Relation between CarS expression and activation of carotenogenesis by stress in Fusarium fujikuroi

Abstract

Fusarium fujikuroi, a model organism for secondary metabolism in fungi, produces carotenoids, terpenoid pigments with antioxidant activity. Previous results indicate that carotenoid synthesis in F. fujikuroi is stimulated by light or by different stress conditions and downregulated by a RING finger protein encoded by carS gene. Here, we have analyzed the effects of three stressors, nitrogen scarcity, heat shock, and oxidative stress. We compared them with the effect of light in the wild type, a carS mutant that overproduces carotenoids, and its complemented strain. The assayed stressors increase the synthesis of carotenoids in the three strains, but mRNA levels of structural genes of carotenogenesis, carRA and carB, are only enhanced in the presence of a functional carS gene. In the wild-type strain, the four conditions affect in different manners the mRNA levels of carS: greater in the presence of light, without significant changes in nitrogen starvation, and with patent decreases after heat shock or oxidative stress, suggesting different activation mechanisms. The spores of the carS mutant are more resistant to H₂O₂ than those of the wild type; however, the mutant shows a greater H₂O₂ sensitivity at the growth level, which may be due to the participation of CarS in the regulation of genes with catalase domains, formerly described. A possible mechanism of regulation by heat stress has been found in the alternative splicing of the intron of the carS gene, located close to its 3' end, giving rise to the formation of a shorter protein. This action could explain the inducing effect of the heat shock, but not of the other inducing conditions, which may involve other mechanisms of action on the CarS regulator, either transcriptionally or post-transcriptionally.

Keywords: CarS; alternative splicing; heat shock; intron; light; nitrogen starvation; oxidative stress; photoinduction.

FIGURE 1

Carotenoid biosynthesis in Fusarium. Chemical modifications in each step are highlighted in color. The dashed arrow summarizes three sequential desaturations. The car genes encoding the enzymes in charge of each reaction are indicated. The orange area indicates the steps in charge of the genes of the car cluster. In the top on the right, it is depicted a simplified model for the effects of the four inducing conditions mentioned in the text (White arrows indicate positive effects on transcription of the car genes and grey bars indicate negative effects on carS transcription).

FIGURE 2

Effect of light and carS gene mutation on carotenoid production and expression of car genes. (A) Relative carotenoid content in the wild type (WT), carS mutant (SG39), and the complemented mutant (SG256). Absolute carotenoid contents are shown in Supplementary Table S1. Carotenoids were analyzed in mycelia incubated for 24 h after the 1-h illumination in the Petri dishes or without illumination. (B) Transcript levels of carRA, carB, and carS genes. The three strains were cultivated for 3 days in shake flasks with DG medium at 30°C in darkness, 25 ml of the cultures were transferred to Petri dishes and were illuminated with white light for 1 hour or incubated in darkness. Levels of mRNA were determined by RT-qPCR and referred to the levels in the wild type grown in the dark, taken as 1.

FIGURE 3

Effect of nitrogen availability and carS mutation on carotenoid production and expression of car genes. (A) Relative carotenoid production or without treatment (control) in the wild type (WT), carS mutant (SG39), and the complemented mutant (SG256) under two different nitrogen concentrations. (B) Transcript levels of carRA, carB, and carS genes at two nitrogen concentrations. The three strains were grown in DG medium for 3 days at 30°C in darkness with 3 g/L⁻¹ NaNO₃ (High N) or with 0.625 g/L⁻¹ NaNO₃ (Low N). Levels of mRNA were determined by RT-qPCR and referred to the levels in the wild type grown in High N, taken as 1. The Petri dishes were incubated for 24 h more before extraction of carotenoids.

FIGURE 4

Effect of heat shock on carotenoid production and expression of car genes. (A) Relative carotenoid production in the wild type (WT), carS mutant (SG39), and the complemented mutant (SG256) measured 1 day after 1 hour of heat shock. (B) Expression of carRA, carB and carS genes after 1-h heat shock. The three strains were grown in DG medium for 3 days at 30°C in darkness and incubated for 1 hour at 42°C. RNA was extracted from samples taken after the heat shock and carotenoids from samples incubated 24 h at 30°C after the heat shock. Controls for RNA were incubated 1 hour at 30°C. Controls for carotenoids were incubated 25 hours at 30°C. Levels of mRNA were determined by RT-qPCR and referred to the levels in the wild type grown without heat shock, taken as 1.

FIGURE 5

Effect of hydrogen peroxide on growth of the wild type (WT), carS mutant (SG39), and the complemented mutant (SG256). (A) Growth of surface colonies on media with different H₂O₂ concentrations. Strains were grown for 4 days in DG agar medium and then 1-mm² pieces of mycelia were transferred to DG agar with the indicated H₂O₂ concentrations. Pictures were taken after 3 days of cultivation at 30°C in darkness. (B) Growth from drops containing serial dilutions of spores of the three strains on DG agar with different H₂O₂ concentrations, incubated for 3 days at 30°C in the dark. (C) Germination of spores exposed to hydrogen peroxide. Spores were incubated with different concentrations of H₂O₂ for 30 min in agitation in darkness. Then the spores were washed twice and spread on DG plates to count number of colonies.

FIGURE 6

Effect of oxidative stress and carS gene mutation on carotenoid production and expression of car genes. (A) Relative carotenoid accumulation after hydrogen peroxide treatment for 24 h in the wild type (WT), carS mutant (SG39), and the complemented mutant (SG256). (B) Expression levels of carRA, carB and carS genes after 1 hour incubation with H₂O₂. Control: samples without treatment. Culture conditions are as those for Figure 2. Levels of mRNA were determined by RT-qPCR and referred to the levels in the wild type grown without hydrogen peroxide, taken as 1.

FIGURE 7

Effect of stressing conditions on splicing of carS mRNA. (A) Isoforms of carS mRNA according to RNA-seq reads. Red and blue bars indicate the mutually exclusive exons fused to the large pale blue exon depending on the splicing event. Arrows indicate primers used in the PCR assays. Stop codons resulting from each mRNA are indicated. Above: intron retention. Middle: splicing of a 168-bp intron. Below: splicing of a 336-bp intron. Only the relevant carS segment is displayed. (B) Three predicted isoforms of the CarS carboxy end according to the three intron maturation alternatives: intron retention (isoform A), 168-bp intron splicing (isoform B), and 336-bp intron splicing (isoform C). (C) PCR test of alternative splicing of carS gene in response to stress. Samples of 25 ml of 3-day-old cultures incubated at 30°C in the dark were transferred to Petri dishes and kept under the indicated stress for 1 h in the dark (D) or under illumination (L). Nitrogen starved mycelia (labelled as Nitrogen) were grown for 3 days in medium with 0.625 g/L⁻¹ NaNO₃ before the transfer to Petri dishes. Oxidative stress was generated incubating mycelia with 16 mM H₂O₂ for 1 h in the Petri dishes. M, Size markers; G, Genomic DNA; C, Control without cDNA. Molecular interpretation of the PCR products in the three mRNA alternatives is depicted on the schemes on the right. Densitometry analysis of the indicated bands is shown below. Intensity of each band is represented with a different color: green, 575-bp band (intron retention); red, 407-bp band (168-bp spliced intron); blue, 239-bp band (336-bp spliced intron).

FIGURE 8

Effect of temperature on mRNA splicing. (A) PCR tests of the effect of temperature on splicing in carS mRNA in response to temperature. Cultures transferred to Petri dishes were incubated for 1 hour at 30°C in the dark for adaptation and incubated afterwards for an additional hour at the indicated temperature. The correspondence of the bands with the alternative splicing events is shown on the right scheme. In this case, the higher distance between the primers results in larger PCR products compared to Figure 7. Densitometry analysis of the indicated bands is shown on the right. Intensity of each band is represented with a different color: green, 976-bp band (intron retention); red, 808-bp band (168-bp spliced intron); blue, 639-bp band (336-bp spliced intron). (B) Effect of temperature on the splicing of the second intron of carB gene, which does not exhibit alternative splicing according to RNA-seq data. In the three electrophoresis pictures: M, Size markers; G, Genomic DNA; C, Control without cDNA.