A Novel Type Pathway-Specific Regulator and Dynamic Genome Environments of a Solanapyrone Biosynthesis Gene Cluster in the Fungus Ascochyta rabiei

Abstract

Secondary metabolite genes are often clustered together and situated in particular genomic regions, like the subtelomere, that can facilitate niche adaptation in fungi. Solanapyrones are toxic secondary metabolites produced by fungi occupying different ecological niches. Full-genome sequencing of the ascomycete Ascochyta rabiei revealed a solanapyrone biosynthesis gene cluster embedded in an AT-rich region proximal to a telomere end and surrounded by Tc1/Mariner-type transposable elements. The highly AT-rich environment of the solanapyrone cluster is likely the product of repeat-induced point mutations. Several secondary metabolism-related genes were found in the flanking regions of the solanapyrone cluster. Although the solanapyrone cluster appears to be resistant to repeat-induced point mutations, a P450 monooxygenase gene adjacent to the cluster has been degraded by such mutations. Among the six solanapyrone cluster genes (sol1 to sol6), sol4 encodes a novel type of Zn(II)2Cys6 zinc cluster transcription factor. Deletion of sol4 resulted in the complete loss of solanapyrone production but did not compromise growth, sporulation, or virulence. Gene expression studies with the sol4 deletion and sol4-overexpressing mutants delimited the boundaries of the solanapyrone gene cluster and revealed that sol4 is likely a specific regulator of solanapyrone biosynthesis and appears to be necessary and sufficient for induction of the solanapyrone cluster genes. Despite the dynamic surrounding genomic regions, the solanapyrone gene cluster has maintained its integrity, suggesting important roles of solanapyrones in fungal biology.

FIG 1

Solanapyrone biosynthesis gene cluster and its genomic environment. Sliding-window analysis of the nucleotide composition was conducted with the chromosome end harboring the genes of the solanapyrone biosynthesis gene cluster. The GC content (in percent; top, black line), RIP index I (number of ApT residues/number of TpA residues; bottom, blue line), and RIP index II [(number of CpA residues + number of TpG residues)/(number of ApC residues + number of GpT residues; bottom, red line] were calculated in a 200-bp window, which was slid in 50-bp increments across the terminal 100 kb from the telomere end. The x axis shows the distance from the left edge of the window. The (TTAGGG)₉ telomere repeats were found in the rightmost region. A schematic diagram of the arrangement and orientation of open reading frames (O1 to O10; red arrows) and pseudogenes (P1 to P8; blue arrows) is shown in the middle. The shaded area indicates the region harboring the solanapyrone cluster genes (sol1 to sol6). The relative positions and details of the gene content in the 100-kb region are listed in Table 1. The horizontal solid line in the top panel shows the 50% GC mark, while the dashed line in the bottom panel shows a RIP index value of 1.

FIG 2

Degeneracy of transposable elements and ORF10 located near the solanapyrone gene cluster. (A) Sliding-window analyses of the nucleotide composition were conducted for the full-length functional Molly transposon from Stagonospora nodorum (1,866 bp; GenBank accession number AJ488502) and pseudogenes (P4 to P6) found in the subtelomeric region (P4 and P5, 1,866 bp; P6, 1,867 bp), including the 5′ and 3′ TA insertion sites, terminal inverted repeats, and putative transposase. Values for GC content (in percent; black line), RIP index I (number of ApT residues/number of TpA residues; blue line), and RIP index II [(number of CpA residues + number of TpG residues)/(number of ApC residues + number of GpT residues; red line] are shown. These values were calculated across a 200-bp window, which was slid in 20-bp increments across the full-length elements. Horizontal solid lines, the 50% GC mark; dashed lines, a RIP index value of 1. (B) Sliding-window analyses of the nucleotide composition of ORF10 plus its 5′ and 3′ flanking regions (2,029 bp in total) (top) and the homologous P450 gene (2,020 bp, GenBank accession number XP_007784573) (bottom). The black box below each graph indicates the position of the coding regions of the sequences. ORF10 with a 5′ flanking region (5′F) and a 3′ flanking region (3′F) and the corresponding homologous region in the P450 gene are shaded for comparison.

FIG 3

Structural organization of the typical C₆ zinc cluster transcription factor (Gal4) and the Sol4 protein. C₆ zinc cluster TFs consist of a highly conserved N-terminal zinc cluster DNA-binding domain consisting of six cysteines coordinating two zinc ions (Zn; purple), immediately followed by a coiled-coil region (C; green) that involves protein-protein interactions, a large C-terminal domain dubbed the MHR (blue) that regulates transcriptional activities, and an acidic activation domain (Ac; red) in the distal C-terminal end. Note that the Sol4 protein lacks the characteristic N-terminal zinc cluster DNA-binding domain of the C₆ zinc cluster TF family but contains the MHR, followed by a coiled-coil region.

FIG 4

Lack of solanapyrone production in Δsol4 mutant culture. (A) Overlaid base peak ion chromatograms of equivalent quantities of culture extracts of the AR628 WT strain (black line) and its Δsol4 mutant (red line), indicated by the observed m/z value and retention time (in minutes, as indicated in parentheses) for each compound at the peak. Peaks corresponding to solanapyrones A and C (which eluted at 4.19 and 4.03 min, respectively, in the WT culture extract) are not found in the Δsol4 mutant. (B) Mean dry weight of the WT strain and the Δsol4 mutant. Mycelial mats were dried and weighed when the cultures were harvested and were analyzed for solanapyrone production 18 days after inoculation. Error bars are standard deviations (n = 28 flasks).

FIG 5

Colony morphology of WT and Δsol4 mutant strains. (A) Colony morphology of AR628 and AR21 WT strains and their corresponding Δsol4 mutants at 2, 4, and 8 weeks after incubation on PDA. For 8-week-old cultures, strains were grown from the left-hand edge of the plates. Note that the restricted colony size for the WT strains is due to the production and accumulation of solanapyrones. (B) (Left) Normal sporulation of both the WT strains and Δsol4 mutants at 3 weeks after incubation on PDA. The mass of spores oozing out from the pycnidia was visible on the colony surface of both strains. (Right) Inhibition of mycelial growth and the toxic effect of solanapyrone on mycelial growth are visible at the colony margins in the wild-type strain but not in the Δsol4 mutant strains. See panel A for the strains shown in the four panels in each column. (C) Growth curves of the WT strains and their corresponding Δsol4 mutants on PDA. Colony diameters were measured at 3-day intervals. Error bars are standard deviations (n = 5). The initial growth rates were similar among the WT and Δsol4 mutant strains in the first 2 weeks, but the growth rate became lower in the WT strain due to inhibition by solanapyrones.

FIG 6

Transcriptional activation of solanapyrone cluster genes by the sol4 gene. The expression levels of the solanapyrone cluster genes by the WT strain and the Δsol4 mutant grown on PDA (A) and in the WT strain and two independent sol4-overexpressing strains (4OE2-2 and 4OE6-2) grown in CM broth upon induction with polygalacturonic acid (B) were analyzed by RT-PCR. The expected band sizes of mRNA (lower bands) and pre-mRNA/genomic DNA (gDNA) (upper bands) are indicated (the numbers to the left and right of the gels are in base pairs). Similar results were obtained from three independent experiments. Lanes M, 100-bp DNA ladder. Since all the primer pairs were designed to amplify flanking exons, including one intron, the absence of the upper band in the Actin1 reference gene (A) indicates that there was no genomic DNA contamination.

FIG 7

Pathway-specific control of the sol4 gene for solanapyrone cluster genes. The expression levels of solanapyrone cluster genes (sol1 to sol6), flanking open reading frames (ORF2, ORF3, and ORF10), and other secondary metabolite-related genes (PKS1 and PKS2) in the AR628 WT strain and its Δsol5 and Δsol4 mutants are shown. Total RNA was extracted from a 9-day-old colony grown on potato-dextrose agar. Quantitative RT-PCR was performed, and the levels of gene expression were normalized by the Actin1 expression levels in the respective samples. The relative gene expressions levels were log₂ transformed. Error bars are standard deviations from three biological replicates. Asterisks, no transcripts were detected from the samples due to the specific deletion of the respective genes.

FIG 8

Synteny blocks shared between the A. rabiei contig harboring the solanapyrone biosynthesis gene cluster and scaffolds of related Dothideomycetes. The sequences of Didymella exigua scaffold 6 (top) and Leptosphaeria maculans supercontig 1 (bottom) were compared pairwise to the sequence of the contig of A. rabiei, and only the 835-kb regions showing synteny with the A. rabiei contig are presented. For the A. rabiei contig, the GC content across the entire contig (835 kb) is provided, and the subtelomeric region (100 kb) harboring the solanapyrone biosynthesis gene cluster is highlighted in a gray box. Note that L. maculans supercontig 1 showed mesosynteny with the A. rabiei contig, while the D. exigua is largely macrosyntenic to the A. rabiei contig, owing to its close taxonomic relationship with A. rabiei (teleomorph, Didymella rabiei).