The Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1 of Fusarium fujikuroi Controls Development, Secondary Metabolism and Pathogenicity

The heterothallic ascomycete Fusarium fujikuroi is a notorious rice pathogen causing super-elongation of plants due to the production of terpene-derived gibberellic acids (GAs) that function as natural plant hormones. Additionally, F. fujikuroi is able to produce a variety of polyketide- and non-ribosomal peptide-derived metabolites such as bikaverins, fusarubins and fusarins as well as metabolites from yet unidentified biosynthetic pathways, e.g. moniliformin. The key enzymes needed for their production belong to the family of polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) that are generally known to be post-translationally modified by a Sfp-type 4′phosphopantetheinyl transferase (PPTase). In this study we provide evidence that the F. fujikuroi Sfp-type PPTase FfPpt1 is essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins, but not moniliformin as shown by analytical methods. Concomitantly, targeted Ffppt1 deletion mutants reveal an enhancement of terpene-derived metabolites like GAs and volatile substances such as α-acorenol. Pathogenicity assays on rice roots using fluorescent labeled wild-type and Ffppt1 mutant strains indicate that lysine biosynthesis and iron acquisition but not PKS and NRPS metabolism is essential for establishment of primary infections of F. fujikuroi. Additionally, FfPpt1 is involved in conidiation and sexual mating recognition possibly by activating PKS- and/or NRPS-derived metabolites that could act as diffusible signals. Furthermore, the effect on iron acquisition of Ffppt1 mutants led us to identify a previously uncharacterized putative third reductive iron uptake system (FfFtr3/FfFet3) that is closely related to the FtrA/FetC system of A. fumigatus. Functional characterization provides evidence that both proteins are involved in iron acquisition and are liable to transcriptional repression of the homolog of the Aspergillus GATA-type transcription factor SreA under iron-replete conditions. Targeted deletion of the first Fusarium homolog of this GATA-type transcription factor-encoding gene, Ffsre1, strongly indicates its involvement in regulation of iron homeostasis and oxidative stress resistance

Apicidin F: characterization and genetic manipulation of a new secondary metabolite gene cluster in the rice pathogen Fusarium fujikuroi.

The fungus F. fujikuroi is well known for its production of gibberellins causing the 'bakanae' disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding ("Api-box") motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants

Proposed biosynthetic pathway of apicidin F.

<p>Based on our data, we postulated a biosynthetic pathway for apicidin F. Noteworthy, catalyzation steps which are marked with a “?” have not been confirmed experimentally. The NRPS as key enzyme incorporates four different amino acids to produce apicidin F. However, most of the amino acids are non-proteinogenic and therefore, have to be modified by other enzymes of the cluster. l-Phenylalanine is the only proteinogenic amino acid and directly used by Apf1. l-Pipecolic acid is the second precursor of Apf1 and is epimerized to D-pipecolic acid, probably by Apf1 activity, prior to incorporation. It is proposed that lysine is converted to Δ¹-pyrroline-5-carboxylate (P5C). A P5C reductase catalyzes the transformation to proline. This enzyme could also convert Δ¹-pyrroline-6-carboxylate (P6C) to l-pip <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. In our studies, we could demonstrate that deletion of the P5C reductase-encoding gene <i>APF3</i> led to the incorporation of proline instead of pip resulting in the production of apicidin J. We claim, therefore, that this enzyme is responsible for the conversion of P6C into l-pip. The NRPS itself has an epimerization domain for the epimerization of l-pip into d-pip. Furthermore, Apf1 incorporates <i>N</i>-methoxy-l-tryptophan. We suggest that one of the P450 oxidases (Apf7 or Apf8) <i>N</i>-oxidizes l-tryptophan and then the <i>O</i>-methyltransferase Apf6 is able to catalyze the methylation of the hydroxy group. The fourth amino acid that is incorporated by Apf1 is l-2-aminooctanedioic acid. It is predicted that the fatty acid synthase-encoding gene <i>APF5</i> is involved in the synthesis of the octanoic acid backbone by fixing one acetyl-CoA unit and three malonyl-CoA units <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. Then one of the P450 oxidases may oxidize this backbone to 2-oxooctanoic acid. The aminotransferase Apf4 is predicted to catalyze the exchange of the keto group with an amino group. The next step would be the oxidation of 2-aminooctanoic acid by one of the P450 oxidases (Apf7 or Apf8). For <i>F. semitectum</i>, it could be shown that deletion of <i>aps7</i> led to the production of apicidin E (lacks the keto group in comparison to apicidin) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Jin1" target="_blank">[14]</a>. We suggest that the last step is the oxidation of 2-amino-8-hydroxyoctanoic acid to 2-aminooctanedioic acid by the FAD-dependent monooxygenase Apf9 because deletion of the corresponding gene led to the production of apicidin K (lacks the acid group and has a hydroxyl group instead).</p

Regulation of the apicidin F cluster.

<p>(A) The pH regulator PacC seems to be an activator of the apicidin F genes. The WT and Δ<i>PACC</i> were grown for three days under optimal conditions (60 mM glutamine, gln). The cultures were harvested and after washing, the mycelium was shifted into new flasks containing 60 mM gln adjusted to an ambient pH of 4 or 8, respectively. After 2 h the cultures were harvested again. (B) The nitrogen regulators AreB and glutamine synthetase (GS) are activators of the apicidin F gene expression. The WT, Δ<i>AREA</i> and Δ<i>AREB</i> and the gln auxotroph mutant Δ<i>GLN1</i> were grown for three days in 60 mM gln. (C) The WT, Δ<i>VEL1</i>, Δ<i>VEL2</i> and Δ<i>LAE1</i> were grown for three days in 60 mM gln. <i>APF6</i> and <i>APF9</i> were used as probes for all Northern blot analyses.</p

Deletion of the cluster genes <i>APF3</i> and <i>APF9</i> revealed new analogs of the apicidin F biosynthetic pathway.

<p>(A) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of <i>APF3</i> (Δ<i>APF3</i>) and <i>APF9</i> (Δ<i>APF9</i>) grown in ICI with 60 mM glutamine for three days. Shown are the extracted ion chromatograms for the [M+H]⁺-ion of proline apicidin F (apicidin J, 632.3079±0.0032, left) and for the [M+H]⁺-ion the Δ<i>APF9</i>-product (apicidin K, 632.3443±0.0032, right). The axes are normalized to the WT-level. (B) Structures of the two identified products: apicidin J and apicidin K.</p

The transcription factor (TF) Apf2 contains a basic DNA binding domain, four ankyrin repeats and is localized in the nucleus.

<p>(A) ClustalW alignment with amino acids of <i>Cochliobolus carbonum</i> ToxE (AFO38874), <i>Fusarium semitectum</i> Aps2 (GQ331953) and <i>Fusarium fujikuroi</i> Apf2 (FFUJ_00012). Identical amino acids are highlighted in grey, the positions of the domains are highlighted in either orange (basic DNA binding domain) or green (four ankyrin repeats) and based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336-Pedley1" target="_blank">[55]</a>. (B) The TF was fused to green fluorescent protein (GFP) at the C-terminus. The Δ<i>APF2</i> mutant was used as background. The two strains were grown for one day in 60 mM glutamine. Size of scale bars is indicated. A supplemental figure with controls is depicted in Fig. S2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>.</p

Mutation of the putative “Api-box” motif in the promoter region of <i>APF1</i> (NRPS) and <i>APF11</i> (transporter) resulted in reduced production of apicidin F.

<p>(A) Bioinformatic searches revealed an eight-base-pair motif with the consensus sequence 5′-TGACGTGA-3′ that was found in all promoters of the apicidin F cluster except in the promoter region of the transcription factor (TF)-encoding gene itself. In our study, we created two mutants with point mutations in the <i>APF1</i>/<i>APF11</i> promoter (P-mut1 and P-mut2, for the strategy see Fig. S3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>). (B) Biosynthesis of apicidin F was monitored with HPLC-HRMS. After growth for three days in 60 mM glutamine, the cultures of the WT and the two mutants P-mut1 and P-mut2 were harvested. Apicidin F was extracted from lyophilized mycelium. 10 µL of a 1 µg/mL apicidin solution (internal standard) were added to 90 µL of the sample. For the calculation, the peak area of apicidin F [M+H]⁺ (646.3235±0.0032) was divided with that of apicidin [M+H]⁺ (624.3756±0.0032). Product formation was normalized to the WT level. Experiment was performed in a triplicate.</p

Apicidin F cluster genes are expressed under high amounts of glutamine (gln) from the second to the third day.

<p>(A) The WT was grown in four nitrogen conditions, 6 and 60 mM gln and 6 and 120 mM NaNO₃ for three days. After harvesting, RNA was isolated from the mycelium. <i>APF1</i> and <i>APF9</i> were used as probes. (B) The WT was grown from the second to the fifth day (d) in 60 mM glutamine. Northern blot analysis was performed with the extracted RNA. <i>APF6</i> and <i>APF9</i> were used as probes.</p

Over-expression of the transcription factor-encoding gene <i>APF2</i> (OE::<i>APF2</i>) is able to overcome the nitrogen regulation of apicidin F.

<p>(A) HPLC-DAD measurement of the extracted mycelium of the WT and the OE::<i>APF2</i> mutant after three days. Both strains were grown in four nitrogen conditions, 6 and 60 mM glutamine (gln) and 6 and 120 mM NaNO₃. Apicidin F was measured at a wavelength of 280 nm. (B) Northern blot analyses of the WT and the OE::<i>APF2</i> mutant. Same conditions were used as for the HPLC measurements. <i>APF2</i> and <i>APF9</i> were taken as probes. (C) HPLC-DAD measurement of the extracted mycelium of the WT and the OE::<i>APF2</i> mutant after seven days in 6 and 120 mM NaNO₃. Product formation was assessed in triplicates and normalized to the WT level.</p

Influence of single <i>APF</i> gene deletions on the expression of the remaining genes and the production of apicidin F in these mutants.

<p>(A) The single gene deletions have no impact on the expression of the remaining genes. Only deletion of <i>APF2</i> (TF) resulted in down-regulation of all genes except of <i>APF3</i>. For this experiment, the WT and the single deletion mutants of the apicidin F gene cluster were grown for three days in 60 mM glutamine (gln). After harvesting, a northern blot was performed and hybridized with indicated probes <i>APF1</i>, <i>APF2</i>, <i>APF3</i>, <i>APF6</i>, <i>APF9</i> and <i>APF11</i>. (B) HPLC-HRMS-chromatograms of the culture filtrates of the WT and the single deletion mutants of the <i>APF</i> gene cluster grown in ICI with 60 mM gln for three days. Shown are the extracted ion chromatograms for the [M+H]⁺-ion of apicidin F (646.3235±0.0032), the axes are normalized to the WT-level. The deletion mutant of the transporter-encoding gene (Δ<i>APF11</i>) still produces WT-levels of apicidin F. Δ<i>APF3</i> produces apicidin F as well but in a decreased manner. Analysis of the mycelium extracts led to comparable results (Fig. S10 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103336#pone.0103336.s001" target="_blank">File S1</a>).</p