Cross-species hybridization with Fusarium verticillioides microarrays reveals new insights into Fusarium fujikuroi nitrogen regulation and the role of AreA and NMR

Abstract

In filamentous fungi, the GATA-type transcription factor AreA plays a major role in the transcriptional activation of genes needed to utilize poor nitrogen sources. In Fusarium fujikuroi, AreA also controls genes involved in the biosynthesis of gibberellins, a family of diterpenoid plant hormones. To identify more genes responding to nitrogen limitation or sufficiency in an AreA-dependent or -independent manner, we examined changes in gene expression of F. fujikuroi wild-type and DeltaareA strains by use of a Fusarium verticillioides microarray representing approximately 9,300 genes. Analysis of the array data revealed sets of genes significantly down- and upregulated in the areA mutant under both N starvation and N-sufficient conditions. Among the downregulated genes are those involved in nitrogen metabolism, e.g., those encoding glutamine synthetase and nitrogen permeases, but also those involved in secondary metabolism. Besides AreA-dependent genes, we found an even larger set of genes responding to N starvation and N-sufficient conditions in an AreA-independent manner. To study the impact of NMR on AreA activity, we examined the expression of several AreA target genes in the wild type and in areA and nmr deletion and overexpression mutants. We show that NMR interacts with AreA as expected but affects gene expression only in early growth stages. This is the first report on genome-wide expression studies examining the influence of AreA on nitrogen-responsive gene expression in a genome-wide manner in filamentous fungi.

FIG. 1.

Functional distribution of nitrogen-regulated genes. (A) Pie chart representing the functions of genes upregulated by nitrogen addition. (B) Pie chart representing the functions of genes repressed by nitrogen addition. The division of genes into several functional categories was accomplished following the MIPS functional database catalogue (http://mips.gsf.de/proj/funcatDB). met., metabolism.

FIG. 2.

Genes with expression levels affected by nitrogen addition. (A) Northern blot experiments showing genes upregulated by glutamine addition. (B) Northern blot experiments showing genes downregulated by glutamine addition. The WT was grown for 5 days in synthetic ICI medium containing 1.8 g/liter glutamine and then shifted into ICI medium without nitrogen (no N). After 4 h, glutamine was added to one half of the flasks to a final concentration of 9.2 g/liter (Gln), and the mycelia were harvested after 2 h. Probes used for hybridization are listed in Table 2. 28S and 18S rRNA was used as the loading control.

FIG. 3.

Most nitrogen-repressed genes are independent of AreA. (A) Venn diagram demonstrating the share of AreA in the regulation of nitrogen metabolite repression. The set of genes downregulated in the ΔareA mutant under nitrogen starvation conditions was compared with the set of genes repressed by the addition of glutamine to the media. “Classic” AreA target genes are genes repressed by nitrogen addition and downregulated in the ΔareA mutant. (B) Comparison of the distribution of AreA-dependent and -independent genes into the functional categories shown in Fig. 1. Gray bars show genes repressed by nitrogen addition and downregulated in the ΔareA mutant; white bars show genes repressed by nitrogen in an AreA-independent manner. The values for the gray bars and the white bars add up to the total number of nitrogen-repressed genes. met., metabolism.

FIG. 4.

Expression pattern of nitrogen-regulated and AreA-dependent genes. (A) Nitrogen-repressed genes not affected by the areA deletion. (B) Genes upregulated in the ΔareA mutant under starvation conditions. (C) Genes downregulated in the areA mutant. The categories (Cat.) are as shown in Table 2. Strains and culture conditions were the same as described for Fig. 1. Probes used for hybridization are listed in Table 2. 28S and 18S rRNA was used as the loading control.

FIG. 5.

Nitrate reductase activity is affected in areA and nmr mutant strains. All strains were grown in media containing 9.2 g/liter l-glutamine as the nitrogen source with (+) or without (−) potassium chlorate (10 mM). Pictures were taken after 2 (medium without chlorate) or 4 (chlorate medium) days of incubation at 28°C. OE::areA, areA overexpression mutant (areA transcription regulated by the glnA promoter); OE::nmr, nmr overexpression mutant (nmr regulated by the glnA promoter).

FIG. 6.

Yeast two-hybrid experiment showing that NMR interacts with the C terminus of AreA. Transformants were dropped in 10-fold dilution steps as indicated on SD medium without tryptophan and leucine (SD −W −L) and on SD without tryptophan, leucine, and histidine and with the addition of 50 mM 3-amino triazole (SD −W −L −H + 3-AT). The decreasing concentration is indicated by a triangle. The positive control was transformation with pBD-WT and pAD-WT (Stratagene). AD, transformation with pAD-GAL4-2.1 (Stratagene); AD-Nmr, transformation with pAD-Nmr, containing the full-length nmr cDNA; BD, transformation with pBD-GAL4 Cam (Stratagene); BD-AreA, transformation with pBD-AreA, containing the full-length areA cDNA; BD-AreA-ZF, transformation with pBD-AreA-ZF, containing the C-terminal ZF domain of AreA.

FIG. 7.

Expression of AreA target genes in areA and nmr mutant strains. Abbreviations for strains are the same as used in Fig. 5. Strains were incubated as described for Fig. 2. Sodium nitrate (NO₃) was added to a final concentration of 10 mM. Probes used for hybridization are listed in Table 2 except the gene coding for the nitrate reductase, niaD (CAA62232), and the gene coding for the bifunctional ent-copalyl diphosphate/ent-kaurene synthase, cps/ks (Q9UVY5).

FIG. 8.

The partial deregulation of AreA target genes in the Δnmr mutant is time dependent. areA and nmr mutant strains (Fig. 5) were grown for 24 h and 48 h, respectively, in synthetic ICI medium containing 0.9 g/liter l-glutamine as the nitrogen source. Probes used for hybridization are listed in Table 2, except the gene coding for the geranylgeranyldiphosphate synthase, ggs2 (CAA75568), and the gene coding for the bifunctional ent-copalyl diphosphate/ent-kaurene synthase, cps/ks (Q9UVY5).