HmbC, a Protein of the HMG Family, Participates in the Regulation of Carotenoid Biosynthesis in Fusarium fujikuroi

Abstract

In the fungus Fusarium fujikuroi, carotenoid production is up-regulated by light and down-regulated by the CarS RING finger protein, which modulates the mRNA levels of carotenoid pathway genes (car genes). To identify new potential regulators of car genes, we used a biotin-mediated pull-down procedure to detect proteins capable of binding to their promoters. We focused our attention on one of the proteins found in the screening, belonging to the High-Mobility Group (HMG) family that was named HmbC. The deletion of the hmbC gene resulted in increased carotenoid production due to higher mRNA levels of car biosynthetic genes. In addition, the deletion resulted in reduced carS mRNA levels, which could also explain the partial deregulation of the carotenoid pathway. The mutants exhibited other phenotypic traits, such as alterations in development under certain stress conditions, or reduced sensitivity to cell wall degrading enzymes, revealed by less efficient protoplast formation, indicating that HmbC is also involved in other cellular processes. In conclusion, we identified a protein of the HMG family that participates in the regulation of carotenoid biosynthesis. This is probably achieved through an epigenetic mechanism related to chromatin structure, as is frequent in this class of proteins.

Keywords: HmbC protein; carS gene; carotenoids; high-mobility group; protoplast formation; pull-down assay.

Figure 1

Transcript levels (FPKM) of the genes encoding the proteins with HMG domains described in Table 1. (A) Expression of genes with relatively high transcription levels. (B) Genes with low transcription levels. (C) Data for the genes carRA and carB are included as examples of strong activations by light and by the carS mutation. WT: Wild type, SG39: carS mutant. Red bars: one h illumination. Data retrieved from the datasets of a formerly realized RNA-Seq study on the effects of light and CarS protein on the F. fujikuroi transcriptome [23]. Only statistically significant differences, according to Student’s t test, are indicated: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 2

Effect of hmbC deletion on carotenoid production. (A) Agar cultures of wild type and four transformants grown for 7 days in the dark at 30 °C. (B) Content of carotenoids in the wild type (WT), the ectopic transformant SG322, and the ΔhmbC transformants SG323 and SG324 grown in the dark. Strains were cultured for 7 days on minimal agar medium. Right graph: Absorption spectra of the acetone extracts used in the carotenoid analysis. (C) Results with the same cultures incubated under continuous illumination. Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 3

Effect of hmbC deletion on carotenoid production and car gene expression in submerged conditions. (A) Samples of 7-day-old submerged cultures of wild type (WT), and ΔhmbC transformants SG323 and SG324 grown in the dark at 30 °C. (B) Carotenoid content of the strains under the conditions shown in panel A. The ectopic SG322 transformant was also included in the analysis. (C) Transcript levels of the carRA, carB, and carS genes in 3-day-old cultures of the wild type and ΔhmbC strains at the same conditions. Only significant differences are indicated according to Student’s t test: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Protoplast formation in the ΔhmbC mutants and control strains. (A) Representative microscopy images of the production of protoplasts of the wild type and ΔhmbC mutant SG323 after 8 h in enzymatic solution. 100 mL of Darken medium were inoculated with a small mycelium plug and incubated for 3 days at 30 °C stirring at 200 rpm. Then, 100 mL of fresh ICI medium was inoculated with 1 mL of the previous culture, and it was incubated overnight at 30 °C. The mycelium was vacuum filtered through sterile filter paper and placed into 25 mL of enzyme solution. Yellow arrowheads indicate visible protoplasts in the photographs. (B) Protoplast formation after 8 h. The number of wild type protoplasts was taken as 1. The results show average and standard deviation from two independent experiments. Statistical identifiers of Student’s t test: ** p < 0.01, *** p < 0.001.

Figure 5

Effect of 1.2 M sorbitol on the morphology of colonies and hyphae at colony borders. Left column: aspect of representative colonies in sorbitol-supplemented medium. Wrinkles on the colony surface are more prominent in the control strains. Central column: differences in the density and appearance of colony edges can also be seen under a stereoscopic microscope with backlighting. Right column: microscope photographs of hyphae at colony edges. Curvy hyphae, indicated by arrows, are less evident in the ΔhmbC transformants. The bars below each column correspond to 1 mm.