The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast

Abstract

The BcWCL1 protein is a blue-light photoreceptor from the fungus Botrytis cinerea. This protein has a central role in B. cinerea circadian regulation and is an ortholog to WC-1 from Neurospora crassa. The BcWCL1 and WC-1 proteins have similar protein domains, including a LOV (Light Oxygen Voltage) domain for light sensing, two PAS (Per Arnt Sim) domains for protein-protein interaction, and a DNA binding domain from the GATA family. Recently, the blue-light response of BcWCL1 was demonstrated in a version without PAS domains (BcWCL1^PAS∆). Here, we demonstrated that BcWCL1^PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1^PAS∆ enables transcriptional activation as a single component in yeast. By using chimeric transcription factors and the luciferase reporter gene, we assessed the transcriptional activity of different fragments of the N-terminal and C-terminal regions of BcWCL1^PAS∆, identifying a functional transcriptional activation domain (AD) in the N-terminal region that belongs to the 9aaTAD family. Finally, we determined that the transcriptional activation levels of BcWCL1^PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1^PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.

Keywords: Botrytis cinerea; blue light; photoreceptor; transcriptional activation; yeast.

Figure 1

Protein domains and predicted structure of NcWC-1 and BcWCL1. (a) Protein domains in NcWC-1, BcWCL1, and BcWCL1^PAS∆. (b) AlphaFold predicted structure of NcWC-1, (c) BcWCL1, and (d) BcWCL1^PAS∆, respectively. In all panels, PAS (Per Arnt Sim) domains are shown green, and LOV (Light Oxygen Voltage) domains are shown in purple. Abbreviation: AD, Activation Domain; NLS, Nuclear Localization Sequence; GATA, DNA binding domain from the GATA-type transcription factor family; aa, amino acidic residue.

Figure 2

BcWCL1^PAS∆ protein self-dimerizes and activates transcription upon blue-light stimulation. (a) BcWCL1^PAS∆ protein fused to Gal4-DBD or Gal4-AD was assayed using a yeast two-hybrid architecture and the luciferase reporter. Protein–protein interaction was measured as luciferase expression under three illumination conditions: BL, constant blue light; BLP, a single blue-light pulse of 2 h duration; and DD, constant darkness (full data set in Figures S1 and S2). (b) Luciferase expression measured under BLP condition for the protein–protein interaction shown in panel (a). (c) BcWCL1^PAS∆ protein fused to Gal4-DBD and assayed as a single component. (d) Luciferase expression measured under BLP condition for the single component shown in panel (c). In panels (b,d), luciferase expression is shown as luminescence in arbitrary units (a.u.) divided by the optical density (OD_600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.

Figure 3

The N-terminal region of BcWCL1^PAS∆ protein is necessary for self-dimerization and transcriptional activation upon blue-light stimulation. (a) The N-terminal region of BcWCL1^PAS∆ protein was dissected by removing three fragments: amino acids 1–120 (sn1-BcWCL1^PAS∆), amino acids 121–240 (sn2-BcWCL1^PAS∆), and amino acids 241–365 (sn3-BcWCL1^PAS∆). (b) Shorter versions of BcWCL1^PAS∆ were assayed for protein–protein interaction using a yeast two-hybrid architecture and the luciferase reporter. Luciferase expression was assayed under three illumination conditions: BL, constant blue light; BLP, a single blue-light pulse of 2 h duration; and DD, constant darkness (full data set in Figures S3 and S4). (c) Luciferase expression measured under BLP condition for the protein–protein interaction shown in panel (b). (d) BcWCL1^PAS∆ protein fused to Gal4-DBD and assayed as a single component. (e) Luciferase expression measured under BLP condition for the single components shown in panel (d). In panels (c,e), luciferase expression is shown as luminescence in arbitrary units (a.u.) divided by the optical density (OD_600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.

Figure 4

Fragment 2 (F2) inside the N-terminal region of BcWCL1^PAS∆ is a functional transcriptional activation domain in yeast. (a) The full N-terminal region, three fragments of the N-terminal region (F1, F2, and F3), and one fragment of the C-terminal region (F4) of BcWCL1^PAS∆ were fused to the Gal4-DBD, generating chimeric transcription factors. Transcriptional activity was measured as luciferase expression under constant darkness (shown in this figure) and constant blue-light conditions (full data set in Figures S5 and S7). (b) Luciferase expression for the chimeric transcription factors shown in panel (a). (c) Deletion of the 9aaTAD motif in the chimeric transcription factors carrying F2 and the full N-terminal region of the BcWCL1^PAS∆ protein. (d) Luciferase expression for the chimeric transcription factors shown in panel (c). In panels (b,d), luciferase expression is shown as luminescence in arbitrary units (a.u.) divided by the optical density (OD_600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.

Figure 5

Activation Domain (AD) in fragment 2 (F2) of BcWCL1^PAS∆ promotes high levels of transcriptional activation in yeast. (a) Gal4 and p65 ADs were fused to the Gal4-DBD, generating chimeric transcription factors. Transcriptional activity was measured as luciferase expression under constant darkness condition. (b) Luciferase expression for the chimeric transcription factors shown in panel (a). Luciferase expression is shown as luminescence in arbitrary units (a.u.) divided by the optical density (OD_600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.