Transient Expression Research Articles

Atypical RXLR effectors are involved in Phytophthora cactorum pathogenesis

Abstract RXLR effectors are pathogenic factors secreted from oomycetes to manipulate the immunity of the host. Typical RXLR effectors contain an RXLR-dEER motif at the N-terminus, whereas atypical RXLRs show variations on this motif. The oomycete Phytophthora cactorum is known to infect over 200 plant species, resulting in significant agricultural economic losses. Although genome-wide identification and functional analyses of typical RXLRs from P. cactorum have been performed, little is known of atypical PcaRXLRs. Here, we identified RXLRs, both typical and atypical, in P. cactorum and compared them with those of other oomycete pathogens. Fewer RXLRs were identified in P. cactorum compared with other Phytophthora species, possibly due to fewer duplication events of RXLRs. In contrast, the percentage of atypical RXLRs was higher in P. cactorum than in other species, suggesting significant roles in P. cactorum pathogenesis. Analysis of RXLR gene expression showed that most were transcribed, suggesting their functionality. Transient expression of two atypical RXLRs in Nicotiana benthamiana showed that they induced necrosis dependent on host SGT1 and HSP90. Furthermore, two additional atypical RXLRs suppressed the defense response in N. benthamiana and promoted P. cactorum infection. These results demonstrate the vital role of atypical RXLRs in P. cactorum and provide valuable information on their evolutionary patterns and interactions with host plants.

Exploring the dual roles of sec-dependent effectors from Candidatus Liberibacter asiaticus in immunity of citrus plants.

The three SDEs of CLas were expressed in citrus leaves by AuNPs-PEI mediated transient expression system, and promoted the proliferation of CLas and inhibited citrus immunity. Huanglongbing (HLB) is the most severe bacterial disease of citrus caused by Candidatus Liberibacter asiaticus (CLas). CLas suppress host immune responses and promote infection by sec-dependent effectors (SDEs), thus insight into HLB pathogenesis is urgently needed to develop effective management strategies. In this study, we focused on the roles of SDE4310, SDE4435 and SDE4955 in citrus. We found that the expression of SDE4310, SDE4435 and SDE4955 to increase with increasing citrus immune genes CsPR1, CsPR2, CsPR5, CsNPR1, CsRBOHD, CsMAP3K and CsBIK1, suggesting that the level of citrus immunity could be judged by the expression of SDE. To further explore the relationship between these three SDEs and citrus immunity, we established a transient expression system in citrus leaves, using gold nanoparticle-polyethyleneimine (AuNPs-PEI) to deliver recombinant plasmid containing SDE4310, SDE4435 or SDE4955 respectively into citrus leaves. Results showed that SDE4310, SDE4435 and SDE4955 were successfully expressed in citrus leaves using this transient expression system, and found that SDE4310, SDE4435 and SDE4955 could promote the CLas proliferation by decreasing the immune gene expression of the citrus. Additionally, we used AuNPs-PEI to deliver siRNA4310 to citrus cells, significantly reducing the expression of SDE4310 within 3days. Although the suppression of SDE4310 expression did not inhibit the CLas proliferation, it increased the expression level of CsPR1, CsNPR1 and CsBIK1. This is also the first time that AuNPs-PEI has been found to be able to deliver exogenous plasmids into citrus cells and express the target protein, providing a new method for future studies on citrus HLB.

Lateral olivocochlear neurons modulate cochlear responses to noise exposure

The sense of hearing originates in the cochlea, which detects sounds across dynamic sensory environments. Like other peripheral organs, the cochlea is subjected to environmental insults, including loud, damage-inducing sounds. In response to internal and external stimuli, the central nervous system directly modulates cochlear function through olivocochlear neurons (OCNs), which are located in the brainstem and innervate the cochlear sensory epithelium. One population of OCNs, the lateral olivocochlear (LOC) neurons, target spiral ganglion neurons (SGNs), the primary sensory neurons of the ear. LOCs alter their transmitter expression for days to weeks in response to noise exposure (NE), suggesting that they could tune SGN excitability over long time periods in response to auditory experience. To examine how LOCs affect auditory function after NE, we characterized OCN transcriptional profiles and found transient LOC-specific gene expression changes after NE, including upregulation of multiple neuropeptide-encoding genes. Next, by generating intersectional mouse lines that selectively target LOCs, we chemogenetically ablated LOCs and assayed auditory responses at baseline and after NE. Compared to controls, mice with reduced LOC innervation showed greater NE-induced functional deficits 1 d later and had worse auditory function after a 2-wk recovery period. The number of remaining presynaptic puncta at the SGN synapse with inner hair cells did not differ between control and LOC-ablated animals, suggesting that the primary role of LOCs after NE is likely not to protect but instead to compensate, ensuring that SGN function is enhanced during periods of need.

The Fusarium graminearum Effector Protease FgTPP1 Suppresses Immune Responses and Facilitates Fusarium Head Blight Disease.

Most plant pathogens secrete effector proteins to circumvent host immune responses, thereby promoting pathogen virulence. One such pathogen is the fungus Fusarium graminearum, which causes Fusarium Head Blight (FHB) disease on wheat and barley. Transcriptomic analyses revealed that F. graminearum expresses many candidate effector proteins during early phases of the infection process, some of which are annotated as proteases. However, the contributions of these proteases to virulence remains poorly defined. Here, we characterize a F. graminearum endopeptidase, FgTPP1 (FGSG_11164), that is highly upregulated during wheat spikelet infection and is secreted from fungal cells. To elucidate the potential role of FgTPP1 in F. graminearum virulence, we generated FgTPP1 deletion mutants (ΔFgtpp1) and performed FHB infection assays. Deletion of FgTPP1 reduced the virulence of F. graminearium as assessed by spikelet bleaching. Infection with wild-type F. graminearum induced full bleaching in about 50% of the spikes at 10-11 days post infection, while this fraction was reduced to between 18 and 27% when using ΔFgtpp1 mutants. Transient expression of green fluorescent protein (GFP)-tagged FgTPP1 revealed that FgTPP1 localizes, in part, to chloroplasts and attenuates chitin-mediated activation of mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species production, and cell death induced by an autoactive disease resistance protein when expressed in planta. Notably, the FgTPP1 protein is conserved across the Ascomycota phylum, suggesting it may be a core effector among ascomycete plant pathogens. These properties make FgTPP1 an ideal candidate for decoy substrate engineering, with the goal of engineering resistance to FHB.

Functional Studies on the LiAG1 Gene of Lilium ‘Ice Pink Queen’ in Flower Development

Lily (Lilium Asiatica Hybrida) is a globally known perennial herbaceous bulbous flower, popular for its large, colourful flowers and high economic and ornamental value. However, pollen generation is a severe issue that reduces the cosmetic value of lilies. In this study, the MADS transcription factor LiAG1 was isolated and identified from the Lilium Asiatica Hybrida ‘Ice Pink Queen’, a male-sterile variety obtained through several years of hybridisation in our laboratory. qRT-PCR revealed that LiAG1 expression was greater in lily anthers, especially during the half-opening stage. The transient expression in tobacco demonstrated that LiAG1 was located in the nucleus. In the ‘Ice Pink Queen’ lily, suppression of LiAG1 using TRV-VIGS (tobacco-rattle-virus-mediated virus-induced gene silencing) resulted in the disappearance of most of the tapetum layer and the absence of the microsporangia. Overexpression of LiAG1 in transgenic Arabidopsis and tobacco resulted in narrower and more involute leaves, plant dwarfing, earlier blooming, and better pollen viability. Overall, our results suggested that LiAG1 might play an important role in flower development, especially anther development, of Lilium Asiatica Hybrida ‘Ice Pink Queen’.

Improving the N-glycosylation occupancy of plant-produced IgG1 by engineering the amino acid environment at Asn297

Monoclonal antibodies are crucial recombinant biopharmaceuticals, with N-glycosylation at Asn297 essential for their functionality. Plants are increasingly used for antibody production, achieving high expression levels and enabling glycoengineering to produce homogenous human-like N-glycan structures. However, plant-produced human IgG1 often shows significant underglycosylation with potential adverse effects for immune functions and stability. This study addressed this limitation of the widely used plant-based expression platform Nicotiana benthamiana by employing protein engineering to enhance N-glycosylation occupancy in plant-produced IgG1. This was achieved through an amino acid mutation near the conserved glycosylation site in the CH2 domain of the heavy chain. The transient expression of trastuzumab and SARS-CoV-2 neutralizing IgG1 antibody COVA2-15 in N. benthamiana, with mutations such as Y300L, resulted in a notable improvement in glycosylation occupancy. While the structural integrity and monodispersity of the IgG1 variant remained unaltered, an improvement in thermal stability was observed. Furthermore, functional assays showed that antigen binding and human hFcRn interaction were unaffected, while FcγRIIIa binding affinity increased. These findings demonstrate the potential of protein-engineering to enhance the quality and functionality of plant-produced IgG1 antibodies, making them comparable to mammalian-produced counterparts.

JrMYB44 is required for the accumulation of polyphenols and contributes to drought tolerance in Juglans regia

Juglans regia, an important economic tree species, is planted all over the world, and drought is one of the crucial factors limiting its growth and development. The various polyphenol content in walnut plants constitutes one of the material bases for the differences in stress resistance among various germplasms. However, the molecular mechanism underlying stress response mediated by polyphenol -dependent pathways remains unclear. v-Myb avian myeloblastosis viral oncogene homolog (MYB) protein of transcription factors play important regulatory roles in the process of plant stress responses. Previously, we identified JrMYB44 could be involved in osmotic stress response in walnut. In this study, we confirmed that the drought resistance of four walnut cultivars (‘Chandler’, ‘Xiangling’, ‘Xilin2’ and ‘Xifu1’) is positively correlated with the accumulation of polyphenols. The content and component changes of polyphenols in JrMYB44 overexpression (OE) and suppression (SE) lines in both walnut and Arabidopsis thaliana demonstrated that JrMYB44 positively regulated polyphenols accumulation. The variation of JrMYB44 expression and polyphenol levels under drought treatment indicated significant correlation between JrMYB44-induced drought tolerance and polyphenol accumulation, which was involved in reactive oxidative species (ROS) balance. The differentially expressed genes (DEGs) between OE and WT implied that JrMYB44 could positively activate downstream genes to participate in the drought stress response. Yeast one-hybrid (Y1H), transient GUS expression assay and dual-luciferase reporter assay (DLR) confirmed that JrMYB44 could recognize downstream JrWRKY7 and JrDREB2A, two transcription factors previously reported to be involved in drought response. Meanwhile, it was confirmed by Y2H, GST-pull down and luciferase complementation imaging assay (LCI) that JrMYB44 could interact with JrMYC2 and JrDof1, another two previously reported potential drought response regulators. Collectively, these results indicated that JrMYB44 could activate JrWRKY7, JrDREB2A and interact with JrMYC2 and JrDof1 to promote walnut polyphenol accumulation and improve drought resistance in a ROS dependent manner.

Spatiotemporal control of translation in live zebrafish embryos via photoprotected mRNAs

Translation of mRNA into protein is a fundamental process and tightly controlled during development. Several mechanisms acting on the mRNA level regulate when and where an mRNA is expressed. To explore the effects of conditional and transient gene expression in a developing organism, it is vital to experimentally enable abrogation and restoration of translation. We recently developed the FlashCaps technology allowing preparation of translationally muted mRNAs and their controlled activation by light. Here, we validate its functionality in vivo. We demonstrate that translation of FlashCap-eGFP-mRNA can be triggered in zebrafish embryos with spatiotemporal control. The injected FlashCap-mRNA is stable for hours and remains muted. Light-mediated activation up to 24 h post fertilization produces visible amounts of eGFP and can be restricted to distinct parts of the embryo. This methodology extends the toolbox for vertebrate models by enabling researchers to locally activate mRNA translation at different timepoints during development.

SmERF6 Promotes the Expression of Terpenoid Pathway in Salvia officinalis and Improves the Production of High Value Abietane Diterpenes, Carnosol and Carnosic acid.

Carnosol (CO) and carnosic acid (CA) are pharmaceutically important diterpenes predominantly produced in members of Lamiaceae, Salvia officinalis (garden sage), Salvia fruticosa and Rosmarinus officinalis. Nevertheless, availability of these compounds in plant system is very low. In an effort to improve the in planta content of these diterpenes in garden sage, SmERF6 (Salvia miltiorrhiza Ethylene Responsive Factor 6) transcription factor was expressed heterologously. Bai et al. (2018) proved that SmERF6 binds to the promoter regions of Copalyl pyrophosphate synthase (CPS) and Kaurene synthase like (KSL) genes, and improves transcription, thereby, augmenting ferruginol levels, a common precursor for abietane diterpenes in Salvia genus, moreover, transgenic hairy roots of S. miltiorrhiza displayed four fold improved tanshinone content. In our study, heterologous transient expression of SmERF6 in S. officinalis exhibited inter-specific activity in promoting differential accumulation of diterpenes. Overexpression studies showed elevation in the levels of CO (2-fold) and CA (5-fold). Further, in infiltrated leaves levels of ferruginol (50%) and CA derivatives (rosmanol, epirosmanol, methyl carnosic acid) were significantly upregulated along with the other signature terpenes. Finally, stable transgenic lines of S. officinalis developed through Agrobacterium mediated in planta genetic transformation accumulated significant amounts of CO (4-folds), CA (3-folds) as compared to wild plants. Overall, the present study is the first report on improving the content of pharmaceutically important diterpenes in S. officinalis by overexpressing pathway specific transcription factors. The current findings showed convincing evidence for the concept of improving specialized metabolite(s) content in medicinal plants by manipulating the expression of transcriptional regulators.

Development and evaluation of a recombinant monoclonal human antibody with virus-neutralizing activity against the F glycoprotein of respiratory syncytial virus

Introduction. Respiratory syncytial virus (RSV) is the most common pathogen causing lower respiratory tract infections in children. RSV also poses a serious threat to the elderly and immunocompromised patients. Developing a therapy based on recombinant human antibodies to block the RSV fusion (F) glycoprotein is urgent to reduce the incidence of RSV infections and prevent associated complications. Aim. To design plasmid vectors for efficient production of the recombinant monoclonal antibody FM1 in a eukaryotic expression system targeting the RSV fusion (F) glycoprotein and to evaluate its activity against RSV subtypes A and B in vitro. Materials and methods. Constructs encoding the recombinant antibody FM1 were designed using genetic engineering. Recombinant antibodies were produced in the CHO-K1 cell line through transient expression. Antibody specimens were purified from the culture supernatant using affinity chromatography, with a modified protein A as the ligand. The virus-neutralizing activity of the antibody was evaluated in a microneutralization assay using several RSV strains on a Vero cell monolayer culture. Results. We developed a two-plasmid vector system to produce the recombinant FM1 antibody targeting the RSV F glycoprotein, using CHO cells as transient producers. The antibody was successfully produced, purified, and characterized, with its biological activity confirmed. The FM1 antibody demonstrated enhanced virus-neutralizing activity against reference and seasonal RSV strains of subtypes A and B compared to the control drug palivizumab. Conclusion. A recombinant FM1 antibody-based drug could address the import substitution challenge for protective measures against RSV infection. The authors are currently developing a stable FM1 producer clone with high productivity and viability and investigating the therapeutic efficacy of this antibody in a sublethal RSV infection mouse model.

Advances in Gene Therapy for Rare Diseases: Targeting Functional Haploinsufficiency Through AAV and mRNA Approaches.

Most rare diseases (RDs) encompass a diverse group of inherited disorders that affect millions of people worldwide. A significant proportion of these diseases are driven by functional haploinsufficiency, which is caused by pathogenic genetic variants. Currently, most treatments for RDs are limited to symptom management, emphasizing the need for therapies that directly address genetic deficiencies. Recent advancements in gene therapy, particularly with adeno-associated viruses (AAVs) and lipid nanoparticle-encapsulated messenger RNA (mRNA), have introduced promising therapeutic approaches. AAV vectors offer durable gene expression, extensive tissue tropism, and a safety profile that makes them a leading choice for gene delivery; however, limitations remain, including packaging size and immune response. In contrast, mRNA therapeutics, formulated in LNPs, facilitate transient protein expression without the risk of genomic integration, supporting repeated dosing and pharmacokinetic control, though with less long-term expression than AAVs. This review analyzes the latest developments in AAV and mRNA technologies for rare monogenic disorders, focusing on preclinical and clinical outcomes, vector design, and delivery challenges. We also address key regulatory and immunological considerations impacting therapeutic success. Together, these advancements in AAV and mRNA technology underscore a new era in RD treatment, providing innovative tools to target the genetic root of these diseases and expanding therapeutic approaches for patients who currently face limited medical options.

Transcriptomic analysis reveals the crucial role of YABBY genes family in hormonal induced parthenocarpy in Cucumis sativus L.

BackgroundThe plant-specific YABBY transcription factor family plays several activities, including responding to abiotic stress, establishing dorsoventral polarity, and developing lateral organs. Cucumis sativus L. commonly referred to as cucumber and one of the first vegetable crops with a fully sequenced genome.ResultsIn this work, we examined the application of NAA, CPPU, and GA4 + 7 to inflict parthenocarpy in the cucumber ZK line. The expression pattern of YABBY genes throughout fruit development and performed a genomic census of cucumber (Cucumis sativus L.). Based on peptide classification, we discovered eight CsYABBY genes and divided them into five subfamilies. Similarities in motif composition and exon-intron structure were also observed. The cis-elements and gene ontology (GO) analysis revealed the involvement of CsYABBY genes in vegetative growth and the transition of vegetative to the reproductive phase. The expression analysis revealed the differential expression response to NAA, CPPU, and GA4 + 7. In particular, the CsYABBY1 was induced sharply by NAA and CPPU but not GA4 + 7. The transient expression of CsCRC disclosed that it is localized in the nucleus.ConclusionThese findings point to the possibility that CsYABBY1 and CsCRC may positively affect fruit development and could be utilized to generate parthenocarpic cucumber fruits.

Efficient gene delivery admitted by small metabolites specifically targeting astrocytes in the mouse brain.

The development of efficient and targeted methods for delivering DNA invivo has long been a major focus of research. In this study, we introduce a gene delivery approach admitted by small metabolites (gDAM) for the efficient and targeted delivery of naked DNA into astrocytes in the adult brains of mice. gDAM uses a straightforward combination of DNA and small metabolites, including glycine, L-proline, L-serine, L-histidine, D-alanine, Gly-Gly, and Gly-Gly-Gly, to achieve astrocyte-specific delivery of naked DNA, resulting in transient and robust gene expression in these cells. Using gDAM, we successfully co-deliver the PiggyBac transposon and the CRISPR-Cas9 system to induce long-term overexpression of the oncogene EGFRvIII and knockout of tumor suppressor genes Nf1, Pten, and Trp53 in astrocytes, leading to the development of astrocyte-derived gliomas in immunocompetent mice. Furthermore, gDAM facilitates the delivery of naked DNA to peripheral glioma astrocytes. The overexpression of interferon-β and granulocyte-macrophage colony-stimulating factor in these peripheral glioma astrocytes significantly prolongs the overall survival of mice bearing 73C glioma cells. This approach offers a new perspective on developing gene delivery systems that specifically target astrocytes to meet the varied needs of both research and gene therapy. The innovative strategy behind gDAM is expected to provide fresh inspiration in the quest for DNA delivery to other tissues, such as skeletal muscle and skin.

Rubus idaeus RiACS1 Gene Is Involved in Ethylene Synthesis and Accelerates Fruit Ripening in Solanum lycopersicum

Raspberry is a berry whose fruit is not tolerant to storage; breeding varieties with extended storage time and high comprehensive quality are significant for raspberries in cold regions. 1-Aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) is a limiting enzyme in the ethylene synthesis process, which plays essential roles in fruit ripening and softening in plants. In this study, the RiACS1 gene in raspberry (Rubus idaeus L.) variety ‘Polka’ was cloned. The RiACS1 gene overexpression vector was constructed and transformed into tomato plants using the Agrobacterium tumefaciens infection method to verify its function in their reproductive development. The RiACS1 gene, with a total length of 1476 bp, encoded a protein with 491 amino acids. The subcellular localization analysis of the RiACS1 protein in the tobacco transient expression system revealed that the RiACS1-GFP fusion protein was mainly located in the nucleus. Compared with the control, the flowering time and fruit color turning time of transgenic strains were advanced, and the fruit hardness was reduced. Overexpression of RiACS1 increased the activity of ACC synthase, ethylene release rate, and respiration rate during the transchromic phase. It changed the substance content, increased the content of vitamin C and anthocyanin in the fruit ripening process, and decreased the content of chlorophyll and titrable acid at the maturity stage. In addition, RiACS1 increased the relative expression levels of ethylene synthesis-related genes such as SlACS4, SlACO3, and SlACO1 in the fruit ripening process, while it decreased the expression levels of SlACS2 at the maturity stage. These results suggested that the RiACS1 gene could promote early flowering and fruit ripening in tomato plants. This study provided a basis for further modifying raspberry varieties using molecular biology techniques.

The Construction of Heterothallic Strains of Komagataella kurtzmanii Using the I-SceI Meganuclease.

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated 'Y-727-2(alpha)' and 'Y-727-9(a)', which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement.

Enhanced Production of Rebaudioside D and Rebaudioside M through V155T Substitution in the Glycosyltransferase UGT91D2 from Stevia rebaudiana.

Steviol glycosides (SGs) are noncaloric natural sweeteners found in the leaves of stevia (Stevia rebaudiana). These diterpene glycosides are biosynthesized by attaching varying numbers of monosaccharides, primarily glucose, to steviol aglycone. Rebaudioside (Reb) D and Reb M are highly glucosylated SGs that are valued for their superior sweetness and organoleptic properties, yet they are present in limited quantities in stevia leaves. This study aims to improve the substrate preference and catalytic efficiency of UDP-sugar-dependent glycosyltransferase UGT91D2 from stevia, which acts as a bottleneck in the biosynthesis of Reb D and Reb M. We modeled the structure of UGT91D2 and substituted two amino acid residues, Y134 and V155, which are located near the glycosyl acceptor and donor, respectively. Expression of the UGT91D2V155T in budding yeast significantly enhanced the production of Reb D and Reb M. Furthermore, transient expression in Nicotiana benthamiana revealed that the V155T substitution improved the glucosylation activity of UGT91D2, suggesting that this substitution enhances UDP-glucose binding and reduces side reactions involving nonglucose donors. By coexpressing multiple stevia UGT genes in N. benthamiana, we successfully produced highly glucosylated SGs from steviol. Our results provide insights into the substrate specificity of UGT91D2 and contribute to the engineering of SG biosynthesis.

Epibrassinolide Regulates Lhcb5 Expression Though the Transcription Factor of MYBR17 in Maize.

Photosynthesis, which is the foundation of crop growth and development, is accompanied by complex transcriptional regulatory mechanisms. Research has established that brassinosteroids (BRs) play a role in regulating plant photosynthesis, with the majority of research focusing on the physiological level and regulation of rate-limiting enzymes in the dark reactions of photosynthesis. However, studies on their effects on maize photosynthesis, specifically on light-harvesting antenna proteins, have yet to be conducted. The peripheral light-harvesting antenna protein Lhcb5 is crucial for capturing and dissipating light energy. Herein, by analyzing the transcriptomic data of maize seedling leaves treated with 24-epibrassinolide (EBR) and verifying them using qPCR experiments, we found that the MYBR17 transcription factor may regulate the expression of the photosynthetic light-harvesting antenna protein gene. Further experiments using protoplast transient expression and yeast one-hybrid tests showed that the maize transcription factor MYBR17 responds to EBR signals and binds to the promoter of the light-harvesting antenna protein Lhcb5, thereby upregulating its expression. These results were validated using an Arabidopsis mybr17 mutant. Our results offer a theoretical foundation for the application of BRs to enhance the photosynthetic efficiency of maize.

The Implication and Advancement of CRISPR Genome Editing in Microalgae and Cyanobacteria

This review paper provides an overview of recent achievements and future prospects of CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-Cas9) genome editing in microalgae and cyanobacteria. The different types of CRISPR systems and Plasmid-based approaches, RNP-based Cas9 expression, transient and stable expression of Cas9 and sgRNA, and various other techniques for targeted gene editing have been reviewed. The paper also highlights the achievements of CRISPR-Cas9 genome editing in different cyanobacteria and algae species. Additionally, the challenges of off-target effects and potential solutions have been discussed. The paper concludes future prospects of CRISPR-Cas9 genome editing in microalgae and cyanobacteria, including gene stacking, markerless genome editing, and curing of episomes.

Type IV collagen expression is regulated by Notch3-mediated Notch signaling during angiogenesis.

Angiogenesis, the process of new blood vessel formation, involves endothelial cell proliferation and migration, accompanied by the remodeling of the extracellular matrix (ECM). Type IV collagen, a major ECM component, plays a critical role in vascular basement membrane regeneration, influencing cell polarity, migration, and survival. This study examines the regulatory role of Notch signaling, mediated by Notch3, in type IV collagen expression using TIG-1 fibroblasts and a co-culture angiogenesis model with human umbilical vein endothelial cells (HUVECs). Using small interfering RNA (siRNA) to suppress Notch3 expression, we observed a significant reduction in COL4A1 gene expression, which encodes the α1 chain of type IV collagen. Conversely, transient expression of the Notch3 intracellular domain (NICD3) activated Notch signaling, resulting in increased COL4A1 expression. In the co-culture model, pre-treatment of TIG-1cells with Notch signaling inhibitors, including siNotch3 and DAPT, decreased the number of α1(IV)-positive TIG-1 fibroblasts adjacent to HUVECs. This reduction highlights the essential role of Notch3-mediated signaling in promoting type IV collagen expression during angiogenesis. Our findings suggest that Notch signaling regulates type IV collagen expression levels, supporting basement membrane formation and vascular maturation. These results provide insight into the molecular mechanisms of angiogenesis, potentially contributing to therapeutic strategies targeting vascular-related pathologies.

High Aspect Ratio Polymer Nanocarriers for Gene Delivery and Expression in Plants.

Plant genetic engineering methods are critical for food security and biofuel production and to enable molecular farming. Here, we elucidated how polymeric high aspect ratio nanocarriers can enable DNA delivery to Nicotiana benthamiana plants and transient expression. We demonstrated that a nanocarrier with 20 nm width, 80 nm length, and a polymer-to-DNA ratio of N/P = 3.0 afforded the most efficient DNA delivery and expression among the parameter space investigated. Additionally, we showed that polymer-DNA complexes with a moderate positive charge of ∼14 mV favored penetration through the cell wall and membranes with the assistance of cell wall degrading enzymes. Together, these results establish a narrow window of aspect ratios and charges of the nanocarrier-DNA complex that enables DNA delivery to plants using polymeric nanocarriers. This fundamental nanocarrier structure-function relationship informs the design of soft-material nanocarriers for nucleic acid delivery in plant cells to facilitate a wide range of plant biotechnology applications.