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Rhombic-spot disease, caused mainly by Neostagonosporella sichuanensis , significantly impacts the yield and quality of fishscale bamboo (Phyllostachys heteroclada). Xylanases are essential for pathogenic fungi infection, yet their specific functions in the physiology and pathogenicity of N. sichuanensis remain unclear. Here, we characterized three xylanase proteins with glycosyl hydrolase 11 domains from the N. sichuanensis SICAUCC 16–0001 genome and examined the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed specific xylanase activity in vitro and induced cell death in Nicotiana benthamiana , independent of their enzymatic function. Both proteins possessed signal peptides and were confirmed as secreted proteins using a yeast secretion system. Subcellular localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and nucleus and can trigger cell death in N. benthamiana through Agrobacterium tumefaciens -mediated transient transformation. qRT-PCR results showed notable upregulation of Nsxyn1 and Nsxyn2 during infection, with Nsxyn1 exhibiting an 80-fold increase at 15 days post-inoculation. Deletion of Nsxyn1 and Nsxyn2 in N. sichuanensis impaired xylan degradation, adaptation to osmotic and oxidative stress, and pathogenic full virulence. Deletion of Nsxyn1 notably slowed fungal growth and reduced spore production, whereas only a reduction in microconidial production was observed in Nsxyn2 mutants. Complementation of Nsxyn1 and Nsxyn2 only partially restored these phenotypic defects in the ∆ Nsxyn1 and ∆ Nsxyn2 mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute to N. sichuanensis virulence and induced plant defense responses, providing new insights into the function of xylanases in the interaction between fishscale bamboo and N. sichuanensis. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this work was to rapidly produce in plats two recombinant antigens (RBDw-Fc and RBDo-Fc) containing the receptor binding domain (RBD) of the spike (S) protein from SARS-CoV-2 variants Wuhan and Omicron as fusion proteins to the Fc portion of a murine IgG2a antibody constant region (Fc). Results: The two recombinant antigens were expressed in Nicotiana benthamiana plants, engineered to avoid the addition of N-linked plant-typical sugars, through vacuum agroinfiltration and showed comparable purification yields (about 35 mg/kg leaf fresh weight). Conclusions: Their Western blotting and Coomassie staining evidenced the occurrence of major in planta proteolysis in the region between the RBD and Fc, which was particularly evident in RBDw-Fc, the only antigen bearing the HRV 3C cysteine protease recognition site. The two RBD N-linked glycosylation sites showed very homogeneous profiles free from plant-typical sugars, with the most abundant glycoform represented by the complex sugar GlcNAc4Man3. Both antigens were specifically recognised in Western Blot analysis by the anti-SARS-CoV-2 human neutralizing monoclonal antibody J08-MUT and RBDw-Fc was successfully used in competitive ELISA experiments for binding to the angiotensin-converting enzyme 2 receptor to verify the neutralizing capacity of the serum from vaccinated patients. Both SARS-Cov-2 antigens fused to a murine Fc region were rapidly and functionally produced in plants with potential applications in diagnostics. [ABSTRACT FROM AUTHOR]

Background: Bouquet is a crucial characteristic indicative of wine quality that develops during the aging stage. The cytochrome P450 VvCYP76F14 multi-functionally catalyzes linalool into (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, which are direct precursors for wine bouquet. Wine bouquet was closely related to VvCYP76F14 activities. Method: The VvCYP76F14 genes were cloned from five wine grape varieties using a homologous cloning method. The variation in residues of VvCYP76F14s were assessed by multiple alignment of amino acid sequences. Functional studies were implemented by in vitro enzyme activity and transient over-expression systems. Results: D299T variation was observed in VvCYP76F14s of 'Yantai 2-2-08', 'Yantai 2-2-19', and 'Yantai 2-3-37' offspring lines, which was correlated with the decreased content of wine bouquet precursors of (E)-8-hydroxylinalool, (E)-8-oxolinalool, and (E)-8-carboxylinalool, respectively. Notably, the key amino acid residue D299 was located at the phase 0 intron positions of VvCYP76F14 genes isolated from distinct wine grape varieties or offspring lines, respectively. Notably, VvCYP76F14s of the 'Yantai2-2-08', 'Yantai 2-2-19', and 'Yantai 2-3-37' mutant lines exhibited lower in vitro enzymatic activities than those of 'L35' and 'Merlot'. In addition, the transient expression of VvCYP76F14 cloned from 'L35' and 'Merlot' restored the levels of wine bouquet precursors in berries of three D299T mutant lines, respectively, whereas VvCYP76F14 cloned from D299T mutant lines failed. Conclusions: D299T variation was observed in three offspring lines and D299T mutation in VvCYP76F14 led to the decrease in wine bouquet precursor contents. VvCYP76F14 was implicated in the regulation of wine bouquet precursors in wine grapes. [ABSTRACT FROM AUTHOR]

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques. [ABSTRACT FROM AUTHOR]

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in Nicotiana benthamiana leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agronomica Sinica is the property of Crop Science Society of China and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The flavonoid diosmin (diosmetin 7-O-rutinoside) is used as a therapeutic agent for disorders of the blood vessels such as hemorrhoids and varicose veins. Diosmin is commercially produced using semi-synthetic methods involving the oxidation of hesperidin, the most abundant flavonoid in citrus fruits. However, this method produces byproducts that are toxic to the environment, and new sustainable methods to produce diosmin are required. Here, we used a synthetic biology approach to produce diosmin without generating toxic byproducts through reconstitution of the diosmin biosynthetic pathway in Nicotiana benthamiana. We first established that N. benthamiana leaves co-infiltrated with all seven genes in the flavonoid biosynthesis pathway produced high levels of luteolin, a precursor of diosmetin. We then compared the activity of modification enzymes such as methyltransferases, glucosyltransferases, and rhamnosyltransferases in Escherichia coli and in planta and selected genes encoding enzymes with the highest activity for producing diosmetin, diosmetin 7-O-glucoside, and diosmin, respectively. Finally, we reconstructed the entire diosmin biosynthetic pathway using three constructs containing ten genes encoding enzymes in this pathway, from phenylalanine ammonia lyase to rhamnosyltransferase. N. benthamiana leaves transiently co-expressing all these genes yielded 37.7 µg diosmin per gram fresh weight. To our knowledge, this is the first report of diosmin production in a heterologous plant system without the supply of a precursor. Successful production of diosmin in N. benthamiana opens new avenues for producing other commercially important flavonoids using similar platforms. [ABSTRACT FROM AUTHOR]

Aluminium (Al3+) toxicity in acidic soils poses a significant challenge for crop cultivation and reduces crop productivity. The primary defense mechanism against Al3+ toxicity involves the activation of organic acid secretion. In this study, responses of 9 Vigna mungo cultivars to Al3+ toxicity were investigated, with a particular emphasis on the root system and crucial genes involved in Al3+ tolerance using molecular cloning and expression analysis. Sensitive blackgram-KM2 cultivars exposed to 100-µM Al3+ toxicity for 72 h exhibited a root-growth inhibition of approximately 66.17%. Significant loss of membrane integrity and structural deformative roots were found to be the primary symptoms of Al3+ toxicity in blackgram. MATE (Multidrug and Toxic Compound Extrusion) and ALS3 (Aluminium Sensitive 3) genes were successfully cloned from a sensitive blackgram cv KM2 with phylogenetic analysis revealing their evolutionary relationship to Vigna radiata and Glycine max. The MATE gene is mainly localized in the plasma membrane, and highly expressed under Al3+, thus suggesting its role in transports of citrate-Al3+ complexes, and detoxifying Al3+ within plant cells. In addition, ALS3 was also induced under Al3+ toxicity, which codes the UDP-glucose transporter and is required for the maintenance of ions homeostasis. In summary, this study highlights the understanding of Al3+ toxicity and underlying molecular mechanisms linked to the efflux of organic acid in blackgram, ultimately aiding the framework for the development of strategies to enhance the resilience of blackgram and other pulse crops in Al-rich soils. [ABSTRACT FROM AUTHOR]

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a deadly, highly contagious disease in both domestic pigs and wild boar. With mortality up to 100%, the disease has been making a serious impact on the swine industry worldwide. Because no effective antiviral treatment has been observed, proactive prevention such as vaccination remains the key to controlling the outbreak. In the pursuit of expediting vaccine development, our current work has made the first report for heterologous production of the viral outer envelope glycoprotein CD2v extracellular domain (CD2v ED), a proposed promising vaccine antigen candidate in the "green" synthetic host Nicotiana benthamiana. Protein oligomerization strategies were implemented to increase the immunogenicity of the target antigen. Herein, the protein was expressed in oligomeric forms based on the C-terminally fused GCN4pII trimerization motif and GCN4pII_TP oligomerization motif. Quantitative western blot analysis showed significantly higher expression of trimeric CD2v ED_GCN4pII with a yield of about 12 mg/100 g of fresh weight, in comparison to oligomeric CD2v ED_GCN4pII_TP, revealing the former is the better choice for further studies. The results of purification and size determination by size exclusion chromatography (SEC) illustrated that CD2v ED_GCN4pII was successfully produced in stable oligomeric forms throughout the extraction, purification, and analysis process. Most importantly, purified CD2v ED_GCN4pII was demonstrated to induce both humoral and cellular immunity responses in mice to extents equivalent to those of the live attenuated vaccine ASFV-G-∆I177L, suggesting it as the potential subunit vaccine candidate for preventing ASFV. [ABSTRACT FROM AUTHOR]

Transplastomic tobacco plants expressing a reporter gene egfp and selectable marker gene aadA as part of a bicistronic operon were obtained using the bioballistics method. Features of egfp gene expression were studied in two groups of transplastomic plants: seed progeny obtained from self-pollination and plants obtained as a result of two successive regeneration cycles from the leaves of the original transformants. A comparative fluorimetric analysis of the recombinant protein accumulation in groups of transplastomic plants and nuclear transformants was carried out. It was found that the amount of recombinant eGFP protein accumulated in the leaves of transplastomic plants was unexpectedly low and did not exceed the level established for nuclear transformants. Real-time PCR results showed that the low level of recombinant eGFP accumulation was not associated with low levels of transgene expression or the presence of nontransgenic copies of the plastid genome in chloroplasts. This is most likely due to limitations imposed at the level of translation of recombinant proteins in chloroplasts. [ABSTRACT FROM AUTHOR]

The large body of data on plant gene expression accumulated through comparative studies directs the efforts of researchers to study the subtle mechanisms of the influence of target genes and, as a result, to develop relatively simple and, at the same time, effective approaches to understand the physiological role of protein products of a gene. Numerous studies have convincingly demonstrated the effectiveness of transient expression strategies for characterizing plant gene functions. The objectives of the review were (1) to review the advantages and limitations of various plant transient expression systems and methods used to elucidate the roles of gene products, (2) to summarize current knowledge on the use of transient expression approaches to understand the subtle mechanisms underlying gene function, and (3) to describe advances in efficient transient expression of plant genes. The review discusses the main and critical steps of each of the methods of transient gene expression in plants, their areas of application, and the main results obtained using plant objects and their contribution to our knowledge of the subtle mechanisms of gene functions underlying plant growth and development, including elucidation of the mechanisms regulating complex metabolic pathways. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract [Objective] As a peony breeding material, Paeonia delavayi has abundant flower color resources. Studying the regulatory effects of its MYB transcription factors on flower color will benefit molecular breeding of peony flower color. [Methods] Using the flowers of yellow and red P . delavayi as materials for hand sectioning. Based on the transcriptomic data of P . delavayi, a MYB transcription factor coding gene PdMYB57 was obtained through sequence alignment. Function of this gene was verified through gene cloning, phylogenetic tree construction, qRT-PCR, transient expression, and HPLC. [Results] PdMYB57 gene has an ORF of 798 bp, encoding an unstable hydrophilic protein of 265 amino acids. PdMYB57 was clustered with the Arabidopsis SG6 subfamily, as well as MYB transcription factors in P . qiui PqMYB113, P . suffruticosa PsMYB57/PsMYB58, and Vitis vinifera VvMYBA1/VvMYBA2. PdMYB57 had a R2R3 conserved domain [R/K] Px [P/A/R] xx [F/Y] motif. PdMYB57 gene was highly expressed in sepals of red P . delavayi and leaves of yellow P . delavayi. HPLC analysis showed that tobacco leaves transiently expressed PdMYB57 gene contained cyanidin-3-O-rutinoside (Cy3R). [Conclusion] PdMYB57 gene encodes an R2R3-MYB transcription factor. PdMYB57 gene expression is tissue-specific and promotes anthocyanin synthesis in plants.

Currently transient expression is one of the preferred plant-based technologies for recombinant protein manufacturing, particularly in respect to pharmaceutically relevant products. Modern hybrid transient expression systems combine the features of Agrobacterium tumefaciens and viral vectors. However, host plant reaction to Agrobacterium-mediated delivery of gene of interest can negatively affect foreign protein accumulation. In this study, we investigated whether the modulation of plant immune response through knockdown of the Nb-SABP2 and Nb-COI1 N. benthamiana genes could improve recombinant protein yield. In plants, the SABP2 and COI1 proteins are involved in the salicylic acid and jasmonic acid metabolism, respectively. We exemplified the utility of this approach with the green fluorescence (GFP) and b nerve growth factor (bNGF) proteins: compared to the tobacco mosaic virus (TMV)-based vector the Nb-SABP2 and Nb-COI1-suppressed plants provided an increased recombinant protein accumulation. We also show that this strategy is extendable to the expression systems utilizing potato virus X (PVX) as the vector backbone: the enhanced amounts of bNGF were detected in the Nb-SABP2 and Nb-COI1-depleted leaves co-infiltrated with the PVX-bNGF. These findings suggest that modulating host plant reaction to agrodelivery of expression vectors could be useful for improving transient foreign protein production in N. benthamiana. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are often engineered at the sequence level for improved clinical performance yet are rarely evaluated prior to candidate selection for their "developability" characteristics, namely expression, which can necessitate additional resource investments to improve the manufacturing processes for problematic mAbs. A strong relationship between primary sequence and expression has emerged, with slight differences in amino acid sequence resulting in titers differing by up to an order of magnitude. Previous work on these "difficult‐to‐express" (DTE) mAbs has shown that these phenotypes are driven by post‐translational bottlenecks in antibody folding, assembly, and secretion processes. However, it has been difficult to translate these findings across cell lines and products. This work presents a systematic approach to study the impact of sequence variation on mAb expression at a larger scale and under more industrially relevant conditions. The analysis found 91 mutations that decreased transient expression of an IgG1κ in Chinese hamster ovary (CHO) cells and revealed that mutations at inaccessible residues, especially those leading to decreases in residue hydrophobicity, are not favorable for high expression. This workflow can be used to better understand sequence determinants of mAb expression to improve candidate selection procedures and reduce process development timelines. [ABSTRACT FROM AUTHOR]

Introduction: Brown rot is the most important fungal disease affecting stone fruit and it is mainly caused by Monilinia fructicola, M. laxa and M. fructigena. Monilinia spp. are necrotrophic plant pathogens with the ability to induce plant cell death by the secretion of different phytotoxic molecules, including proteins or metabolites that are collectively referred to as necrotrophic effectors (NEs). Methods: We exploited the genomes of M. fructicola, M. laxa and M. fructigena to identify their common group of secreted effector proteins and tested the ability of a selected set of effectors to induce cell death in Nicotiana benthamiana, Solanum lycopersicum and Prunus spp. leaves. Results: Fourteen candidate effector genes of M. fructicola, which displayed high expression during infection, were transiently expressed in plants by agroinfiltration using a modified Tobacco Rattle Virus (TRV)-based expression system. Some, but not all, effectors triggered leaf discoloration or cell death in N. benthamiana and S. lycopersicum, which are non-hosts for Monilinia and in Prunus spp., which are the natural hosts. The effector MFRU_030g00190 induced cell death in almost all Prunus genotypes tested, but not in the Solanaceous plants, while MFRU_014g02060, which is an ortholog to BcNep1, caused necrosis in all plant species tested. Conclusion: This method provides opportunities for screening Prunus germplasm with Monilinia effector proteins, to serve as a tool for identifying genetic loci that confer susceptibility to brown rot disease. [ABSTRACT FROM AUTHOR]

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture. [ABSTRACT FROM AUTHOR]

We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plantbased production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a genesilencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants. [ABSTRACT FROM AUTHOR]

Summary: Agroinfiltration of Nicotiana benthamiana is routinely used in plant science and molecular pharming to transiently express proteins of interest. Here, we discuss four phenomena that should be avoided to improve transient expression. Immune responses can be avoided by depleting immune receptors and employing pathogen‐derived effectors; transcript degradation by using silencing inhibitors or RNA interference machinery mutants; endoplasmic reticulum stress by co‐expressing chaperones; and protein degradation can be avoided with subcellular targeting, protease mutants and co‐expressing protease inhibitors. We summarise the reported increased yields for various recombinant proteins achieved with these approaches and highlight remaining challenges to further improve the efficiency of this versatile protein expression platform. [ABSTRACT FROM AUTHOR]

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana–Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA in vitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid‐ and jasmonic acid‐signalling marker genes, reactive oxygen species accumulation, and BAX‐mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence‐related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector‐mediated Foc TR4 pathogenesis. [ABSTRACT FROM AUTHOR]

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Northwest A & F University - Natural Science Edition is the property of Editorial Department of Journal of Northwest A&F University (Natural Science Edition) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Refolding based Bimolecular Fluorescence Complementation (BiFC) has emerged as an important in vivo technique to identify protein interactions. Significant improvements have been made to enhance the detection capacities of BiFC, however less attention has been paid to the detection of expression levels of proteins. Here we demonstrate development and validation of an improved method to identify protein interactions that incorporates an expression control based on bicistronic expression of the protein of interest and a fluorescent protein separated by a self-cleaving peptide. This method gives robust identification of positive interactions and more reliably identifies absence of interactions. We also show an earlier identified non-interacting pair in yeast two-hybrid (Y2H) to be interacting in vivo. [ABSTRACT FROM AUTHOR]

Rhombic-spot disease, caused mainly by Neostagonosporella sichuanensis, significantly impacts the yield and quality of fishscale bamboo (Phyllostachys heteroclada). Xylanases are essential for pathogenic fungi infection, yet their specific functions in the physiology and pathogenicity of N. sichuanensis remain unclear. Here, we characterized three xylanase proteins with glycosyl hydrolase 11 domains from the N. sichuanensis SICAUCC 16–0001 genome and examined the function of Nsxyn1 and Nsxyn2. Purified Nsxyn1 and Nsxyn2 proteins displayed specific xylanase activity in vitro and induced cell death in Nicotiana benthamiana, independent of their enzymatic function. Both proteins possessed signal peptides and were confirmed as secreted proteins using a yeast secretion system. Subcellular localization revealed that Nsxyn1 and Nsxyn2 localized in both the cytoplasm and nucleus and can trigger cell death in N. benthamiana through Agrobacterium tumefaciens-mediated transient transformation. qRT-PCR results showed notable upregulation of Nsxyn1 and Nsxyn2 during infection, with Nsxyn1 exhibiting an 80-fold increase at 15 days post-inoculation. Deletion of Nsxyn1 and Nsxyn2 in N. sichuanensis impaired xylan degradation, adaptation to osmotic and oxidative stress, and pathogenic full virulence. Deletion of Nsxyn1 notably slowed fungal growth and reduced spore production, whereas only a reduction in microconidial production was observed in Nsxyn2 mutants. Complementation of Nsxyn1 and Nsxyn2 only partially restored these phenotypic defects in the ∆Nsxyn1 and ∆Nsxyn2 mutants. These findings suggest that Nsxyn1 and Nsxyn2 contribute to N. sichuanensis virulence and induced plant defense responses, providing new insights into the function of xylanases in the interaction between fishscale bamboo and N. sichuanensis.

The development and creation of a new generation vaccines based on recombinant proteins that assemble into virus-like particles (VLPs), as well as recombinant proteins in the form of nanoparticles, are promising directions in modern biotechnology. Due to their large size (20–200 nm) and multiplicity of viral antigenic determinants on the surface, VLPs can stimulate strong humoral and cellular immune responses. The main types of VLPs, as well as the features and disadvantages of the main expression systems used for their biosynthesis, are considered in this review. The main focus was on plant expression systems that ensure the biosynthesis of a target recombinant protein from a DNA matrix integrated into the nuclear or chloroplast genomes of a plant (stable expression) or from a matrix for temporary production of the target product (transient expression). Various approaches for increasing the yield of VLP-forming recombinant proteins, including fusion with a transit peptide that directed the protein into the chloroplast, were discussed. The possibility of accumulation of recombinant proteins expressed in plants and intended for creation of VLP-vaccines in another type of nanoparticle, protein bodies, using specific signal sequences was also considered. [ABSTRACT FROM AUTHOR]

The brown macroalga Saccharina japonica (Laminariales, Phaeophyta) is the most productive cultured seaweed in the world. In order to improve the biosafety of transgenic kelp, it is necessary to develop endogenous constitutive promoters, replacing those of virus origin. In this study, the housekeeping actin gene from S. japonica (SjACT) was found to contain three exons and two introns, representing a unique actin gene structure pattern in brown algae. Additionally, the 5′ upstream region was obtained using genome walking, and fused to the reporter gene lacZ or EGFP to construct promoter-detective vectors. Using an established genetic transformation system, kelps in different life-cycle stages were transformed. The detection results showed that, in the diploid sporophyte stage, the transient expression from the lacZ gene could be observed in the frond, stipe, or holdfast of kelps, indicating a manner of being non-tissue-specific. And, in the haploid gametophyte stage of S. japonica, the fluorescence of the expressed EGFP were detected in vivo in gametophyte cells of both genders. These results indicate that the promoter of the SjACT gene (pSjACT) functions in a constitutive manner and is expected to be a key endogenous element in the genetic manipulation of kelps. [ABSTRACT FROM AUTHOR]

Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect of Agrobacterium‐infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in our Agrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium‐mediated transformation methods in other plant species. [ABSTRACT FROM AUTHOR]

Banana, a globally popular fruit, is widely cultivated in tropical and sub-tropical regions. After fruit harvest, remaining banana plant materials are low-value byproducts, mostly composted or used as fibre or for food packaging. As an aim to potentially increase farmer income, this study explored underutilised banana biomass as a novel plant tissue for production of a high-value product. Protein scFvTG130 used in this study, is an anti-toxoplasma single chain variable fragment antibody that can be used in diagnostics and neutralising the Toxoplasma gondii pathogen. Using detached banana leaves, we investigated the factors influencing the efficacy of a transient expression system using reporter genes and recombinant protein, scFvTG130. Transient expression was optimal at 2 days after detached banana leaves were vacuum infiltrated at 0.08 MPa vacuum pressure for a duration of 3 min with 0.01% (v/v) Tween20 using Agrobacterium strain GV3101 harbouring disarmed virus-based vector pIR-GFPscFv TG130. The highest concentration of anti-toxoplasma scFvTG130 antibody obtained using detached banana leaves was 22.8 µg/g fresh leaf tissue. This first study using detached banana leaf tissue for the transient expression of a recombinant protein, successfully demonstrated anti-toxoplasma scFvTG130 antibody expression, supporting the potential application for other related proteins using an underutilised detached banana leaf tissue. • Post-harvest banana biomass serves as a novel high value protein production system. • Detached leaves remain viable and express recombinant proteins for several days. • Transient anti-toxoplasma ScFv antibody expression was optimal at two days. • Levels of 22.8 µg/g ScFv protein achieved in fresh leaf tissue. • This is a first report using detached banana leaf tissue for protein expression. [ABSTRACT FROM AUTHOR]

Summary: Transient expression in Nicotiana benthamiana offers a robust platform for the rapid production of complex secondary metabolites. It has proven highly effective in helping identify genes associated with pathways responsible for synthesizing various valuable natural compounds. While this approach has seen considerable success, it has yet to be applied to uncovering genes involved in anthocyanin biosynthetic pathways. This is because only a single anthocyanin, delphinidin 3‐O‐rutinoside, can be produced in N. benthamiana by activation of anthocyanin biosynthesis using transcription factors. The production of other anthocyanins would necessitate the suppression of certain endogenous flavonoid biosynthesis genes while transiently expressing others. In this work, we present a series of tools for the reconstitution of anthocyanin biosynthetic pathways in N. benthamiana leaves. These tools include constructs for the expression or silencing of anthocyanin biosynthetic genes and a mutant N. benthamiana line generated using CRISPR. By infiltration of defined sets of constructs, the basic anthocyanins pelargonidin 3‐O‐glucoside, cyanidin 3‐O‐glucoside and delphinidin 3‐O‐glucoside could be obtained in high amounts in a few days. Additionally, co‐infiltration of supplementary pathway genes enabled the synthesis of more complex anthocyanins. These tools should be useful to identify genes involved in the biosynthesis of complex anthocyanins. They also make it possible to produce novel anthocyanins not found in nature. As an example, we reconstituted the pathway for biosynthesis of Arabidopsis anthocyanin A5, a cyanidin derivative and achieved the biosynthesis of the pelargonidin and delphinidin variants of A5, pelargonidin A5 and delphinidin A5. [ABSTRACT FROM AUTHOR]

Andrographis paniculata (Burm. f.) Nees is an important medicinal plant known for its bioactive compound andrographolide. NAC transcription factors (NAM, ATAF1/2, and CUC2) play a crucial role in secondary metabolite production, stress responses, and plant development through hormonal signaling. In this study, a putative partial transcript of three NAC family genes (ApNAC83, ApNAC21 22 and ApNAC02) was used to isolate full length genes using RACE. Bioinformatics analyses such as protein structure prediction, cis-acting regulatory elements, and gene ontology analysis were performed. Based on in silico predictions, the diterpenoid profiling of the plant's leaves (five-week-old) and the real-time PCR-based expression analysis of isolated NAC genes under abscisic acid (ABA) treatment were performed. Additionally, the expression analysis of isolated NAC genes under MeJA treatment and transient expression in Nicotiana tabacum was performed. Full-length sequences of three members of the NAC transcription factor family, ApNAC83 (1102 bp), ApNAC21 22 (996 bp), and ApNAC02 (1011 bp), were isolated and subjected to the promoter and gene ontology analysis, which indicated their role in transcriptional regulation, DNA binding, ABA-activated signaling, and stress management. It was observed that ABA treatment leads to a higher accumulation of andrographolide and 14-deoxyandrographolide content, along with the upregulation of ApNAC02 (9.6-fold) and the downregulation of ApNAC83 and ApNAC21 22 in the leaves. With methyl jasmonate treatment, ApNAC21 22 expression decreased, while ApNAC02 increased (1.9-fold), with no significant change being observed in ApNAC83. The transient expression of the isolated NAC genes in a heterologous system (Nicotiana benthamiana) demonstrated their functional transcriptional activity, leading to the upregulation of the NtHMGR gene, which is related to the terpene pathway in tobacco. The expression analysis and heterologous expression of ApNAC21 22 and ApNAC02 indicated their role in andrographolide biosynthesis. [ABSTRACT FROM AUTHOR]

Currently transient expression is one of the preferred plant-based technologies for recombinant protein manufacturing, particularly in respect to pharmaceutically relevant products. Modern hybrid transient expression systems combine the features of Agrobacterium tumefaciens and viral vectors. However, host plant reaction to Agrobacterium-mediated delivery of gene of interest can negatively affect foreign protein accumulation. In this study, we investigated whether the modulation of plant immune response through knockdown of the Nb-SABP2 and Nb-COI1 N. benthamiana genes could improve recombinant protein yield. In plants, the SABP2 and COI1 proteins are involved in the salicylic acid and jasmonic acid metabolism, respectively. We exemplified the utility of this approach with the green fluorescence (GFP) and β nerve growth factor (βNGF) proteins: compared to the tobacco mosaic virus (TMV)-based vector the Nb-SABP2 and Nb-COI1-suppressed plants provided an increased recombinant protein accumulation. We also show that this strategy is extendable to the expression systems utilizing potato virus X (PVX) as the vector backbone: the enhanced amounts of βNGF were detected in the Nb-SABP2 and Nb-COI1-depleted leaves co-infiltrated with the PVX-βNGF. These findings suggest that modulating host plant reaction to agrodelivery of expression vectors could be useful for improving transient foreign protein production in N. benthamiana.

IntroductionBrown rot is the most important fungal disease affecting stone fruit and it is mainly caused by Monilinia fructicola, M. laxa and M. fructigena. Monilinia spp. are necrotrophic plant pathogens with the ability to induce plant cell death by the secretion of different phytotoxic molecules, including proteins or metabolites that are collectively referred to as necrotrophic effectors (NEs).MethodsWe exploited the genomes of M. fructicola, M. laxa and M. fructigena to identify their common group of secreted effector proteins and tested the ability of a selected set of effectors to induce cell death in Nicotiana benthamiana, Solanum lycopersicum and Prunus spp. leaves.ResultsFourteen candidate effector genes of M. fructicola, which displayed high expression during infection, were transiently expressed in plants by agroinfiltration using a modified Tobacco Rattle Virus (TRV)-based expression system. Some, but not all, effectors triggered leaf discoloration or cell death in N. benthamiana and S. lycopersicum, which are non-hosts for Monilinia and in Prunus spp., which are the natural hosts. The effector MFRU_030g00190 induced cell death in almost all Prunus genotypes tested, but not in the Solanaceous plants, while MFRU_014g02060, which is an ortholog to BcNep1, caused necrosis in all plant species tested.ConclusionThis method provides opportunities for screening Prunus germplasm with Monilinia effector proteins, to serve as a tool for identifying genetic loci that confer susceptibility to brown rot disease.

Abstract Citrus canker is a bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that affects the citrus industry worldwide. Hrp pili subunits (HrpE), an essential component of Type III secretion system (T3SS) bacteria, play a crucial role in the pathogenesis of Xcc by transporting effector proteins into the host cell and causing canker symptoms. Therefore, development of antibodies that block HrpE can suppress disease progression. In this study, a specific scFv detecting HrpE was developed using phage display technique and characterized using sequencing, ELISA, Western blotting, and molecular docking. In addition, a plant expression vector of pCAMBIA-scFvH6 was constructed and agroinfiltrated into Nicotiana tabacum cv. Samson leaves. The hypersensitive response (HR) in the leaves of transformed and non-transformed plants was evaluated by inoculating leaves with Xcc. After three rounds of biopanning of the phage library, a specific human scFv antibody, named scFvH6, was identified that showed high binding activity against HrpE in ELISA and Western blotting. Molecular docking results showed that five intermolecular hydrogen bonds are involved in HrpE-scFvH6 interaction, confirming the specificity and high binding activity of scFvH6. Successful transient expression of pCAMBIA-scFvH6 in tobacco leaves was verified using immunoassay tests. The binding activity of plant-produced scFvH6 to detect HrpE in Western blotting and ELISA was similar to that of bacterial-produced scFvH6 antibody. Interestingly, tobacco plants expressing scFvH6 showed a remarkable reduction in HR induced by Xcc compared with control plants, so that incidence of necrotic lesions was significantly higher in non-transformed controls (≥ 1.5 lesions/cm2) than in the plants producing scFvH6 (≤ 0.5 lesions/cm2) after infiltration with Xcc inoculum. Our results revealed that the expression of scFvH6 in tobacco leaves can confer resistance to Xcc, indicating that this approach could be considered to provide resistance to citrus bacterial canker disease.

Abstract The Solanaceae family includes the largest flowering crops such as tomatoes, potatoes, and eggplants. Consumer demand has led to massive development of plants in the Solanum genus, and many different Solanum varieties are now available on the market. The recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based genome editing have allowed laboratories and smaller crop production companies to utilize the technology in various crops. The traditional transformation method in crops involves the use of Agrobacterium, which is considered the most efficient method for introducing exogenous genetic materials in target plants. The Agrobacterium-mediated transformation method has been also established in the Solanaceae family, enabling CRISPR/Cas-based genome editing in crops like tomatoes, potatoes, and eggplants. However, the Agrobacterium-mediated approach inevitably accompanies the insertion of exogenous DNA into the plant genome and often causes the formation of chimera that require further propagation steps. Alternatively, the CRISPR/Cas components can be introduced into protoplasts in the form of DNA for transient expression or a mixture of protein and RNA to avoid genomic insertion of foreign materials. The protoplast transformation approach involves processes including protoplast preparation, transfection, and regeneration, which require a comprehensive understanding and greater technical mastery of the tissue culture phase. Here we highlight the current research advances in protoplast transformation and discuss how to optimize the procedures of protoplast isolation, transfection, and regeneration for efficient and reproducible CRISPR/Cas-based genome editing in the genus Solanum.

Anisodus luridus, a perennial herb belonging to the genus Anisodus of the Solanaceae family, is an important Tibetan medicinal plant that produces pharmaceutical tropane alkaloids (TAs) including hyoscyamine and scopolamine. Its high yield of hyoscyamine makes A. luridus a valuable plant source for commercially producing TAs. In this study, we conduct homologous gene research across transcriptome data of different tissues together with functionally tested sequences in Atropa belladonna as a reference and identify 13 candidate genes for TAs biosynthesis in A. luridus. The results show that these 13 TAs biosynthesis genes identified in A. luridus were highly conserved in terms of sequence similarity and gene expression patterns compared to A. belladonna, suggesting that the two species may share the same biosynthetic pathway for TAs biosynthesis. Furthermore, scopolamine was detected in Nicotiana benthamiana leaves when these 13 enzymes were co-expressed in N. benthamiana, which confirmed that these 13 TAs biosynthesis genes are involved in the biosynthesis of TAs. The results of our study not only systematically elucidate the tropane alkaloid biosynthetic pathway of A. luridus, but also realize the de novo synthesis of TAs in N. benthamiana for the first time. It is now possible to make N. benthamiana a potential source for TAs production through synthetic biology techniques.

Agroinfiltration-mediated transient expression is widely used in plant gene functional studies. In this study, the functionality of a metabolic engineering construct containing multiple expression cassettes for betalain biosynthesis genes was validated via agroinfiltration in petunia flower petals. The effects of transcription factors that upregulate anthocyanin biosynthesis and a gene that regulates cellular pH were also demonstrated on petunia petals by agroinfiltration. We also showed that flower petal agroinfiltration is a quick approach to test gene editing components, e.g. different guide RNAs. In general, the agroinfiltration-mediated transient transformation in flower petals is a great tool for testing flowering colour–related trait genes, as well as for optimising gene editing components. [ABSTRACT FROM AUTHOR]

Plant viral movement proteins (MPs) serve a critical role in facilitating intercellular transport, pivotal for propagating infections from afflicted cells to their neighboring counterparts. Leveraging MPs within transient expression systems holds the potential to augment the expanse of protein expression zones. In this investigation, we present evidence showcasing the synergistic effects of fusing a transient expression system vector with the movement protein of tomato mosaic virus (ToMV MP), thus broadening the scope of protein expression within plant stem tissues. Through agroinfiltration, we achieved transient expression of green fluorescent protein (GFP) in tomato stem tissues, which was subsequently confirmed via immunoblotting with an anti-GFP antibody. Quantitative analysis unveiled that the co-expression of ToMV MP alongside GFP led to an over twofold increase in GFP production within stem tissues distal from the primary infection site. This research underscores the efficacy of combining ToMV MP with transient expression systems as a potent strategy for achieving transient expression of target genes within tomato plants. [ABSTRACT FROM AUTHOR]

We studied the influence of heterologous signal peptides in the β-chains of glycoprotein hormones on the biosynthesis of these hormones in a transiently transfected culture of Chinese hamster ovary cells CHO S. When the natural signal peptides of the β-chains were replaced with the heterologous signal peptide of human serum albumin, cell productivity was increased 2–2.5 times for human luteinizing hormone, human chorionic gonadotropin, and human thyroid-stimulating hormone, but not for human follicle-stimulating hormone. No significant increase in cell productivity was observed for human azurocidin signal peptide and human glycoprotein hormone α-chain signal peptide. The used approach allows quick assessing the effect of heterologous signal peptides on the biosynthesis of heterodimeric proteins of various classes. [ABSTRACT FROM AUTHOR]

Transient transformation is a notably efficient and expeditious method for obtaining foreign gene expression products, making it a prominent avenue for investigating functional genes in pasture. The present study aimed to explore the non-in vitro transient transformation technique of Lotus japonicus, a leguminous herbage, to analyze functional genes in the future. This was achieved by evaluating the expression products of GUS reporter gene and the bioactivity of functional gene. The results indicated that the transient expression system was successfully optimized as follows: the utilization of parameters, including an OD600 absorption of 0.8 for the bacterium, a vacuum pressure of 12 Kpa, and an infiltration time of 15 min, yielded a transient transformation rate of 57.78%. The GUS recombinant protein demonstrated optimal accumulation on the 8th day, while the functional evaluation and utilization of the exogenous gene were observed within a time frame of 2–12 days. The entire branch-leaf explant demonstrated an efficient expression of the GUS recombinant protein within a 12-day timeframe while maintaining viability. This finding suggests that this system not only facilitates the extended analysis of gene function but also accurately reflects the regulatory capabilities of the entire living plant. Furthermore, the results confirmed the viability of the transient transformation system by demonstrating improved tolerance to high temperature, drought, and salt stress in Lotus japonicus expressing foreign LjHsp70-1 gene. The successful establishment of this transient expression system offers a valuable way and framework for investigating the functional characteristics of vital genes in Lotus japonicus or other microphyllous plants.Key Message: In this high-efficiency gene transient expression system for Lotus japonicus, the duration of exogenous gene expression reaches 12 days by using the branch-leaf explant for genetic transformation. [ABSTRACT FROM AUTHOR]

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Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has underscored the need for rapid and cost- effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development. [ABSTRACT FROM AUTHOR]

Abstract Agrobacterium‐mediated transient expression methods are widely used to study gene function in both model and non‐model plants. Using a dual‐luciferase assay, we quantified the effect of Agrobacterium‐infiltration parameters on the transient transformation efficiency of Catharanthus roseus seedlings. We showed that transformation efficiency is highly sensitive to seedling developmental state and a pre‐ and post‐infiltration dark incubation and is less sensitive to the Agrobacterium growth stage. For example, 5 versus 6 days of germination in the dark increased seedling transformation efficiency by seven‐ to eight‐fold while a dark incubation pre‐ and post‐infiltration increased transformation efficiency by five‐ to 13‐fold. Agrobacterium in exponential compared with stationary phase increased transformation efficiency by two‐fold. Finally, we quantified the variation in our Agrobacterium‐infiltration method in replicate infiltrations and experiments. Within a given experiment, significant differences of up to 2.6‐fold in raw firefly luciferase (FLUC) and raw Renilla luciferase (RLUC) luminescence occurred in replicate infiltrations. These differences were significantly reduced when FLUC was normalized to RLUC values, highlighting the utility of including a reference reporter to minimize false positives. Including a second experimental replicate further reduced the potential for false positives. This optimization and quantitative validation of Agrobacterium infiltration in C. roseus seedlings will facilitate the study of this important medicinal plant and will expand the application of Agrobacterium‐mediated transformation methods in other plant species.

This study used the DNA of Bacillus amyloliquefaciens Ba168 as a template to amplify the flagellin BP8-2 gene and ligate it into the fusion expression vector pCAMBIA1300-35S-EGFP after digestion for the construction of the expression vector pCAMBIA1300-EGFP-BP8-2. Next, using Nicotiana benthamiana as receptor material, transient expression was carried out under the mediation of Agrobacterium tumefaciens C58C1. Finally, the transient expression and subcellular localisation of flagellin BP8-2 protein were analysed using the imaging of co-transformed GFP under laser confocal microscopy. The results showed that flagellin BP8-2 was localised in the cell membrane and nucleus, and the RT-PCR results showed that the BP8-2 gene could be stably expressed in tobacco leaf cells. Furthermore, there was stronger antiviral activity against tobacco mosaic virus (TMV) infection in Nicotiana glutinosa than in BP8-2 and ningnanmycin, with an inhibitory effect of 75.91%, protective effect of 77.45%, and curative effect of 68.15%. TMV movement and coat protein expression were suppressed, and there was a high expression of PR-1a, PAL, and NPR1 in BP8-2-treated tobacco leaf. These results suggest that flagellin BP8-2 inhibits TMV by inducing resistance. Moreover, BP8-2 has low toxicity and is easily biodegradable and eco-friendly. These results further enrich our understanding of the antiviral mechanisms of proteins and provide alternatives for controlling viral diseases in agriculture.

A wide range of virus-like particles (VLPs) is extensively employed as carriers to display various antigens for vaccine development to fight against different infections. The plant-produced truncated variant of the hepatitis E virus (HEV) coat protein is capable of forming VLPs. In this study, we demonstrated that recombinant fusion proteins comprising truncated HEV coat protein with green fluorescent protein (GFP) or four tandem copies of the extracellular domain of matrix protein 2 (M2e) of influenza A virus inserted at the Tyr485 position could be efficiently expressed in Nicotiana benthamiana plants using self-replicating vector based on the potato virus X genome. The plant-produced fusion proteins in vivo formed VLPs displaying GFP and 4M2e. Therefore, HEV coat protein can be used as a VLP carrier platform for the presentation of relatively large antigens comprising dozens to hundreds of amino acids. Furthermore, plant-produced HEV particles could be useful research tools for the development of recombinant vaccines against influenza.

The COVID-19 pandemic has underscored the need for rapid and cost-effective diagnostic tools. Serological tests, particularly those measuring antibodies targeting the receptor-binding domain (RBD) of the virus, play a pivotal role in tracking infection dynamics and vaccine effectiveness. In this study, we aimed to develop a simple enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibodies, comparing two plant-based platforms for diagnostic reagent production. We chose to retain RBD in the endoplasmic reticulum (ER) to prevent potential immunoreactivity issues associated with plant-specific glycans. We produced ER-retained RBD in two plant systems: a stable transformation of BY-2 plant cell culture (BY2-RBD) and a transient transformation in Nicotiana benthamiana using the MagnICON system (NB-RBD). Both systems demonstrated their suitability, with varying yields and production timelines. The plant-made proteins revealed unexpected differences in N-glycan profiles, with BY2-RBD displaying oligo-mannosidic N-glycans and NB-RBD exhibiting a more complex glycan profile. This difference may be attributed to higher recombinant protein synthesis in the N. benthamiana system, potentially overloading the ER retention signal, causing some proteins to traffic to the Golgi apparatus. When used as diagnostic reagents in ELISA, BY2-RBD outperformed NB-RBD in terms of sensitivity, specificity, and correlation with a commercial kit. This discrepancy may be due to the distinct glycan profiles, as complex glycans on NB-RBD may impact immunoreactivity. In conclusion, our study highlights the potential of plant-based systems for rapid diagnostic reagent production during emergencies. However, transient expression systems, while offering shorter timelines, introduce higher heterogeneity in recombinant protein forms, necessitating careful consideration in serological test development.