Pyrrocidine, a molecular off switch for fumonisin biosynthesis

Abstract

Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides' protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.

Fig 1. Pyrrocidine A and B elicit differential gene expression.

(A) Heatmaps showing transcription levels of 4510 differentially expressed genes upon exposure to pyrrocidine A (PA, 5μg/mL) or pyrrocidine B (PB, 20μg/mL). The Y-axis represents genes that are clustered and colored by z-score. See colored key. The X-axis shows the 3 biological replicates of each treatment. (B) Venn diagram shows the number of genes with altered expression due to pyrrocidine A and/or B exposure. Each circle represents up- or down-regulation by pyrrocidine A or B, which is denoted by up/down arrows and A/B, respectively. Intersected regions represent genes regulated in both treatments, while the direction of regulation may vary.

Fig 2. Functional catalog of pyrrocidine-responsive genes.

Function enrichment analysis was conducted with MIPS FunCat Server (http://mips.helmholtz-muenchen.de/funcatDB/) [14]. The five club shapes correspond to the following MIPS functional categories: metabolism (01), cellular transport (20), cellular communication/signal transduction mechanism (30), cell rescue, defense and virulence (32), and interaction with the environment (34). The numbers of genes functionally enriched in the different categories are included, and genes with multifunctional enrichment are shown in overlapping regions. Of 525 pyrrocidine-responsive genes, 196 genes were functionally enriched in these five different categories after applying a filter of p-value < 0.05. FvABC3 is one of nine genes that functionally span four different enriched categories (01, 20, 32, 34), and FvZBD1 is functionally enriched only in metabolism along with another 80 genes.

Fig 3. qRT-PCR results validated the pyrrocidine B induced up-regulation of FvABC3 and FvZBD1, and that induction of FvABC3 was independent of ΔFvZEAR.

qRT-PCR was performed with M-3125 exposed or not exposed to 20 μg/mL pyrrocidine B for a final hour of growth after 47 hours in 2 mL of PDB. The Y-axis of the box plots shows the -ΔCt value, which was calculated by subtracting the Ct value of the β-tubulin reference gene from the Ct value of each gene of interest. Expression of FvABC3 in (A) wild-type M-3125 and (B) the ΔFvZEAR-1 mutant. (C) Induction of FvZBD1 in wild type upon exposure to pyrrocidine B. Three biological replicates, each with 3 technical replicates were assessed. All nine data points of each treatment are included in the box plots. The range of -ΔCt values is shown with error bars, and each box indicates first and third quartile. Mean and median are marked with an “×” and a line in each of the boxes.

Fig 4. Deletion of FvABC3 in F. verticillioides increased its sensitivity to pyrrocidine B.

Strains were monitored for 100 hours in PDB medium amended with pyrrocidine B at 10 μg/mL. OD₆₀₀ measurements taken every 2 hours were plotted (mean ± standard deviation). FRC M-3125 serves as the control (black curve). Two ΔFvABC3 deletion mutants are shown in light and dark blue, and 2 complemented strains in light and dark green. Corresponding growth inhibition phenotypes of the ΔFvABC3 deletion mutants are highlighted in the honeycomb plate after 100-hour incubation.

Fig 5. FvZBD1 is adjacent to the well-characterized FUM cluster.

Genes are represented by colored arrows with labels. The direction of arrows denotes the orientation of genes. A zoom-in section of the FvZBD1 locus indicates the deletion occurs at 646 bp downstream of FvZNF1 and 999 bp upstream of FUM21 when generating ΔFvZBD1 mutants. FvZBD1 is replaced with the hygromycin resistance cassette (HRC).

Fig 6. Deletion of FvZBD1 in F. verticillioides significantly enhanced virulence on maize seedlings.

Fifty Silver Queen maize seeds were inoculated with 10⁴/mL conidial suspensions for each of the different F. verticillioides strains prior to planting. Seeds treated with sterile water served as a control. Plants were grown for 14 days before measuring their heights and assessing numbers of germinated seeds. Three biological replicates were conducted. Results are shown from one representative trial. The other two trials showed the same overall trends. (A) Histogram showing the mean height of seedlings. Numbers on the X-axis correspond to the following treatments: 1, sterile water control; 2, M-3125; 3, ΔFvZBD1-1; 4, ΔFvZBD1-2; 5, ΔFvZBD1-1::C-1; 6, ΔFvZBD1-2::C-1; 7, FvZBD1-Ect. Statistical analysis was conducted with the two-tailed Mann Whitney Wilcoxon test (***, p-value < 0.001). (B) Visualization of seedling growth among treatments. Numbers on the pots correspond to those in (A). (C) Two-dimensional visualization of seedling growth among different treatments. Each dot represents a technical replicate of a particular treatment. Total height (cm) of all seedlings per pot is denoted on the Y-axis, and the X-axis shows the number of germinated seeds per pot.

Fig 7. Deletion of FvZBD1 dramatically increased fumonisin production in GYAM liquid cultures.

Two milliliters of GYAM liquid medium in snap-cap tubes were inoculated with 10⁴ spores of each strain and cultured in the dark at 27°C, 250 rpm for 7 days. Fumonisin concentrations were determined by LC-MS and normalized to the vacuum-desiccated fungal mass weight, as indicated on the Y-axis. The experiment was conducted three times, with 3 technical replicates each. The two trials showed similar patterns, and one representative trial is plotted. Statistical differences (p-value < 0.05) were estimated with the two-tailed Mann Whitney Wilcoxon test and denoted with lower case letters for each fumonisin group. Strains sharing the same letters are not significantly different. FB1/FB2/FB3 represent fumonisin B1/B2/B3.

Fig 8. Pyrrocidine B reduces fumonisin production in a dose-dependent manner.

Wild-type Fusarium verticillioides (FRC M-3125) was grown for 3 days in 3 mL potato dextrose broth (PDB) in a snap-cap, round bottom tube incubated at 27°C with shaking at 250 rpm. From this culture, 10 μL was inoculated into each of 21 replicate tubes containing 3 mL fresh PDB. These were incubated for 24 hrs as before, after which the following seven treatments were applied to triplicate cultures: No treatment control, DMSO control, 0.5, 1.0, 2.5, 5.0, and 10 μg/mL pyrrocidine B. The cultures were incubated as above for 4 days and then extracted and analyzed for FB1, FB2, and FB3. The experiment was conducted a minimum of three times, with similar results obtained from all experiments. Data from a single trial are presented.

Fig 9. ΔFvZBD1 mutants, but not wild type, displayed uniform colony margins on GYAM plates.

(A) Seven-day-old GYAM agar cultures displayed different growth phenotypes of M-3125 (left) and ΔFvZBD1-1 (right), both shown from the front view. (B) Growth phenotypes of nine-day-old GYAM cultures for the F. verticillioides strains with both front and reverse views (left and right halves, respectively, of the split images). Genotypes are labeled as shown.

Fig 10. Illustration of biological antagonism between two maize seed endophytes, F. verticillioides and S. zeae.

F. verticillioides is primarily confined to the pedicel of the maize kernel, while S. zeae is more frequently isolated from embryonic tissue. Pyrrocidine A and B, two lactam-containing antibiotics produced by S. zeae, are postulated to contribute to an allelopathic antagonism between the two fungi. The chemical structures of pyrrocidines differ by a double (red circle) or single bond within lactam ring, resulting in higher toxicity of pyrrocidine A than B. Detailed analyses were carried out for an ABC transporter encoding gene, FvABC3, and a putative zinc-binding dehydrogenase encoding gene, FvZBD1. We hypothesize that FvABC3 facilitates persistence of F. verticillioides in maize seeds when encountering pyrrocidines, while the pyrrocidines induce the expression of FvZBD1 in F. verticillioides, a gene negatively impacting the production of fumonisins and virulence in maize seedlings. FvZBD1 is the most highly induced gene in both pyrrocidine A and B treatments, and its impact on fumonisin production provides evidence for allelochemical activity and response.