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

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 (BcWCL1PAS∆). Here, we demonstrated that BcWCL1PAS∆ is capable of self-dimerization through its N-terminal region upon blue-light stimulation. Interestingly, we observed that BcWCL1PAS∆ 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 BcWCL1PAS∆, 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 BcWCL1PAS∆ AD are comparable to those obtained with commonly used ADs in eukaryotic cells (Gal4 and p65). In conclusion, the BcWCL1PAS∆ protein self-dimerized and activated transcription in a blue-light-dependent fashion, opening future applications of this photoreceptor in yeast optogenetics.


Introduction
Botrytis cinerea is one of the most common phytopathogens in the world [1,2]. This fungus responds to light stimulation, promoting conidiation and inhibition of sclerotia formation [3][4][5][6][7]. The B. cinerea capacity to respond the light cues is based on photoreceptors, which are specialized proteins capable of light-sensing [8]. Interestingly, the B. cinerea genome encodes 11 photoreceptors that potentially can sense light of different wavelengths [9][10][11]. The set of B. cinerea photoreceptors includes: two cryptochromes (BcCRY1 and BcCRY2) for UV light detection, four proteins containing LOV (Light Oxygen Voltage) domains (BcWCL1, BcVVD, BcLOV3, and BcLOV4) and responding to blue light, two rhodopsin-like proteins (BOP1 and BOP2) for green light perception, and three phytochromes (BcPHY1, BcPHY2, and BcPHY3) for red light sensing [10]. Among these photoreceptors, LOV-containing photoreceptors are very interesting, since they use FMN or FAD molecules as a cofactor, which are abundant molecules in eukaryotic cells [12,13]. In

Deletion of PAS Domains in BcWCL1 Does Not Affect the Overall Protein Structure
Previously, we demonstrated that deletion of the PAS domains in BcWCL1 (BcWCL1 PAS∆ ) uncover the blue-light response of this protein [25]. In addition, we also observed that BcWCL1 PAS∆ is capable of transcriptional activation in yeast upon blue-light stimulation [25]. In order to discard a structural effect on BcWCL1 protein due to the deletion of PAS domains, we assess the overall protein structure of BcWCL1 and BcWCL1 PAS∆ using AlphaFold2 [28]. The results showed that the deletion of PAS domains did not alter the structure of the LOV domain in BcWCL1 PAS∆ (Figure 1), which is consistent with the light response of this protein. We also extracted from the AlphaFold database [29] the predicted structure for NcWC-1 (Figure 1), N. crassa ortholog to BcWCL1, showing a similar globular structure for PAS and LOV domains compared to BcWCL1 ( Figure 1B,C). Interestingly, AlphaFold was not able to predict the structure in extensive regions of NcWC-1, BcWCL1, and BcWCL1 PAS∆ ( Figure 1B-D), including the N-terminal and C-terminal regions. Importantly, the N-terminal and C-terminal regions of NcWC-1 have putative transcriptional activation domains (ADs), where the former has been experimentally validated [23]. Thus, the N-terminal and C-terminal regions of NcWCL1 and BcWCL1 proteins could be intrinsically disordered, considering that different ADs such as p53 and NF-κB (p65) have been reported as unfolded [30,31]. In conclusion, a potential AD in the N-terminal region of BcWCL1 (or BcWCL1 PAS∆ ) could explain the light-mediated transcriptional activation observed in yeast. Altogether, the deletion of PAS domains in BcWCL1 did not affect the LOV domain structure; however, the predicted structure does not explain the light-dependent transcriptional activation observed in this protein.

Deletion of PAS Domains in BcWCL1 Does Not Affect the Overall Protein Structure
Previously, we demonstrated that deletion of the PAS domains in BcWCL1 (BcWCL1 PAS∆ ) uncover the blue-light response of this protein [25]. In addition, we also observed that BcWCL1 PAS∆ is capable of transcriptional activation in yeast upon blue-light stimulation [25]. In order to discard a structural effect on BcWCL1 protein due to the deletion of PAS domains, we assess the overall protein structure of BcWCL1 and BcWCL1 PAS∆ using AlphaFold2 [28]. The results showed that the deletion of PAS domains did not alter the structure of the LOV domain in BcWCL1 PAS∆ (Figure 1), which is consistent with the light response of this protein. We also extracted from the AlphaFold database [29] the predicted structure for NcWC-1 (Figure 1), N. crassa ortholog to BcWCL1, showing a similar globular structure for PAS and LOV domains compared to BcWCL1 ( Figure 1B,C). Interestingly, AlphaFold was not able to predict the structure in extensive regions of NcWC-1, BcWCL1, and BcWCL1 PAS∆ ( Figure 1B-D), including the N-terminal and C-terminal regions. Importantly, the N-terminal and C-terminal regions of NcWC-1 have putative transcriptional activation domains (ADs), where the former has been experimentally validated [23]. Thus, the N-terminal and C-terminal regions of NcWCL1 and BcWCL1 proteins could be intrinsically disordered, considering that different ADs such as p53 and NF-κB (p65) have been reported as unfolded [30,31]. In conclusion, a potential AD in the N-terminal region of BcWCL1 (or BcWCL1 PAS∆ ) could explain the light-mediated transcriptional activation observed in yeast. Altogether, the deletion of PAS domains in BcWCL1 did not affect the LOV domain structure; however, the predicted structure does not explain the light-dependent transcriptional activation observed in this protein.

BcWCL1 PAS∆ Self-Dimerizes and Activates Transcription in Response to Light
Initially, we confirmed the BcWCL1 PAS∆ capacity of light-dependent transcriptional activation in yeast using a Y2H strategy. Hence, we generated a version of BcWCL1 PAS∆ fused to the Gal4 DNA binding domain (DBD) and AD, BcWCL1 PAS∆ -Gal4-DBD and

BcWCL1 PAS∆ Self-Dimerizes and Activates Transcription in Response to Light
Initially, we confirmed the BcWCL1 PAS∆ capacity of light-dependent transcriptional activation in yeast using a Y2H strategy. Hence, we generated a version of BcWCL1 PAS∆ fused to the Gal4 DNA binding domain (DBD) and AD, BcWCL1 PAS∆ -Gal4-DBD and BcWCL1 PAS∆ -Gal4-AD, respectively. These proteins were assayed for light-mediated protein-protein interaction and transcriptional activation using the luciferase reporter gene (Figure 2A and full data set in Figure S1). The combination of BcWCL1 PAS∆ -Gal4-DBD and BcWCL1 PAS∆ -Gal4-AD results in a strong luciferase expression upon a blue-light pulse (BLP) of 2 h duration ( Figure 2B). By contrast, luciferase expression decreased when BcWCL1 PAS∆ -Gal4-DBD was individually assayed ( Figure 2C,D), and was absent when BcWCL1 PAS∆ -Gal4-AD was the sole component ( Figure S2). Therefore, our results support the supposition that BcWCL1 PAS∆ is capable of transcriptional activation in yeast, acting as a single component that responds to blue light and, in addition, self-dimerizes upon blue-light stimulation. BcWCL1 PAS∆ -Gal4-AD, respectively. These proteins were assayed for light-mediated protein-protein interaction and transcriptional activation using the luciferase reporter gene (Figure 2A and full data set in Figure S1). The combination of BcWCL1 PAS∆ -Gal4-DBD and BcWCL1 PAS∆ -Gal4-AD results in a strong luciferase expression upon a blue-light pulse (BLP) of 2 h duration ( Figure 2B). By contrast, luciferase expression decreased when BcWCL1 PAS∆ -Gal4-DBD was individually assayed ( Figure 2C,D), and was absent when BcWCL1 PAS∆ -Gal4-AD was the sole component ( Figure S2). Therefore, our results support the supposition that BcWCL1 PAS∆ is capable of transcriptional activation in yeast, acting as a single component that responds to blue light and, in addition, self-dimerizes upon bluelight stimulation. We further investigate the molecular mechanism of BcWCL1 PAS∆ light-dependent self-dimerization in yeast. To this end, we dissected the N-terminal region of this protein to generate a set of shorter versions of BcWCL1 PAS∆ ( Figure 3A). The BcWCL1 PAS∆ N-terminal region was shortened by removing three sections of similar size: the first 120 amino acids (sn1-BcWCL1 PAS∆ ), a further 120 amino acids for a total remotion of 240 amino acids (sn2-BcWCL1 PAS∆ ), and finally 365 amino acids corresponding to the entire N-terminal region (sn3-BcWCL1 PAS∆ ). These versions were fused to Gal4-DBD and Gal4-AD and then assayed in a Y2H configuration ( Figure 3B). The results showed that removing the first 120 amino acids of the N-terminal decreased the light-mediated protein-protein interaction between the sn1-BcWCL1 PAS∆ versions compared to the full N-terminal protein version (compare luciferase expression in Figures 2B and 3C). Furthermore, the deletion of amino acids 240-365 of the N-terminal region (sn2-BcWCL1 PAS∆ and sn3-BcWCL1 PAS∆ versions, respectively) completely abolished the light-mediated protein-protein interaction We further investigate the molecular mechanism of BcWCL1 PAS∆ light-dependent self-dimerization in yeast. To this end, we dissected the N-terminal region of this protein to generate a set of shorter versions of BcWCL1 PAS∆ ( Figure 3A). The BcWCL1 PAS∆ N-terminal region was shortened by removing three sections of similar size: the first 120 amino acids (sn1-BcWCL1 PAS∆ ), a further 120 amino acids for a total remotion of 240 amino acids (sn2-BcWCL1 PAS∆ ), and finally 365 amino acids corresponding to the entire N-terminal region (sn3-BcWCL1 PAS∆ ). These versions were fused to Gal4-DBD and Gal4-AD and then assayed in a Y2H configuration ( Figure 3B). The results showed that removing the first 120 amino acids of the N-terminal decreased the light-mediated protein-protein interaction between the sn1-BcWCL1 PAS∆ versions compared to the full N-terminal protein version (compare luciferase expression in Figures 2B and 3C). Furthermore, the deletion of amino acids 240-365 of the N-terminal region (sn2-BcWCL1 PAS∆ and sn3-BcWCL1 PAS∆ versions, respectively) completely abolished the light-mediated protein-protein interaction ( Figures  3C and S3). Then, we assayed each shorter version of BcWCL1 PAS∆ as single components ( Figure 3D,E), observing luciferase expression only for sn1-BcWCL1 PAS∆ upon blue-light stimulation ( Figure 3E and full data set in the Figure S4). Interestingly, the removal of the first 120 amino acids (sn1-BcWCL1 PAS∆ ) improves the light response of the protein compared to the full N-terminal protein version ( Figure 3E). Altogether, amino acids 1-120 of the N-terminal region are important for BcWCL1 PAS∆ self-dimerization upon bluelight stimulation. In addition, amino acids 121-240 of the N-terminal region are necessary for the light-dependent self-dimerization and transcriptional activation in yeast. Therefore, the results suggest the presence of a functional AD between residues 121 and 240 of the BcWCL1 PAS∆ N-terminal region. ( Figures 3C and S3). Then, we assayed each shorter version of BcWCL1 PAS∆ as single components ( Figure 3D,E), observing luciferase expression only for sn1-BcWCL1 PAS∆ upon blue-light stimulation ( Figure 3E and full data set in the Figure S4). Interestingly, the removal of the first 120 amino acids (sn1-BcWCL1 PAS∆ ) improves the light response of the protein compared to the full N-terminal protein version ( Figure 3E). Altogether, amino acids 1-120 of the N-terminal region are important for BcWCL1 PAS∆ self-dimerization upon blue-light stimulation. In addition, amino acids 121-240 of the N-terminal region are necessary for the light-dependent self-dimerization and transcriptional activation in yeast. Therefore, the results suggest the presence of a functional AD between residues 121 and 240 of the BcWCL1 PAS∆ N-terminal region.

The Transcriptional Activation Domain of BcWCL1 PAS∆ Is Localized in the N-Terminal Region between Amino Acids 121-240
In order to demonstrate that the N-terminal region of BcWCL1 PAS∆ contains a functional AD in yeast, we used a strategy based on the development of chimeric transcription

The Transcriptional Activation Domain of BcWCL1 PAS∆ Is Localized in the N-Terminal Region between Amino Acids 121-240
In order to demonstrate that the N-terminal region of BcWCL1 PAS∆ contains a functional AD in yeast, we used a strategy based on the development of chimeric transcription factors [32][33][34][35]. In this strategy, the Gal4-DBD (amino acids 1-149) was fused to the full N-terminal region of BcWCL1 PAS∆ and three previously delimited fragments of this region: F1 (amino acids 1-120), F2 (amino acids 121-240), and F3 (amino acids 241-365) ( Figure 4A). A fourth fragment (F4) from the C-terminal region (amino acids 971-1137) of BcWCL1 PAS∆ was also assayed ( Figure 4A). The functionality of the chimeric transcription factors was tested using the luciferase reporter ( Figure 4A). As a result, the full N-terminal region and F2 of BcWCL1 PAS∆ showed transcriptional activation of the luciferase reporter ( Figure 4B), which was independent of blue-light stimulation ( Figure S5), consistent with the absence of the LOV domain in the chimeric transcription factors. Interestingly, the F2 showed higher transcriptional activity compared to the full N-terminal region (Figure 4B), suggesting a potential inhibitory effect of F1 over F2 (see discussion section). Therefore, we conclude that BcWCL1 PAS∆ contains a functional AD between amino acids 121 and 240 of the BcWCL1 PAS∆ N-terminal region. factors [32][33][34][35]. In this strategy, the Gal4-DBD (amino acids 1-149) was fused to the full N-terminal region of BcWCL1 PAS∆ and three previously delimited fragments of this region: F1 (amino acids 1-120), F2 (amino acids 121-240), and F3 (amino acids 241-365) ( Figure  4A). A fourth fragment (F4) from the C-terminal region (amino acids 971-1137) of BcWCL1 PAS∆ was also assayed ( Figure 4A). The functionality of the chimeric transcription factors was tested using the luciferase reporter ( Figure 4A). As a result, the full N-terminal region and F2 of BcWCL1 PAS∆ showed transcriptional activation of the luciferase reporter ( Figure 4B), which was independent of blue-light stimulation ( Figure S5), consistent with the absence of the LOV domain in the chimeric transcription factors. Interestingly, the F2 showed higher transcriptional activity compared to the full N-terminal region ( Figure 4B), suggesting a potential inhibitory effect of F1 over F2 (see discussion section). Therefore, we conclude that BcWCL1 PAS∆ contains a functional AD between amino acids 121 and 240 of the BcWCL1 PAS∆ N-terminal region.

Transcriptional Activity of the BcWCL1 Activation Domain Is Comparable to Other Eukaryotic Activation Domains
Finally, we compared the transcriptional activity of the BcWCL1 PAS∆ AD (F2) to that of other eukaryotic ADs such as Gal4 and p65. To evaluate this, we developed chimeric transcription factors carrying the Gal4-DBD fused to Gal4 and p65 ADs ( Figure 5A). Then, we compared the transcriptional activity of each chimeric transcription factor by measuring the luciferase reporter gene expression ( Figure 5A). Interestingly, the chimeric transcription factor with region F2 of BcWCL1 PAS∆ showed higher levels of luciferase expression compared to Gal4 and p65 ADs (Figures 5B and S8). Furthermore, the results confirm that our approach based on chimeric transcription factors is a viable strategy to assess transcriptional activation in yeast. In conclusion, the BcWCL1 PAS∆ AD promotes a strong transcriptional activation in yeast, which, combined with its light-mediated self-dimerization, supports future applications of this photoreceptor in the development of novel optogenetic systems.  Then, we analyzed the N-terminal region of BcWCL1 PAS∆ for the presence of a 9aaTAD amino acids motif, which is commonly recognized by the eukaryotic transcriptional machinery, and it is present in ADs of transcription factors such as Gal4 and p65 [36]. Interestingly, the analysis revealed the presence of two 9aaTAD motifs in the N-terminal region of BcWCL1 PAS∆ , which are located inside F1 and F2, respectively ( Figure S6). As we observed no transcriptional activity for F1 in our previous chimeric transcription factor assays ( Figure 4B), we explored the functionality of the 9aaTAD motif inside the transcriptionally active F2. Thus, we generated chimeric transcription factors without the 9aaTAD motif for the F2 and the full N-terminal region of BcWCL1 PAS∆ ( Figure 4C). These chimeric transcription factors were assayed for luciferase expression (Figure 4C), observing a decreased transcriptional activity of the luciferase reporter compared to the wild-type versions ( Figures 4D and S7). Interestingly, the reduction observed in F2 suggests that the 9aaTAD motif is important for transcriptional activation, but additional amino acids inside F2 are also necessary for the transcriptional response. In conclusion, the BcWCL1 PAS∆ contains a functional AD in yeast, which belongs to the 9aaTAD family of ADs, and whose transcriptional activity is between amino acids 121 and 240 of the BcWCL1 PAS∆ N-terminal region.

Transcriptional Activity of the BcWCL1 PAS∆ Activation Domain Is Comparable to Other Eukaryotic Activation Domains
Finally, we compared the transcriptional activity of the BcWCL1 PAS∆ AD (F2) to that of other eukaryotic ADs such as Gal4 and p65. To evaluate this, we developed chimeric transcription factors carrying the Gal4-DBD fused to Gal4 and p65 ADs ( Figure 5A). Then, we compared the transcriptional activity of each chimeric transcription factor by measuring the luciferase reporter gene expression ( Figure 5A). Interestingly, the chimeric transcription factor with region F2 of BcWCL1 PAS∆ showed higher levels of luciferase expression compared to Gal4 and p65 ADs (Figures 5B and S8). Furthermore, the results confirm that our approach based on chimeric transcription factors is a viable strategy to assess transcriptional activation in yeast. In conclusion, the BcWCL1 PAS∆ AD promotes a strong transcriptional activation in yeast, which, combined with its light-mediated selfdimerization, supports future applications of this photoreceptor in the development of novel optogenetic systems.

Discussion
The blue-light sensing capacity of BcWCL1 was previously demonstrated in a version without PAS domains (BcWCL1 PAS∆ ) [25]. Here, we showed that BcWCL1 PAS∆ selfdimerized upon blue-light stimulation through its N-terminal region (Figure 3). Furthermore, we demonstrate that the N-terminal region of BcWCL1 PAS∆ contains a functional AD (fragment 2 or F2, amino acids 121-240) in yeast ( Figure 4). Interestingly, our experiments suggest that fragment 1 (F1, amino acid 1-120) has an inhibitory effect on the transcriptional activation of F2. This is supported by the results obtained using a shorter version of BcWCL1 PAS∆ (sn1-BcWCL1 PAS∆ ) where F1 was removed, showing an increased light-mediated dimerization and transcriptional activation compared to the BcWCL1 PAS∆ protein ( Figure 3E). Therefore, the inhibitory effect of F1 is light-dependent, where the conformational change (by light) in the LOV domain releases F2 from F1 inhibition and activates transcription. The chimeric transcription factor results also support this conclusion, when F2 was assayed individually (without LOV domain and F1 region), we observed that F2 transcriptional activation is now light-independent and higher than the full N-terminal region (Figures 4 and S5). In general, our findings in BcWCL1 PAS∆ are in agreement with previously reported results in NcWC-1, where a functional AD (amino acids 100-200) was described in the N-terminal region of this protein [23]. In addition, a putative AD has been described in the C-terminal region of NcWC-1 [21,22], which was not detected in our experiments with the chimeric transcription factor carrying the C-terminal region of BcWCL1 PAS∆ (Figure 4). Therefore, protein functionality at the N-terminal region of BcWCL1 correlates with its ortholog protein (NcWC-1).
The chimeric transcription factor strategy used in this work was previously implemented to discover ADs from bacteria [33,35] and in the development of synthetic transcription factors [32,34]. This approach allowed us to dissect the N-terminal region of BcWCL1 PAS∆ , confirming an AD into the F2 that belongs to the 9aaTAD family ( Figure 4). Finally, the chimeric transcription factors strategy was validated using the Gal4 and p65 ADs, confirming that BcWCL1 PAS∆ AD promotes a stronger activation of the luciferase reporter compared to Gal4 and p65 ADs ( Figure 5). Therefore, BcWCL1 PAS∆ is a blue-light photoreceptor that contains a functional AD in yeast, where blue light exerts BcWCL1 PAS∆ self-dimerization and transcriptional activation through its N-terminal region.
The BcWCL1 PAS∆ protein has unique characteristics that promote its application in yeast optogenetics; notably, the presence of a LOV domain sensitive to blue light, selfdimerization upon blue-light stimulation through its N-terminal region, and the presence of an AD in the N-terminal region (amino acids 121-240). Importantly, the deletion of the first 120 amino acids in the N-terminal region improves the light response of the protein as a single component (Figure 3), supporting the application of this shorter version (sn1-BcWCL1 PAS∆ ) in yeast optogenetics. Furthermore, luciferase expression activated by BcWCL1 PAS∆ delf-dimerization is higher under constant BL than in BLP condition ( Figures S1-S4), showing a similar behavior compared to the FUN-LOV (FUNgal Light Oxygen Voltage) optogenetic switch [27,37]. In this sense, different single-component optogenetic systems have been developed in yeast, which self-dimerizes and activates transcription upon light illumination [38,39]. The blue-light photoreceptor vivid (VVD) from N. crassa contains a LOV domain and self-dimerizes upon blue-light stimulation through its N-terminal region [40]. This photoreceptor was linked to the Gal4 or LexA DBDs, and fused to the Gal4-AD, developing a single-component optogenetic system for light-controlled gene expression that has been implemented in different biological platforms, including yeast [38], mammalian cells [41,42], Zebrafish, and Drosophila [43]. Similarly, the EL222 protein is a blue-light photoreceptor and transcription factor from Erythrobacter litoralis, which self-dimerizes upon blue-light stimulation through its LOV domain, binding a target promoter region and activating transcription [44][45][46]. Thus, EL222 has been fused to the VP16 AD and used for light-activated gene expression in different chassis, including yeast [39,47], mammalian cells [48], and Zebrafish [49]. Therefore, singlecomponent optogenetic systems have a wide range of potential applications in different model organisms.
Altogether, we have demonstrated that the N-terminal region of BcWCL1 PAS∆ is necessary for self-dimerization and transcriptional activation upon blue-light stimulation. Future experiments should address the possibility of deleting the BcWCL1 PAS∆ protein region between the LOV domain and the C-terminal end. This could reduce the protein size, favoring the utilization of BcWCL1 PAS∆ as a single-component optogenetic system for light-controlled gene expression.

Yeast Strains and Culture Conditions
All the experiments were carried out in the BY4741 yeast strain (MATa; his3∆1; leu2∆0; met15∆0; ura3∆0). This strain was maintained in YPDA (2% glucose, 2% peptone, 1% yeast extract, and 2% agar) at 30 • C. The BY4741 strain was transformed with different plasmids (Section 2.4) and grown in Synthetic Complete (SC) medium (0.67% yeast nitrogen base without amino acids, 2% glucose, 0.2% dropout mix) without the corresponding amino acids for auxotrophic selection. All the yeast strains used and generated in this work are listed in Supplementary Table S1.

Protein Structure Analysis
Protein domains in NcWC-1 and BcWCL1 were analyzed using the InterPro Scan search tool [50]. In addition, the 3D structure prediction of BcWCL1 and BcWCL1 PAS∆ was performed using AlphaFold2 [28] under default settings and using the ColabFold server [51]. In the case of NcWC-1, the 3D structure was extracted from the AlphaFold protein structure database (UniProt: Q01371) [29]. Based on these analyses, three fragments of similar size were defined in the N-terminal region of BcWCL1 PAS∆ : F1 (amino acids 1-120), F2 (amino acids 121-240), and F3 (amino acids 241-365). The full N-terminal region of BcWCL1 PAS∆ was also analyzed (amino acids 1-365). A fourth fragment (F4: amino acids 971-1137) was defined in the C-terminal region of BcWCL1 PAS∆ . All fragments and the full N-terminal region were systematically removed from BcWCL1 PAS∆ or used to generate chimeric transcription factors (Section 4.3).
The protein sequence of BcWCL1 was scanned for transcriptional ADs carrying the 9aaTAD motif using the online tool described by [52]. This 9aaTAD motif is present in ADs of different eukaryotic transcription factors, including Gal4 (yeast) and p65 (NF-kappa B, animal cells) [52,53]. The prediction of the 9aaTAD motif in BcWCL1 was used to generate chimeric transcription factors without the 9aaTAD motif (Section 4.3).

Genetic Constructs Design and Plasmid Construction
All genetic constructs were designed using the Benchling online platform for molecular biology (https://www.benchling.com/; accessed on 1 March 2022). For light-dependent protein-protein interaction assays, plasmids were constructed based on the components of the FUN-LOV optogenetic switch [27]. For this, pRS423 and pRS425 plasmids were used as backbones for cloning each construct under the ADH1 promoter control and ADH2 transcriptional terminator [25,54]. The BcWCL1 PAS∆ protein was fused to the Gal4-DBD and cloned into the pRS423 plasmid as previously described [25]. In addition, the BcWCL1 PAS∆ protein was also fused to Gal4-AD and cloned into the pRS425 plasmid. The different versions of BcWCL1 PAS∆ carrying deletions in the N-terminal (full region or fragments) were fused to Gal4-BDB (amino acids 1-149) or Gal4-AD (amino acids 747-881) and cloned into pRS423 and pRS425 plasmids, respectively.
In the chimeric transcription factor assays, the genetic constructs were generated by fusing the Gal4-DBD (amino acids 1-149) to each N-terminal fragment (F1, F2, and F3), the full N-terminal region, and the C-terminal region (F4). For this, pRS423 plasmid was used as the backbone for cloning each chimeric protein under the ADH1 promoter control and ADH2 transcriptional terminator [25,54].
In the light-dependent protein-protein interaction and chimeric transcription factor assays, the genetic elements included in the genetic constructs were PCR amplified using Phusion Flash High Fidelity Master mix (Thermo Scientific, Waltham, Massachusetts, USA). The PCR reactions were carried out using primers with 50 bp of overhang between adjacent elements of the genetic construct. This enabled genetic construct assembly using Yeast Recombinational Cloning (YRC) as described by [55]. Briefly, PCR fragments were co-transformed with linearized versions of the pRS423 or pRS425 plasmids using the standard Lithium acetate transformation protocol [56]. Then, YRC-assembled plasmids were extracted from yeast using Zymoprep Yeast Plasmid Miniprep II (Zymo Research, Irvine, California, USA) and used for E. coli transformation. Next, YRC-assembled plasmids were confirmed by bacterial colony PCR using GoTaq (Promega, Madison, Wisconsin, USA) under the manufacturer's instructions. Finally, YRC assembled plasmid were confirmed by sequencing using the Macrogen Sanger sequencing service (Macrogen, Seoul, Republic of Korea). Plasmids used and generated in this work are listed in the Supplementary Table S2. Primers utilized for plasmids assembly are listed in the Supplementary Table S3.

Protein-Protein Interaction and Transcriptional Activity
In protein-protein interaction and chimeric transcription factor assays, the firefly luciferase reporter gene was used to measure transcriptional activity [57]. The luciferase reporter is optimized for in vivo transcriptional measurements in yeast cells, carrying the ARE and PEST sequences for its mRNA and protein degradation, respectively [57]. This destabilized version of the firefly luciferase has 20-30 min of half-life, enabling realtime measurements of transcriptional activity in yeast [57]. The luciferase reporter was controlled by the 5XGAL1 synthetic promoter as previously described [25,27]. These assays were carried out using a Synergy H1M plate reader (Agilent, Santa Clara, California, USA) for simultaneous measurements of luminescence (in arbitrary units; a.u.) and optical density (OD) of the yeast cells. Briefly, yeast cultures were grown overnight in a 96-well plate with 200 µL of SC medium at 30 • C. The next day, a new 96-well plate with optical bottom carrying 285 µL of SC medium and supplemented with 1 mM of luciferin was inoculated with 15 µL of overnight cultures. This plate was incubated in the plate reader for 24 h at 25 • C, allowing the measurements of luminescence (Lum) and OD at 600 nm (OD 600nm ) every 10 min with 30 s of shaking prior to data acquisition [25,54].
The protein-protein interaction assays were performed under three illumination conditions: constant darkness (DD), constant blue light (BL), and a single blue-light pulse (BLP) of 2 h duration [25,37,54]. In DD experiments, the plate reader was programmed for continuous kinetics (Gen5 software, Agilent, Santa Clara, California, USA), where the 96-well plate was incubated inside the equipment under DD at 25 • C, measuring Lum and OD 600 mn every 10 min. In BL experiments, the plate reader was programmed for discontinuous kinetics (Gen5 software, Agilent, Santa Clara, California, USA), incubating the 96-well plate outside the equipment at room temperature (25 • C) and enabling its illumination by a LED illumination system. This illumination system provides blue light at 466 nm with an intensity of 24 µmol m 2 s −1 [37,54]. Finally, in BLP experiments, the plate reader was programmed for discontinuous kinetics, where the 96-well plate was incubated for 7 h inside the equipment at 25 • C under DD, measuring Lum and OD 600 mn every 10 min. Then, the 96-well plate was incubated for 2 h outside of the equipment for blue-light illumination at room temperature (25 • C). After the 2 h illumination pulse, the 96well plate was incubated inside the equipment at 25 • C for 15 h under DD conditions. In BL and BLP experiments, when incubation was carried out outside the plate reader, the 96 well plate was automatically moved inside the equipment every 10 min for the acquisition of Lum and OD 600nm . After data acquisition, the 96-well plate was automatically moved outside the plate reader for illumination. The chimeric transcription factor assays were performed under DD and BL conditions, using the same experimental setup (continuous and discontinuous kinetics) described above. All the experiments were performed in six biological replicates during 24 h.
Data of Lum and OD 600nm were normalized by dividing Lum by OD (Lum/OD 600nm ) for each time point. All data sets were analyzed using the GraphPad Prism software version 9.5.1.

Conclusions
In conclusion, BcWCL1 PAS∆ protein self-dimerized upon blue-light stimulation through its N-terminal region, where the section between amino acids 121 and 240 (Fragment 2 or F2) is necessary for self-dimerization. As a single component, the BcWCL1 PAS∆ protein is capable of light-dependent transcriptional activation through the F2 region in yeast. The F2 region contains a functional AD that belongs to the 9aaTAD family, and whose transcriptional activity strength is higher than classical ADs such as Gal4 and p65. Therefore, the BcWCL1 PAS∆ version of the BcWCL1 photoreceptor is a candidate for the development of a new single-component optogenetic system in yeast.