Cross-Species Hybridization with Fusarium verticillioides Microarrays Reveals New Insights into Fusarium fujikuroi Nitrogen Regulation and the Role of AreA and NMR
2008
The rice pathogenic fungus Fusarium fujikuroi belongs to the Gibberella fujikuroi species complex, which consists of about 60 species of genus Fusarium section Liseola of monophyletic origin (45, 47, 48, 85). Most Fusarium species are plant pathogens, with both wide and narrow host ranges represented. Fusarium species are also well known for synthesizing an extensive variety of sometimes toxic secondary metabolites. The synthesis of gibberellins (GAs) (56, 79) and the red pigment bikaverin (3, 36) in F. fujikuroi has been well studied. Although both secondary metabolites are nitrogen-free compounds, their biosynthesis is strictly repressed by nitrogen (reviewed in reference 80). For the GA biosynthetic genes, we have recently shown that AreA directly regulates their expression by binding to specific double GATA sequence elements in their promoters (39). These are the first examples of an AreA-dependent regulation of secondary metabolites that do not have any nitrogen in their structure. For the bikaverin biosynthetic genes, the role of AreA is not as clear and it seems not to act via direct binding to the promoters of bikaverin genes (P. Wiemann and B. Tudzynski, unpublished data).
AreA is a member of the GATA-type transcription factor family and a functional homologue of two positively acting nitrogen regulators in Saccharomyces cerevisiae, Gln3 (40) and Gat1 (Nil1) (69). AreA homologues have been isolated from several filamentous fungi and include the Aspergillus nidulans AreA (34), Neurospora crassa NIT2 (21), Penicillium chrysogenum NreA (26), Fusarium oxysporum Fnr1 (17), and Magnaporthe grisea Nut1 (20). Most of our knowledge on the molecular mechanism of AreA-dependent nitrogen regulation derives from studies of A. nidulans and N. crassa, where AreA activity is negatively affected by binding to the nitrogen metabolite regulators NmrA and NMR1, respectively, when rich nitrogen sources are available. The deletion of the nmr gene in either fungus resulted in a significant derepression of AreA target genes under nitrogen-sufficient conditions (2, 51). In contrast, for F. fujikuroi, the NMR homologue was shown to play only a minor role in nitrogen regulation, at least in the regulation of GA biosynthesis genes, despite fully complementing the N. crassa nmr-1 mutant (39). Therefore, we postulate that F. fujikuroi must contain additional regulatory proteins that affect AreA activity and nitrogen metabolite repression of its target genes. These additional proteins may be involved in protein interactions with AreA or in the poorly understood processes that modulate AreA activity via the rapid degradation of the areA transcript in response to intracellular ammonium and glutamine (7, 41). In A. nidulans, AreA accumulates in the nucleus during nitrogen starvation and is exported from the nucleus and complexes with NmrA when rich nitrogen sources are added to starving mycelia (78). In S. cerevisiae, the target of rapamycin (TOR) kinase cascade regulates the cellular response to the nutrient status of the cell. The TOR kinase is active under conditions of nitrogen sufficiency and is inactivated by nitrogen starvation conditions or by the addition of rapamycin. The transcriptional activators Gln3 and Gat1 (Nil1) are phosphorylated in a TOR-dependent manner under nitrogen-sufficient conditions and are restricted to the cytoplasm after complexing with the regulator Ure2. Nitrogen starvation or the addition of rapamycin inhibits TOR, resulting in the dephosphorylation of the transcriptional activators, their release from Ure2, and their translocation to the nucleus, where they activate their target genes (reviewed in references 13 and 60).
Recently, we have shown that the TOR protein kinase in F. fujikuroi plays a role in the regulation of genes involved in GA and bikaverin biosynthesis, ammonium transport, ribosome biogenesis, protein synthesis, and autophagy (76). In contrast to what is seen for the yeast model system S. cerevisiae, the inhibition of TOR by rapamycin resulted in only partial derepression of AreA-regulated nitrogen metabolite repression genes, which suggests that there must be additional factors which inactivate AreA under conditions of nitrogen availability (76).
One strategy to learn more about the role AreA may play as a global nitrogen regulator is to compare the expression profiles of the wild-type (WT) and areA mutant strains after growth in nitrogen-limited and -sufficient conditions. Microarrays are a powerful tool to investigate the genome-wide regulation of genes. The recent availability of genomic data from Fusarium verticillioides, a close relative of F. fujikuroi that is estimated to share ∼93% nucleotide identity, provides an opportunity to explore gene expression changes in F. fujikuroi by a cross-species microarray analysis. Microarrays have already been successfully used for cross-species hybridization between different fungi. S. cerevisiae microarrays successfully identified differentially expressed genes of Pichia pastoris (63), N. crassa microarrays identified developmentally regulated genes and mating-type-dependent genes of Sordaria macrospora (46, 54), and finally, an A. niger macroarray identified growth-phenotype-related genes of Aspergillus oryzae (73).
The aim of this study was to discover a set of genes in F. fujikuroi which are significantly affected by nitrogen availability by cross-species hybridization using an oligonucleotide microarray derived from F. verticillioides expressed sequence tag (EST) data. By comparing the expression patterns of the WT and the areA mutant under conditions of nitrogen starvation and sufficiency, we were able to differentiate between AreA-dependent and non-AreA-dependent nitrogen-repressed and nitrogen-induced genes. The expression pattern of representative genes from each expression group was confirmed by Northern blot analysis and helped to provide an overview of the role AreA plays in nitrogen metabolite repression. Furthermore, we show that nitrogen-regulated genes can be differentiated into rapamycin (TOR)-dependent and -independent genes, confirming our previous suggestion (76) that TOR controls mainly AreA-independent genes (e.g., ribosome biogenesis and translation control genes) and only partially affects the expression of AreA target genes.
To further examine the role of NMR in nitrogen metabolism in F. fujikuroi, we compared the expressions of the newly identified, larger set of AreA target genes in the WT, nmr deletion, and nmr-overexpressing mutants and in areA deletion and areA-overexpressing strains. Although initial experiments did not indicate a significant effect of either deletion or overexpression of nmr on the expression of AreA target genes, we found that NMR has an inhibitory effect on AreA during early growth phases, as AreA target genes were upregulated in the areA mutant during the first 24 h of growth. We demonstrated by a yeast two-hybrid approach that AreA interacts with NMR, as has been shown previously for N. crassa and A. nidulans (35, 51), and that this interaction probably affects AreA activity mainly in early stages of growth.
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