Figure 1.
Phylogenetic and phenotypic characteristics of F. fujikuroi.
A: Maximum likelihood tree showing phylogenetic relationships of F. fujikuroi and other species representing the Asian, African and American clades of the Gibberella fujikuroi complex (GFC), as well as F. oxysporum, F. graminearum and F. solani. The midpoint rooted tree is based on concatenated nucleotide sequences of 28 genes involved in primary metabolism that are highly homologous in different fusaria. Branches show bootstrap values (%), scale bar indicates amino acid substitutions per site. B: Phenotypic characteristics: a) Variation in pigmentation of the wild-type F. fujikuroi grown in a liquid medium containing (from top to bottom) 60 mM glutamine (fusarins), 6 mM NaNO3 (fusarubins) and 6 mM glutamine (bikaverin); b) Perithecia resulting from a sexual cross of two isolates of F. fujikuroi with opposite mating types. Fusarubins account for the dark color of perithecia [15]; c) F. fujikuroi grown on complete medium and regeneration medium; d) fluorescent microscopy image of the DsRed-labeled F. fujikuroi wild type penetrating rice root cells during infection; e) Microscopic image of microconidial chain on KCl agar medium; f) characteristic symptoms of bakanae disease due to wild-type-infected rice seedlings (left) compared to the GA-deficient mutant (right).
Table 1.
Comparative genome statistics.
Figure 2.
Whole genome comparison of F. fujikuroi withF. verticillioides.
Dotplot of F. fujikuroi chromosomes and scaffolds against F. verticillioides calculated using MUMer [121] highlights overall collinearity. Orthologous DNA is represented by red dots, inverted segments are shown as blue dots. Inset magnifies F. fujikuroi chromosome XII, which has no homologue in the F. verticillioides scaffold set. The missing subtelomeric regions of chromosome IV in F. fujikuroi are highlighted by vertical purple lines. Dots that are located above or below the line indicating collinearity represent largely repetitive DNA.
Figure 3.
Characterization of F. fujikuroi chromosomes and variation in acetylation and methylation statues of histone H3.
A: Information for chromosomes I, V and VIII is shown as examples of the 12 F. fujikuroi chromosomes (seeFigure S3 for additional chromosomes). For each chromosome, the position of the centromere is shown at the top; below this in descending order are: GC content, location of SM biosynthetic gene clusters, acetylation and methylation states of histone H3, and changes in gene expression. Variation in histone H3 modification statues indicates chromosomal regions in which genes are expressed (H3K9ac and H3K4me2) or silent (H3K9me3). “Δ expression up” indicates a more than twofold increase in gene expression during growth of F. fujikuroi in nitrogen-rich medium, whereas “Δ expression down” indicates an at least twofold decrease in gene expression. SM biosynthetic gene cluster locations are indicated by arrows labeled with the PKS, NRPS or TC (DTC means diterpene cyclase; STC means sesquiterpene cyclase) gene in each cluster (see Table 4 and Table S4). For the same analyses of other F. fujikuroi chromosomes, see Figure S3. B: Immunocytological analysis of histone acetylation and methylation in F. fujikuroi. Detection of specific histone markers was performed with H3K9me3 and H3K9ac-specific antibodies. DNA was counterstained with DAPI. H3K9me3 is significantly enriched in heterochromatin that forms several chromocenters, while H3K9ac is evenly distributed in the nuclei (scale bar = 5 µm).
Table 2.
Occurrence of selected gene families and other genetic elements in genome sequences of seven Fusarium species.
Figure 4.
Comparison of GA biosynthetic gene clusters in Fusarium genome sequences.
Arrows that are the same color represent genes, or gene sets, that have closely related homologues in two or more species/isolates. Light blue arrows represent GA biosynthetic genes. Black arrows represent genes that do not have closely related homologues in the GA cluster region of other species/isolates. Ψ indicates a pseudogene. For those that are available, gene/protein designations are indicated next to or below species names. FOXB and FOXG designations are for F. oxysporum isolates Fo5176 and Fol 4287, respectively.
Table 3.
Presence of SM gene clusters and production of the concomitant chemical products under standard laboratory conditions.
Figure 5.
Relative expression of CPS/KS gene in rice and maize roots.
Rice and maize roots were infected with Fusarium fujikuroi spores and every 2 days RNA was isolated from three or five plants and used in real time PCR analysis. The expression levels were obtained using the delta-delta Ct and were normalized against three reference genes encoding a related actin (FFUJ_05652), a GDP-mannose transporter (FFUJ_07710) and ubiquitin (FFUJ_08398). The expression levels of CPS/KS at 4 days in maize was arbitrarily set as 1, and all other expression levels were reported relative to it.
Figure 6.
Infection of roots of rice plants by GA-producing wild-type (IMI58289) and GA-deficient (SG139) strains of F. fujikuroi.
Rice plants were co-cultivated with either strain of F. fujikuroi for 7 days at 28°C and 80% humidity. Images of the corresponding whole plants following the incubation period are shown in Figure 1Bf. 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 F. fujikuroi. A: 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/C: 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 Figure 1Bd) as well as in intercellular spaces. White lines in the images are scale bars corresponding to 200 µm (B) or 50 µm (C). D: 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.
Figure 7.
Comparison of the fumonisin biosynthetic gene (FUM) cluster in F. fujikuroi, F. verticillioides, F. oxysporum and Aspergillus niger.
Horizontal arrows that are the same color represent genes, or gene sets, that have closely related homologues in two or more fungi. Blue horizontal arrows represent FUM genes, and the numbers in them correspond to FUM gene designations rather than designations from genome databases. Ψ indicates a pseudogene. Gene content and order is well conserved in the Fusarium species but less conserved in A. niger.
Figure 8.
Comparison of the PKS19 (left) and PKS8 (right) clusters in genome sequences of Fusarium.
Horizontal arrows that are the same color represent genes, or gene sets, that have closely related homologues in two or more fungi. The putative PKS19 cluster is embedded within an AT-rich region, consists of six genes (FFUJ_12235–12250; horizontal green arrows), and among the species examined is unique to F. fujikuroi. However, synteny of genes corresponding to PKS19 flanking genes in F. fujikuroi is highly conserved in F. mangiferae, F. verticillioides, F. circinatum and F. oxysporum. The vertical red arrows indicate the genomic location corresponding to the location of the PKS19 cluster in F. fujikuroi. The putative PKS8 cluster consists of three genes (green horizontal arrows). Only a remnant of PKS8 is present in F. fujikuroi, whereas the intact PKS8 cluster is present in the other species examined. The synteny of the PKS8 cluster-flanking genes is partially conserved among the fusaria examined. For those that are available, gene designations are indicated below species names.
Figure 9.
Comparison of genes flanking the putative 11-gene NRPS31 cluster in F. fujikuroi with homologous regions in genome sequences of other Fusarium species.
Horizontal arrows that are the same color represent genes, or gene sets, that have closely related homologues in two or more fungi. Exceptions are indicated by green arrows, which represent NRPS31-cluster genes. Blue arrows represent NRPS31 cluster-flanking genes, and yellow arrows represent genes that do not have closely related homologues in the NRPS31 cluster-flanking region of F. fujikuroi. The vertical red arrows indicate the genomic location corresponding to the location of the NRPS31 cluster in F. fujikuroi. For those that are available, gene designations are indicated below species names.
Table 4.
Expression patterna of the secondary metabolite biosynthetic gene clusters under four growth conditions.
Figure 10.
Changes in levels of selected proteins encoded by SM biosynthetic genes in F. fujikuroi as determined by comparative (−N/+N) quantitative proteomics.
A: 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: 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.
Figure 11.
Location of the GA biosynthetic gene cluster on F. fujikuroi chromosome V as well as levels of histone modifications and gene expression within and flanking the cluster.
Histone marks are described in the legend to Figure 3. Expression data were derived from microarray experiments in low (6 mM glutamine) and high (60 mM glutamine) nitrogen and are plotted as the changes in log2 expression values in high-nitrogen medium compared to low nitrogen medium. H3K9ac and gene expression are correlated, as both are decreased under high nitrogen conditions.
Figure 12.
Location of the bikaverin biosynthetic gene cluster on F. fujikuroi chromosome V as well as levels of histone modifications and gene expression within and flanking the cluster.
Histone marks are described in the legend to Figure 3. Expression data were derived from microarray experiments in low and high nitrogen and are plotted as the changes in log2 expression values in high-nitrogen medium compared to low-nitrogen medium. H3K9ac and gene expression are correlated, as both are decreased under high nitrogen conditions.
Figure 13.
Location of the NRPS/APS biosynthetic gene cluster on F. fujikuroi chromosome I, levels of histone modifications and gene expression within and flanking the cluster, and production of metabolites following overexpression of cluster genes APS2 and APS8.
A: Synteny between the apicidin gene cluster in F. semitectum [105] and the apicidin-like gene cluster in F. fujikuroi. B: Histone modifications and gene expression in and flanking the cluster. Histone marks are described in the legend to Figure 3. Expression data were derived from microarray experiments in low and high nitrogen and are plotted as the changes in log2 expression values in high-nitrogen medium compared to low-nitrogen medium. H3K9ac and gene expression are overall correlated, as both are increased under high nitrogen conditions. In some genes increased H3K4me2 was observed, also suggesting transcription. C: Chemical analysis of the product of the unique PKS19 gene cluster. The traces show the extracted ion chromatograms for [C34H48O6N5+H]+ (first line) and [C35H42O7N5+H]+ (second to fourth line) determined by HPLC-FTMS of an apicidin standard (first line) and of culture fluids from F. fujikuroi IMI58289, OE::APS8 and the OE::APS2/OE::APS8 mutant. D: UV spectra of apicidin and the apicidin-like compound. The similar spectra suggest a structural similarity.
Figure 14.
Functional characterization of the PKS19 cluster, a putative polyketide biosynthetic gene cluster that is unique to F. fujikuroi.
(A) Position and organization of the PKS19 gene cluster on F. fujikuroi chromosome VIII, GC content, distribution of active histone marks, and gene expression in the PKS19 cluster. Histone marks are described in the legend for Figure 3. Expression data are from microarray analysis of wild-type F. fujikuroi (strain IMI58289). Values are log2 change in expression in a high versus low-nitrogen medium. H3K9ac and gene expression are correlated, as both are increased in the high-nitrogen medium. Some genes exhibited increased levels of H3K4me2 in the high-nitrogen medium, which also suggests transcription. (B) Chemical analysis of the SM product(s) of the PKS19 cluster. The traces show the combined extracted ion chromatograms for metabolites with molecular formulas [C12H16O4+H]+, [C12H18O5+H]+ and [C12H18O4+H]+ determined by HPLC-FTMS of culture fluids from F. fujikuroi strains: IMI58289, wild-type strain; OE::TF, a strain over-expressing the transcription factor encoded by FFUJ_12242; OE::PKS19, a strain over-expressing the PKS19 gene FFUJ_12239; and OE::TF/OE::PKS19, a strain over-expressing both FFUJ-12242 and FFUJ_12239. (C) Northern blot analysis of PKS19 cluster genes in strains IMI58289 (WT), OE::TF, OE::PKS19 and OE::TF/OE::PKS19. (D) UV spectra of metabolites corresponding to peaks 1 through 4 from chromatograms shown in C. The similar spectra of the metabolites suggest structural similarity.
Figure 15.
Relative expression of PKS19 and APS1 genes in maize and rice roots.
Maize and rice roots were infected with Fusarium fujikuroi spores, and every 2 days RNA was isolated from three or five plants and used in real time PCR analysis. The expression levels were obtained using the delta-delta Ct and were normalized against three reference genes encoding a related actin (FFUJ_05652), a GDP-mannose transporter (FFUJ_07710) and ubiquitin (FFUJ_08398). The expression levels of PKS19 (A) at 4 days in maize was arbitrarily set as 1, and all other expression levels were reported relative to it. In the case of the APS1 gene (B), the expression levels of this gene at 4 days in maize was arbitrarily set as 1, and all other expression levels were reported relative to it.