GS-dependent and independent sensing of different nitrogen sources.

<p>Transcription of NCR-sensitive and -insensitive genes in response to different nitrogen sources in the wild type and the Δ<i>gln1</i> strain. <b>A</b>. The wild type (WT) and the Δ<i>gln1</i> strain were grown for 3 days in ICI submerse cultures with 6 mM glutamine as nitrogen source. After 3 days glutamine (gln), NH₄NO₃ or NaNO₃ (NO₃^-) were added to a total concentration of 60 mM in case of gln and NH₄NO₃, and 120 mM in case of NaNO₃. An equal volume of H₂O was added as control. The mycelia were harvested 2 hours after nitrogen addition (short term exposure), and total RNA was used for northern analysis. <i>18S</i> rRNA was visualized as a loading control. <b>B</b>. The wild-type (WT) and the Δ<i>gln1</i> mutant were grown for five days on CM agar with 100 mM glutamine and with or without 50 mM KClO₃. If AreA is active the nitrate reductase-encoding gene <i>niaD</i> is expressed, and the nitrate reductase reduces KClO₃ to the toxic KClO₂. In the wild type, high glutamine levels repress the expression of <i>areA</i> and <i>niaD</i>, while the growth of the Δ<i>gln1</i> mutant is restricted due to an active AreA and subsequent expression of <i>niaD</i> leading to accumulation of toxic KClO₂. <b>C</b>. The wild-type (WT) and the Δ<i>gln1</i> strains were grown for 3 days (long term exposure) in ICI submerse cultures with 6 mM glutamine (control; only water was added) or 6 mM glutamine and additionally 54 mM glutamine (gln), 54 mM ammonium tartrate (NH₄⁺), 108 mM NaNO₃ (NO₃^-) or 108 mM glutamate (Glu) as nitrogen source. Total RNA (15 µg) was used for northern analysis and hybridized with probes as indicated. <i>18S</i> rRNA was visulaized as a loading control. Abbreviations: <i>mepB</i> and <i>mepC</i>, ammonium transporter genes. </p

Characterization of heterologous Δ<i>gln1</i> complementation mutants.

<p><b>A</b>. Plate assays comparing the growth of the wild type (WT), the Δ<i>gln1</i> mutantand different complemented transformants. Complete medium (CM) contained 18 mM glutamine, while the minimal medium (CD) contained no additional nitrogen source. Pictures were taken after 3 days of cultivation. <b>B</b>. Pictures of the wild type (WT), the Δ<i>gln1</i> mutant and the heterologous GS complementants grown for 3 days in ICI submerse cultures with either 6 mM glutamine (gln) (GA- and bikaverin-inducing (red coloration) conditions) or 60 mM glutamine as nitrogen source. <b>C</b>. Total RNA was isolated from mycelia grown for 5 days in ICI submerse cultures with 6 mM glutamine and used for northern blot analysis. <i>18S</i> rRNA was visualized as a loading control. Abbreviations: <i>aap1</i> and <i>aap8</i>, amino acid transporter genes; <i>cipC</i>, <i>ddr48</i> and the cross-pathway control gene <i>cpc</i> were found to be GS-target genes [47]; <i>mepB</i>, ammonium transporter gene. </p

The <i>gln1</i> gene expression and GS protein levels are positively regulated by AreA.

<p>Regulation of <i>gln1</i> gene expression and GS protein levels in <i>F</i>. <i>fujikuroi</i>. <b>A</b>. The wild-type (WT), Δ<i>areA</i>, Δ<i>meaB</i> and Δ<i>nmr</i> strains were grown for 3 days in ICI submerse culture with either 6 mM or 60 mM glutamine as nitrogen source. Total RNA was used for northern analysis using the genomic gln1 fragment as probe. <i>18S</i> rRNA was visualized as a loading control. <b>B</b>. Total protein extract was used for western analysis using the polyclonal anti-TbGS antibodies and HRP-conjugated anti-rabbit secondary antibodies. Ponceau staining as loading control.</p

Impact of the <i>F. fujikuroi</i> GS on amino acid and carbon metabolism.

<p><b>A</b>. Amino acid measurement of the wild-type, the Δ<i>gln1</i> mutant and three complemented strains carrying point-mutated <i>gln1</i> gene copies. The strains were grown for 3 days in ICI medium with 6 mM glutamine as nitrogen source. After 3 days the mycelia were harvested, lyophilized and used for amino acid analysis. <b>B</b>. Transcription of nitrogen and carbon metabolism genes. The wild type, the Δ<i>gln1</i> and the three deregulated mutants were grown for 3 days in ICI medium with 6 mM glutamine as N-source. The nitrogen-starved mycelia were harvested and the total RNA was used for northern analysis. <i>18S</i> rRNA was visualized as a loading control. Abbreviations: <i>gdhA</i>, NADH-dependent glutamate dehydrogenase; <i>gdhB</i>, NAD⁺-dependent glutamate dehydrogenase; <i>gltA</i>, glutamine oxoglutarate aminotransferase (GOGAT); <i>pro4</i>; glutamate-5-kinase; <i>gpd1</i>, glyceraldehyde-3-phosphate dehydrogenase gene; <i>pki1</i>, pyruvate kinase. <b>C</b>. The wild type and D60/S62 mutant strains were grown for 3 days in ICI medium containing 6 mM glutamine. Mycelia were harvested 30 minutes after addition of H₂O, 60 mM glutamine (gln), 60 mM ammonium tartrate (NH₄⁺) or potassium glutamate (glu) and hybridized with the indicated probes. <i>18S</i> rRNA was visualized as a loading control.</p

Characterization of site-directed Δ<i>gln1</i> complementation mutants.

<p><b>A</b>. Plate assays comparing the growth of the wild type (WT), the Δ<i>gln1</i> mutant and different transformants complemented with a point-mutated copy of <i>gln1</i>. Complete medium (CM) contained 18 mM glutamine, while the minimal medium (CD) contained no additional nitrogen source. Pictures were taken after 3 days of cultivation. <b>B</b>. Pictures of the wild type (WT), the Δ<i>gln1</i> mutant and the point-mutated GS complementants (see Figure 4) grown for 3 days in ICI medium with either 6 mM (GA- and bikaverin-inducing (red coloration) conditions) or 60 mM glutamine as nitrogen source. <b>C</b>. Total RNA was isolated from mycelia grown for 5 days in ICI medium with 6 mM glutamine and used for northern blot analysis. <i>18S</i> rRNA was visualized as a loading control. Abbreviations: see legend of Figure 3.D. Total protein extract of the mycelia from <b>B</b> was used for western analysis using the polyclonal anti-TbGS antibodies and HRP-conjugated anti-rabbit secondary antibodies. Ponceau staining as loading control. <b>E</b>. Total RNA (Northern blot analysis) and protein (Western blot analysis) was isolated from mycelia of the wild type, the Δ<i>gln1</i> mutant and the transformants complemented with the three point-mutated copies of <i>gln1</i> which restore secondary metabolism but not glutamine formation. The strains were grown for 3 days in ICI medium with 6 mM glutamine.</p

A Sensing Role of the Glutamine Synthetase in the Nitrogen Regulation Network in Fusarium fujikuroi

Contains fulltext : 125173.pdf (publisher's version ) (Open Access)In the plant pathogenic ascomycete Fusarium fujikuroi the synthesis of several economically important secondary metabolites (SM) depends on the nitrogen status of the cells. Of these SMs, gibberellin and bikaverin synthesis is subject to nitrogen catabolite repression (NCR) and is therefore only executed under nitrogen starvation conditions. How the signal of available nitrogen quantity and quality is sensed and transmitted to transcription factors is largely unknown. Earlier work revealed an essential regulatory role of the glutamine synthetase (GS) in the nitrogen regulation network and secondary metabolism as its deletion resulted in total loss of SM gene expression. Here we present extensive gene regulation studies of the wild type, the Deltagln1 mutant and complementation strains of the gln1 deletion mutant expressing heterologous GS-encoding genes of prokaryotic and eukaryotic origin or 14 different F. fujikuroi gln1 copies with site-directed mutations. All strains were grown under different nitrogen conditions and characterized regarding growth, expression of NCR-responsive genes and biosynthesis of SM. We provide evidence for distinct roles of the GS in sensing and transducing the signals to NCR-responsive genes. Three site directed mutations partially restored secondary metabolism and GS-dependent gene expression, but not glutamine formation, demonstrating for the first time that the catalytic and regulatory roles of GS can be separated. The distinct mutant phenotypes show that the GS (1) participates in NH4 (+)-sensing and transducing the signal towards NCR-responsive transcription factors and their subsequent target genes; (2) affects carbon catabolism and (3) activates the expression of a distinct set of non-NCR GS-dependent genes. These novel insights into the regulatory role of the GS provide fascinating perspectives for elucidating regulatory roles of GS proteins of different organism in general

Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen Fusarium fujikuroi Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.Keywords: Functional characterization, Species complex, Gibberellin biosynthetic pathway, Aspergillus nidulans, Mass spectrometry, Polyketide synthase genes, Neurospora crassa, Red pigment bikaverin, Mango malformation disease, Fumonisin productio

Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen <i>Fusarium fujikuroi</i> Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

<div><p>The fungus <i>Fusarium fujikuroi</i> causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of <i>F. fujikuroi</i> that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the <i>F. fujikuroi</i> genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus <i>Fusarium</i>. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to <i>F. fujikuroi</i>, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (<i>PKS19</i>) and another that includes a non-ribosomal peptide synthetase gene (<i>NRPS31</i>) are unique to <i>F. fujikuroi</i>. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered <i>F. fujikuroi</i> strains overexpressing cluster genes. <i>In planta</i> expression studies suggest a specific role for the <i>PKS19</i>-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of <i>F. fujikuroi</i> as a rice pathogen.</p></div

Expression pattern^a of the secondary metabolite biosynthetic gene clusters under four growth conditions.

a<p>+++, >90% of the genes belonging to the cluster are expressed under the condition indicated. ++, 50–90% of the genes belonging to the cluster are expressed under the condition indicated. +, 25–50% of the genes belonging to the cluster are expressed under the condition indicated. −, 0–25% of the genes belonging to the cluster are expressed under the condition indicated.</p>b<p>DTC and STC indicate diterpene synthase and sesquiterpene synthase, respectively.</p><p>Key enzymes of which the respective product is known are indicated in bold letters and the respective metabolites are listed; n/k indicates that the corresponding metabolite is not yet known. Red labeled key enzymes and corresponding metabolites are <i>Fusarium fujikuroi</i>-specific.</p

Whole genome comparison of <i>F. fujikuroi</i> with<i>F. verticillioides</i>.

<p>Dotplot of <i>F. fujikuroi</i> chromosomes and scaffolds against <i>F. verticillioides</i> calculated using MUMer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Delcher1" target="_blank">[121]</a> highlights overall collinearity. Orthologous DNA is represented by red dots, inverted segments are shown as blue dots. Inset magnifies <i>F. fujikuroi</i> chromosome XII, which has no homologue in the <i>F. verticillioides</i> scaffold set. The missing subtelomeric regions of chromosome IV in <i>F. fujikuroi</i> are highlighted by vertical purple lines. Dots that are located above or below the line indicating collinearity represent largely repetitive DNA.</p