Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays

Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays Abdelaziz Ramadan, Kaho Oka, Kenji Miura This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4897984/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2024 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Improved yields of recombinant proteins (RP) are necessary for protein production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein expression systems. With improved protein yield, lettuce ( Lactuca sativa ) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting LsRDR1 and LsRDR6 using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of LsRDR1 and LsRDR6 with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down LsRDR6 and LsRDR1 , but not LsRDR2 , at both 3 and 5 days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3 dpi and 5 dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3 dpi and 5 dpi, respectively. To confirm these results, co-infiltration with Bet V 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production. Transient expression Tsukuba system Lettuce RDR1 RDR6 Recombinant proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction RNA-mediated gene silencing, or RNA interference (RNAi), is a vital and conserved system for regulating gene expression in prokaryotes and eukaryotes (Felden and Paillard 2017 ). In plants, it also acts as a defense mechanism against viruses and other non-viral pathogens (Lopez-Gomollon and Baulcombe 2022 ). Post-transcriptional gene silencing (PTGS) occurs through Dicer-like enzymes (DCL) that process double-stranded RNAs into small interfering RNA duplexes (siRNAs), which can move from cell to cell to produce systemic silencing, and RNA-dependent RNA polymerases (RDRs) that act on single-stranded RNA bound with siRNA to produce double-stranded RNA (Zamore 2004 ; Melnyk et al. 2011 ). Finally, Argonaute (AGO) and siRNA form an RNA-induced silencing complex (RISC), which leads to the destruction of homologous RNAs (Nakanishi 2016 ). To counteract host RNAi defenses, many viruses with the “arm-race” concept encode viral suppressors of RNA silencing (VSRs), like p1, p10, p19, P21, P24, HCPro, and others which inhibit key steps in the RNAi system and have multifunctional roles in viral processes (Burgyán and Havelda 2011 ; Lopez-Gomollon and Baulcombe 2022 ). VSRs primarily suppress RNA-silencing pathways through various mechanisms, including binding to dsRNA and inhibiting components of the host RNAi machinery, such as RISC and DNA methylation enzymes (Csorba et al. 2015 ). The introduction of foreign DNA into plant genomes through Agrobacterium tumefaciens has revolutionized plant biotechnology for many applications, including genetic engineering and crop improvement, gene function studies, plant-microbe interactions, and recombinant protein production (Gelvin 2003 ; Tzfira and Citovsky 2006 ). The immune response of plants, including the gene silencing system toward Agrobacterium itself and foreign DNA that usually contains viral elements, such as the Cauliflower Mosaic virus 35S (CaMV 35S) promoter, limits the expression of recombinant proteins (RPs) inside plants (Zipfel et al. 2006 ; Kawazu et al. 2019 ). Nicotiana benthamiana , a close relative of tobacco, has emerged as a prominent host plant for the production of various biologically active molecules and RPs. The ability of Nicotiana benthemiana to produce RPs is correlated with a natural mutation within the gene encoding RNA-dependent RNA polymerase 1, Rdr1 (Bally et al. 2015 , 2018 ). Similarly, a knockout mutation in the RDR6 gene in N. benthemiana plants resulted in the accumulation of a larger amount of recombinant green fluorescent proteins compared to the wild type (Matsuo and Atsumi 2019 ). In addition, in Arabidopsis thaliana , a mutation in RDR6 substantially improves RP expression (von Schaewen et al. 2018 ). These data suggested a strong correlation between gene silencing and RP expression. Lettuce has emerged as a promising alternative for the expression of RP, offering advantages such as oral delivery and the potential for large-scale production (Clarke et al. 2017 ) with various products, including vaccine antigens, antibodies, and therapeutic proteins, being successfully expressed in lettuce (Chan and Daniell 2015 ). However, the RP yield of lettuce remained relatively low compared to N. benthemiana (Yamamoto et al. 2018 ). In contrast to the gene silencing mechanism of Nicotiana benthemiana which is easily suppressed by different viral suppressors (Arzola et al. 2011 ), lettuce was unresponsive to viral suppressors of gene silencing, such as P19 (from tomato bushy stunt virus, TBSV), P1/HcPro (from turnip mosaic virus, TuMV), or P1/HcPro (from tobacco etch virus, TEV) (Wroblewski et al. 2005 ; Simmons and VanderGheynst 2007 ), possibly because of the intricate interplay between various viral suppressors and the defense mechanisms of lettuce, which is poorly understood. In this study, we focused on the RDR gene family in lettuce and, using phylogenetic analysis, classified them according to their homology with RDR members of the Arabidopsis. When we utilized RNAi technology for suppressing the gene silencing mechanism in lettuce Salinas, by designing two unique RNAi that target highly conserved regions between both LsRDR1 and LsRDR6, the expression of both genes was specifically reduced. Using RNAi-1 as a representative in combination with the EGFP gene in the Tsukuba system vector, EGFP expression was enhanced significantly in both Salinas lettuce and commercially available lettuce. To validate the efficiency of RNAi-1, the expression of another therapeutic protein, Bet v 1, was assessed in Salinas lettuce, revealing a significant increase in its expression. This study revealed a new potential for the enhancement of RP production in lettuce. Materials and methods Plant materials and growth conditions Lettuce Salinas seeds (Watanabe Seeds Co., Chiba, Japan) were planted in loose rock wool in plug trays until germination. Seedlings were then transplanted to plastic pots with soil suitable for vegetables (one plant/pot) and irrigated with a high-nitrogen fertilizer (15-6-6). Seedlings were grown under a photoperiodic condition (16-h light/8-h dark cycle at 100 µmol m − 2 s − 1 ) at 23 ◦C in a plant growth chamber. N. benthamiana plants were used to express EGFP for standard curve preparation. N. benthamiana seeds were planted in loose rockwool. Seedlings were grown under a photoperiodic condition (16-h light/8-h dark cycle of white light 100 µmol m − 2 s − 1) at 23 ◦C in a plant growth chamber. Primers The primers used for polymerase chain reaction (PCR) and quantitative real-time PCR (q-RT-PCR) are listed in Supplementary Table 1. Phylogenetic tree and protein domain analysis Information on lettuce RNA-dependent RNA polymerases (RDR) sequences was obtained using genome V.11 from the gene search tool of the National Center for Biotechnology Information (NCBI). Subsequently, using the NGPhylogeny (FastME/OneClick) online tool ( https://ngphylogeny.fr ), the ORF sequences of the Arabidopsis and lettuce RDRs were aligned using MAFFT Alignment (Katoh and Standley 2013 ; Lemoine et al. 2019 ). The phylogenetic tree was visualized using PRESTO (Phylogenetic tReE visualization, https://ngphylogeny.fr/data/displaytree ). The domain structures of all lettuce and Arabidopsis RDRs were checked using the SMART (Simple Modular Architecture Research Tool) database ( http://smart.embl-heidelberg.de ) (Letunic and Bork 2018 ; Letunic et al. 2021 ). Multiple sequence alignment of lettuce RDRs amino acids was carried out using ClustalW, available at https://www.genome.jp/tools-bin/clustalw . Subsequently, the alignment data were visualized using the SnapGene Viewer. Cloning into the Tsukuba system vector Tsukuba system vector pTKB3 previously described in(Omori et al. 2023 ) was used for cloning of RNAi and EGFP inserts. Alignment between the DNA sequences of LsRDR1 and LsRDR6 was performed and visualized using ApE-A plasmid editor v3.1.2. Two sequences, 120 bp for RNAi-1 and 100 bp for RNAi-2, exhibiting 75% and 76% similarity, respectively, between LsRDR1 and LsRDR6, were used in reverse orientation and separated by the ChsA intron. (Dafny-Yelin et al. 2007 ). The corresponding DNA sequence was synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) and cloned into Sal1-digested pTKB3 using an In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). Bet v 1 vector was constructed and expressed in a previous study (Yamada et al. 2020 ).All vectors used pTKB3 only (E), pTKB3-RNAi (R), pTKB3-EGFP and pBYR2HS-HF-Betv1 were transformed into A. tumefaciens strain GV3101 using by electroporation and then used for plant infiltration. Transient expression of proteins in lettuce Preparation of Agrobacterium tumefaciens suspension and transient expression in lettuce were performed as previously described (Yamamoto et al. 2018 ) with modifications. A. tumefaciens GV3101 containing the binary vector was grown in modified YEB media (6 g/L of yeast extract, 5 g/L of trypone, 5 g/L of sucrose, and 2 mM MgSO4) with antibiotics (100 mg/L of kanamycin, 30 mg/L gentamycin, and 30 mg/L of rifampin) for 2 days at 28°C. Then, 2-day cultures were diluted 100 times in the same modified YEB with antibiotics, 10 mM MES (pH 5.6), and 20 µM acetosyringone, and grown for 18–24 h at 28°C on a rotary shaker at 140 rpm. The optical density OD600 was adjusted to approximately 1 for agrobacterium harboring empty vector or RNAi vector and 0.5 in the case of EGFP or Bet v 1. Then, 55 g/L of sucrose and 200 µM acetosyringone were added to the bacterial culture and the suspension was incubated for 1 h at 22°C. After incubation, 2,4-dichlorophenoxyacetic acid and Tween-20 were added to the final concentrations of 100 µg/mL and 0.005%, respectively, and the suspension was used for syringe-infiltration in 2-month-old lettuce Salinas leaves Supplementary Fig. 2a. In case of suspension mixture of different agrobacterium, doubled OD 600 values was prepared then equal volumes were mixed. After syringe infiltration, the lettuce leaves were rinsed with water. Water was then removed with paper towels, and the lettuce was incubated for 3–5 days at 22°C under a 16-h light and 8-h dark photoperiod. Green leaf lettuce with roots was obtained commercially from a local grocery store, as shown in Supplementary Fig. 1b, rinsed with distilled water, and the water was removed using paper towels. After syringe infiltration, the lettuce heads were rinsed with water. Water was then removed with paper towels, and the lettuce was incubated for 3–5 days at 22°C under a 16-h light and 8-h dark photoperiod. RNA extraction and q-RT-PCR Total RNA was extracted from 100 mg leaf samples of lettuce using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. cDNA synthesis and real-time PCR were performed as previously described (Abdellatif et al., 2023 ). An amount of 2 µg of RNA was used to synthesize cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for real-time PCR are listed in Supplementary Table 1. THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was used for RT-PCR amplification and detection with a 7900HT real-time PCR system (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Relative transcript abundance was calculated using the comparative CT method, as described previously (Miura et al. 2007 ), after normalization of raw signals with the housekeeping transcript abundance of an actin gene. Protein extraction, coomassie brilliant blue (CBB), and western blotting (WB) analysis. Soluble proteins were prepared as described previously (Miura et al. 2012 ), with some modifications. Briefly, Plant leaves (100 mg) were ground by bead beating using a Cell Destroyer PS-1000 (Pro Sense, Inc., Tokyo, Japan) at 2,500 rpm for 15 s after freezing in liquid nitrogen. Then, 1 mL of lysis buffer [50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.2 mM sodium orthovanadate, 100 mM NaF, 10% glycerol, 0.2% Triton X-100, 5 mM DTT, and 1× protein inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan)] was added. The powdered leaves and lysis buffer were completely mixed by bead-beating and incubated on ice with shaking for 1 h. The samples were spun and the liquid solution was used as the soluble protein extract. Ten microliters of sample was boiled with 4x sample buffer and then subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB), and images were captured using WSE-6300 LuminoGraph III (ATTO). For immunoblot analysis, crude extracts were 10-fold diluted (except for Bet v 1, samples were not diluted) with Milli-Q and boiled with 4x sample buffer, then, 10 µl of sample solution was applied. Proteins were transferred onto a PVDF membrane (Amersham Hybond P PVDF, GE Healthcare). The blot was probed with the anti-GFP monoclonal antibody GF28R (Thermo Fisher Scientific) or anti-DYKDDDDK monoclonal antibody (Wako) and detected by Luminata Forte Western HRP substrate (Millipore) using a WSE-6300 LuminoGraph III (ATTO, Tokyo, Japan). The CBB band intensities and western blotting band intensities were calculated using ImageJ software. Fluorescent measurement and EGFP quantification 50 µl of soluble protein fractions were added in 96 well plates where each sample was added in one well surrounded by empty wells. The fluorescence of EGFP was detected at 488 nm excitation and 510 nm emission using a Varioskan Lux Multimode microplate reader, and measurements were analyzed using Skanlet software 7.0.1 (Thermo Fisher Scientific, Waltham, MA, USA). In order to quantify EGFP in leaf extract using fluorescence signals, we combined two standard curves, one was created using band intensities obtained by ImageJ of known concentrations of Bovine serum albumin (BSA) to obtain the concentrations of EGFP extracts from N. benthemiana . The second was created using EGFP of known concentrations from the first curve and their fluorescence signals were measured by Varioskan Lux multimode microplate reader. Using the last standard curve, fluorescence signals of any protein extract of EGFP could approximately refer to the concentration of EGFP. Results Phylogenetic analysis of RDR gene family in lettuce To identify the RDR family genes in lettuce, we queried the lettuce genomic protein database (version 11) using the protein sequences of both Arabidopsis RDR1 and RDR6. This search led us to identify five genes (Supplementary Table 2) that showed a significant match with Arabidopsis proteins. To elucidate the relationships between lettuce and Arabidopsis RDR genes, we constructed a phylogenetic tree based on the protein sequences of these genes using the online tool NGphylogeny. The tree revealed two main clusters: cluster 1 included both lettuce and Arabidopsis RDR6, in addition to RDR 1 and 2 of both lettuce and Arabidopsis, whereas cluster w included other RDRs (Fig. 1 a). In contrast to Lettuce RDRs 1, 2, and 6, which were very close to their corresponding Arabidopsis proteins, both lettuce RDR5a and b were separated from Arabidopsis RDRs 3, 4, and 5 (Fig. 1 a). Similar to the phylogenetic tree, domain analysis using SMART (Fig. 1 b) showed the first cluster with an RNA-directed RNA polymerase (RdRP) domain in addition to the RNA recognition motif (RRM); the second cluster that included RDRs 3, 4, and 5 contained only the RdRP domain. Protein alignment using ClustalW revealed the canonical amino acid motif DLDGD, which is characteristic of RDRs of the 1st cluster and the atypical amino acid motif DFDGD, which is characteristic of RDRs of the 2nd cluster (Fig. 1 c). These results reflect the accuracy of blasting and phylogenetic analysis, which classifies RDR genes based on differences in amino acid sequences, motifs, and domains. Knockdown of LsRDR1 and LsRDR6 by a single RNAi To knockdown both LsRDR1 and LsRDR6 using single RNAi, two RNAi were constructed (Fig. 2 a and Supplementary Fig. 1) and cloned into pTKB3 vector (Fig. 2 b). The transcript levels of both LsRDR1 and LsRDR6 were significantly reduced in the presence of either RNAi-1 or RNAi-2 compared to those in control vector at both 3 dpi (Fig. 2 c) and 5 dpi (Fig. 2 d). However, at OD 600 = 0.5 no significant difference was observed except for LsRDR6 at 3 dpi, silencing was significant at both OD 600 = 1 and 2. At 3 dpi, OD 600 = 1 of RNAi-1 and RNAi-2 exhibited a reduction in the transcription of LsRDR1 by 61.6% and 51.8%, respectively and of LsRDR6 by 65.64% and 54.3%, respectively. RNAi-1 and RNAi-2 at OD 600 = 2 reduced the transcription of LsRDR1 by 77.8% and 72%, respectively and of LsRDR6 by 84.3% and 66.9%, respectively. While at 5 dpi, OD 600 = 1 of RNAi-1 and RNAi-2 exhibited a reduction in the transcription of LsRDR1 by 58.1% and 48.9% respectively and of LsRDR6 by 46.9% and 38.2%, respectively. RNAi-1 and RNAi-2 at OD 600 = 2 reduced the transcription of LsRDR1 by 76% and 74.2%, respectively and of LsRDR6 by 73.3% and 70.2%, respectively. Notably, the transcript of LsRDR2 , the closest member of the phylogenetic tree, was not affected at both 3 and 5 dpi and at all OD values tested, indicating that both RNAi used were specific to LsRDR1 and LsRDR6. Statistical analysis was performed between EV, RNAi-1 and RNAi-2 samples of the same OD. Since RNAi-1 reduced transcripts of LsRDR1 and LsRDR6 more, compared with RNAi-2, RNAi-1 was used for the subsequent analysis. Co-infiltration with LsRDR1,6-RNAi enhanced the expression of EGFP in lettuce Salinas To evaluate the effect of RNAi-1 which we hereafter called it LsRDR1,6-RNAi on protein expression capacity, lettuce Salinas leaves co-infiltrated with a mixture of A. tumefaciens harboring pTKB3-RNAi and pTKB3-EGFP were brighter than those infiltrated with a mix of A. tumefaciens harboring the pTKB3 empty vector (Fig. 3 a). The fluorescence signals of total soluble protein extracts was measured using Varioskan Lux multimodal microplate reader (Fig. 3 c). Quantitative fluorescence analysis revealed that the signal significantly increased about 3 folds. Image using blue light and an SC-52 filter (Fujifilm), also showed a clear enhancing effect of LsRDR1,6-RNAi (Fig. 3 b). We then quantified the EGFP yield using a combination of the conventional BSA quantification of EGFP extract from Nicotiana benthemiana (Supplementary Fig. 4a), standard curve of BSA band intensities (Supplementary Fig. 4b), fluorescence signals of samples with known EGFP concentrations (Supplementary Fig. 4c) and a standard curve between known concentrations of EGFP and fluorescence signals (Supplementary Fig. 4d). Using this approach, the EGFP protein yield of protein extract from lettuce leaves was measured and showed an increase from approx. 0.18 mg/gFW to 0.9 mg/gFW in the presence of LsRDR1,6-RNAi which is equivalent to a 4.85-fold increase. To confirm the fluorescence data, the soluble protein fractions were analyzed by CBB and WB (Fig. 3 e and g, respectively). The bands were more intense in the case of the co-infiltration of EGFP + LsRDR1,6-RNAi vectors, and analysis of the CBB and WB bands using ImageJ software revealed an increase of approximately 1.5- and 2-fold, respectively (Fig. 3 f and h, respectively). The quantitative transcript level of EGFP was significantly increased (1.58-fold) confirming the previous data (Fig. 3 i). Also, the LsRDR1,6-RNAi efficiency was confirmed in co-infiltration assays by reducing the transcription of LsRDR1 and LsRDR6 by 53.3% and 50.5% respectively (Fig. 3 j and k). At 5dpi, the fluorescence was still brighter, the WB band intensities were stronger and the protein yield of EGFP increased 3.66-fold from 0.3 mg/gFW to 1.13 mg/gFW in the presence of LsRDR1,6-RNAi (Supplementary Fig. 3a-c). Also, the significant enhancement and silencing of LsRDR1 and LsRDR6 were maintained (Supplementary Fig. 3d-f). The data presented here revealed that the efficiency of LsRDR1,6-RNAi was sustained during 5 dpi with slight changes from the levels at 3 dpi. LsRDR1,6-RNAi also stimulates EGFP expression in commercial lettuce To test the applicability of LsRDR1,6-RNAi on different lettuce cultivars, commercially available lettuce from the local market (Supplementary Fig. 2b) co-infiltrated with a mixture of A. tumefaciens harboring pTKB3-LsRDR1,6-RNAi and pTKB3-EGFP showed a brighter and wider expression of EGFP compared with the control (Fig. 4 a) at 3 dpi. The fluorescence of total soluble protein extracts were higher 1.5-folds in the presence of LsRDR1,6-RNAi (Fig. 4 b). Quantification of protein yield using fluorescence signals revealed an increase from 0.11 mg/gFW to 0.27 mg/gFW in the presence of LsRDR1,6-RNAi (Fig. 4 c) which is equivalent to 2.35-fold increase. CBB and WB analyses of soluble protein extracts exhibited more intense bands for EGFP co-expressed with LsRDR1,6-RNAi (Fig. 4 d and f), and quantitative analysis of the corresponding bands using ImageJ software revealed 1.4- and 1.6-fold increases, respectively (Fig. 4 e and g, respectively). At 5dpi, the fluorescence was also still brighter, the WB band intensities were stronger and the protein yield of EGFP increased 2.33-fold from 0.25 mg/gFW to 0.6 mg/gFW in the presence of RNAi-1 (Supplementary Fig. 3g-i). The data presented here revealed that the protein expression level in this cultivar is lower than that of Salinas lettuce. Co-infiltration with LsRDR1,6-RNAi enhanced the expression of Bet v 1 in lettuce Salinas To conclude our study, we used a therapeutic protein Bet v 1 to test its expression in lettuce and whether its expression level can be enhanced by RNAi-1. At 5 dpi, total soluble protein extracts of lettuce Salinas leaves co-infiltrated with a mixture of A. tumefaciens harboring pTKB3-RNAi-1 and pBYR2HS-HF-Betv1 showed more accumulation of Bet v 1 compared with the control (Fig. 5 a). Quantitative analysis of band intensities revealed an increase of about 2.64-fold of Bet v 1 in the presence of RNAi-1 compared with that in the presence of empty vector (Fig. 5 b). Notably, the WB samples were not diluted since the expression level was much lower compared with EGFP. Also, Bet v 1 was not detectable through CBB stain and therefore not quantified. Discussion Taken together, it is now feasible to efficiently suppress the gene silencing mechanism in lettuce using LsRDR1,6-RNAi, which precisely downregulates both LsRDR1 and LsRDR6 genes. Genes expected to play roles related to pathogenicity and RP production in lettuce in a similar manner as in Arabidopsis and Nicotiana benthemiana . In this study, we observed that OD 600 = 2 achieved a silencing level approaching 80% (Fig. 2 c and d). However, we primarily relied on OD 600 = 1 to minimize potential stress associated with excessively high OD, especially in the presence of OD 600 = 0.5 of EGFP. The enhancement of EGFP expression in the presence of LsRDR1,6-RNAi slightly decreased at 5 dpi (Supplementary Fig. 3c and i), which may be attributed to the counter-silencing of the lettuce immune response. Another possible explanation is the saturation of EGFP expression in cells by 5 dpi, leading to a diminished observable effect of LsRDR1,6-RNAi. In the case of Bet v 1, a 2.64-fold increase was clearly detected at 5 dpi (Fig. 5 a and b), likely due to its lower baseline expression in lettuce compared to EGFP, which supports the saturation hypothesis. Additionally, it was clear that the expression of EGFP and the effect of LsRDR1,6-RNAi was lower in commercial lettuce compared with Salinas lettuce (Fig. 3 d and 4 c). It is important to note that the commercial lettuce used in this study exhibited difficulties in infiltration, possibly due to its advanced age compared to the Salinas lettuce or due to the difference in the amount of wax on leaf surface. This factor likely contributed to the lower expression levels of both EGFP and LsRDR1,6-RNAi in the commercial lettuce relative to Salinas. The expression levels of recombinant proteins in various plant hosts, including commercial lettuce and N. benthemiana were compared previously (Yamamoto et al. 2018 ) showing that commercial lettuce demonstrated 1/10 of the expression potential of N. benthamiana (0.37 mg/gFW in lettuce and 3.7 mg/gFW in N. benthamiana ) when EGFP was expressed. However, in the current study, we observed a significant increase in EGFP expression levels in both commercial and Salinas lettuce varieties 0.6 mg/gFW and 1.12 mg/gFW, getting closer to the levels observed in N. benthamiana . In a prior study (Yamada et al. 2020 ), Bet v 1 expression in Nicotiana benthamiana reached approximately 1 mg/g fresh weight, which was notably lower than the expression levels observed for EGFP. These results led us to expect a similarly low expression of Bet v 1 in lettuce. Even though we aimed to evaluate the effectiveness of our RNAi approach in enhancing the expression of various recombinant proteins, which seems to be efficient in different cultivars of lettuce. Suppression of the lettuce gene silencing mechanism did not occur using three different viral suppressors: P19 (from tomato bushy stunt virus (TBSV)), P1/HcPro (from turnip mosaic virus, TuMV), or P1/HcPro (from tobacco etch virus, TEV) (Wroblewski et al. 2005 ; Simmons and VanderGheynst 2007 ). The efficiency of LsRDR1,6-RNAi was demonstrated by the decreased transcription of these genes in lettuce Salinas (Fig. 2 c), along with increased expression of EGFP in two different lettuce cultivars (Figs. 3 and 4 ). This implies that this approach may also be applicable to other lettuce cultivars. Phylogenetic analysis of RDR gene families was conducted in several plant species including rice (5 members; (Kapoor et al. 2008 )), maize (7 members; (Qian et al. 2011 )), tomato (6 members; (Bai et al. 2012 )) in addition to Arabidopsis (6 members; (Willmann et al. 2011 )) but was done here in lettuce for the first time (Fig. 1 ). LsRDR1,2 and 6 shared a common canonical amino acid DLDGD motif, whereas other RDRs shared an atypical DFDGD motif (Fig. 1 c), similar to other species such as Arabidopsis and Musa acuminata (Ahmed et al. 2021 ). In Arabidopsis, RDR6 , Suppressor of gene silencing 3 (SGS3), and RNA-directed DNA methylation 12 (RDM12) work together as part of the RNA-silencing pathway to regulate gene expression and suppress the activity of invasive nucleic acids, such as viral nucleic acids and T-DNA (Hua et al. 2021 ). SGS3 is a cofactor of RDR6 that enhances its ability to generate dsRNAs. It also stabilizes dsRNA intermediates and recruits other components of the RNA-silencing machinery, such as AGO proteins, to facilitate target mRNA cleavage or translational repression. RDM12 is a component of the RNA-directed DNA methylation (RdDM) pathway, which is a branch of the RNA-silencing pathway responsible for establishing DNA methylation and transcriptional gene silencing at target loci. RDM12 interacts with RDR6 and SGS3 to promote the production of secondary siRNAs, which guide the DNA methylation and silencing of homologous genomic sequences. Their coordinated actions contribute to the robustness and specificity of RNA silencing-mediated defense mechanisms in plants. In lettuce, the endogenous polyubiquitin promoter was shown to enhance RP expression much more than the CaMV35S promoter, owing to excessive methylation in lettuce, which was more than 10 times the methylation in Arabidopsis(Kawazu et al. 2019 ). Another report demonstrated that the use of the endogenous lettuce LsU6-10 promoter was more efficient for gene editing mediated by CRISPR-Cas9 than a similar promoter from Arabidopsis (Riu et al. 2023 ). These reports indicate that foreign DNA may not be compatible with lettuce, and that utilizing endogenous elements enhances RP expression by circumventing the plant silencing mechanism. Studying additional elements such as SGS3 and RDM12 in lettuce could help us better understand how gene silencing works and provide insights into improving recombinant protein expression. In conclusion, this study marks a significant step toward enhancing lettuce RP expression using RNAi technology. It would be worthwhile to investigate the duration for which LsRDR1,6-RNAi maintains its silencing effect and test its efficacy on various lettuce varieties. Also, it is important to explore the effects of stable mutants of both LsRDR1 and LsRDR6 generated via CRISPR-Cas9 on lettuce phenotypes, disease susceptibility, and protein expression efficiency. In addition, a deeper understanding of why viral suppressors do not succeed in overcoming the silencing mechanism of lettuce is required, and more diverse suppressors should be tested. The findings here not only contribute valuable and new insights into the silencing mechanism in lettuce, but also pave the way for future investigations on the preparation of a new plant model for RP production. Abbreviations Recombinant protein RP RNA interference RNAi RNA-dependent RNA Polymerase RDR Post-transcriptional gene silencing PTGS pTKB3 empty vector E Transcriptional gene silencing TGS pTKB3 with RNAi R Dicer-like enzymes DCL Days post infiltration Dpi small interfering RNA siRNA RNAi of LsRDR1 and LsRDR6 LsRDR1,6-RNAi RNA-induced silencing complexes RISC Coomassie brilliant blue CBB microRNAs miRNA Western blotting WB viral suppressors of RNA silencing VSR Agronaute AGO Hairpin RNA hpRNA Enhanced green fluorescent protein EGFP RNA-directed DNA methylation 12 RDM12 Suppressor of gene silencing 3 SGS3 Optical density OD Declarations Acknowledgment The authors wish to thank Ms. Yuriko Nagai, Ms. Yumiko Iguchi, and Ms. Yuri Nemoto at the University of Tsukuba for their technical support. Author contribution K.M. designed the study; A.R. and K.O. performed the experiments and data analyses; A.R. wrote the manuscript; and K.M. revised the manuscript. All authors contributed to the article and approved the submission of this manuscript. All authors read and agreed to the published version of the manuscript. Funding This study was supported by the Program on Open Innovation Platform with Enterprise, Research Institute, and Academia, Japan Science and Technology Agency (JST-OPERA, JPMJOP1851). Data availability The datasets generated and analyzed in the current study are available from the corresponding author upon reasonable request. Conflict of interest The authors declare no competing interests. References Abdellatif IMY, Yuan S, Yoshihara S, et al (2023) Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations. Int J Mol Sci 24(2):1560. https://doi.org/10.3390/ijms24021560 Ahmed FF, Hossen MI, Sarkar MAR, et al (2021) Genome-wide identification of DCL, AGO and RDR gene families and their associated functional regulatory elements analyses in banana (Musa acuminata). 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The small letters represent the sense and anti-sense RNAi target sequences; the uppercase letters represent the sequence of ChsA intron. Supplementary Fig. 2 Images for lettuce Salinas and the commercial lettuce used. (a) Lettuce Salinas grown in laboratory (b) Commercial lettuce with roots purchased from a local grocery store. Supplementary Fig. 3 The effect of LsRDR1,6-RNAi on EGFP in Salinas and commercial. Lettuce at 5 dpi. (a) and (g) The fluorescence of protein extracts of Salinas and commercial lettuce leaves, respectively, was detected at 5 dpi using blue light and ultraviolet-absorbing filter Fujifilm SC-52. (b) and (h) is WB of 1 mlsamples of soluble protein extracts of Salinas and commercial lettuce leaves, respectively, and detected by anti-GFP antibodies. (c) and (i) Protein yield values in milligram/gram fresh weight were calculated using fluorescent signal values of protein extracts of Salinas and commercial lettuce leaves, respectively, by using standard curves. (d-f) The transcription levels of EGFP, LsRDR1 and LsRDR6, respectively, using co-infiltrated samples. All data represent the mean and standard error values of 4-6 replications. Statisticallysignificant differences are represented by one asterisk for p < 0.05 and two asterisks for p < 0.01 based on unpaired Student’s t-test. Supplementary Fig. 4 Standard curves for quantifying EGFP using fluorescent signals. (a) CBB stain of 3 replicates of EGFP protein extracts expressed in N. benthamiana (b) The band intensities were calculated using imageJ and then used for standard curve formation between band intensities and concentrations of BSA. (c) Fluorescence signals of different concentrations of EGFP protein extracts determined by BSA standard curve. (d) The fluorescence signals obtained by Varioskan Lux multimode microplate reader of known EGFP concentrations were used to create a standard curve. 240813RamadanetalSupplementaryTables.docx Supplementary Table 1 Primers used in the study Supplementary Table 2 List of probable RNA-dependent RNA polymerases in lettuce Salinas Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2024 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Accept 09 Sep, 2024 Reviewers agreed at journal 08 Sep, 2024 Reviewers invited by journal 08 Sep, 2024 Editor assigned by journal 07 Sep, 2024 First submitted to journal 01 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4897984","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":351176585,"identity":"9432f025-7669-4c58-9aad-09703a735e98","order_by":0,"name":"Abdelaziz Ramadan","email":"","orcid":"","institution":"University of Tsukuba: Tsukuba Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Abdelaziz","middleName":"","lastName":"Ramadan","suffix":""},{"id":351176586,"identity":"62a66570-76ac-4af0-a957-97ee35fd2407","order_by":1,"name":"Kaho Oka","email":"","orcid":"","institution":"University of Tsukuba: Tsukuba Daigaku","correspondingAuthor":false,"prefix":"","firstName":"Kaho","middleName":"","lastName":"Oka","suffix":""},{"id":351176587,"identity":"198cdede-4096-4f60-9b81-af514a6ed32b","order_by":2,"name":"Kenji Miura","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0003-1262-2176","institution":"University of Tsukuba","correspondingAuthor":true,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Miura","suffix":""}],"badges":[],"createdAt":"2024-08-12 06:21:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4897984/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4897984/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00299-024-03324-6","type":"published","date":"2024-09-24T15:57:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":64747513,"identity":"ab65e11a-7cb6-40df-9957-515bb95f8677","added_by":"auto","created_at":"2024-09-18 09:59:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1597779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of lettuce and Arabidopsis RNA-dependent RNA polymerases (RDRs). \u003c/strong\u003e(a) The phylogenetic tree was constructed utilizing the NGphyogony tool using six members of Arabidopsis RDR genes (1–6) and five members of lettuce RDR (1–3, 5a, and 5b). (b) The protein domains of Arabidopsis and lettuce RDRs were retrieved from the SMART database. RdRP and RRM are the RNA-directed RNA polymerase domain and RNA recognition motif, respectively. (c) The alignment of the amino acid sequences made by Clustalw of the five lettuce RDRs referring to the conserved amino acid DL/FDGD signature (red rectangle). The alignment data were visualized using SnapGene Viewer.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/27a8cefb59b9a38ff9f9f974.png"},{"id":64746828,"identity":"330a30d8-fb88-46bb-ba19-de914af871a8","added_by":"auto","created_at":"2024-09-18 09:51:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2163228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNA interference (RNAi)–mediated knockdown of both LsRDR1 and LsRDR6. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eDNA sequences of \u003cem\u003eLsRDR1\u003c/em\u003eand \u003cem\u003eLsRDR6\u003c/em\u003e were aligned together with RNAi-1 and RNAi-2 and visualized using ApE- A plasmid editor v3.1.2. The target sequences were highlighted by blue rectangles. (b) Schematic representation of the T-DNA regions of the plasmids pTKB3-EGFP and pTKB3-RNAi used for protein expression and RDR gene silencing respectively. 35S-p×2, CaMV 35S promoter with the double-enhancer element; TMVΩ, 5′-leader sequence (called Ω) of tobacco mosaic virus (TMV); HSPter, heat shock protein gene terminator; Ext3′, tobacco extensin gene 3′ element; LIR and SIR, long intergenic region and short intergenic region of the bean yellow dwarf virus (Beyda) genome, respectively; C1/C2, BeYDV ORFs C1 and C2 encoding replication initiation protein (Rep) and RepA, respectively; LB and RB, left and right borders of the T-DNA region, respectively; Nos-p and Nos-t, NOS promoter and terminator, respectively; p19, a gene-silencing suppressor gene from tomato bushy stunt virus and ChsA, Chalcone synthase A intron from Petunia hybrida used for the formation of hpRNA flanked with reverse orientations of RNAi target sequences. (c) and (d) The transcription levels of different RDR genes at 3 dpi \u0026nbsp;and 5 dpi, respectively, in the leaves of Salinas lettuce infiltrated with Agrobacterium harboring RNAi vectors, RNAi-1 (R1) and RNAi-2 (R2) against those with empty vector (E) at different OD values. Data represent the mean and standard error values of 6-12 replications. Means indicated by different small letters are significantly different, based on an ANOVA with post hoc Tukey’s HSD (P \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/be8fb56aa90198dc0a61c0af.png"},{"id":64746833,"identity":"40f8391d-4d5a-4225-9ef5-8765b153fef7","added_by":"auto","created_at":"2024-09-18 09:51:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":7033547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of the expression of RDR genes enhanced the expression of EGFP in Salinas lettuce at 3 dpi.\u003c/strong\u003e (a) EGFP fluorescence in 6 Salinas lettuce leaves was detected at 3 dpi using blue light and ultraviolet-absorbing filter Fujifilm SC-52. The dotted rectangle represents the location where samples were taken for protein extraction. (E) represents EGFP co-expressed with empty vector while (R) represents EGFP co-expressed with LsRDR1,6-RNAi. Scale bar, 1 cm. (b) The fluorescence of protein extracts was detected using blue light and ultraviolet-absorbing filter Fujifilm SC-52. (c) Fluorescent signals of EGFP were quantitatively estimated using a Varioskan Lux multimode microplate reader. Data represent the mean and standard error values of six replications. (d) Protein yield values in milligram/gram fresh weight were calculated using fluorescent signal values and standard curves. (e) CBB staining for undiluted samples of 10 ml total soluble proteins extracts of Salinas lettuce leaves infiltrated with \u003cem\u003eAgrobacterium\u003c/em\u003econtaining EGFP+E “yellow dotted rectangle” and Agrobacterium containing EGFP+RNAi “red dotted rectangle. The band seen at 55 kDa in a CBB staining gel corresponds to a large subunit of Rubisco and represents a loading control. (f) CBB band intensities were quantified using ImageJ software. (g) Western blot (WB) analysis was performed using 1 ml samples of total soluble proteins and detected using anti-GFP antibodies. (h) WB band intensities were quantified using ImageJ software. (j) The transcription levels of EGFP of Salinas lettuce leaves harvested at 3 dpi. (j) and (k) The transcription levels of LsRDR1 and LsRDR6, respectively, using samples used in co-infiltration assays. All data represent the mean and standard error values of six replications. Statistically significant differences are represented by one asterisk for p \u0026lt; 0.05 and two asterisks for p \u0026lt; 0.01 based on unpaired Student’s t-test.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/637065b9137c8ee7edc2b9a6.png"},{"id":64747514,"identity":"7c76b350-e751-4b56-b2c7-e56f28031982","added_by":"auto","created_at":"2024-09-18 09:59:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4792225,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKnockdown of the expression of RDR genes increased the expression of EGFP in commercial lettuce at 3 dpi. \u003c/strong\u003e(a) EGFP fluorescence in 3 representative commercial lettuce leaves was detected at 3 dpi using blue light and ultraviolet-absorbing filter Fujifilm SC-52. The dotted rectangle represents the location where samples were taken for protein extraction. (E) represents EGFP co-expressed with empty vector while (R) represents EGFP co-expressed with LsRDR1,6-RNAi. Scale bar, 1 cm. \u0026nbsp;(b) Fluorescent signals of EGFP were quantitatively estimated using a Varioskan Lux multimode microplate reader. (c) Protein yield values in milligram/gram fresh weight were calculated using fluorescent signal values and standard curves. (d) and (e) are CBB–stained 10 mlsamples of soluble proteins and quantification values of band intensities obtained using ImageJ software, respectively. (f) and (g) are WB of 1 ml samples of soluble proteins and quantification values of band intensities obtained using ImageJ software, respectively. All data represent the mean and standard error values of six replications. Statistically significant differences are represented by one asterisk for p \u0026lt; 0.05 and two asterisks for p \u0026lt; 0.01 based on unpaired Student’s t-test.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/761c8fdf0d76611de174be6a.png"},{"id":64746831,"identity":"1e8aa975-606d-471b-b917-eaec54952539","added_by":"auto","created_at":"2024-09-18 09:51:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1046860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effect of LsRDR1,6 on the expression of Bet v 1 in Salinas lettuce at 5 dpi. \u003c/strong\u003e(a)\u003cstrong\u003e \u003c/strong\u003eWB analysis was performed using 10 ml samples of total soluble proteins extracted at 5 dpi from 4 different leaves and detected usinganti-DYKDDDDK. The CBB band seen at 55 kDa corresponds to a large subunit of Rubisco and represents a loading control. (b) WB band intensities were quantified using ImageJ software. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll data represent the mean and standard error values of four replications. Statisticallysignificant differences are represented by one asterisk for p \u0026lt; 0.05 based on unpaired Student’s t-test.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/631073965385f3fd04658b5d.png"},{"id":65628158,"identity":"9eb30e9d-327d-4a69-a37b-a68f33cfd0e3","added_by":"auto","created_at":"2024-09-30 16:18:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10561884,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/8084a90c-8123-4565-9a7f-b338de63d70d.pdf"},{"id":64746835,"identity":"50328578-2e4e-4bca-9e60-4c2ad0762886","added_by":"auto","created_at":"2024-09-18 09:51:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3591046,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 1 Sequence of RNAi constructs used in this study. \u003c/strong\u003eThe small letters represent the sense and anti-sense RNAi target sequences; the uppercase letters represent the sequence of ChsA intron.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 2 Images for lettuce Salinas and the commercial lettuce used. \u003c/strong\u003e(a) Lettuce Salinas grown in laboratory (b) Commercial lettuce with roots purchased from a local grocery store.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 3 The effect of LsRDR1,6-RNAi on EGFP in Salinas and commercial. Lettuce at 5 dpi. \u003c/strong\u003e(a) and (g) The fluorescence of protein extracts of Salinas and commercial lettuce leaves, respectively, was detected at 5 dpi using blue light and ultraviolet-absorbing filter Fujifilm SC-52. (b) and (h) is WB of 1 mlsamples of soluble protein extracts of Salinas and commercial lettuce leaves, respectively, and detected by anti-GFP antibodies. (c) and (i) Protein yield values in milligram/gram fresh weight were calculated using fluorescent signal values of protein extracts of Salinas and commercial lettuce leaves, respectively, by using standard curves. (d-f) The transcription levels of EGFP, LsRDR1 and LsRDR6, respectively, using co-infiltrated samples. All data represent the mean and standard error values of 4-6 replications. Statisticallysignificant differences are represented by one asterisk for p \u0026lt; 0.05 and two asterisks for p \u0026lt; 0.01 based on unpaired Student’s t-test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. 4 Standard curves for quantifying EGFP using fluorescent signals. \u003c/strong\u003e(a) CBB stain of 3 replicates of EGFP protein extracts expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e (b) The band intensities were calculated using imageJ and then used for standard curve formation between band intensities and concentrations of BSA. (c) Fluorescence signals of different concentrations of EGFP protein extracts determined by BSA standard curve. (d) The fluorescence signals obtained by Varioskan Lux multimode microplate reader of known EGFP concentrations were used to create a standard curve.\u003c/p\u003e","description":"","filename":"240812Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/1817090336747dd0588cd20c.pdf"},{"id":64746830,"identity":"79b45ef6-ba38-4f9e-82f6-d3b4e9c21dc0","added_by":"auto","created_at":"2024-09-18 09:51:06","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1 \u003c/strong\u003ePrimers used in the study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Table 2 \u003c/strong\u003eList of probable RNA-dependent RNA polymerases in lettuce Salinas\u003c/p\u003e","description":"","filename":"240813RamadanetalSupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4897984/v1/4fa0b6042c95e8e8e57b872d.docx"}],"financialInterests":"","formattedTitle":"Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRNA-mediated gene silencing, or RNA interference (RNAi), is a vital and conserved system for regulating gene expression in prokaryotes and eukaryotes (Felden and Paillard \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In plants, it also acts as a defense mechanism against viruses and other non-viral pathogens (Lopez-Gomollon and Baulcombe \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Post-transcriptional gene silencing (PTGS) occurs through Dicer-like enzymes (DCL) that process double-stranded RNAs into small interfering RNA duplexes (siRNAs), which can move from cell to cell to produce systemic silencing, and RNA-dependent RNA polymerases (RDRs) that act on single-stranded RNA bound with siRNA to produce double-stranded RNA (Zamore \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Melnyk et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Finally, Argonaute (AGO) and siRNA form an RNA-induced silencing complex (RISC), which leads to the destruction of homologous RNAs (Nakanishi \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To counteract host RNAi defenses, many viruses with the \u0026ldquo;arm-race\u0026rdquo; concept encode viral suppressors of RNA silencing (VSRs), like p1, p10, p19, P21, P24, HCPro, and others which inhibit key steps in the RNAi system and have multifunctional roles in viral processes (Burgy\u0026aacute;n and Havelda \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Lopez-Gomollon and Baulcombe \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). VSRs primarily suppress RNA-silencing pathways through various mechanisms, including binding to dsRNA and inhibiting components of the host RNAi machinery, such as RISC and DNA methylation enzymes (Csorba et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe introduction of foreign DNA into plant genomes through \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e has revolutionized plant biotechnology for many applications, including genetic engineering and crop improvement, gene function studies, plant-microbe interactions, and recombinant protein production (Gelvin \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Tzfira and Citovsky \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The immune response of plants, including the gene silencing system toward \u003cem\u003eAgrobacterium\u003c/em\u003e itself and foreign DNA that usually contains viral elements, such as the Cauliflower Mosaic virus 35S (CaMV 35S) promoter, limits the expression of recombinant proteins (RPs) inside plants (Zipfel et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Kawazu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). \u003cem\u003eNicotiana benthamiana\u003c/em\u003e, a close relative of tobacco, has emerged as a prominent host plant for the production of various biologically active molecules and RPs. The ability of \u003cem\u003eNicotiana benthemiana\u003c/em\u003e to produce RPs is correlated with a natural mutation within the gene encoding RNA-dependent RNA polymerase 1, Rdr1 (Bally et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similarly, a knockout mutation in the RDR6 gene in \u003cem\u003eN. benthemiana\u003c/em\u003e plants resulted in the accumulation of a larger amount of recombinant green fluorescent proteins compared to the wild type (Matsuo and Atsumi \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition, in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, a mutation in RDR6 substantially improves RP expression (von Schaewen et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). These data suggested a strong correlation between gene silencing and RP expression.\u003c/p\u003e \u003cp\u003eLettuce has emerged as a promising alternative for the expression of RP, offering advantages such as oral delivery and the potential for large-scale production (Clarke et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) with various products, including vaccine antigens, antibodies, and therapeutic proteins, being successfully expressed in lettuce (Chan and Daniell \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the RP yield of lettuce remained relatively low compared to \u003cem\u003eN. benthemiana\u003c/em\u003e (Yamamoto et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In contrast to the gene silencing mechanism of \u003cem\u003eNicotiana benthemiana\u003c/em\u003e which is easily suppressed by different viral suppressors (Arzola et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), lettuce was unresponsive to viral suppressors of gene silencing, such as P19 (from tomato bushy stunt virus, TBSV), P1/HcPro (from turnip mosaic virus, TuMV), or P1/HcPro (from tobacco etch virus, TEV) (Wroblewski et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Simmons and VanderGheynst \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), possibly because of the intricate interplay between various viral suppressors and the defense mechanisms of lettuce, which is poorly understood.\u003c/p\u003e \u003cp\u003eIn this study, we focused on the \u003cem\u003eRDR\u003c/em\u003e gene family in lettuce and, using phylogenetic analysis, classified them according to their homology with RDR members of the Arabidopsis. When we utilized RNAi technology for suppressing the gene silencing mechanism in lettuce Salinas, by designing two unique RNAi that target highly conserved regions between both LsRDR1 and LsRDR6, the expression of both genes was specifically reduced. Using RNAi-1 as a representative in combination with the EGFP gene in the Tsukuba system vector, EGFP expression was enhanced significantly in both Salinas lettuce and commercially available lettuce. To validate the efficiency of RNAi-1, the expression of another therapeutic protein, Bet v 1, was assessed in Salinas lettuce, revealing a significant increase in its expression. This study revealed a new potential for the enhancement of RP production in lettuce.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and growth conditions\u003c/h2\u003e \u003cp\u003eLettuce Salinas seeds (Watanabe Seeds Co., Chiba, Japan) were planted in loose rock wool in plug trays until germination. Seedlings were then transplanted to plastic pots with soil suitable for vegetables (one plant/pot) and irrigated with a high-nitrogen fertilizer (15-6-6). Seedlings were grown under a photoperiodic condition (16-h light/8-h dark cycle at 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) at 23 ◦C in a plant growth chamber.\u003c/p\u003e \u003cp\u003e \u003cem\u003eN. benthamiana\u003c/em\u003e plants were used to express EGFP for standard curve preparation. \u003cem\u003eN. benthamiana\u003c/em\u003e seeds were planted in loose rockwool. Seedlings were grown under a photoperiodic condition (16-h light/8-h dark cycle of white light 100 \u0026micro;mol m\u0026thinsp;\u0026minus;\u0026thinsp;2 s\u0026thinsp;\u0026minus;\u0026thinsp;1) at 23 ◦C in a plant growth chamber.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePrimers\u003c/h2\u003e \u003cp\u003eThe primers used for polymerase chain reaction (PCR) and quantitative real-time PCR (q-RT-PCR) are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree and protein domain analysis\u003c/h2\u003e \u003cp\u003eInformation on lettuce RNA-dependent RNA polymerases (RDR) sequences was obtained using genome V.11 from the gene search tool of the National Center for Biotechnology Information (NCBI). Subsequently, using the NGPhylogeny (FastME/OneClick) online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngphylogeny.fr\u003c/span\u003e\u003cspan address=\"https://ngphylogeny.fr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the ORF sequences of the Arabidopsis and lettuce RDRs were aligned using MAFFT Alignment (Katoh and Standley \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lemoine et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The phylogenetic tree was visualized using PRESTO (Phylogenetic tReE visualization, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngphylogeny.fr/data/displaytree\u003c/span\u003e\u003cspan address=\"https://ngphylogeny.fr/data/displaytree\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The domain structures of all lettuce and Arabidopsis RDRs were checked using the SMART (Simple Modular Architecture Research Tool) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Letunic and Bork \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Letunic et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Multiple sequence alignment of lettuce RDRs amino acids was carried out using ClustalW, available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/tools-bin/clustalw\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/tools-bin/clustalw\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Subsequently, the alignment data were visualized using the SnapGene Viewer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCloning into the Tsukuba system vector\u003c/h2\u003e \u003cp\u003eTsukuba system vector pTKB3 previously described in(Omori et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) was used for cloning of RNAi and EGFP inserts. Alignment between the DNA sequences of \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e was performed and visualized using ApE-A plasmid editor v3.1.2. Two sequences, 120 bp for RNAi-1 and 100 bp for RNAi-2, exhibiting 75% and 76% similarity, respectively, between LsRDR1 and LsRDR6, were used in reverse orientation and separated by the ChsA intron. (Dafny-Yelin et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The corresponding DNA sequence was synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA) and cloned into Sal1-digested pTKB3 using an In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). Bet v 1 vector was constructed and expressed in a previous study (Yamada et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).All vectors used pTKB3 only (E), pTKB3-RNAi (R), pTKB3-EGFP and pBYR2HS-HF-Betv1 were transformed into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV3101 using by electroporation and then used for plant infiltration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTransient expression of proteins in lettuce\u003c/h2\u003e \u003cp\u003ePreparation of \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e suspension and transient expression in lettuce were performed as previously described (Yamamoto et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) with modifications. \u003cem\u003eA. tumefaciens\u003c/em\u003e GV3101 containing the binary vector was grown in modified YEB media (6 g/L of yeast extract, 5 g/L of trypone, 5 g/L of sucrose, and 2 mM MgSO4) with antibiotics (100 mg/L of kanamycin, 30 mg/L gentamycin, and 30 mg/L of rifampin) for 2 days at 28\u0026deg;C. Then, 2-day cultures were diluted 100 times in the same modified YEB with antibiotics, 10 mM MES (pH 5.6), and 20 \u0026micro;M acetosyringone, and grown for 18\u0026ndash;24 h at 28\u0026deg;C on a rotary shaker at 140 rpm. The optical density OD600 was adjusted to approximately 1 for agrobacterium harboring empty vector or RNAi vector and 0.5 in the case of EGFP or Bet v 1. Then, 55 g/L of sucrose and 200 \u0026micro;M acetosyringone were added to the bacterial culture and the suspension was incubated for 1 h at 22\u0026deg;C. After incubation, 2,4-dichlorophenoxyacetic acid and Tween-20 were added to the final concentrations of 100 \u0026micro;g/mL and 0.005%, respectively, and the suspension was used for syringe-infiltration in 2-month-old lettuce Salinas leaves Supplementary Fig.\u0026nbsp;2a. In case of suspension mixture of different agrobacterium, doubled OD\u003csub\u003e600\u003c/sub\u003e values was prepared then equal volumes were mixed. After syringe infiltration, the lettuce leaves were rinsed with water. Water was then removed with paper towels, and the lettuce was incubated for 3\u0026ndash;5 days at 22\u0026deg;C under a 16-h light and 8-h dark photoperiod.\u003c/p\u003e \u003cp\u003eGreen leaf lettuce with roots was obtained commercially from a local grocery store, as shown in Supplementary Fig.\u0026nbsp;1b, rinsed with distilled water, and the water was removed using paper towels. After syringe infiltration, the lettuce heads were rinsed with water. Water was then removed with paper towels, and the lettuce was incubated for 3\u0026ndash;5 days at 22\u0026deg;C under a 16-h light and 8-h dark photoperiod.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and q-RT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 100 mg leaf samples of lettuce using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer\u0026rsquo;s instructions. cDNA synthesis and real-time PCR were performed as previously described (Abdellatif et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). An amount of 2 \u0026micro;g of RNA was used to synthesize cDNA using a high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The primers used for real-time PCR are listed in Supplementary Table\u0026nbsp;1. THUNDERBIRD SYBR qPCR Mix (Toyobo, Osaka, Japan) was used for RT-PCR amplification and detection with a 7900HT real-time PCR system (Applied Biosystems/Thermo Fisher Scientific, Waltham, MA, USA). Relative transcript abundance was calculated using the comparative CT method, as described previously (Miura et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), after normalization of raw signals with the housekeeping transcript abundance of an actin gene.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein extraction, coomassie brilliant blue (CBB), and western blotting (WB) analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSoluble proteins were prepared as described previously (Miura et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), with some modifications. Briefly, Plant leaves (100 mg) were ground by bead beating using a Cell Destroyer PS-1000 (Pro Sense, Inc., Tokyo, Japan) at 2,500 rpm for 15 s after freezing in liquid nitrogen. Then, 1 mL of lysis buffer [50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.2 mM sodium orthovanadate, 100 mM NaF, 10% glycerol, 0.2% Triton X-100, 5 mM DTT, and 1\u0026times; protein inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan)] was added. The powdered leaves and lysis buffer were completely mixed by bead-beating and incubated on ice with shaking for 1 h. The samples were spun and the liquid solution was used as the soluble protein extract. Ten microliters of sample was boiled with 4x sample buffer and then subjected to SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB), and images were captured using WSE-6300 LuminoGraph III (ATTO). For immunoblot analysis, crude extracts were 10-fold diluted (except for Bet v 1, samples were not diluted) with Milli-Q and boiled with 4x sample buffer, then, 10 \u0026micro;l of sample solution was applied. Proteins were transferred onto a PVDF membrane (Amersham Hybond P PVDF, GE Healthcare). The blot was probed with the anti-GFP monoclonal antibody GF28R (Thermo Fisher Scientific) or anti-DYKDDDDK monoclonal antibody (Wako) and detected by Luminata Forte Western HRP substrate (Millipore) using a WSE-6300 LuminoGraph III (ATTO, Tokyo, Japan). The CBB band intensities and western blotting band intensities were calculated using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFluorescent measurement and EGFP quantification\u003c/h2\u003e \u003cp\u003e50 \u0026micro;l of soluble protein fractions were added in 96 well plates where each sample was added in one well surrounded by empty wells. The fluorescence of EGFP was detected at 488 nm excitation and 510 nm emission using a Varioskan Lux Multimode microplate reader, and measurements were analyzed using Skanlet software 7.0.1 (Thermo Fisher Scientific, Waltham, MA, USA).\u003c/p\u003e \u003cp\u003eIn order to quantify EGFP in leaf extract using fluorescence signals, we combined two standard curves, one was created using band intensities obtained by ImageJ of known concentrations of Bovine serum albumin (BSA) to obtain the concentrations of EGFP extracts from \u003cem\u003eN. benthemiana\u003c/em\u003e. The second was created using EGFP of known concentrations from the first curve and their fluorescence signals were measured by Varioskan Lux multimode microplate reader. Using the last standard curve, fluorescence signals of any protein extract of EGFP could approximately refer to the concentration of EGFP.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic analysis of RDR gene family in lettuce\u003c/h2\u003e \u003cp\u003eTo identify the RDR family genes in lettuce, we queried the lettuce genomic protein database (version 11) using the protein sequences of both Arabidopsis RDR1 and RDR6. This search led us to identify five genes (Supplementary Table\u0026nbsp;2) that showed a significant match with Arabidopsis proteins. To elucidate the relationships between lettuce and Arabidopsis RDR genes, we constructed a phylogenetic tree based on the protein sequences of these genes using the online tool NGphylogeny. The tree revealed two main clusters: cluster 1 included both lettuce and Arabidopsis RDR6, in addition to RDR 1 and 2 of both lettuce and Arabidopsis, whereas cluster w included other RDRs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). In contrast to Lettuce RDRs 1, 2, and 6, which were very close to their corresponding Arabidopsis proteins, both lettuce RDR5a and b were separated from Arabidopsis RDRs 3, 4, and 5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Similar to the phylogenetic tree, domain analysis using SMART (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb) showed the first cluster with an RNA-directed RNA polymerase (RdRP) domain in addition to the RNA recognition motif (RRM); the second cluster that included RDRs 3, 4, and 5 contained only the RdRP domain. Protein alignment using ClustalW revealed the canonical amino acid motif DLDGD, which is characteristic of RDRs of the 1st cluster and the atypical amino acid motif DFDGD, which is characteristic of RDRs of the 2nd cluster (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). These results reflect the accuracy of blasting and phylogenetic analysis, which classifies RDR genes based on differences in amino acid sequences, motifs, and domains.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eKnockdown of\u003c/b\u003e \u003cb\u003eLsRDR1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eLsRDR6\u003c/b\u003e \u003cb\u003eby a single RNAi\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo knockdown both \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e using single RNAi, two RNAi were constructed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and Supplementary Fig.\u0026nbsp;1) and cloned into pTKB3 vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The transcript levels of both \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e were significantly reduced in the presence of either RNAi-1 or RNAi-2 compared to those in control vector at both 3 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec) and 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). However, at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5 no significant difference was observed except for \u003cem\u003eLsRDR6\u003c/em\u003e at 3 dpi, silencing was significant at both OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 and 2. At 3 dpi, OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 of RNAi-1 and RNAi-2 exhibited a reduction in the transcription of \u003cem\u003eLsRDR1\u003c/em\u003e by 61.6% and 51.8%, respectively and of \u003cem\u003eLsRDR6\u003c/em\u003e by 65.64% and 54.3%, respectively. RNAi-1 and RNAi-2 at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2 reduced the transcription of \u003cem\u003eLsRDR1\u003c/em\u003e by 77.8% and 72%, respectively and of \u003cem\u003eLsRDR6\u003c/em\u003e by 84.3% and 66.9%, respectively. While at 5 dpi, OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 of RNAi-1 and RNAi-2 exhibited a reduction in the transcription of \u003cem\u003eLsRDR1\u003c/em\u003e by 58.1% and 48.9% respectively and of \u003cem\u003eLsRDR6\u003c/em\u003e by 46.9% and 38.2%, respectively. RNAi-1 and RNAi-2 at OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2 reduced the transcription of \u003cem\u003eLsRDR1\u003c/em\u003e by 76% and 74.2%, respectively and of \u003cem\u003eLsRDR6\u003c/em\u003e by 73.3% and 70.2%, respectively. Notably, the transcript of \u003cem\u003eLsRDR2\u003c/em\u003e, the closest member of the phylogenetic tree, was not affected at both 3 and 5 dpi and at all OD values tested, indicating that both RNAi used were specific to \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6.\u003c/em\u003e Statistical analysis was performed between EV, RNAi-1 and RNAi-2 samples of the same OD. Since RNAi-1 reduced transcripts of \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e more, compared with RNAi-2, RNAi-1 was used for the subsequent analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCo-infiltration with LsRDR1,6-RNAi enhanced the expression of EGFP in lettuce Salinas\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of RNAi-1 which we hereafter called it LsRDR1,6-RNAi on protein expression capacity, lettuce Salinas leaves co-infiltrated with a mixture of \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring pTKB3-RNAi and pTKB3-EGFP were brighter than those infiltrated with a mix of \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring the pTKB3 empty vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The fluorescence signals of total soluble protein extracts was measured using Varioskan Lux multimodal microplate reader (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Quantitative fluorescence analysis revealed that the signal significantly increased about 3 folds. Image using blue light and an SC-52 filter (Fujifilm), also showed a clear enhancing effect of LsRDR1,6-RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). We then quantified the EGFP yield using a combination of the conventional BSA quantification of EGFP extract from \u003cem\u003eNicotiana benthemiana\u003c/em\u003e (Supplementary Fig.\u0026nbsp;4a), standard curve of BSA band intensities (Supplementary Fig.\u0026nbsp;4b), fluorescence signals of samples with known EGFP concentrations (Supplementary Fig.\u0026nbsp;4c) and a standard curve between known concentrations of EGFP and fluorescence signals (Supplementary Fig.\u0026nbsp;4d). Using this approach, the EGFP protein yield of protein extract from lettuce leaves was measured and showed an increase from approx. 0.18 mg/gFW to 0.9 mg/gFW in the presence of LsRDR1,6-RNAi which is equivalent to a 4.85-fold increase. To confirm the fluorescence data, the soluble protein fractions were analyzed by CBB and WB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee and g, respectively). The bands were more intense in the case of the co-infiltration of EGFP\u0026thinsp;+\u0026thinsp;LsRDR1,6-RNAi vectors, and analysis of the CBB and WB bands using ImageJ software revealed an increase of approximately 1.5- and 2-fold, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ef and h, respectively). The quantitative transcript level of EGFP was significantly increased (1.58-fold) confirming the previous data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei). Also, the LsRDR1,6-RNAi efficiency was confirmed in co-infiltration assays by reducing the transcription of \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e by 53.3% and 50.5% respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ej and k). At 5dpi, the fluorescence was still brighter, the WB band intensities were stronger and the protein yield of EGFP increased 3.66-fold from 0.3 mg/gFW to 1.13 mg/gFW in the presence of LsRDR1,6-RNAi (Supplementary Fig.\u0026nbsp;3a-c). Also, the significant enhancement and silencing of \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e were maintained (Supplementary Fig.\u0026nbsp;3d-f). The data presented here revealed that the efficiency of LsRDR1,6-RNAi was sustained during 5 dpi with slight changes from the levels at 3 dpi.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLsRDR1,6-RNAi also stimulates EGFP expression in commercial lettuce\u003c/h2\u003e \u003cp\u003eTo test the applicability of LsRDR1,6-RNAi on different lettuce cultivars, commercially available lettuce from the local market (Supplementary Fig.\u0026nbsp;2b) co-infiltrated with a mixture of \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring pTKB3-LsRDR1,6-RNAi and pTKB3-EGFP showed a brighter and wider expression of EGFP compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea) at 3 dpi. The fluorescence of total soluble protein extracts were higher 1.5-folds in the presence of LsRDR1,6-RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Quantification of protein yield using fluorescence signals revealed an increase from 0.11 mg/gFW to 0.27 mg/gFW in the presence of LsRDR1,6-RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) which is equivalent to 2.35-fold increase. CBB and WB analyses of soluble protein extracts exhibited more intense bands for EGFP co-expressed with LsRDR1,6-RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed and f), and quantitative analysis of the corresponding bands using ImageJ software revealed 1.4- and 1.6-fold increases, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee and g, respectively). At 5dpi, the fluorescence was also still brighter, the WB band intensities were stronger and the protein yield of EGFP increased 2.33-fold from 0.25 mg/gFW to 0.6 mg/gFW in the presence of RNAi-1 (Supplementary Fig.\u0026nbsp;3g-i). The data presented here revealed that the protein expression level in this cultivar is lower than that of Salinas lettuce.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCo-infiltration with LsRDR1,6-RNAi enhanced the expression of Bet v 1 in lettuce Salinas\u003c/h2\u003e \u003cp\u003eTo conclude our study, we used a therapeutic protein Bet v 1 to test its expression in lettuce and whether its expression level can be enhanced by RNAi-1. At 5 dpi, total soluble protein extracts of lettuce Salinas leaves co-infiltrated with a mixture of \u003cem\u003eA. tumefaciens\u003c/em\u003e harboring pTKB3-RNAi-1 and pBYR2HS-HF-Betv1 showed more accumulation of Bet v 1 compared with the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Quantitative analysis of band intensities revealed an increase of about 2.64-fold of Bet v 1 in the presence of RNAi-1 compared with that in the presence of empty vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Notably, the WB samples were not diluted since the expression level was much lower compared with EGFP. Also, Bet v 1 was not detectable through CBB stain and therefore not quantified.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTaken together, it is now feasible to efficiently suppress the gene silencing mechanism in lettuce using LsRDR1,6-RNAi, which precisely downregulates both \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e genes. Genes expected to play roles related to pathogenicity and RP production in lettuce in a similar manner as in Arabidopsis and \u003cem\u003eNicotiana benthemiana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we observed that OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2 achieved a silencing level approaching 80% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec and d). However, we primarily relied on OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1 to minimize potential stress associated with excessively high OD, especially in the presence of OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.5 of EGFP. The enhancement of EGFP expression in the presence of LsRDR1,6-RNAi slightly decreased at 5 dpi (Supplementary Fig.\u0026nbsp;3c and i), which may be attributed to the counter-silencing of the lettuce immune response. Another possible explanation is the saturation of EGFP expression in cells by 5 dpi, leading to a diminished observable effect of LsRDR1,6-RNAi. In the case of Bet v 1, a 2.64-fold increase was clearly detected at 5 dpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and b), likely due to its lower baseline expression in lettuce compared to EGFP, which supports the saturation hypothesis. Additionally, it was clear that the expression of EGFP and the effect of LsRDR1,6-RNAi was lower in commercial lettuce compared with Salinas lettuce (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). It is important to note that the commercial lettuce used in this study exhibited difficulties in infiltration, possibly due to its advanced age compared to the Salinas lettuce or due to the difference in the amount of wax on leaf surface. This factor likely contributed to the lower expression levels of both EGFP and LsRDR1,6-RNAi in the commercial lettuce relative to Salinas. The expression levels of recombinant proteins in various plant hosts, including commercial lettuce and \u003cem\u003eN. benthemiana\u003c/em\u003e were compared previously (Yamamoto et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) showing that commercial lettuce demonstrated 1/10 of the expression potential of \u003cem\u003eN. benthamiana\u003c/em\u003e (0.37 mg/gFW in lettuce and 3.7 mg/gFW in \u003cem\u003eN. benthamiana\u003c/em\u003e) when EGFP was expressed. However, in the current study, we observed a significant increase in EGFP expression levels in both commercial and Salinas lettuce varieties 0.6 mg/gFW and 1.12 mg/gFW, getting closer to the levels observed in \u003cem\u003eN. benthamiana\u003c/em\u003e. In a prior study (Yamada et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Bet v 1 expression in Nicotiana benthamiana reached approximately 1 mg/g fresh weight, which was notably lower than the expression levels observed for EGFP. These results led us to expect a similarly low expression of Bet v 1 in lettuce. Even though we aimed to evaluate the effectiveness of our RNAi approach in enhancing the expression of various recombinant proteins, which seems to be efficient in different cultivars of lettuce.\u003c/p\u003e \u003cp\u003eSuppression of the lettuce gene silencing mechanism did not occur using three different viral suppressors: P19 (from tomato bushy stunt virus (TBSV)), P1/HcPro (from turnip mosaic virus, TuMV), or P1/HcPro (from tobacco etch virus, TEV) (Wroblewski et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Simmons and VanderGheynst \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The efficiency of LsRDR1,6-RNAi was demonstrated by the decreased transcription of these genes in lettuce Salinas (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec), along with increased expression of EGFP in two different lettuce cultivars (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This implies that this approach may also be applicable to other lettuce cultivars.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis of RDR gene families was conducted in several plant species including rice (5 members; (Kapoor et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2008\u003c/span\u003e)), maize (7 members; (Qian et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)), tomato (6 members; (Bai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2012\u003c/span\u003e)) in addition to Arabidopsis (6 members; (Willmann et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)) but was done here in lettuce for the first time (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). LsRDR1,2 and 6 shared a common canonical amino acid DLDGD motif, whereas other RDRs shared an atypical DFDGD motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec), similar to other species such as Arabidopsis and \u003cem\u003eMusa acuminata\u003c/em\u003e (Ahmed et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn Arabidopsis, \u003cem\u003eRDR6\u003c/em\u003e, Suppressor of gene silencing 3 (SGS3), and RNA-directed DNA methylation 12 (RDM12) work together as part of the RNA-silencing pathway to regulate gene expression and suppress the activity of invasive nucleic acids, such as viral nucleic acids and T-DNA (Hua et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). SGS3 is a cofactor of RDR6 that enhances its ability to generate dsRNAs. It also stabilizes dsRNA intermediates and recruits other components of the RNA-silencing machinery, such as AGO proteins, to facilitate target mRNA cleavage or translational repression. RDM12 is a component of the RNA-directed DNA methylation (RdDM) pathway, which is a branch of the RNA-silencing pathway responsible for establishing DNA methylation and transcriptional gene silencing at target loci. RDM12 interacts with RDR6 and SGS3 to promote the production of secondary siRNAs, which guide the DNA methylation and silencing of homologous genomic sequences. Their coordinated actions contribute to the robustness and specificity of RNA silencing-mediated defense mechanisms in plants. In lettuce, the endogenous polyubiquitin promoter was shown to enhance RP expression much more than the CaMV35S promoter, owing to excessive methylation in lettuce, which was more than 10 times the methylation in Arabidopsis(Kawazu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Another report demonstrated that the use of the endogenous lettuce LsU6-10 promoter was more efficient for gene editing mediated by CRISPR-Cas9 than a similar promoter from Arabidopsis (Riu et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These reports indicate that foreign DNA may not be compatible with lettuce, and that utilizing endogenous elements enhances RP expression by circumventing the plant silencing mechanism. Studying additional elements such as SGS3 and RDM12 in lettuce could help us better understand how gene silencing works and provide insights into improving recombinant protein expression.\u003c/p\u003e \u003cp\u003eIn conclusion, this study marks a significant step toward enhancing lettuce RP expression using RNAi technology. It would be worthwhile to investigate the duration for which LsRDR1,6-RNAi maintains its silencing effect and test its efficacy on various lettuce varieties. Also, it is important to explore the effects of stable mutants of both \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e generated via CRISPR-Cas9 on lettuce phenotypes, disease susceptibility, and protein expression efficiency. In addition, a deeper understanding of why viral suppressors do not succeed in overcoming the silencing mechanism of lettuce is required, and more diverse suppressors should be tested. The findings here not only contribute valuable and new insights into the silencing mechanism in lettuce, but also pave the way for future investigations on the preparation of a new plant model for RP production.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRecombinant protein \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; RP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RNA interference \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; RNAi\u003c/p\u003e\n\u003cp\u003eRNA-dependent RNA Polymerase \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Post-transcriptional gene silencing \u0026nbsp; \u0026nbsp; PTGS\u003c/p\u003e\n\u003cp\u003epTKB3 empty vector \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; E \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Transcriptional gene silencing \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;TGS\u003c/p\u003e\n\u003cp\u003epTKB3 with RNAi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;R \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Dicer-like enzymes \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; DCL\u003c/p\u003e\n\u003cp\u003eDays post infiltration \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Dpi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;small interfering RNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;siRNA\u003c/p\u003e\n\u003cp\u003eRNAi of LsRDR1 and LsRDR6 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; LsRDR1,6-RNAi \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; RNA-induced silencing complexes \u0026nbsp; \u0026nbsp;\u0026nbsp;RISC\u003c/p\u003e\n\u003cp\u003eCoomassie brilliant blue \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;CBB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; microRNAs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; miRNA\u003c/p\u003e\n\u003cp\u003eWestern blotting \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;WB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; viral suppressors of RNA silencing \u0026nbsp; \u0026nbsp;VSR\u003c/p\u003e\n\u003cp\u003eAgronaute \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; AGO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hairpin RNA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;hpRNA\u003c/p\u003e\n\u003cp\u003eEnhanced green fluorescent protein \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;EGFP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;RNA-directed DNA methylation 12 \u0026nbsp;RDM12\u003c/p\u003e\n\u003cp\u003eSuppressor of gene silencing 3 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;SGS3 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Optical density \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; OD\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank Ms. Yuriko Nagai, Ms. Yumiko Iguchi, and Ms. Yuri Nemoto at the University of Tsukuba for their technical support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eK.M. designed the study; A.R. and K.O. performed the experiments and data analyses; A.R. wrote the manuscript; and K.M. revised the manuscript. All authors contributed to the article and approved the submission of this manuscript. All authors read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Program on Open Innovation Platform with Enterprise, Research Institute, and Academia, Japan Science and Technology Agency (JST-OPERA, JPMJOP1851).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe datasets generated and analyzed in the current study are available from the corresponding author upon reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdellatif IMY, Yuan S, Yoshihara S, et al (2023) Stimulation of Tomato Drought Tolerance by PHYTOCHROME A and B1B2 Mutations. 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Cell 125:749\u0026ndash;760. https://doi.org/10.1016/j.cell.2006.03.037\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Transient expression, Tsukuba system, Lettuce, RDR1, RDR6, Recombinant proteins","lastPublishedDoi":"10.21203/rs.3.rs-4897984/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4897984/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eImproved yields of recombinant proteins (RP) are necessary for protein production efficiency and ease of purification. Achieving high yield in non-tobacco plants will enable diverse plants to be used as hosts in transient protein expression systems. With improved protein yield, lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e) could take the lead as a plant host for RP production. Therefore, this study aimed to improve RP production in lettuce var. Salinas by designing a single RNA interference (RNAi) construct targeting \u003cem\u003eLsRDR1\u003c/em\u003eand \u003cem\u003eLsRDR6\u003c/em\u003e using the Tsukuba system vector. Two RNAi constructs, RNAi-1 and RNAi-2, targeting common regions of \u003cem\u003eLsRDR1\u003c/em\u003e and \u003cem\u003eLsRDR6\u003c/em\u003e with 75% and 76% similarity, respectively, were employed to evaluate simultaneous gene silencing. Quantitative transcription analysis demonstrated that both RNAi constructs effectively knocked down \u003cem\u003eLsRDR6\u003c/em\u003e and \u003cem\u003eLsRDR1\u003c/em\u003e, but not \u003cem\u003eLsRDR2\u003c/em\u003e, at both 3 and 5 days post-infiltration (dpi), with RNAi-1 exhibited slightly higher efficiency. Based on the protein yield, co-expression of RNAi-1 with enhanced green fluorescent protein (EGFP) increased EGFP expression by approximately 4.9-fold and 3.7-fold at 3 dpi and 5 dpi, respectively, compared to control. A similar but slightly lower increase (2.4-fold and 2.33-fold) was observed in commercial lettuce at 3 dpi and 5 dpi, respectively. To confirm these results, co-infiltration with Bet V 1, a major allergen from birch pollen, resulted in a 2.5-fold increase in expression in Salinas lettuce at 5 dpi. This study marks a significant advancement in enhancing transient protein production in lettuce, elevating its potential as a host for recombinant protein production.\u003c/p\u003e","manuscriptTitle":"Silencing of RDR1 and RDR6 genes by a single RNAi enhances lettuce's capacity to express recombinant proteins in transient assays","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-18 09:51:01","doi":"10.21203/rs.3.rs-4897984/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2024-09-09T09:33:05+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-09-08T23:08:01+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-08T23:06:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-07T07:42:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2024-09-02T01:43:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7d4e780e-2646-4541-9359-97e6af8f5392","owner":[],"postedDate":"September 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:11:30+00:00","versionOfRecord":{"articleIdentity":"rs-4897984","link":"https://doi.org/10.1007/s00299-024-03324-6","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2024-09-24 15:57:01","publishedOnDateReadable":"September 24th, 2024"},"versionCreatedAt":"2024-09-18 09:51:01","video":"","vorDoi":"10.1007/s00299-024-03324-6","vorDoiUrl":"https://doi.org/10.1007/s00299-024-03324-6","workflowStages":[]},"version":"v1","identity":"rs-4897984","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4897984","identity":"rs-4897984","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}