We employed the pDT1 vector as the foundational framework (Figure 1A) (He Z. et al., 2016). The 4×Myc tag within the Plant Cassette I was substituted with 1×Myc, accompanied by the incorporation of flexible and rigid linkers on the flanks (GGGGSGPPPG and PAPAPGGGGS) (Robinson and Sauer, 1998; Zhao et al., 2008; Reddy Chichili et al., 2013; Li et al., 2016). In the Plant Cassette II, the UBQ10 promoter was replaced with the 35S promoter, augmented by the inclusion of an Omega (Ω) sequence to enhance transcription efficiency. Additionally, the 3×HA tag was substituted with 1×Flag, again accompanied by flexible and rigid linkers on the both flanks (GGGGSGPPPG and PAPAPGGGGS). The N- and C-terminal fragments of various fluorescent proteins were inserted into the Plant Cassette I and Plant Cassette II, respectively. The coding sequences (CDSs) for these fluorescent proteins, including EYFP, mVenus, mRFP1Q66T, and mCherry, were synthesized by Tsingke (Beijing, China). The sequences of the fluorescent proteins can be found in Supplementary File S1. The codons for the fluorescent proteins used in the BiFC vectors generated in this study were optimized for plant expression and designed to avoid rare plant codons to ensure efficient protein expression in plants. EYFP was split between amino acids 155 and 156, yielding EYFP-N155 and EYFP-C156. Similarly, mVenus was divided between amino acids 211 and 212, resulting in mVenus-N211 and mVenus-C212. mRFP1Q66T was split between amino acids 168 and 169, leading to mRFP1Q66-N168 and mRFP1Q66-C169. Lastly, mCherry was divided between amino acids 159 and 160, producing mCherry-N159 and mCherry-C160. The BiFC vectors created in this study are named as follows: pDTQ26 (for EYFP BiFC), pDTQ18/28/29 (for mVenus BiFC), pDTQ21/31 (for mRFP1Q66T BiFC), and pDTQ23/33 (for mCherry BiFC).

All plasmids used in this study were generated using In-Fusion Cloning methods (https://www.takarabio.com/products/cloning/in-fusion-cloning). The sequences subcloned into plasmids were verified by Sanger sequencing. The coding sequences (CDSs) of BIC2 and CRY2 were amplified either from Arabidopsis cDNA or from previous plasmids using PCR. The purified PCR products were subsequently subcloned into pDTQ29, pDTQ31, and pDTQ33 BiFC vectors, digested by Spe I/Stu I through in-fusion. For pDTQ26 and pDTQ28, Xma I/Stu I digestion was employed; pDTQ18 used Xma I/Mfe I digestion, pDTQ21 and pDTQ23 utilized Spe I/Mfe I digestion. Additionally, the luciferase gene was individually integrated into each corresponding vector to serve as a negative control. All restriction enzymes were purchased from New England Biolabs.

BiFC assays in N. benthamiana were conducted following previously described methods (Chen et al., 2021). For transient expression, Agrobacterium strains (AGL0) carrying the BiFC plasmids were infiltrated into four-week-old N. benthamiana leaves at an OD600 of 0.5. The infiltrated N. benthamiana plants were kept in dark conditions overnight and then transferred to white light for 48 h. However, for studying CRY2 photobodies, the injected leaves were incubated in the dark for 2 h before microscopic imaging. Subsequently, samples were collected for microscopic imaging.

The seedlings of one-week-old Kitaake, a rice variety frequently employed in research and laboratory settings, were selected for protoplast isolation and transfection experiments (He F. et al., 2016; Wang Q. et al., 2021). In summary, the chosen leaf sections (∼1 g) were cut into 0.5 mm strips with a sharp razor, and all strips were immediately transferred into a 10 mL enzyme solution (10 mM MES (cat # 4,432-31-9, Sigma-Aldrich, United States) KOH, pH 5.7, 3% (w/v) Cellulase ‘Onozuka’R-10 (cat # 181005-02, Yakult Honsha, Japan), 1.5% (w/v) MacerozymeR-10 (cat # 171208-02, Yakult Honsha, Japan), 10 mM CaCl₂ (cat # 10035-04-8, Sigma-Aldrich, United States), 0.1% BSA (cat # 9048-46-8, Sigma-Aldrich, United States) (w/v) and 0.6 M Mannitol (cat # 69-65-8, Sigma-Aldrich, United States). After vacuum treatment in the dark for 30 min, enzymatic digestion was carried out by gentle shaking (40 rpm/min) at 28°C in dark conditions for 5 h. The enzymatic hydrolysis was then halted by adding 10 mL of W5 buffer (154 mM NaCl (cat # 7647-14-5, Sigma-Aldrich, United States), 125 mM CaCl₂, 5 mM KCl (cat # 7447-40-7, Sigma-Aldrich, United States), 2 mM MES KOH, pH 5.7), filtered through a 150–250 mesh stainless steel screen. The filtrate was centrifuged at 100 g for 2 min. The pelleted protoplasts were resuspended with W5 buffer and kept at 4°C in the dark for 30 min. Subsequently, the protoplasts were collected by centrifugation at 100 g for 2 min. The harvested protoplasts were resuspended in 1 mL MMG solution (0.6 M Mannitol, 15 mM MgCl₂ (cat # 7791-18-6, Sigma-Aldrich, United States), 4 mM MES KOH, pH 5.7) for subsequent polyethylene glycol 4,000(PEG4000, cat # 25322-68-3, Sigma-Aldrich, United States)-mediated transfection.

PEG-mediated transfection was performed as described with some modifications (Wang Q. et al., 2021). Briefly, after mixing 150 μL of freshly isolated protoplasts with 15 μg plasmid DNA, 165 μL of newly prepared 40% PEG solution was added, and the tubes were inverted several times to mix the contents. Following a 10–20 min incubation in the dark, 1 mL W5 solution was added slowly and mixed well by gently inverting the tubes. The protoplasts were collected by centrifugation at 100 g for 2 min and then resuspended in 1 mL W5 solution. This step was repeated three times. Finally, the tubes were incubated in the dark at room temperature for 18–24 h.

Microscopic images were captured using a Leica TCS SP8X DLS confocal laser scanning microscope equipped with a HC PL APO CS2 639/1.40 OIL objective. After 3 days of infiltration, the leaf disk was cut off for imaging. The corresponding wavelength was selected to scan and observe the fluorescence of the respective color. EYFP and mVenus were excited by a 514 nm Ar/ArKr laser with a wavelength of 520–550 nm. mRFP1Q66T and mCherry were excited by a 561 nm white light laser with a wavelength of 610–625 nm. Wavelength scans of three regions of interest from three different infiltrated leaves were used for statistical analyses. All images were captured by Leica.

Western blot analysis was performed to determine the expression levels of the two different proteins co-expressed in N. benthamiana. For each assay, approximately 0.2 g of 3-day infiltrated N. benthamiana leaves were used. The infiltrated leaves were ground in liquid nitrogen, and the protocol referenced previous studies (Qu et al., 2020; Chen et al., 2021) was followed. In brief, total proteins were extracted from the samples using extraction buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 1% Triton X-100 (v/v), 10% glycerol (v/v), and 1× Protease inhibitor cocktail (LOT 63675100, Roche, Germany). Primary antibodies used in this study included Anti-Flag (dilution ratio = 1:1,500, no. F1804; Sigma-Aldrich, United States), and anti-BIC2 antibodies. Secondary antibodies used were anti-Mouse-HRP (dilution ratio = 1:1,500, cat # 31430, Thermo Fisher Scientific, United States) and anti-Rabbit-HRP (dilution ratio = 1:15,000, cat # 31460, Thermo Fisher Scientific, United States). Western blots were detected using the Odyssey CLx Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, United States).

Protein-protein interactions (PPI) play a crucial role in cellular signal transduction. The bimolecular fluorescence complementation (BiFC) assay provides a rapid and intuitive method to investigate the localization and interactions of target proteins within living cells. In the conventional approach, the N-terminal and C-terminal fragments of the fluorescent protein are typically expressed using two separate vectors, which depends on the efficiency of co-transformation into the same cells. To overcome these challenges, we have developed novel dual-transgenic BiFC vectors, known as pDTQs, derived from the previously published pDT1 vector (Figure 1A) (He Z. et al., 2016). The pDTQ vectors have two independent Plant Cassettes (Plant Cassette I and Plant Cassette II), which enable the sequential expression of two proteins within a single vector. When we initially used pDT1 for dual expression, we observed very low protein expression from Plant Cassette II. We suspected that this might be due to the weak promoter. To address this issue, we first modified pDT1 by replacing the promoter in Plant Cassette II with cauliflower mosaic virus 35S promoter (Figure 1A). We also introduced a long linker, composed of a flexible and rigid linker, between the target and the fluorescent proteins (Figure 1B). These long linkers are designed to facilitate the recombination of the fused fluorescent proteins into their active forms. Additionally, we selected four different excitation wavelengths and monomer-type fluorescent proteins for BiFC analysis, including EYFP, mVenus, mRFP1Q66T, and mCherry (Fan et al., 2008; Feng et al., 2017), which improve the versatility of the BiFC system for studying protein interactions. mRFP1Q66T, an enhanced monomeric red fluorescent protein with improved photostability (Jach et al., 2006). The N- and C-terminal fragments of the same fluorescent proteins were inserted into the Plant Cassette I and Plant Cassette II, respectively, in the same pDTQ vector. The resulting BiFC vectors have been designated as pDTQ26 (for EYFP BiFC), pDTQ18/28/29 (for mVenus BiFC), pDTQ21/31 (for mRFP1Q66T BiFC), and pDTQ23/33 (for mCherry BiFC), with splitted fluorescent proteins fused in varying orientations.

Next, we assessed the efficiency of the pDTQs system by investigating the oligomerization of Arabidopsis CRY2 and CRY2-BIC2 interactions in N. benthamiana. Arabidopsis cryptochrome 2 (CRY2) has been previously reported to undergo blue light-dependent homodimerization (Wang et al., 2016). BIC2 (Blue light inhibitors of Cryptochromes) has been found to interact with photo-activated CRY2 to inhibit CRY2 photo-oligomerization (Wang et al., 2016; Ma et al., 2020). CRY2-CRY2 or CRY2-BIC2 were cloned into the eight pDTQ vectors. The luciferase (LUC) gene was introduced into pDTQs as negative controls. Transient expression of the pDTQ vectors was carried out following a previously published method (Chen et al., 2021).

To evaluate the efficiency of the pDTQ26 vector for EYFP-based BiFC analysis, the coding sequence (CDS) of CRY2 or luciferase (LUC) was inserted into the Plant Cassette I via Xma I, and the CDS of CRY2, BIC2 or LUC was inserted into the Plant Cassette II using Stu I. LUC was used as a negative control. BiFC transient expression experiments were conducted in tobacco by injecting Agrobacterium strains carrying the indicated nEYFP and cEYFP plasmids. Fluorescence signals were examined 48 h post-transfection using a confocal microscope. Strong fluorescence signals were detected in the nucleus when nEYFP-CRY2/CRY2-cEYFP and nEYFP-BIC2/CRY2-cEYFP were injected (Figure 2A), indicating the interaction of CRY2-CRY2 and BIC2-CRY2 in plants. Conversely, very few signals were observed in the negative controls (nEYFP-LUC/CRY2-cEYFP, nEYFP-BIC2/LUC-cEYFP, nEYFP-LUC/LUC-cEYFP) (Figure 2A). The number of fluorescent nuclei was quantified for each BiFC pair, with at least three independent injection sites evaluated. The number of fluorescent nuclei in nEYFP-CRY2/CRY2-cEYFP and nEYFP-BIC2/CRY2-cEYFP was significantly higher than that in the negative controls (Figure 2B). Additionally, western blots were performed to assess the protein levels of BIC2, CRY2, and LUC in the transient expression leaves of nEYFP-BIC2/CRY2-cEYFP and nEYFP-BIC2/LUC-cEYFP. Consistent with the observed fluorescence signals, BIC2, CRY2, and LUC proteins were expressed (Supplementary Figure S1A), confirming that the reconstituted fluorescence signals resulted from the expression of BIC2 and CRY2 and their interactions. These results further indicate that the pDTQ26 vector for EYFP BiFC analysis effectively detects protein-protein interactions.

To assess the effectiveness of the mVenus-based BiFC vectors, we inserted the CDS of CRY2, BIC2, or LUC into the Plant Cassette I of pDTQ18/28 or pDTQ29 using Xma I or Spe I, resulting in constructs with nmVenus-CRY2/BIC2/LUC or CRY2/BIC2/LUC-nmVenus. Additionally, we inserted CRY2 or LUC into the Plant Cassette II of pDTQ18 or pDTQ28/29 using Mfe I or Stu I, creating cmVenus-CRY2/LUC or CRY2/LUC-cmVenus. These plasmids were then individually introduced into tobacco leaves. Strong fluorescence signals were observed in the CRY2-CRY2 and BIC2-CRY2 BiFC pairs (Figures 3A–C). The number of fluorescent nuclei in the CRY2-CRY2 and BIC2-CRY2 pairs was significantly higher than in the negative controls, including LUC-CRY2, BIC2-LUC, and LUC-LUC (Figures 3D–F). For each vector, we selected a positive experimental group (BIC2-CRY2) and a negative control group (BIC2-LUC) for western blot analysis. The immunoblot results confirmed the expression of the indicated proteins in the transient expression assays (Supplementary Figure S1B–D). These findings demonstrate that the pDTQ18/28/29 vectors for mVenus BiFC analysis are suitable for detecting protein-protein interactions in plants.

We also explored the use of the red fluorescent protein mRFP1Q66T for BiFC analysis. mRFP1Q66T is known to be monomeric and exhibit increased photostability (Jach et al., 2006). To assess its suitability for BiFC, we split mRFP1Q66T between amino acids 168 and 169, and inserted each N- (amino acids 1–168) and C-terminus (amino acids 169–226) of mRFP1Q66T into the pDTQ vectors, resulting in pDTQ21 and pDTQ31. We then inserted the CDS of CRY2, BIC2, or LUC into the Plant Cassette I of pDTQ21/31 using Spe I, creating constructs with CRY2/BIC2/LUC-nmRFP1Q66T. Additionally, we inserted CRY2 or LUC into the Plant Cassette II of pDTQ21 or pDTQ31 using Mfe I or Stu I, resulting in cmRFP1Q66T-CRY2/LUC or CRY2/LUC-cmRFP1Q66T. Fluorescent RFP signals were detected in the CRY2-CRY2 and BIC2-CRY2 BiFC pairs (Figures 4A,B), indicating successful reconstitution of RFP in plants. However, it is worth noting that the CRY2-CRY2 mRFP1Q66T BiFC signals were relatively lower compared to those of EYFP and mVenus BiFC signals, because of fewer average fluorescent nuclei observed (Figures 4C,D). To confirm protein expression, we conducted western blot analysis for the positive experimental group (BIC2-CRY2) and the negative control group (BIC2-LUC), confirming the protein expression of BIC2, LUC, and CRY2 (Supplementary Figure S1E,F). These results demonstrate that the pDTQ21/31 mRFP1Q66T BiFC vectors can also be effectively used for PPI analysis in plants.

In addition, we explored another red fluorescent protein mCherry for BiFC analysis. To evaluate the efficacy of the mCherry-based BiFC vectors, we inserted the CDS of CRY2, BIC2, or LUC into the Plant Cassette I of pDTQ23/33 using Spe I, resulting in constructs with CRY2/BIC2/LUC-nmCherry. Similarly, we inserted CRY2 or LUC into the Plant Cassette II of pDTQ23 or pDTQ33 using Mfe I or Stu I, generating cmCherry-CRY2/LUC or CRY2/LUC-cmCherry. Fluorescent RFP signals were observed in the CRY2-CRY2 and BIC2-CRY2 BiFC pairs (Figures 5A,B). Similar to the mRFP1Q66T BiFC vectors, the fluorescence signals observed in the CRY2-CRY2 mCherry BiFC pairs were relatively weaker compared to those of EYFP and mVenus BiFC signals (Figures 5C,D). The expression of BIC2, LUC and CRY2 in the positive experimental group (BIC2-CRY2) and a negative control group (BIC2-LUC) were also confirmed by western blot analysis (Supplementary Figure S1G,H). These results indicate that the pDTQ23/33 mCherry BiFC vectors can also be effectively used for protein-protein interaction analysis in plants.

Given that photoactivated CRY2 has been shown to condense into photobodies in the nucleus (Wang et al., 2016; Wang X. et al., 2021), we conducted a more detailed examination of the photobody formation of CRY2-CRY2 BiFC signals. As depicted in Figure 6, distinct nuclear photobodies were readily observed in the pDTQ26/29/31/23 constructs for CRY2-CRY2 pairs. Nevertheless, it remains to be determined whether the photobodies formed by CRY2-CRY2 reconstitution from the pDTQ vectors exhibit liquid-liquid phase separation, a phenomenon reported for many proteins under specific conditions to enhance local concentration and facilitate their biochemical activities (Yu et al., 2009; Hyman et al., 2014; Shin et al., 2017). An intriguing avenue for further exploration is understanding how proteins are recruited into these foci by their interacting partners. Our pDTQ vectors serve as valuable tools for investigating this intriguing question at the visible level. In any case, these results underscore the utility of pDTQ vectors in examining the formation of protein foci within plant cells.

The experimentation using the pDTQ BiFC vectors has demonstrated high efficiency and low background signal in the tobacco transient expression system. To explore the applicability of pDTQ vectors in other plant transient expression systems, particularly in tropical plants, we transiently expressed the BIC2-CRY2 BiFC pair in pDTQ26, pDTQ29, pDTQ31, and pDTQ23 vectors in rice protoplasts. Strong fluorescence signals were observed in the nuclei of rice protoplasts (Figure 7). Indeed, these results suggest that the pDTQ vectors are applicable in the rice transient expression system for testing protein-protein interactions. However, their suitability for other plant species would necessitate further investigation and validation.