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

Influence of FfPpt1 on conidiogenesis and sexual mating recognition.

<p>A: Spores produced of indicated strains per cm² after 10 days of incubation on solidified V8 medium in constant light conditions. Blue: strain IMI58289; red: strain C-1995. FEC: medium was supplemented with 2 µm ferrichrome; contiguous incubation: strains were incubated as described in Methods. B: DIC images of spores produced by the indicated strain. Strains were incubated and spores were collected as described in Methods. C: Representative photographs of sexual crossings of indicated strains as described in Methods.</p

Influence of FfPpt1, FfSre1 and FfFtr3/FfFet3 on lysine biosynthesis, iron homeostasis and oxidative stress.

<p>A: Growth ability of the indicated strains on solidified Czapek Dox (CD) medium supplemented as indicated. Representative pictures were taken after 3 days of incubation at 28°C in the dark. B: Phylograms of ferroxidases and iron permeases from <i>F. fujikuroi</i> (Ff), characterized proteins from <i>F. graminearum</i> (Fg) and <i>A. fumigatus</i> (Af), as well as homologous sequences from <i>F. oxysporum</i> (FOXG) and <i>F. verticillioides</i> (FVEG) obtained from the Broad Institute database were created as described in Methods. Scale bars represent character changes. C and D: Northern blot analysis using indicated genes as probes and rRNA visualization as loading control. The indicated strains were grown as described in Methods. (−); addition of water, (+) addition of FeCl₃ to a final concentration of 1 mm.</p

Involvement of FfPpt1 in PKS- and PKS/NRPS-derived secondary metabolite production and gene regulation.

<p>HPLC-UV chromatograms (bikaverins (510 nm) fusarubins (450 nm) and fusarins (363 nm)) in relative units (mAU) of indicated strains incubated as described in Methods. For HPLC conditions see Methods. Northern blot analyses of indicated strains from the same culture conditions probed with indicated cluster genes and rRNA visualization as loading control.</p

Fluorescence microscopy of Ffppt1 and wild-type strains during rice root infection assays.

<p>Representative fluorescent microscopy pictures of indicated strains in rice root infection assays performed as described in Methods. Gamborg B5 Medium was supplemented as indicated.</p

Effect of Ff<i>ppt1</i> deletion on terpene-derived metabolites.

<p>A: HPLC quantified amounts of GA₃ (red) and the sum of GA₄ and GA₇ (blue) in mg per l culture and mycelium dry weight in mg of indicated strains. Data are given as means and standard deviations of two biological replicates. For cultivation and HPLC conditions see Methods. B: Quantified amounts of α-acorenol (red) and <i>ent</i>-kaurene (blue) of indicated C-1995 strains by GC-MS. Data are given as means and standard deviations of three biological replicates. For cultivation and GC-MS conditions see Methods. C: Northern blot analysis of the first GA cluster genes of the indicated strains. rRNA visualization as loading control.</p

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

Changes in levels of selected proteins encoded by SM biosynthetic genes in <i>F. fujikuroi</i> as determined by comparative (−N/+N) quantitative proteomics.

<p><b>A</b>: Increased (blue) and decreased (yellow) protein levels in response to nitrogen availability. Protein levels shown in columns A and B are data from independent experiments. Values are shown for only proteins quantified in both experiments. A log₂ ratio>0 (−N/+N) indicates an increase in abundance in the low-nitrogen condition; a log₂ ratio<0 indicates a decrease in abundance in the low-nitrogen condition; and a log₂ ratio of zero indicates no changes in protein levels. Boxes with an asterisk indicate, that this protein could only be quantified in one nitrogen condition. The numbers in the far right column indicate how many proteins could be quantified within a cluster; value to the left of the slash is from replicate A, and value after the slash is from replicate B. <b>B</b>: Key to heat map showing Log₂ values that correspond to different shades of blue and yellow. Standard deviation of a ratio is reflected in the size of the blue and yellow boxes.</p

Infection of roots of rice plants by GA-producing wild-type (IMI58289) and GA-deficient (SG139) strains of <i>F. fujikuroi</i>.

<p>Rice plants were co-cultivated with either strain of <i>F. fujikuroi</i> for 7 days at 28°C and 80% humidity. Images of the corresponding whole plants following the incubation period are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g001" target="_blank"><b>Figure 1Bf</b></a>. Both the wild-type strain and GA-deficient mutant were engineered to express the dsRed fluorescent protein. Stars indicate the position of cells that have been penetrated by and are filled with hyphae of <i>F. fujikuroi</i>. <b>A</b>: Microscopic overviews (10-fold magnification) of a root infected with the mutant SG139 (above) or wild-type. Note the absence of invaded cells in the root infected with the GA-deficient mutant. Images are an overlay of images from brightfield and Texas Red filter, which captures fluorescence emitted by the DsRed protein. <b>B/C</b>: 40-fold (B) and 63-fold (C) magnified images of roots infected with GA-deficient mutant (left) or wild-type strain (right). In most cases, hyphae of the mutant strain were observed between cells, in ‘intercellular’ spaces, whereas wild-type hyphae were often observed inside the cells (indicated by stars; see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g001" target="_blank">Figure 1Bd</a>) as well as in intercellular spaces. White lines in the images are scale bars corresponding to 200 µm (B) or 50 µm (C). <b>D</b>: Quantification of penetration events per rice root infected with wild-type or SG139, respectively. In addition, the total number of shots per root taken for analysis as well as penetration events of the mutant compared to the wild-type are shown.</p