikuroi and many other fungi. We have previously shown that the deletion of areA-GF resulted in mutants with significantly reduced gibberellin (GA) production. Here we demonstrate that the expression level of six of the seven GA biosynthesis genes is drastically reduced in mutants lacking areA . Furthermore, we show that, despite the fact that GAs are nitrogen-free diterpenoid compounds, which are not obviously involved in nitrogen metabolism, AREA binds directly to the promoters of the six N-regulated genes. The binding of AREA was analysed in more detail using the promoter of one of the GA-biosynthesis genes encoding the ent -kaurene oxidase ( P450-4 ). Deletion/mutation analysis of the P450-4 promoter fused to the Escherichia coli uidA gene, which encodes b b b b -glucuronidase, allowed the in vivo identification of functional GATA motifs. We have also analysed the nmr gene of G . fujikuroi ( nmr-GF ) which has high similarity to the Neurospora crassa nmr-1 and Aspergillus nidulans nmrA genes, both involved in nitrogen metabolite repression. In contrast to our expectation, deletion of nmr-GF did not result in significant derepression of the GA biosynthesis genes in the presence of ammonium, glutamine or glutamate. Overexpression of the nmr-GF gene fused to the strong promoter of the G. fujikuroi glutamine synthetase ( gs ) gene revealed only a very slight repression of the nitrate reductase ( niaD ) gene, resulting in weak resistance to chlorate. Surprisingly, this effect was only observed in the presence of high amounts of glutamate; cultivation on ammonium failed to induce any resistance to chlorate. Despite the limited effect of gene replacement and overexpression of nmr-GF on the nitrogen metabolism of G. fujikuroi itself, the gene fully restored nitrogen metabolite repression in A. nidulans and N. crassa nmr mutants. Therefore, we postulate that, in contrast to A. nidulans and N. crassa , NMR does not function independently as the main modulator of AREA in G. 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 ®nger DNA-binding domain that is at least 98% identical to the zinc ®nger 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 A mutants, gene replacement experiments were performed using a linearized replacement vector carrying the hygromycin B phosphotransferase (hph) gene. The replacement of the zinc ®nger by the hygromycin cassette resulted in transformants which were unable to utilize nitrogen sources other than ammonium and glutamine, and gave signi®cantly reduced gibberellin production yields. Complementation of such a mutant with the wild-type gene led to the full recovery of gibberellin production.Key words Nitrogen regulation á Gibberella fujikuroi á areA gene á Gibberellin biosynthesis Ó Springer-Verlag 1999 Communicated by C. A. M. J. J. ven den Hondel B. Tudzynski (8) á V. Homann

The rice pathogen, Gibberella fujikuroi, produces large amounts of gibberellins, a group of natural plant hormones, which induce the superelongation (bakanae) disease of rice. Gibberellins are diterpenoid compounds which are synthesized via the isoprenoid pathway. Here we report the isolation and molecular characterization of the geranylgeranyl diphosphate synthase (ggs) gene from G. fujikuroi. Geranylgeranyl diphosphate synthase is a key enzyme in isoprenoid biosynthesis. Southern blot analysis showed that G. fujikuroi has a single copy of the ggs gene, which is not linked to the farnesyl diphosphate synthase gene. This indicates that the genes of the isoprenoid pathway are not clustered in the fungal genome. The ggs gene is not interrupted by an intron and codes for a polypetide of 418 amino acids. Peptide sequence comparison showed a high degree of similarity to the corresponding Neurospora crassa gene (al-3). However, transcription analyses revealed that the ggs gene, in contrast to the analogous N. crassa gene, is not regulated by blue light. Ammonium and glucose did not affect the transcription of the G. fujikuroi ggs gene, indicating that it is not subject to nitrogen and carbon catabolite repression. The G. fujikuroi gene complements a N. crassa al-3 mutant.

Farnesylpyrophosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis. Several classes of essential metabolites, including sterols, quinones, carotenoids and gibberellins, are terpenoids with high biological activity. The structural gene for FPP synthase was isolated from two ascomycete fungi, Neurospora crassa and Gibberella fujikuroi. A comparative analysis of the nucleotide sequences of both FPPS genes revealed the presence of introns at the same positions at the 5' end of the coding regions. Furthermore, the most conserved region of the gene was isolated from two other plant pathogenic fungi, Sphaceloma manihoticola and Claviceps purpurea, by PCR. Sequence analysis showed a high degree of similarity between the deduced proteins of all known FPP synthase genes. In contrast to animals, all analyzed fungi contain a single copy of the gene, although FPP is the precursor for essential sterol and quinone biosynthesis and secondary metabolites, such as gibberellins, as well. Transcription analysis in different light regimes has shown that the FPPS genes in G. fujikuroi and N. crassa are not regulated by light induction.

Salivary calcium plays a vital role in bio-mineralization of dental enamel and exposed dentin. In order to elucidate the yet unknown cellular and molecular mechanisms of calcium secretion in human salivary glands the presence of various relevant plasma membrane transport systems for calcium were investigated. Using an RT-PCR approach, expression of the epithelial calcium channel (CaT-Like), the calcium binding protein (calbindin-2), the endoplasmic reticulum pumps (SERCA-2 and -3), and the plasma membrane calcium ATPases (PMCA-1, -2, and -4), were found in parotid and submandibular glands. Immunohistochemistry revealed that CaT-Like is located in the basolateral plasma membrane of acinar cells; while calbindin-2, SERCA-2 and SERCA-3 were found inside the acinar cells; and PMCA-2 was found in the apical membrane and in the secretory canaliculi between the cells. Based on these findings, we propose the following model of calcium secretion in human salivary glands: (1) calcium enters the acinar cell at the basolateral side via calcium channel CaT-Like (calcium influx); (2) intracellular calcium is taken up into the endoplasmic reticulum by SERCA-2 and possibly SERCA3 or bound to calbindin-2 (intracellular calcium pool); and (3) calcium is secreted by PMCAs at the apical plasma membrane (calcium efflux).