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Key message Enhanced recombinant protein expression was achieved in Salinas lettuce and commercial lettuce by designing a unique RNAi that knockdown the gene-silencing mechanism in transient assays. 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 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.

Background Binding of transcription factors to their target sequences is a primary step in the regulation of gene expression and largely determines gene regulatory networks. Chromatin immunoprecipitation (ChIP) is an indispensable tool used to investigate the binding of DNA-binding proteins (e.g., transcription factors) to their target sequences in vivo. ChIP assays require specific antibodies that recognize endogenous target transcription factors; however, in most cases, such specific antibodies are unavailable. To overcome this problem, many ChIP assays use transgenic plants that express epitope-tagged transcription factors and immunoprecipitate the protein with a tag-specific antibody. However, generating transgenic plants that stably express epitope-tagged proteins is difficult and time-consuming. Results Here, we present a rapid, efficient ChIP protocol using transient expression in Arabidopsis mesophyll protoplasts that can be completed in 4 days. We provide optimized experimental conditions, including the amount of transfected DNA and the number of protoplasts. We also show that the efficiency of our ChIP protocol using protoplasts is comparable to that obtained using transgenic Arabidopsis plants. We propose that our ChIP method can be used to analyze in vivo interactions between tissue-specific transcription factors and their target sequences, to test the effect of genotype on the binding of a transcription factor within a protein complex to its target sequences, and to measure temperature-dependent binding of a transcription factor to its target sequence. Conclusions The rapid and simple nature of our ChIP assay using Arabidopsis mesophyll protoplasts facilitates the investigation of in vivo interactions between transcription factors and their target genes.

Crude protein fractions from Mirabilis jalapa of northern Iran containing Mirabilis Antiviral Protein (MAP) reduced the systemic accumulation of a plant RNA virus in Nicotiana benthamiana . A MAP gene was amplified by RT-PCR from total RNAs isolated from these Iranian M. jalapa plants ( MAP-Tehran ) without the 5´-end signal peptide sequence, to set up an experimental system that would transiently express the protein in the cell cytosol by agroinfiltration and simultaneously trigger a response to study its antiviral effects. The amplified fragment was first cloned into a high-copy plasmid without promoters flanking the cloning site and its nucleotide and amino acid sequences were determined and compared to available MAP sequences. Two putative ribosome inhibitor domains were found in the protein N-terminal and C-terminal regions, respectively. The cloned MAP-Tehran was transferred to binary vectors for agroinfiltration. Only constructs that expressed MAP-Tehran fused to the N- or C- halves of the monomeric red fluorescent protein used for bimolecular fluorescence complementation assays were obtained. In N. benthamiana leaves agroinfiltrated with these constructs, signs of cell plasmolysis became detectable under confocal microscopy at day 3 after infiltration (dai), and from day 4 necrotic lesions were visible. In agroinfiltrated patches at 2–3 dai, before the onset of cell death, reduced accumulation of a co-expressed reporter gene and also of a mechanically inoculated virus were observed. A double-alanine substitution (Glu → Ala, Arg → Ala) in the C-terminal region inhibitor domain of the MAP constructs sufficed to abolish all of these effects. Agroinfiltration of either the necrosis-inducing or non-inducing MAP variants caused no significant effect on the accumulation of an RNA virus inoculated on upper, non-infiltrated leaves.

The study of gene function in roses is hampered by the low efficiency of transformation systems and the long time span needed for the generation of transgenic plants. For some functional analyses, the transient expression of genes would be an efficient alternative. Based on current protocols for the transient expression of genes via the infiltration of Agrobacterium into plant tissues, we developed a transient expression system for rose petals. We used β-glucuronidase (GUS) as a marker gene to optimize several parameters with effects on GUS expression. The efficiency of expression was found to be dependent on the rose genotype, flower age, position of petals within a flower, Agrobacterium strain and temperature of co-cultivation. The highest GUS expression was recorded in petals of the middle whirls of half-bloomed flowers from cultivars of ‘Pariser Charme' and ‘Marvel'.

Summary Drought and Verticillium wilt disease are two main factors that limit cotton production, which necessitates the identification of key molecular switch to simultaneously improve cotton resistance to Verticillium dahliae and tolerance to drought stress. R2R3‐type MYB proteins could play such a role because of their conserved functions in plant development, growth, and metabolism regulation, however, till date a MYB gene conferring the desired resistance to both biotic and abiotic stresses has not been found in cotton. Here, we describe the identification of GhMYB36, a gene encoding a R2R3‐type MYB protein in Gossypium hirsutum, which confers drought tolerance and Verticilium wilt resistance in both Arabidopsis and cotton. GhMYB36 was highly induced by PEG‐simulated drought stress in G. hirsutum. GhMYB36‐silenced cotton plants were more sensitive to both drought stress and Verticillium wilt. GhMYB36 overexpression in transgenic Arabidopsis and cotton plants gave rise to improved drought tolerance and Verticillium wilt resistance. Transient expression of fused GhMYB36‐GFP in tobacco cells was able to localize GhMYB36 in the cell nucleus. In addition, RNA‐seq analysis together with qRT‐PCR validation in transgenic Arabidopsis overexpressing GhMYB36 revealed significantly enhanced PR1 expression. Luciferase interaction assays indicated that GhMYB36 are probably bound to the promoter of PR1 to activate its expression and the interaction, which was further verified by Yeast one hybrid assay. Taken together, our results suggest that GhMYB36 functions as a transcription factor that is involved in drought tolerance and Verticillium wilt resistance in Arabidopsis and cotton by enhancing PR1 expression.

The production of therapeutic and industrial recombinant proteins in plants has advantages over established bacterial and mammalian systems in terms of cost, scalability, growth conditions, and product safety. In order to compete with these conventional expression systems, however, plant expression platforms must have additional economic advantages by demonstrating a high protein production yield with consistent quality. Over the past decades, important progress has been made in developing strategies to increase the yield of recombinant proteins in plants by enhancing their expression and reducing their degradation. Unlike bacterial and animal systems, plant expression systems can utilize not only cell cultures but also whole plants for the production of recombinant proteins. The development of viral vectors and chloroplast transformation has opened new strategies to drastically increase the yield of recombinant proteins from plants. The identification of promoters for strong, constitutive, and inducible promoters or the tissue-specific expression of transgenes allows for the production of recombinant proteins at high levels and for special purposes. Advances in the understanding of RNAi have led to effective strategies for reducing gene silencing and increasing recombinant protein production. An increased understanding of protein translation, quality control, trafficking, and degradation has also helped with the development of approaches to enhance the synthesis and stability of recombinant proteins in plants. In this review, we discuss the progress in understanding the processes that control the synthesis and degradation of gene transcripts and proteins, which underlie a variety of developed strategies aimed at maximizing recombinant protein production in plants.

Previous results showed that expression of the gene encoding the LONG-CHAIN BASE2 (LCB₂) subunit of serine palmitoyltransferase (SPT), designated BcLCB₂, from nonheading Chinese cabbage (Brassica campestris ssp. chinensis) was up-regulated during hypersensitive cell death (HCD) induced by the Phytophthora boehmeriae elicitor PB90. Overexpression of BcLCB₂ in Nicotiana tabacum leaves suppressed the HCD normally initiated by elicitors and PB90-triggered H₂O₂ accumulation. BcLCB₂ also functioned as a suppressor of mouse Bcl-2 associated X (Bax) protein-mediated HCD and cell death caused by Ralstonia solanacearum. BcLCB₂ overexpression suppressed Bax- and oxidant stress-triggered yeast cell death. Reactive oxygen species (ROS) accumulation induced by Bax was compromised in BcLCB₂-overexpressing yeast cells. The findings that NbLCB₂ silencing in Nicotiana benthamiana enhanced elicitor-triggered HCD, combined with the fact that myriocin, a potent inhibitor of SPT, had no effect on Bax-induced programmed cell death, suggested that suppression of cell death was not involved in the dominant-negative effect that resulted from BcLCB₂ overexpression. A BcLCB₂ mutant assay showed that the suppression was not involved in SPT activity. The results suggest that plant HCD and stress-induced yeast cell death might share a common signal transduction pathway involving LCB₂, and that LCB₂ protects against cell death by inhibiting ROS accumulation, this inhibition being independent of SPT activity.

Summary The production of influenza vaccines in plants is achieved through transient expression of viral hemagglutinins (HAs), a process mediated by the bacterial vector Agrobacterium tumefaciens. HA proteins are then produced and matured through the secretory pathway of plant cells, before being trafficked to the plasma membrane where they induce formation of virus‐like particles (VLPs). Production of VLPs unavoidably impacts plant cells, as do viral suppressors of RNA silencing (VSRs) that are co‐expressed to increase recombinant protein yields. However, little information is available on host molecular responses to foreign protein expression. This work provides a comprehensive overview of molecular changes occurring in Nicotiana benthamiana leaf cells transiently expressing the VSR P19, or co‐expressing P19 and an influenza HA. Our data identifies general responses to Agrobacterium‐mediated expression of foreign proteins, including shutdown of chloroplast gene expression, activation of oxidative stress responses and reinforcement of the plant cell wall through lignification. Our results also indicate that P19 expression promotes salicylic acid (SA) signalling, a process dampened by co‐expression of the HA protein. While reducing P19 level, HA expression also induces specific signatures, with effects on lipid metabolism, lipid distribution within membranes and oxylipin‐related signalling. When producing VLPs, dampening of P19 responses thus likely results from lower expression of the VSR, crosstalk between SA and oxylipin pathways, or a combination of both outcomes. Consistent with the upregulation of oxidative stress responses, we finally show that reduction of oxidative stress damage through exogenous application of ascorbic acid improves plant biomass quality during production of VLPs.

The emergence of drug-resistant pathogens poses a serious critical threat to global public health and requires immediate action. Antimicrobial peptides (AMPs) are a class of short peptides ubiquitously found in all living forms, including plants, insects, mammals, microorganisms and play a significant role in host innate immune system. These peptides are considered as promising candidates to treat microbial infections due to its distinct advantages over conventional antibiotics. Given their potent broad spectrum of antimicrobial action, several AMPs are currently being evaluated in preclinical/clinical trials. However, large quantities of highly purified AMPs are vital for basic research and clinical settings which is still a major bottleneck hindering its application. This can be overcome by genetic engineering approaches to produce sufficient amount of diverse peptides in heterologous host systems. Recently plants are considered as potential alternatives to conventional protein production systems such as microbial and mammalian platforms due to their unique advantages such as rapidity, scalability and safety. In addition, AMPs can also be utilized for development of novel approaches for plant protection thereby increasing the crop yield. Hence, in order to provide a spotlight for the expression of AMP in plants for both clinical or agricultural use, the present review presents the importance of AMPs and efforts aimed at producing recombinant AMPs in plants for molecular farming and plant protection so far.

The demand for recombinant proteins in terms of quality, quantity, and diversity is increasing steadily, which is attracting global attention for the development of new recombinant protein production technologies and the engineering of conventional established expression systems based on bacteria or mammalian cell cultures. Since the advancements of plant genetic engineering in the 1980s, plants have been used for the production of economically valuable, biologically active non-native proteins or biopharmaceuticals, the concept termed as plant molecular farming (PMF). PMF is considered as a cost-effective technology that has grown and advanced tremendously over the past two decades. The development and improvement of the transient expression system has significantly reduced the protein production timeline and greatly improved the protein yield in plants. The major factors that drive the plant-based platform towards potential competitors for the conventional expression system are cost-effectiveness, scalability, flexibility, versatility, and robustness of the system. Many biopharmaceuticals including recombinant vaccine antigens, monoclonal antibodies, and other commercially viable proteins are produced in plants, some of which are in the pre-clinical and clinical pipeline. In this review, we consider the importance of a plant- based production system for recombinant protein production, and its potential to produce biopharmaceuticals is discussed.