The flavoproteins CryD and VvdA cooperate with the white collar protein WcoA in the control of photocarotenogenesis in Fusarium fujikuroi

Abstract

Light stimulates carotenoid biosynthesis in the ascomycete fungus Fusarium fujikuroi through transcriptional activation of the structural genes of the pathway carRA, carB, and cart, but the molecular basis of this photoresponse is unknown. The F. fujikuroi genome contains genes for different predicted photoreceptors, including the WC protein WcoA, the DASH cryptochrome CryD and the Vivid-like flavoprotein VvdA. We formerly found that null mutants of wcoA, cryD or vvdA exhibit carotenoid photoinduction under continuous illumination. Here we show that the wild type exhibits a biphasic response in light induction kinetics experiments, with a rapid increase in carotenoid content in the first hours, a transient arrest and a subsequent slower increase. The mutants of the three photoreceptors show different kinetic responses: the wcoA mutants are defective in the rapid response, the cryD mutants are affected in the slower response, while the fast and slow responses were respectively enhanced and attenuated in the vvdA mutants. Transcriptional analyses of the car genes revealed a strong reduction of dark and light-induced transcript levels in the wcoA mutants, while minor or no reductions were found in the cryD mutants. Formerly, we found no change on carRA and carB photoinduction in vvdA mutants. Taken together, our data suggest a cooperative participation of WcoA and CryD in early and late stages of photoinduction of carotenoid biosynthesis in F. fujikuroi, and a possible modulation of WcoA activity by VvdA. An unexpected transcriptional induction by red light of vvdA, cryD and carRA genes suggest the participation of an additional red light-absorbing photoreceptor.

Fig 1. Colonies of the wild type and representative ΔwcoA (SF226), ΔcryD (SF236) and ΔvvdA (SF258) mutants.

The strains were grown for 7 days at 30°C in the dark or under continuous illumination on DGasn medium. The brownish color of the ΔwcoA mutants in the dark and the reddish color of the ΔcryD mutants in the light are due to the production of secondary metabolite pigments unrelated to carotenoids, such as bikaverins and other polyketides.

Fig 2. Effect of the mutation of the photoreceptor genes cryD, wcoA and vvdA on carotenogenesis in the dark versus constant illumination.

A: Total concentration of carotenoids in the wild type and in parallel cultures of the ΔwcoA and ΔcryD mutants grown for 7 days at 30°C on DGasn medium in the dark or under continuous illumination. Data are the means and standard deviations of four determinations from two biological replicates, taking as 1 the value of the wild type under light. Data for ΔvvdA under the same culture conditions were recently published [42]. B: Real-time RT-PCR analyses of the genes carRA and carB in RNA samples of the wild-type and the indicated strains grown for 7 days at 30°C on DGasn medium in the dark or under continuous illumination. Relative expression for each gene was referred to the value in the wild type grown in the dark. Data are the means and standard deviations of six determinations from two biological replicates. Here, and in following figures, the wild type and the mutants for each gene are represented by different colors (brown, wild type; reddish-orange, ΔvvdA; blue, ΔwcoA; green, ΔcryD). Lighter versions of the colors are used for illuminated samples. Statistical analysis for differences between data of wild type and mutants are displayed in S1 Table.

Fig 3. Light-induced carotenogenesis in wild type and mutants of the photoreceptor genes cryD, wcoA and vvdA.

A: Kinetics of carotenoid accumulation after illumination of the wild type, ΔwcoA mutants SF226 and SF229, ΔcryD mutants SF236 and SF237, and ΔvvdA mutants SF256 and SF258. The strains were incubated for three days in the dark on DGasn agar and exposed to white light for the time indicated in abscissae. Each point is the mean and standard deviation of four determinations from two biological replicates. In this and in other figures, overlapping data were separated for better visualization. B: Real-time RT-PCR analyses of the genes carRA (pale bars) and carB (dark bars) in RNA samples of the same strains under the same culture conditions after 48 h illumination. Relative expression for each gene was referred to the value in the wild type grown in the dark. Data are the means and standard deviations of six determinations from two biological replicates.

Fig 4. Effect of light intensity on photoinduction of carotenoid accumulation in the wild type and ΔwcoA mutants SF226 and SF229, ΔcryD mutants SF236 and SF237, and ΔvvdA mutants SF256 and SF258.

The strains were incubated for three days in the dark in DGasn medium (left bars, darker colors) and exposed to 0.07 W m^-2, 0.7 W m^-2 or 7 W m^-2 (brighter colors from left to right) of white light for 6 hours (above) and 48 h (below). Data show means and standard deviations of four determinations from two biological replicates. Statistical analysis for differences between data of wild type and mutants are displayed in S1 Table.

Fig 5. Effect of the vvdA mutation on expression of the photoreceptor genes cryD and wcoA.

Real-time RT-PCR analyses of genes cryD and wcoA in total RNA samples from the wild type (brown squares) and the ΔvvdA mutants SF256 (red circles), SF257 (red rhombs) and SF258 (red triangles) grown for three days in DGasn medium in the dark or after 15 min, 30 min, 1 h, 2 h, or 4 h exposure to 7 W m^-2 white light. Relative levels are referred to the value of the wild type in the dark. Data show means and standard deviations for nine measurements from three biological replicates. Statistical analysis for differences between data of wild type and mutants are displayed in S1 Table.

Fig 6. Effect of the wcoA mutation on expression of genes of the carotenoid pathway.

Real-time RT-PCR analyses of genes carB, carRA and carT in total RNA samples from the wild type (brown squares) and the ΔwcoA mutants SF226 (blue circles) and SF229 (blue triangles) grown for three days in DGasn medium in the dark or after 15 min, 30 min, 1 h, 2 h, or 4 h exposure to 7 W m^-2 white light. Relative levels are referred to the value of the wild type in the dark. Data show means and standard deviations for nine measurements from three biological replicates.

Fig 7. Effect of the cryD mutation on expression of genes of the carotenoid pathway and other light-regulated genes of F. fujikuroi.

Real-time RT-PCR analyses of genes carB, carRA, carT, carO, carX and phr1 in total RNA samples from the wild type (brown squares) and the ΔcryD mutants SF236 (green circles) and SF237 (green triangles) grown for three days in DGasn medium in the dark or after 15 min, 30 min, 1 h, 2 h, or 4 h exposure to 7 W m^-2 white light. Relative levels are referred to the value of the wild type in the dark. Data show means and standard deviations for nine measurements from three biological replicates. Statistical analysis for differences between data of wild type and mutants are displayed in S1 Table. For better comparison, mean values for mRNA levels of genes carB, carRA, carO and carX in wild type and mutants at the point of maximal expression (one hour of illumination) are indicated in the figure.

Fig 8. Effect of light wavelength on photoinduced expression of the photoreceptor genes cryD, wcoA and vvdA, and the carotenogenic gene carRA.

Real-time RT-PCR analyses of genes cryD, wcoA, vvdA and carRA in total RNA samples from the wild type grown for three days in DGasn medium in the dark or after 15 min, 30 min, 1 h, 2 h, or 4 h exposure to white light (black symbols), blue light (blue symbols) or red light (red symbols). Relative levels are referred to the value of the wild type in the dark. Data show means and standard deviations for six measurements from two biological replicates. The inset shows the transmittance spectra of the uncolored, blue and red filters used for illumination in this experiment (adapted from [33]).

Fig 9. A hypothetic model for regulation of carotenogenesis by light in F. fujikuroi.

Circles correspond to proteins and squares to genes. The sizes of the circles represent low or high protein amounts in the corresponding conditions. In the absence of information on PhyA regulation, the same size is used for this protein. Non-activated proteins are indicated in pale colors with black letters and light-activated proteins are indicated in dark colors with white letters. The thickness of the arrows roughly represents predicted intensity of the regulatory effects, while positive and negative signs (arrows and truncated lines) represent inducing or repressing effects. Effect of VvdA could be achieved by protein/protein interactions, while CryD would up-regulate the car genes post-transcriptionally. The final effect on carotenoid biosynthesis is indicated below.