All genetic materials, such as Horseradish latent virus (HRLV), Mirabilis mosaic virus (MMV), Figwort mosaic virus (FMV), and Cauliflower mosaic virus (CaMV) promoter elements and seeds of Nicotiana tabacum and Nicotiana benthamiana were kindly offered by Dr. I. B. Maiti, University of Kentucky, USA. All oligonucleotides were synthesized from Eurofins Scientific, Luxembourg, and all enzymes used in this study, such as T4 DNA ligase, PNK, restriction enzymes, Taq DNA Polymerase, and DNaseI were procured from Thermo Fisher Scientific Inc. (Waltham, MA). Chemicals such as X-Gluc, DEPC, Agarose, 4-methylumbelliferyl-beta-D-glucuronide (MUG), etc., were purchased from Sigma-Aldrich (St. Louis, USA). The SYBR green master mix (GoTaq^® qPCR) was bought from Promega Corporation, Madison, Wisconsin (Cat. No. A6001), the First Strand cDNA Synthesis Kit from Thermo Fisher Scientific Inc. (Cat. No. K1622), and the RNeasy Plant Mini Kit from QIAGEN (Cat. No. 74904), Hilden, Germany. The basic chemicals such as Tris, agar, Luria Broth (LB), Murashige and Skoog (MS) medium media, MES, KCL, MgCl₂, acetosyringone, Kanamycin, Rifampicin, etc., were obtained from HiMedia Laboratories, from Mumbai, India.

The hybrid promoter MuasFuasH17 (in short MFH17) was shuffled using the DNaseI-mediated DNA shuffling method described in Ranjan et al. (2012), with few changes. The Muas, Fuas, and H17 fragments were PCR amplified from pUCMuas, pUCFuas, and pUCH17 clones and gel eluted. Around 5 µg of the isolated fragments, namely Muas, Fuas, and H17, were individually treated with 0.5 U of DNaseI enzyme for 10 minutes. The reaction was stopped by adding 0.5 M EDTA and heat-inactivated for 10 minutes. The three reactions, Muas, Fuas, and H17, were mixed and precipitated using 3 M sodium acetate and 99% cold ethanol. The precipitated fragments were then run on 2% agarose gel, and fragments less than 100 bp were eluted. The eluted fragments were then reannealed together using self-priming PCR (primer-less PCR) having the following parameters: 30 seconds at 95°C (denaturation), 30 seconds at 42°C (annealing) and 60 seconds at 72°C (extension) for 25 cycles. Next, the pool of reannealed fragments was used as a PCR reaction template with MFH17-specific primers named Muas F and HRLV R ( Supplementary Data 2 in Supplementary Data Sheet 1 ). The positive PCR products were gel eluted, cloned into pUC119, and sequenced.

The oligonucleotide blocks-based DNA shuffling was done following the previously published protocol with small changes (Fujishima et al., 2015). Briefly, twelve pairs (sense and anti-sense strand) of oligos from MFH17 promoter with 40-50 bp in size were synthesized with the addition of AG as 5’ overhang in the sense strand and TC as 5’ overhang in the anti-sense strand ( Supplementary Data 2 in Supplementary Data Sheet 1 ). The sense and anti-sense were then annealed in a thermal cycler by heating at 95°C for 5 minutes and gradually decreasing the temperature to 4°C (1°C/min). The annealed product was then phosphorylated individually using Polynucleotide Kinase (PNK) enzyme and incubated for 30 minutes at 37°C. Parallelly, H17min (HRLV-Flt; 48 bp; -45 to +3; Supplementary Data 2 in Supplementary Data Sheet 1 ) oligos were also synthesized, which contained Xba1 and HindIII restriction sites in their 5’ and 3’ region, respectively, and annealed, phosphorylated, and cloned into pUC119 vectors leading to the development of pUCH17min. The previously annealed twelve oligos were mixed in a single tube reaction, ligated using T4 DNA ligase enzyme, and kept overnight at 4°C. After incubation, the 5’ and 3’ adapters (5’ containing EcoRI and 3’ XbaI restriction sites; Supplementary Data 2 in Supplementary Data Sheet 1 ) were added and incubated for another 4-5 hours. Finally, the reaction mixture was cloned into the EcoRI and XbaI sites in the pUCH17min vector. The positive clones were then sequenced and sub-cloned into the plant expression vector pKYLXGUS for further analysis.

The MFH17 promoter contains the H17min sequence (HRLV-Flt; 48 bp; -45 to +3; Supplementary Data 2 in Supplementary Data Sheet 1 ) as the core promoter region, which is the RNA polymerase binding region. This is why, for developing oligonucleotide blocks shuffled promoters, the H17min sequence was used as the minimal promoter instead of the commonly used 35S minimal promoter (Cai et al., 2020; Yang et al., 2024).

All the developed constructs (hybrid, mutant, shuffled) cloned into the pKYLXGUS vector were individually transformed into Agrobacterium tumefaciens strain GV3101 using the freeze-thaw method (Weigel and Glazebrook, 2006). For agroinfiltration in Nicotiana benthamiana, the positive agrobacterium clones were grown overnight in LB Broth containing Rifampicin and Kanamycin antibiotics. The culture was washed twice and suspended in agro-infiltration buffer (50 mM MES, 2 mM Na₃PO₄, 27 mM D-Glucose, and 0.1 mM acetosyringone). The concentration of the agrobacterium culture was kept at OD₆₀₀ 0.1 and infiltrated into the abaxial side of the healthy leaves using a needle-less hypodermic syringe (Mandal et al., 2015). The infiltrated plants were kept in the greenhouse under low-light conditions for 48 hours. The total GUS expression was measured using a fluorometric GUS assay described in Sethi et al. (2022) and Kumari et al. (2024).

For transient analysis in rice seedlings, the positive agrobacterium colonies were grown in LB Broth containing Rifampicin and Kanamycin antibiotics overnight. The culture was then washed and suspended in liquid-infiltration buffer (4g/L Murashige and Skoog (MS) medium, 40 mM KCl, 42 mM MgCl₂, 200 mM sucrose, 200 mM glucose, 0.01% Silwet and 150 µM acetosyringone) and the culture concentration was adjusted to OD₆₀₀ 1.0. Two weeks old, rice seedlings were submerged into the agrobacterium culture and infiltrated for 10 minutes under 0.9 bar pressure inside the vacuum desiccator. The rice seedlings were washed with distilled water and grown in the greenhouse for 48 hours under low-light conditions. After incubation, GUS expression was measured from the whole seedlings using a fluorometric GUS assay.

For GFP expression analysis, the vectors MuasFuasH17pLXGFP (MFH17), 35SpLXGFP (35S), 2X35SpLXGFP (2X35S), and Ctrl (VC) were transformed into A. tumefaciens strain GV3101 and agro-infiltrated into healthy leaves of N. benthamiana in small areas. The plants were kept in the greenhouse under low light for 5 days. After incubation, the leaves were detached, viewed under UV light, and photographed using Gel Doc XR+System (Bio-Rad).

For the development of tobacco transgenic plants, the MuasFuasH17pLXGUS (MFH17), 35SpLXGUS (35S), and Ctrl (VC) plasmids were transformed into Agrobacterium tumefaciens strain LBA4404 and the positive colonies were used in developing transgenic plants using leaf disc from Nicotiana tabacum cv. Samsun by following the previously published protocol (Horsch et al., 1985). Ten plants were generated from the independent callus and grown in the greenhouse, and each plant’s seeds were harvested and used for seed segregation analysis. Briefly, the seeds were dried and grown in an MS-agar medium containing 200 mg/L Kanamycin for twenty-one days. The Kanamycin-resistant (Kan^R) and Kanamycin-sensitive (Kan^S) seedlings were counted and compared with a 3:1 (Kan^R: Kan^S) ratio using a chi-square test. The lines showing the best phenotype and chi-square value of < 1.5 (p ≤ 0.05) were considered “true transgenic” and grown till the T₂ generation (Honda et al., 2002; Patro et al., 2015; Chatterjee et al., 2017). Furthermore, the genomic DNA from the selected lines was isolated and used for gene integration PCR, where the promoter (MFH17), gene (GUS), Kanamycin-resistant gene (nptII), and poly (A) signal (rbcSE9) present in the T-DNA region of pKYLXGUS vector were PCR amplified using their respective primers ( Supplementary Data 2 in Supplementary Data Sheet 1 ).

For the development of Arabidopsis transgenic plants, MuasFuasH17pLXGUS (MFH17), 35SpLXGUS (35S), and Ctrl (VC) plasmids were transformed into Agrobacterium tumefaciens strain GV3101 and floral dip method was used to develop Arabidopsis thaliana (ecotype Columbia) transgenic plants, following a previously published protocol (Zhang et al., 2006). The infected plant’s seeds were harvested, dried, and germinated in an MS-agar medium containing 50 mg/L Kanamycin. Ten Kanamycin-resistant lines were grown, and their seeds were used for segregation analysis, as described above. A single line with the best phenotype and seed segregation ratio was grown until T₂ generation, and their gene integration PCR was done as described above.

The total RNA from 21-day-old seedlings was isolated using an RNeasy Plant Mini Kit following the manufacturer’s protocol. The isolated RNA was quantitated, and the DNA contamination was purified using the DNaseI enzyme. The purified RNA was then used to make a cDNA pool using the First Strand cDNA Synthesis Kit. Next, around 2 µL (20 ng) of cDNA was then used as a template for RT-PCR reaction containing 10 µL SYBR green master mix, 1 µL forward primer, 1 µL reverse primer and 6 µL nuclease-free water in QuantStudio™5 Real-Time PCR System (Applied Biosystems) having following parameters: 95°C for 10 min, followed by 40 cycles of 95°C for 15s, 60°C for 1 min. The GUS transcripts were used as targets, and the Nt18S for Nicotiana tabacum and AtActin for Arabidopsis thaliana housekeeping genes were used in normalizing the data [Chanwala et al. (2024); Khan et al. (2018); Supplementary data 2 in Supplementary Data Sheet 1 ). The data was evaluated using the 2^-ΔΔCT method and represented as fold change (Livak and Schmittgen, 2001).

For detection of GUS activity in different plant tissues, the samples were dipped into an X-Gluc buffer solution containing 50 mM Sodium Phosphate Buffer (pH- 7.0), 0.01% Tween 20, 10 mM EDTA and 0.3% X-Gluc powder and kept at 37°C overnight (12 hours). The sample was then washed, destained in 70% ethanol, and photographed under a Stereomicroscope System (Olympus, SZ61).

All experiments were conducted using three biological replicates, and the average and standard deviation values were utilized for plotting the data. The statistical analysis using the average values was done using the Student’s t-test, where the asterisk ‘*’ sign was used to depict the level of significance (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001).

To develop strong constitutive plant promoters, different fragments from three previously characterized constitutive pararetroviral-based promoters, namely Mirabilis mosaic virus (MMV-Flt), Figwort mosaic virus (FMV-Flt), and Horseradish latent virus (HRLV-Flt) were taken. Two fragments from the HRLV-Flt promoter, namely H17 (250 bp) and H12 (500 bp), were kept as core promoter regions, and three promoter fragments, namely Muas from MMV-Flt, Fuas from FMV-Flt and H17uas from HRLV-Flt were kept as an upstream activation sequence (UAS), functioning as transcriptional enhancers ( Figure 1A ). These three UAS were combined with H17 and H12 core promoter in different combination and ten hybrid promoters were developed, where six were di-hybrids and four were tri-hybrid promoters ( Figure 1B ). The promoter activity analysis of the developed promoter along with H17, H12, 35S, and 2X35S promoters was transiently done in Nicotiana benthamiana leaves, and their GUS activity was measured and represented in Figure 1B .

The result indicated that all the hybrids were strong and highly active in N. benthamiana leaves and showed higher expression than the 35S promoter. The tri-hybrid promoter MuasFuasH17 (in short MFH17), which is a combination of Muas, Fuas, and H17, showed the highest expression, with almost 2 times higher than the 35S promoter and 1.3 times higher than the 2X35S promoter.

The CREs present in the MFH17 promoter were examined using PlantCARE (Lescot et al., 2002) and PLACE (Higo et al., 1999) databases. The sequences were coordinated with numbers, where the transcription start site (TSS) was annotated as +1, and all CREs were numbered accordingly ( Supplementary Data 4 in Supplementary Data Sheet 1 ).

It was interesting to observe that even though MuasFuasH17 (MFH17) and FuasMuasH17 promoters shared the same CREs and same promoter domains, and the only variation between them was the position of Muas and Fuas fragments, their promoter activity had significant differences ( Figure 1B , Supplementary Data 1 in Supplementary Data Sheet 1 ). Therefore, we attempted to shuffle the promoter fragments of the MFH17 promoter more finely using the directed evolution technique of DNA shuffling to further enhance/upgrade the activity of the MFH17 promoter. The general protocol followed four main steps: 1) fragmentation of Muas, Fuas, and H17 promoters by digestion with DNaseI enzyme, 2) reannealing of these fragments using self-priming PCR (primer-less), 3) selecting of reannealed fragments using PCR (with primer) and 4) cloning, sequencing and screening of unique mutants ( Figure 2A ).

Fifty clones were generated using this technique and sequenced, and we observed that the mutational frequency was very low, as most of the generated clones were unchanged or with only minor point mutations. After screening the sequences, we selected eight mutant clones (D1, D2, D3, D4, D5, D6, D7, and D8) based on the position and type of mutation on the regulatory region of the MFH17 promoter. The D1 and D2 mutant clones had four point mutations and three point mutations, respectively, and caused sequence mutation of MYC (-501; T→A) cis-regulatory elements in D1 and sequence mutation of GATA-motif (-603; T→C) and MYC (-501; T→A) in D2. In the case of the D3 mutant, the whole of the Fuas region was deleted, and a few point mutations were generated, which caused ABRE (-159; C→T) cis-regulatory element mutation. The D4, D6, D7, and D8 were deletion mutants with complete deletion of the Fuas region and partial deletions in the Muas and H17 regions, as depicted in Figure 2B (all sequences are provided in Supplementary Data 1 in Supplementary Data Sheet 1 ). The D5 mutant was an insertion mutation with the addition of a complete Fuas region upstream of the MFH17 promoter ( Figure 2B ).

The promoter activity analysis of all the generated mutant clones was done, and the result revealed that all the mutant clones showed decreased activity compared to the MFH17 promoter. The D1 and D2 mutants, which only had a few point mutations, did not show much difference, whereas all the deletion mutants, such as D3, D4, D6, D7, and D8, showed a significant reduction in the overall activity of MFH17 promoter. Interestingly, the D5 mutant clone with an additional insertion of Fuas enhancer upstream of the MFH17 promoter showed slightly lower expression ( Figure 2B ).

We observed that the DNaseI-mediated shuffling did not yield a “true shuffled” sequence but instead led to either point, deletion, or insertion mutations. Therefore, we attempted another DNA shuffling technique called overhang-based oligonucleotide block shuffling (Fujishima et al., 2015), where 40-50 bp of oligonucleotides sequence from MFH17 promoter were synthesized with sense strand containing AG as 5’ overhang and anti-sense strand containing TC as 5’ overhang as shown in Figure 3A . These oligonucleotides were mixed in a single reaction tube and randomly ligated with one another and cloned upstream of H17min promoter ( Figure 3A ). We generated thirty shuffled promoter clones, namely Sh1 to Sh30, and their promoter activity analysis was done.

The result revealed that this technique led to accurate shuffling of DNA sequences, as shown for the sequence of Sh6 ( Supplementary Data 1 in Supplementary Data Sheet 1 ). However, out of the thirty shuffled promoter clones analyzed, most had no expression, and some had weak expressions, such as Sh3 and Sh26. Only the Sh6 shuffled promoter showed moderate expression, around 58% less than the activity of the MFH17 promoter ( Figure 3B ). This might indicate that for proper functioning of a promoter, the regulatory elements must be present in an ideal arrangement. Consequently, the MFH17 promoter is arranged in an optimum arrangement for high-gene expression.

The activity of the MFH17 promoter was further checked in rice seedlings using the GUS reporter gene and compared with 35S and 2X35S promoters. The histochemical staining of rice seedlings showed strong blue coloration in all three promoter-infiltrated seedlings, signifying high gene expression ( Figure 4A ). The total GUS activity was quantified from these seedlings, using fluorometric GUS assay and found that similar to promoter activity in N. benthamiana leaves, the MFH17 promoter had higher expression in rice seedlings than compared to both 35S and 2X35S promoters ( Figure 4B ).

Next the efficiency of MFH17 in driving GFP was analyzed in N. benthamiana leaves. The MFH17, 35S, and 2X35S promoter (driving GFP gene) was agro-infiltrated into a small region of N. benthamiana leaves and photographed. The images showed intense green fluorescence of GFP expression in the infiltrated regions, signifying that the MFH17 promoter could also efficiently drive the GFP reporter gene ( Figure 4C ).

Previous reports have shown the significance of the as-1 element (TGACG) for the activity of a promoter (Khan et al., 2018; Cai et al., 2020). Our recent work also observed that mutating the as-1 element from the Horseradish latent virus sub-genomic transcript promoter led to an almost 75% decrease in its promoter activity (Sherpa et al., 2023). The cis-regulatory element analysis of MFH17 revealed that it contained six as-1 elements ( Supplementary Data 4 in Supplementary Data Sheet 1 ) and were present as pairs in each fragment (Muas, Fuas, and H17) of this promoter ( Figure 5A ). We wanted to check the effect of these as-1 elements pairs in MFH17 promoter activity and therefore did site-directed mutagenesis and generated seven mutant clones, out of which M (as-1 pair mutation in Muas), F (as-1 pair mutation in Fuas), H (as-1 pair mutation in H17) were single pair-mutants, MF (as-1 pair mutations in Muas and Fuas), FH (as-1 pair mutations in Fuas and H17), MH (as-1 pair mutations in Muas and H17) were double pair-mutants and MFH (as-1 pair mutations in Muas, Fuas and H17) was a triple pair-mutant ( Figure 5B ).

The analysis revealed that in the case of the single-pair mutants, the M mutant promoter, which is the most distally present mutation, did not lead to much decrease in the transcriptional activity of the MFH17 promoter, whereas the F and H mutants showed a 21% and 22% decrease, respectively in the activity of MFH17 promoter. The double-pair mutants, MF, MH, and FH, showed more decrease with 39%, 32%, and 42% decline, respectively. The triple-pair mutant MFH promoter showed a 54% decrease in the transcriptional activity of the MFH17 promoter ( Figure 5B ).

To understand the performance of MFH17 in planta and study its tissue specificity, transgenic Nicotiana tabacum cv. Samsun and Arabidopsis thaliana (ecotype Columbia) were developed. Ten independent transgenic lines of tobacco and Arabidopsis containing MFH17 and 35S promoter driving GUS reporter gene were developed, and their seed segregation and phenotypic analyses were performed, as described in Material and Methods. For MFH17 promoter analysis in tobacco, line 2 was selected, and for analysis in Arabidopsis, line 4 was selected ( Supplementary Data 5 in Supplementary Data Sheet 1 ). Additionally, genomic DNA was extracted from these two lines, and gene integration PCR was done, which showed distinct bands of MFH17, GUS, nptII, and rbcSE9 ( Supplementary Data 6 in Supplementary Data Sheet 1 ). These two lines were grown till T₂ generation, and their analysis was done.

From tobacco transgenic plants, twenty-one days old seedlings, forty-five days old leaf, stem, root tissues, and ninety days old flowering tissues (ovary, style, anther, and filament) were stained with X-Gluc solution to observe the tissue-specific expression of MFH17 promoter and compared with 35S promoter transgenic plant and control (VC) plant. The result showed that MFH17 and 35S promoters stained deeply in all the tissues, revealing their strong and constitutive nature ( Figure 6A ). We quantitated and compared the activity of MFH17 and 35S promoters by using twenty-one days old seedlings using GUS fluorometric analysis and found that similar to the previous transient analysis, the MFH17 showed stronger activity than 35S, which was approximately 1.7 times higher ( Figure 6B ). Further, the total uidA (GUS) transcript level from twenty-one days old seedlings using RT-PCR was measured, and it was found that MFH17 transgenic plants had a higher transcript level than compared to the 35S ( Figure 6C ). We also measured the activity of the MFH17 promoter from the leaf, stem, and root tissue and found it had higher expression in the root than in the leaf or stem ( Figure 6D ).

Likewise, in the case of transgenic Arabidopsis plants, twenty-one days old seedlings, forty-five days old leaf, stem, and root tissues, and seventy days old flowering tissues (inflorescence, flower, and pistil) were stained with X-Gluc solution and tissue-specific expression patterns of MFH17 and 35S promoter was observed. The result showed that similar to transgenic tobacco, the MFH17 and 35S promoters stained deeply in all the tissues, showing their strong and constitutive nature ( Figure 7A ). The total promoter activity was quantitated from twenty-one days old seedlings and found that the MFH17 promoter showed 1.6 times higher activity than the 35S promoter ( Figure 7B ). The GUS RNA transcript level was also higher in MFH17 Arabidopsis seedlings than compared to the 35S seedlings ( Figure 7C ). Furthermore, the activity of the MFH17 promoter in leaf, stem, and root tissue from forty-five days old transgenic plants was measured, and similar to the expression in tobacco, the promoter showed higher activity in the root than in the leaf and stem ( Figure 7D ).