Three Genes Involved in Different Signaling Pathways, carS, wcoA, and acyA, Participate in the Regulation of Fusarin Biosynthesis in Fusarium fujikuroi

Abstract

The phytopathogenic fungus Fusarium fujikuroi has a rich secondary metabolism which includes the synthesis of very different metabolites in response to diverse environmental cues, such as light or nitrogen. Here, we focused our attention on fusarins, a class of mycotoxins whose synthesis is downregulated by nitrogen starvation. Previous data showed that mutants of genes involved in carotenoid regulation (carS, encoding a RING finger protein repressor), light detection (wcoA, White Collar photoreceptor), and cAMP signaling (AcyA, adenylate cyclase) affect the synthesis of different metabolites. We studied the effect of these mutations on fusarin production and the expression of the fus1 gene, which encodes the key polyketide synthase of the pathway. We found that the three proteins are positive regulators of fusarin synthesis, especially WcoA and AcyA, linking light regulation to cAMP signaling. Genes for two other photoreceptors, the cryptochrome CryD and the Vivid flavoprotein VvdA, were not involved in fusarin regulation. In most cases, there was a correspondence between fusarin production and fus1 mRNA, indicating that regulation is mainly exerted at the transcriptional level. We conclude that fusarin synthesis is subject to a complex control involving regulators from different signaling pathways.

Keywords: CarS; Fus1 polyketide synthase; Fusarins; White Collar; adenylate cyclase; photoreceptor.

Figure 1

Fusarin production and detection. (A) Cultures of F. fujikuroi wild-type strain FKMC 1995 grown in DG medium with 20 mM (high N) or 4.2 mM of asparagine (low N). (B) Chemical structure of fusarin C. (C) Absorption spectrum of fusarins with a characteristic peak at 350 nm. Presence of fusarins in high-N culture in (A) is indicated by yellow color due to their absorption at 400–550 nm.

Figure 2

The effects of nitrogen concentration and illumination on fusarin production and fus1 gene expression in a F. fujikuroi wild-type strain (WT) and the wcoA⁻ mutants SF226 and SF229. (A,B) Amount of fusarins secreted into the culture media. (C,D) Fusarins accumulated in the mycelia of the WT, SF226, and SF229 strains. (E,F) Effect of wcoA deletion on fus1 mRNA levels in the same strains. (A,C,E) Cultures in darkness. (B,D,F) Cultures under illumination. The effect of wcoA mutation on fus1 mRNA levels was measured by qPCR, using tubulin as an endogenous control. Mycelia were grown in liquid DGasn media with 20 mM (High N) or 4.2 mM of asparagine (Low N) at 150 rpm. Strains were incubated in darkness (left panels) or light (right panels) at 30 °C for 7 days. Filtrates and mycelia were taken from the same cultures. Fusarin data show average and standard deviations from two independent experiments. Expression data represent the average and standard deviation of 4 measurements from 2 independent experiments. Statistically significant differences are indicated with different letters according to the Tukey HSD test for a significance level of α = 0.05.

Figure 3

Amounts of fusarins secreted by a wild-type strain and ΔcryD and ΔvvdA mutants under different nitrogen concentrations and illumination conditions. (A,B) Amount of total fusarins secreted by the wild type (WT) and the ΔcryD mutants SG236 and SG237 into the medium. (C,D) Fusarins produced and secreted into culture medium by the WT and the ΔvvdA mutants SG256 and SG258. The strains were incubated for 7 days at 30 °C without (A,C) or with illumination (B,D). Statistical differences are indicated with different letters according to a Tukey HSD test for a significance level of α = 0.05.

Figure 4

Fusarin production and fus1 expression by the wild type and carS mutants SF114 and SF116, grown under different conditions of nitrogen and illumination. (A,B) Fusarins in the filtrates of 7-day-old cultures at 30 °C in minimal medium with high N (20 mM asparagine) and low N (4.2 mM asparagine) concentrations in the dark (left) and in the light (right). Data show mean and standard deviation values from two independent experiments. (C,D) Relative fus1 mRNA amounts in mycelia from the cultures whose fusarin production levels are shown in the upper graphs. Data represent mean and standard deviation values from 4 measurements from 2 independent experiments. Statistically significant differences are indicated with different letters according to the Tukey HSD test for a significance level of α = 0.05.

Figure 5

Transcript levels of fusarin genes in the wild type IMI 58289, and carS mutant SG39, isolated from this strain. (A) Transcripts per million (TPMs) of the fus1 gene in both strains in the dark and in the light. (B) TPMs of a selection of fusarin cluster genes in the wild type (WT) and carS mutant in the dark and after illumination. Values obtained from the datasets of a former RNA-seq study on the influence of light and the CarS protein on the F. fujikuroi transcriptome [31]. Briefly, mycelia were grown for 3 days in flasks, and 25 mL samples were transferred to Petri dishes for illumination for 1 h or incubation in the dark. WT: wild type. 39: SG39 carS mutant. Statistically significant differences are indicated with different letters according to the Tukey HSD test for a significance level of α = 0.05.

Figure 6

Effect of acyA deletion on fusarin production and fus1 gene expression in cultures with high and low nitrogen concentrations. (A) Fusarin secreted into the culture media; (B) Fusarin accumulated in the mycelia. Cultures of the wild type (WT) and ΔacyA mutants SG271 and SG272 were grown at 30 °C for 7 days in the dark in medium with 20 mM asparagine (High N) or with 4.2 mM asparagine (Low N). Filtrates and mycelia corresponded to the same cultures. Data show the averages and standard deviations from two independent experiments. (C,D) Effect of nitrogen on fus1 mRNA levels in the same strains grown in a high-N medium (C) or low-N medium (D). Data are average and standard deviation values of 4 measurements from 2 independent experiments. The same letters on the bars represent a lack of statistical significance in the differences among the strains on the conditions indicated, according to the Tukey HSD (p > 0.05).