Next Article in Journal
Elucidating the Racemization Mechanism of Aliphatic and Aromatic Amino Acids by In Silico Tools
Next Article in Special Issue
Practical Approaches for the Yeast Saccharomyces cerevisiae Genome Modification
Previous Article in Journal
Trypanosoma cruzi Secreted Cyclophilin TcCyP19 as an Early Marker for Trypanocidal Treatment Efficiency
Previous Article in Special Issue
A New Role for Yeast Cells in Health and Nutrition: Antioxidant Power Assessment
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 24
Issue 15
10.3390/ijms241511874
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Matías Guerrero
1,2,†,
Carlos Ruiz
1,2,†,
Andrés Romero
1,2,
Luka Robeson
1,
Diego Ruiz
1,2 and
Francisco Salinas
1,2,*
1
Laboratorio de Genómica Funcional, Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia 5090000, Chile
2
ANID–Millennium Science Initiative–Millennium Institute for Integrative Biology (iBIO), Santiago 8330025, Chile
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2023, 24(15), 11874; https://doi.org/10.3390/ijms241511874
Submission received: 29 June 2023 / Revised: 20 July 2023 / Accepted: 22 July 2023 / Published: 25 July 2023
(This article belongs to the Special Issue Yeast: Molecular and Cell Biology)
Browse Figures
Versions Notes

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 (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.

1. 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 general, flavin cofactors generate a cysteinyl flavin C4a adduct upon blue light stimulation, generating a conformational change in the LOV domain [14]. These features enable the functional characterization of LOV-based photoreceptors using different biological platforms. For instance, the BcLOV4 photoreceptor has been characterized using two different eukaryotic models: a human cell line (HEK293T) and the budding yeast Saccharomyces cerevisiae, both demonstrating that BcLOV4 binds plasma membrane anionic phospholipids in response to blue light [15]. Therefore, B. cinerea photobiology is an interesting source of photoreceptors with different biological functions, although their molecular characterization has been only partially addressed.
The BcWCL1 protein is a blue light photoreceptor and transcription factor from the GATA-type Zinc family [10,16,17]. Importantly, the BcWCL1 protein is an ortholog to the WC-1 protein from Neurospora crassa (hereafter referred to as NcWC-1), a well-characterized blue light photoreceptor containing a LOV domain [18,19,20]. Furthermore, NcWC-1 is also a transcription factor from the GATA-type Zinc family involved in light response and circadian regulation in N. crassa [18,19,20,21]. Besides the LOV domain and DNA binding domain (DBD), the NcWC-1 protein structure also includes two PAS (Per Arnt Sim) domains (PASB and PASC) involved in protein–protein interaction, and two transcriptional activation domains (AD) in the N-terminal and C-terminal regions, respectively [21,22]. Interestingly, the N-terminal region (amino acids 100–200) of NcWC-1 has been validated as a functional AD in N. crassa [23]. In the circadian context, NcWC-1 interacts with the WC-2 protein (hereafter referred to as NcWC-2) through its PAS domains, forming the White-Collar Complex (WCC) [21,22]. The WCC binds the frq (frequency) promoter, which controls the expression of the negative element in the N. crassa circadian clock, the FRQ protein [19]. In B. cinerea, BcWCL1 plays a similar role in circadian regulation, also forming the WCC in the nucleus and promoting the light-mediated transcriptional activation of several genes, including bcfrq (encoding BcFRQ protein), ortholog to the frq gene in N. crassa [16,17]. Importantly, the deletion of bcwcl1 (encoding BcWCL1) and bcfqr genes impairs virulence and oxidative stress response in B. cinerea [16,24]. Altogether, the importance of BcWCL1 in the B. cinerea circadian clock and pathogenesis is propelling new efforts toward its molecular characterization.
Recently, the blue light response of BcWCL1 has been described using the budding yeast S. cerevisiae as a biological platform [25]. By using the yeast two-hybrid (Y2H) architecture [26], and replacing the photoreceptor components in a previously described optogenetic system [27] with different versions of BcWCL1 and BcWCL2, we assayed the light-dependent protein–protein interaction between these proteins [25]. As expected, BcWCL1 and BcWCL2 interaction is mainly mediated by the PAS domains with or without light stimulation [25]. Interestingly, in the absence of PAS domains in both proteins (BcWCL1PAS∆ and BcWCL2PAS∆), the protein–protein interaction becomes light-dependent [25]. Moreover, BcWCL1PAS∆ was capable of light-mediated transcriptional activation in the absence of an interacting partner [25]. These results opened important possibilities regarding BcWCL1 photobiology: 1, blue light exerts a conformational change in BcWCL1PAS∆ exposing a transcriptional activation domain that is functional in yeast; and 2, BcWCL1PAS∆ self-dimerizes upon blue light stimulation.
In this work, we assess the light-dependent transcriptional activity of BcWCL1PAS∆ in yeast. By using the Y2H strategy and different versions of BcWCL1PAS∆, we demonstrated that BcWCL1PAS∆ self-dimerizes through its N-terminal region upon blue light stimulation. Furthermore, we used different fragments of the BcWCL1PAS∆ protein to generate chimeric transcription factors, allowing us to identify a functional transcriptional AD in the N-terminal region (amino acids 121–240). Finally, we compared the transcriptional activity of the newly-identified AD with respect to commonly-used ADs in eukaryotic cells (Gal4 and p65), observing that the BcWCL1PAS∆-AD can reach higher levels of transcriptional activation in yeast. Altogether, our results support potential applications of the BcWCL1PAS∆ photoreceptor in the development of new optogenetic systems in yeast.

2. Results

2.1. Deletion of PAS Domains in BcWCL1 Does Not Affect the Overall Protein Structure

Previously, we demonstrated that deletion of the PAS domains in BcWCL1 (BcWCL1PAS∆) uncover the blue-light response of this protein [25]. In addition, we also observed that BcWCL1PAS∆ 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 BcWCL1PAS∆ using AlphaFold2 [28]. The results showed that the deletion of PAS domains did not alter the structure of the LOV domain in BcWCL1PAS∆ (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 BcWCL1PAS∆ (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 BcWCL1PAS∆) 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.

2.2. BcWCL1PAS∆ Self-Dimerizes and Activates Transcription in Response to Light

Initially, we confirmed the BcWCL1PAS∆ capacity of light-dependent transcriptional activation in yeast using a Y2H strategy. Hence, we generated a version of BcWCL1PAS∆ fused to the Gal4 DNA binding domain (DBD) and AD, BcWCL1PAS∆-Gal4-DBD and BcWCL1PAS∆-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 BcWCL1PAS∆-Gal4-DBD and BcWCL1PAS∆-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 BcWCL1PAS∆-Gal4-DBD was individually assayed (Figure 2C,D), and was absent when BcWCL1PAS∆-Gal4-AD was the sole component (Figure S2). Therefore, our results support the supposition that BcWCL1PAS∆ 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.
We further investigate the molecular mechanism of BcWCL1PAS∆ 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 BcWCL1PAS∆ (Figure 3A). The BcWCL1PAS∆ N-terminal region was shortened by removing three sections of similar size: the first 120 amino acids (sn1-BcWCL1PAS∆), a further 120 amino acids for a total remotion of 240 amino acids (sn2-BcWCL1PAS∆), and finally 365 amino acids corresponding to the entire N-terminal region (sn3- BcWCL1PAS∆). 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-BcWCL1PAS∆ versions compared to the full N-terminal protein version (compare luciferase expression in Figure 2B and Figure 3C). Furthermore, the deletion of amino acids 240–365 of the N-terminal region (sn2-BcWCL1PAS∆ and sn3-BcWCL1PAS∆ versions, respectively) completely abolished the light-mediated protein–protein interaction (Figure 3C and Figure S3). Then, we assayed each shorter version of BcWCL1PAS∆ as single components (Figure 3D,E), observing luciferase expression only for sn1-BcWCL1PAS∆ upon blue-light stimulation (Figure 3E and full data set in the Figure S4). Interestingly, the removal of the first 120 amino acids (sn1-BcWCL1PAS∆) 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ N-terminal region.

2.3. The Transcriptional Activation Domain of BcWCL1PAS∆ Is Localized in the N-Terminal Region between Amino Acids 121–240

In order to demonstrate that the N-terminal region of BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ contains a functional AD between amino acids 121 and 240 of the BcWCL1PAS∆ N-terminal region.
Then, we analyzed the N-terminal region of BcWCL1PAS∆ 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 BcWCL1PAS∆, 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 BcWCL1PAS∆ (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 (Figure 4D and Figure 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ N-terminal region.

2.4. Transcriptional Activity of the BcWCL1PAS∆ Activation Domain Is Comparable to Other Eukaryotic Activation Domains

Finally, we compared the transcriptional activity of the BcWCL1PAS∆ 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 BcWCL1PAS∆ showed higher levels of luciferase expression compared to Gal4 and p65 ADs (Figure 5B and Figure 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 BcWCL1PAS∆ 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.

3. Discussion

The blue-light sensing capacity of BcWCL1 was previously demonstrated in a version without PAS domains (BcWCL1PAS∆) [25]. Here, we showed that BcWCL1PAS∆ self-dimerized upon blue-light stimulation through its N-terminal region (Figure 3). Furthermore, we demonstrate that the N-terminal region of BcWCL1PAS∆ 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 BcWCL1PAS∆ (sn1-BcWCL1PAS∆) where F1 was removed, showing an increased light-mediated dimerization and transcriptional activation compared to the BcWCL1PAS∆ 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 (Figure 4 and Figure S5). In general, our findings in BcWCL1PAS∆ 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 BcWCL1PAS∆ (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 BcWCL1PAS∆, 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 BcWCL1PAS∆ AD promotes a stronger activation of the luciferase reporter compared to Gal4 and p65 ADs (Figure 5). Therefore, BcWCL1PAS∆ is a blue-light photoreceptor that contains a functional AD in yeast, where blue light exerts BcWCL1PAS∆ self-dimerization and transcriptional activation through its N-terminal region.
The BcWCL1PAS∆ protein has unique characteristics that promote its application in yeast optogenetics; notably, the presence of a LOV domain sensitive to blue light, self-dimerization 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-BcWCL1PAS∆) in yeast optogenetics. Furthermore, luciferase expression activated by BcWCL1PAS∆ 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, single-component optogenetic systems have a wide range of potential applications in different model organisms.
Altogether, we have demonstrated that the N-terminal region of BcWCL1PAS∆ is necessary for self-dimerization and transcriptional activation upon blue-light stimulation. Future experiments should address the possibility of deleting the BcWCL1PAS∆ protein region between the LOV domain and the C-terminal end. This could reduce the protein size, favoring the utilization of BcWCL1PAS∆ as a single-component optogenetic system for light-controlled gene expression.

4. Materials and Methods

4.1. 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.

4.2. 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 BcWCL1PAS∆ 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 BcWCL1PAS∆: F1 (amino acids 1–120), F2 (amino acids 121–240), and F3 (amino acids 241–365). The full N-terminal region of BcWCL1PAS∆ was also analyzed (amino acids 1–365). A fourth fragment (F4: amino acids 971–1137) was defined in the C-terminal region of BcWCL1PAS∆. All fragments and the full N-terminal region were systematically removed from BcWCL1PAS∆ 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).

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 BcWCL1PAS∆ protein was fused to the Gal4-DBD and cloned into the pRS423 plasmid as previously described [25]. In addition, the BcWCL1PAS∆ protein was also fused to Gal4-AD and cloned into the pRS425 plasmid. The different versions of BcWCL1PAS∆ 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.

4.4. 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 real-time 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 (OD600nm) 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 OD600 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 m2 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 OD600 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 96-well 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 OD600nm. 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 OD600nm were normalized by dividing Lum by OD (Lum/OD600nm) for each time point. All data sets were analyzed using the GraphPad Prism software version 9.5.1.

5. Conclusions

In conclusion, BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ version of the BcWCL1 photoreceptor is a candidate for the development of a new single-component optogenetic system in yeast.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241511874/s1.

Author Contributions

Conceptualization, M.G., C.R. and F.S.; methodology, M.G., C.R., A.R., L.R. and D.R.; software, M.G., C.R., A.R. and L.R.; validation, M.G. and C.R..; formal analysis, M.G., C.R., A.R., L.R. and D.R.; investigation, M.G., C.R., A.R., L.R. and D.R.; resources, M.G., C.R., A.R., L.R. and D.R.; data curation, M.G and C.R.; writing—original draft preparation, M.G. and F.S.; writing—review and editing, F.S.; visualization, F.S.; supervision, F.S.; project administration, F.S.; funding acquisition, F.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ANID-Millennium Science Initiative Program-ICN17_022 and ANID-FONDECYT grant number 1210955 to F.S.; and by the ANID-PhD scholarships 21220756 to M.G. and 21210525 to A.R.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data sets supporting reported results are available upon request to the corresponding author.

Acknowledgments

We thank Paulo Canessa and Vicente Rojas for providing us with the plasmid carrying the BcWCL1PAS∆ sequence. We also thank Gonzalo Monsalvez for technical help during this project execution and two friends for reagent donation.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Dean, R.; Van Kan, J.A.; Pretorius, Z.A.; Hammond-Kosack, K.E.; Di Pietro, A.; Spanu, P.D.; Rudd, J.J.; Dickman, M.; Kahmann, R.; Ellis, J.; et al. The Top 10 fungal pathogens in molecular plant pathology. Mol. Plant Pathol. 2012, 13, 414–430. [Google Scholar] [CrossRef] [Green Version]
  2. Veloso, J.; van Kan, J.A.L. Many Shades of Grey in Botrytis-Host Plant Interactions. Trends Plant Sci. 2018, 23, 613–622. [Google Scholar] [CrossRef]
  3. Suzuki, Y.; Kumagai, T.; Oda, Y. Locus of blue and near ultraviolet reversible photoreaction in the stages of conidial development in Botrytis cinerea. J. Gen. Microbiol. 1977, 98, 199–204. [Google Scholar] [CrossRef] [Green Version]
  4. Suzuki, Y.; Oda, Y. Inhibitory Loci of both Blue and near Ultraviolet Lights on Lateral-type Sclerotial Development in Botrytis cinerea. Jpn. J. Phytopathol. 1979, 45, 54–61. [Google Scholar] [CrossRef] [Green Version]
  5. Tan, K.K. Complete reversibility of sporulation by near ultraviolet and blue light in Botrytis cinerea. Trans. Br. Mycol. Soc. 1974, 63, 203–205. [Google Scholar] [CrossRef]
  6. Tan, K.K. Interaction of near-ultraviolet, blue, red, and far-red light in sporulation of Botrytis cinerea. Trans. Br. Mycol. Soc. 1975, 64, 215–222. [Google Scholar] [CrossRef]
  7. Tan, K.K.; Epton, H.A.S. Effect of light on the growth and sporulation of Botrytis cinerea. Trans. Br. Mycol. Soc. 1973, 61, 145–157. [Google Scholar] [CrossRef]
  8. Shcherbakova, D.M.; Shemetov, A.A.; Kaberniuk, A.A.; Verkhusha, V.V. Natural Photoreceptors as a Source of Fluorescent Proteins, Biosensors, and Optogenetic Tools. Annu. Rev. Biochem. 2015, 84, 519–550. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  9. Amselem, J.; Cuomo, C.A.; van Kan, J.A.L.; Viaud, M.; Benito, E.P.; Couloux, A.; Coutinho, P.M.; de Vries, R.P.; Dyer, P.S.; Fillinger, S.; et al. Genomic Analysis of the Necrotrophic Fungal Pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLOS Genet. 2011, 7, e1002230. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  10. Schumacher, J. How light affects the life of Botrytis. Fungal Genet. Biol. 2017, 106, 26–41. [Google Scholar] [CrossRef]
  11. Van Kan, J.A.; Stassen, J.H.; Mosbach, A.; Van Der Lee, T.A.; Faino, L.; Farmer, A.D.; Papasotiriou, D.G.; Zhou, S.; Seidl, M.F.; Cottam, E.; et al. A gapless genome sequence of the fungus Botrytis cinerea. Mol. Plant Pathol. 2017, 18, 75–89. [Google Scholar] [CrossRef] [Green Version]
  12. Losi, A.; Gardner, K.H.; Möglich, A. Blue-Light Receptors for Optogenetics. Chem. Rev. 2018, 118, 10659–10709. [Google Scholar] [CrossRef]
  13. Christie, J.M.; Gawthorne, J.; Young, G.; Fraser, N.J.; Roe, A.J. LOV to BLUF: Flavoprotein Contributions to the Optogenetic Toolkit. Mol. Plant 2012, 5, 533–544. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  14. Zoltowski, B.D.; Vaccaro, B.; Crane, B.R. Mechanism-based tuning of a LOV domain photoreceptor. Nat. Chem. Biol. 2009, 5, 827–834. [Google Scholar] [CrossRef]
  15. Glantz, S.T.; Berlew, E.E.; Jaber, Z.; Schuster, B.S.; Gardner, K.H.; Chow, B.Y. Directly light-regulated binding of RGS-LOV photoreceptors to anionic membrane phospholipids. Proc. Natl. Acad. Sci. USA 2018, 115, E7720–E7727. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Canessa, P.; Schumacher, J.; Hevia, M.A.; Tudzynski, P.; Larrondo, L.F. Assessing the Effects of Light on Differentiation and Virulence of the Plant Pathogen Botrytis cinerea: Characterization of the White Collar Complex. PLoS ONE 2014, 8, e84223. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  17. Schumacher, J. Tools for Botrytis cinerea: New expression vectors make the gray mold fungus more accessible to cell biology approaches. Fungal Genet. Biol. 2012, 49, 483–497. [Google Scholar] [CrossRef] [PubMed]
  18. Ballario, P.; Vittorioso, P.; Magrelli, A.; Talora, C.; Cabibbo, A.; Macino, G. White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein. EMBO J. 1996, 15, 1650–1657. [Google Scholar] [CrossRef]
  19. Froehlich, A.C.; Liu, Y.; Loros, J.J.; Dunlap, J.C. White Collar-1, a circadian blue light photoreceptor, binding to the frequency promoter. Science 2002, 297, 815–819. [Google Scholar] [CrossRef]
  20. He, Q.; Cheng, P.; Yang, Y.; Wang, L.; Gardner, K.H.; Liu, Y. White collar-1, a DNA binding transcription factor and a light sensor. Science 2002, 297, 840–843. [Google Scholar] [CrossRef]
  21. Cheng, P.; Yang, Y.; Wang, L.; He, Q.; Liu, Y. WHITE COLLAR-1, a Multifunctional NeurosporaProtein Involved in the Circadian Feedback Loops, Light Sensing, and Transcription Repression of wc-2. J. Biol. Chem. 2003, 278, 3801–3808. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Ballario, P.; Talora, C.; Galli, D.; Linden, H.; Macino, G. Roles in dimerization and blue light photoresponse of the PAS and LOV domains of Neurospora crassa white collar proteins. Mol. Microbiol. 1998, 29, 719–729. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, B.; Kettenbach, A.N.; Gerber, S.A.; Loros, J.J.; Dunlap, J.C. Neurospora WC-1 Recruits SWI/SNF to Remodel frequency and Initiate a Circadian Cycle. PLOS Genet. 2014, 10, e1004599. [Google Scholar] [CrossRef] [Green Version]
  24. Hevia, M.A.; Canessa, P.; Müller-Esparza, H.; Larrondo, L.F. A circadian oscillator in the fungus Botrytis cinerea regulates virulence when infecting Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 2015, 112, 8744–8749. [Google Scholar] [CrossRef]
  25. Rojas, V.; Salinas, F.; Romero, A.; Larrondo, L.F.; Canessa, P. Interactions between Core Elements of the Botrytis cinerea Circadian Clock Are Modulated by Light and Different Protein Domains. J. Fungi 2022, 8, 486. [Google Scholar] [CrossRef]
  26. Fields, S.; Song, O.-k. A novel genetic system to detect protein–protein interactions. Nature 1989, 340, 245–246. [Google Scholar] [CrossRef]
  27. Salinas, F.; Rojas, V.; Delgado, V.; López, J.; Agosin, E.; Larrondo, L.F.; Idnurm, A. Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast. mBio 2018, 9, e00626-18. [Google Scholar] [CrossRef] [Green Version]
  28. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  29. Varadi, M.; Anyango, S.; Deshpande, M.; Nair, S.; Natassia, C.; Yordanova, G.; Yuan, D.; Stroe, O.; Wood, G.; Laydon, A.; et al. AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2021, 50, D439–D444. [Google Scholar] [CrossRef]
  30. Gao, M.; Yang, J.; Liu, S.; Su, Z.; Huang, Y. Intrinsically Disordered Transactivation Domains Bind to TAZ1 Domain of CBP via Diverse Mechanisms. Biophys. J. 2019, 117, 1301–1310. [Google Scholar] [CrossRef]
  31. Wells, M.; Tidow, H.; Rutherford, T.J.; Markwick, P.; Jensen, M.R.; Mylonas, E.; Svergun, D.I.; Blackledge, M.; Fersht, A.R. Structure of tumor suppressor p53 and its intrinsically disordered N-terminal transactivation domain. Proc. Natl. Acad. Sci. USA 2008, 105, 5762–5767. [Google Scholar] [CrossRef] [PubMed]
  32. Brent, R.; Ptashne, M. A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 1985, 43, 729–736. [Google Scholar] [CrossRef] [PubMed]
  33. Ruden, D.M.; Ma, J.; Li, Y.; Wood, K.; Ptashne, M. Generating yeast transcriptional activators containing no yeast protein sequences. Nature 1991, 350, 250–252. [Google Scholar] [CrossRef]
  34. Sadowski, I.; Ma, J.; Triezenberg, S.; Ptashne, M. GAL4-VP16 is an unusually potent transcriptional activator. Nature 1988, 335, 563–564. [Google Scholar] [CrossRef]
  35. Ma, J.; Ptashne, M. A new class of yeast transcriptional activators. Cell 1987, 51, 113–119. [Google Scholar] [CrossRef] [PubMed]
  36. Piskacek, M.; Havelka, M.; Rezacova, M.; Knight, A. The 9aaTAD Transactivation Domains: From Gal4 to p53. PLoS ONE 2016, 11, e0162842. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Figueroa, D.; Baeza, C.; Ruiz, D.; Inzunza, C.; Romero, A.; Toro, R.; Salinas, F. Expanding the molecular versatility of an optogenetic switch in yeast. Front. Bioeng. Biotechnol. 2022, 10, 1029217. [Google Scholar] [CrossRef]
  38. Xu, X.; Du, Z.; Liu, R.; Li, T.; Zhao, Y.; Chen, X.; Yang, Y. A Single-Component Optogenetic System Allows Stringent Switch of Gene Expression in Yeast Cells. ACS Synth. Biol. 2018, 7, 2045–2053. [Google Scholar] [CrossRef]
  39. Zhao, E.M.; Zhang, Y.; Mehl, J.; Park, H.; Lalwani, M.A.; Toettcher, J.E.; Avalos, J.L. Optogenetic regulation of engineered cellular metabolism for microbial chemical production. Nature 2018, 555, 683–687. [Google Scholar] [CrossRef]
  40. Zoltowski, B.D.; Schwerdtfeger, C.; Widom, J.; Loros, J.J.; Bilwes, A.M.; Dunlap, J.C.; Crane, B.R. Conformational Switching in the Fungal Light Sensor Vivid. Science 2007, 316, 1054–1057. [Google Scholar] [CrossRef] [Green Version]
  41. Wang, X.; Chen, X.; Yang, Y. Spatiotemporal control of gene expression by a light-switchable transgene system. Nat. Methods 2012, 9, 266–269. [Google Scholar] [CrossRef] [PubMed]
  42. Ma, Z.; Du, Z.; Chen, X.; Wang, X.; Yang, Y. Fine tuning the LightOn light-switchable transgene expression system. Biochem. Biophys. Res. Commun. 2013, 440, 419–423. [Google Scholar] [CrossRef] [PubMed]
  43. Qian, Y.; Li, T.; Zhou, S.; Chen, X.; Yang, Y. A Single-Component Optogenetic Gal4-UAS System Allows Stringent Control of Gene Expression in Zebrafish and Drosophila. ACS Synth. Biol. 2023, 12, 664–671. [Google Scholar] [CrossRef] [PubMed]
  44. Nash, A.I.; McNulty, R.; Shillito, M.E.; Swartz, T.E.; Bogomolni, R.A.; Luecke, H.; Gardner, K.H. Structural basis of photosensitivity in a bacterial light-oxygen-voltage/helix-turn-helix (LOV-HTH) DNA-binding protein. Proc. Natl. Acad. Sci. USA 2011, 108, 9449–9454. [Google Scholar] [CrossRef] [PubMed]
  45. Rivera-Cancel, G.; Motta-Mena, L.B.; Gardner, K.H. Identification of Natural and Artificial DNA Substrates for Light-Activated LOV–HTH Transcription Factor EL222. Biochemistry 2012, 51, 10024–10034. [Google Scholar] [CrossRef]
  46. Zoltowski, B.D.; Motta-Mena, L.B.; Gardner, K.H. Blue Light-Induced Dimerization of a Bacterial LOV–HTH DNA-Binding Protein. Biochemistry 2013, 52, 6653–6661. [Google Scholar] [CrossRef] [Green Version]
  47. Rullan, M.; Benzinger, D.; Schmidt, G.W.; Milias-Argeitis, A.; Khammash, M. An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation. Mol. Cell 2018, 70, 745–756.e746. [Google Scholar] [CrossRef]
  48. Motta-Mena, L.B.; Reade, A.; Mallory, M.J.; Glantz, S.; Weiner, O.D.; Lynch, K.W.; Gardner, K.H. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat. Chem. Biol. 2014, 10, 196–202. [Google Scholar] [CrossRef]
  49. Reade, A.; Motta-Mena, L.B.; Gardner, K.H.; Stainier, D.Y.; Weiner, O.D.; Woo, S. TAEL: A zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control. Development 2017, 144, 345–355. [Google Scholar] [CrossRef] [Green Version]
  50. Jones, P.; Binns, D.; Chang, H.Y.; Fraser, M.; Li, W.; McAnulla, C.; McWilliam, H.; Maslen, J.; Mitchell, A.; Nuka, G.; et al. InterProScan 5: Genome-scale protein function classification. Bioinformatics 2014, 30, 1236–1240. [Google Scholar] [CrossRef] [Green Version]
  51. Mirdita, M.; Schütze, K.; Moriwaki, Y.; Heo, L.; Ovchinnikov, S.; Steinegger, M. ColabFold: Making protein folding accessible to all. Nat. Methods 2022, 19, 679–682. [Google Scholar] [CrossRef] [PubMed]
  52. Piskacek, S.; Gregor, M.; Nemethova, M.; Grabner, M.; Kovarik, P.; Piskacek, M. Nine-amino-acid transactivation domain: Establishment and prediction utilities. Genomics 2007, 89, 756–768. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Piskacek, M.; Havelka, M.; Rezacova, M.; Knight, A. The 9aaTAD Is Exclusive Activation Domain in Gal4. PLoS ONE 2017, 12, e0169261. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Romero, A.; Rojas, V.; Delgado, V.; Salinas, F.; Larrondo, L.F. Modular and Molecular Optimization of a LOV (Light–Oxygen–Voltage)-Based Optogenetic Switch in Yeast. Int. J. Mol. Sci. 2021, 22, 8538. [Google Scholar] [CrossRef]
  55. Oldenburg, K.R.; Vo, K.T.; Michaelis, S.; Paddon, C. Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 1997, 25, 451–452. [Google Scholar] [CrossRef] [Green Version]
  56. Gietz, R.D.; Schiestl, R.H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2007, 2, 31–34. [Google Scholar] [CrossRef]
  57. Rienzo, A.; Pascual-Ahuir, A.; Proft, M. The use of a real-time luciferase assay to quantify gene expression dynamics in the living yeast cell. Yeast 2012, 29, 219–231. [Google Scholar] [CrossRef] [Green Version]
Ijms 24 11874 g001 550
Figure 1. Protein domains and predicted structure of NcWC-1 and BcWCL1. (a) Protein domains in NcWC-1, BcWCL1, and BcWCL1PAS∆. (b) AlphaFold predicted structure of NcWC-1, (c) BcWCL1, and (d) BcWCL1PAS∆, 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 1. Protein domains and predicted structure of NcWC-1 and BcWCL1. (a) Protein domains in NcWC-1, BcWCL1, and BcWCL1PAS∆. (b) AlphaFold predicted structure of NcWC-1, (c) BcWCL1, and (d) BcWCL1PAS∆, 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.
Ijms 24 11874 g001
Ijms 24 11874 g002 550
Figure 2. BcWCL1PAS∆ protein self-dimerizes and activates transcription upon blue-light stimulation. (a) BcWCL1PAS∆ 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) BcWCL1PAS∆ 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 (OD600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.
Figure 2. BcWCL1PAS∆ protein self-dimerizes and activates transcription upon blue-light stimulation. (a) BcWCL1PAS∆ 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) BcWCL1PAS∆ 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 (OD600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.
Ijms 24 11874 g002
Ijms 24 11874 g003 550
Figure 3. The N-terminal region of BcWCL1PAS∆ protein is necessary for self-dimerization and transcriptional activation upon blue-light stimulation. (a) The N-terminal region of BcWCL1PAS∆ protein was dissected by removing three fragments: amino acids 1–120 (sn1-BcWCL1PAS∆), amino acids 121–240 (sn2-BcWCL1PAS∆), and amino acids 241–365 (sn3-BcWCL1PAS∆). (b) Shorter versions of BcWCL1PAS∆ 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) BcWCL1PAS∆ 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 (OD600nm) 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 BcWCL1PAS∆ protein is necessary for self-dimerization and transcriptional activation upon blue-light stimulation. (a) The N-terminal region of BcWCL1PAS∆ protein was dissected by removing three fragments: amino acids 1–120 (sn1-BcWCL1PAS∆), amino acids 121–240 (sn2-BcWCL1PAS∆), and amino acids 241–365 (sn3-BcWCL1PAS∆). (b) Shorter versions of BcWCL1PAS∆ 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) BcWCL1PAS∆ 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 (OD600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.
Ijms 24 11874 g003
Ijms 24 11874 g004 550
Figure 4. Fragment 2 (F2) inside the N-terminal region of BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 (OD600nm) 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 BcWCL1PAS∆ 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 (OD600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.
Ijms 24 11874 g004
Ijms 24 11874 g005 550
Figure 5. Activation Domain (AD) in fragment 2 (F2) of BcWCL1PAS∆ 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 (OD600nm) 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 BcWCL1PAS∆ 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 (OD600nm) of the yeast cells. The average of six biological replicates with the standard deviation is shown as the color-shaded region.
Ijms 24 11874 g005
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Guerrero, M.; Ruiz, C.; Romero, A.; Robeson, L.; Ruiz, D.; Salinas, F. The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast. Int. J. Mol. Sci. 2023, 24, 11874. https://doi.org/10.3390/ijms241511874

AMA Style

Guerrero M, Ruiz C, Romero A, Robeson L, Ruiz D, Salinas F. The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast. International Journal of Molecular Sciences. 2023; 24(15):11874. https://doi.org/10.3390/ijms241511874

Chicago/Turabian Style

Guerrero, Matías, Carlos Ruiz, Andrés Romero, Luka Robeson, Diego Ruiz, and Francisco Salinas. 2023. "The N-Terminal Region of the BcWCL1 Photoreceptor Is Necessary for Self-Dimerization and Transcriptional Activation upon Light Stimulation in Yeast" International Journal of Molecular Sciences 24, no. 15: 11874. https://doi.org/10.3390/ijms241511874

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop