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C-repeat binding factor (CBF) transcription factors can activate the expression of a series of cold regulation-related genes, thereby improving the cold resistance of plants. However, no detailed information is known about the biological functions of CBF proteins in yellowhorn (Xanthoceras sorbifolium). In this study, a total of 59 CBF gene family members were identified in five yellowhorn cultivars (WF18, Zhongshi 4, Jinguanxipei 2021, Zhong Guan NO.2 , and XsoG11) , revealing their intraspecific structural and functional diversity, with 8 core genes present in all cultivars. Phylogenetic and motif analyses highlighted conserved features and species-specific adaptations. Gene duplication events revealed that tandem duplicates are major factors involved in the expansion of this gene family in yellowhorn. Expression profiling under stress conditions demonstrated the involvement of these genes in stress responses. Of particular interest was Xg11_CBF11 , which showed strong induction by low-temperature stress. Overexpression of Xg11_CBF11 in Arabidopsis thaliana was performed to validate its cold resistance function. The wild-type and T2 transgenic A. thaliana plants were subjected to low-temperature stress at 4°C for 0, 24, and 48 h, and physiological indexes related to antioxidant enzyme activity, photosynthesis, and cell membrane permeability were determined by comparative test. The results were as follows: the POD and SOD activities of transgenic lines were significantly higher than those of wild-type lines, indicating Xg11_CBF11 improved the adaptability of A. thaliana to low-temperature; The increase of relative conductivity and malondialdehyde, the decrease of chlorophyll content in transgenic lines were smaller than those of wild-type lines, indicating Xg11_CBF11 enhanced the resistance of A. thaliana to low-temperature stress. These results implied that Xg11_CBF11 has a positive regulatory effect on A. thaliana 's response to low-temperature stress. [ABSTRACT FROM AUTHOR]

[Display omitted] IL-33 is a type 2 inflammatory cytokine that is elevated in the esophageal epithelium of eosinophilic esophagitis (EoE) subjects. We previously developed a mouse model of EoE dependent on constitutive overexpression of IL-33 from the esophageal epithelium (EoE33). Our objective was to develop an inducible, IL-33–dependent model of EoE and examine induction of EoE-associated pathology. We utilized a tetracycline-inducible system to express IL-33 in the esophagus by generating 2 transgenic mice. The first (iSophagus) expresses a reverse tetracycline transactivator from the esophageal epithelium. The second (TRE33) features a tetracycline response element driving expression of IL-33. When crossed, these mice generate an inducible model of EoE (iEoE33). Mice were administered doxycycline-infused chow for up to 2 weeks. Cytokines were assessed by ELISA or bead-based multiplex analysis. T cells were assessed by flow cytometry. Pathology was assessed by histology and immunohistochemistry for IL-33, eosinophil peroxidase, CD4, and Ki-67. iEoE33 was treated with steroids and crossed with IL-13−/− mice. Doxycycline-treated iEoE33 mice demonstrated expression of IL-33 in the esophageal epithelium, and esophageal pathology including eosinophilia, CD4+ cell infiltrate, basal zone hyperplasia, and dilated intercellular spaces. These findings became pronounced on day 7 of induction, were accompanied by weight loss and esophageal thickening, and were steroid responsive and IL-13 dependent. Inducible IL-33 expression in the esophageal epithelium elicited features pathognomonic of EoE. iEoE33 enables investigation of EoE disease mechanisms as well as initiation, progression, and resolution. [ABSTRACT FROM AUTHOR]

Human adenovirus (HAdV)-based oncolytic vectors, which are designed to preferentially replicate in and kill cancer cells, have shown modest efficacy in human clinical trials in part due to poor viral distribution throughout the tumor mass. Previously, we showed that expression of the p14 fusion-associated small transmembrane (FAST) fusogenic protein could enhance oncolytic HAdV efficacy and reduce tumor growth rate in a human xenograft mouse model of cancer. We now explore whether co-expression of the adenovirus death protein (ADP) with p14 FAST protein could synergize to further enhance oncolytic vector efficacy. ADP is naturally encoded within the early region 3 (E3) of HAdV, a region which is frequently removed from HAdV-based vectors, and functions to enhance cell lysis and progeny release. We evaluated a variety of approaches to achieve optimal expression of the two proteins, the most efficient method being insertion of an expression cassette within the E3 deletion, consisting of the coding sequences for p14 FAST protein and ADP separated by a self-cleaving peptide derived from the porcine teschovirus-1 (P2A). However, the quantities of p14 FAST protein and ADP produced from this vector were reduced approximately 10-fold compared to a similar vector-expressing only p14 FAST protein and wildtype HAdV, respectively. Compared to our original oncolytic vector-expressing p14 FAST protein alone, reduced expression of p14 FAST protein and ADP from the P2A construct reduced cell-cell fusion, vector spread, and cell-killing activity in human A549 adenocarcinoma cells in culture. These studies show that a self-cleaving peptide can be used to express two different transgenes in an armed oncolytic HAdV vector, but also highlight the challenges in maintaining adequate transgene expression when modifying vector design. [ABSTRACT FROM AUTHOR]

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a crucial role in the viral immune processes of organisms, with STAT being a key transcription factor downstream in this pathway. The STAT gene of Bombyx mori has two splicing forms, named BmSTAT-S and BmSTAT-L. This study compared the effects of the two splicing forms on Bombyx mori nucleopolyhedrovirus (BmNPV) infection through cell-level interference and further explored whether BmSTAT participates in the immune response to BmNPV infection via transgenic intervention at the individual level. Our research results indicated that BmNPV upregulates the expression of BmSTAT-S and BmSTAT-L in Bombyx mori BmE cells and larvae. Furthermore, BmE cells with interference of BmSTAT-S or BmSTAT-L displayed significantly higher expression levels of the viral gene GP41 and increased viral fluorescence compared to the control group after 48 h of infection with BmNPV. Then, we constructed transgenic silkworms with genetic interference, and the results showed that both the transgenic silkworms with systemic interference and midgut-specific interference of the two splice forms of BmSTAT exhibited significantly reduced survival rates and increased viral replication numbers after infection with BmNPV. The above results indicated that the BmSTAT gene is involved in the immune response of Bombyx mori to BmNPV and these findings lay the foundation for further research on the mechanism of JAK/STAT signaling pathway involvement in BmNPV infection. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) is a Dependoparvovirus with a ssDNA ~4.7 kb genome in a ~25 nm icosahedral capsid structure. AAV genomes encode nine known functional proteins from two open reading frames between two inverted terminal repeats (ITRs). In recombinant AAV vectors for gene therapy use, the AAV genome is replaced with a transgene of interest flanked by ITRs and subsequently packaged within an AAV capsid made up of three viral structural proteins (VP1, VP2, and VP3) in an approximate 1:1:10 ratio, respectively. The AAV capsid, particularly VP3, has traditionally been ascribed to capsid-cellular receptor binding. However, AAV9 VP1/VP2 exhibits a capsid-promoter interaction that can alter neuronal cellular tropism in the rat and non-human primate central nervous system. This capsid-promoter interaction is altered by AAV9EU (AAV9 with six glutamates inserted at aa139) which exhibits a significant reduction in nuclear transgene DNA, a decrease in neuronal transduction, and a reduction in vivo relative transgene mRNA levels. AAV9EU has six amino acid insertions in VP1, VP2, and MAAP (membrane-associated accessory protein), but no combination of VP with MAAP recapitulated the AAV9EU in vivo phenotype. Surprisingly, AAV9 produced in the absence of MAAP9 exhibits an increase in relative transgene levels. While co-infusing two AAV9 vectors, differing only in transgene and MAAP9 presence during production, exhibit a significantly increased in vivo transgene fluorescence intensity by fivefold of both transgenes. Together, an MAAP9-related activity acts both in cis and in trans to increase AAV9 transgene mRNA levels and AAV9 transgene protein levels in vivo. IMPORTANCE Recombinant adeno-associated viruses (AAVs) are used extensively in clinical gene therapy for treating a range of tissues and pathologies in humans. In particular, AAV9 occupies a prominent position in central nervous system (CNS) gene therapy given its central role in ongoing clinical trials and an FDA-approved therapeutic. Despite its widespread use, recent studies have identified unique roles for the AAV capsid in in vivo transgene expression; for example, interior-facing capsid residues of AAV VP1 and VP2 modulate cellular transgene expression in vivo. The following experiments identified that the AAV9 MAAP protein exerts a significant influence on in vivo transgene expression. This finding could further explain how AAV can remain latent after infection in vivo. Together, these studies provide novel functional insights that highlight the importance of further understanding basic AAV biology. [ABSTRACT FROM AUTHOR]

Functional genomics are needed for helminths to facilitate basic and transformational studies to improve global public health. This review charts the recent advances in CRISPR genetic engineering for flatworm parasites. Schistosomes were genome edited with Cas9 and Cas12a for knockout (KO) mutation and transgene knock-in (KI). Approaches for Schistosoma mansoni were adapted for Schistosoma japonicum and the liver fluke, Opisthorchis viverrini. KO of a liver fluke growth factor confirmed a role in biliary disease and malignancy. Cas12a- and Cas13a-based diagnosis was established in regions endemic for schistosomiasis and opisthorchiasis. Genome safe harbor sites have been predicted in S. mansoni using computational genome annotation and chromatin structure analysis and targeted for KI with GFP. Derivation of heritably transgenic loss- or gain-of function lines is the next milestone. CRISPR genome editing is actively used for schistosomes and other flukes. The ability to genetically manipulate these flatworms enables deeper investigation of their (patho)biological nature. CRISPR gene knockout (KO) demonstrated that a liver fluke growth mediator contributes to disease progression. Genome safe harbor sites have been predicted in Schistosoma mansoni and targeted for transgene insertion. CRISPR-based diagnosis has been demonstrated for infection with schistosomes and Opisthorchis viverrini. This review charts the progress, and the state of play, and posits salient questions for the field to address. Derivation of heritably transgenic loss-of-function or gain-of-function lines is the next milestone. [ABSTRACT FROM AUTHOR]

Introduction: Plants are inherently connected with the microbiome, which plays a crucial role in regulating various host plant biological processes, including immunity, nutrient acquisition, and resistance against abiotic and biotic stresses. Many factors affect the interaction between plants and microbiome. Methods and results: In this study, microbiome samples were collected from five niches (bulk soil, rhizoplane, root endosphere, phylloplane, and leaf endosphere) across four developmental stages (seedling, flowering, podding, and maturity) of various soybean varieties. Composition and structure of bacterial and fungal communities were analyzed using 16S rRNA gene and ITS (Internally Transcribed Spacer) region amplicon sequencing. It was observed that both niches and developmental stages significantly impact on the assembly and composition of soybean microbiome. However, variety, presence of a transgene, and glyphosate application had minimal effects on microbial communities. The dominant microbiome varied across the five niches, with most containing beneficial microbial communities capable of promoting plant growth or increasing disease resistance. Types and abundance of the dominant microbes affected network stability, potentially resulting in functional changes in different ecological niches. Conclusion: This study provides theoretical evidence for microbial protection of plants against diseases and demonstrates that systematic analysis of the composition and diversity of soybean microbiomes can contribute to the development of biological control technologies. [ABSTRACT FROM AUTHOR]

Background: Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. Results: The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. Conclusions: These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications. [ABSTRACT FROM AUTHOR]

Abstract Adeno-associated virus (AAV) vectors can mediate long-term expression of immunogenic transgenes in vivo through transduction of tolerogenic cells in the liver. Tissue-targeted AAV vectors allow transduction of non-hepatic cells, but this necessitates development of strategies to minimize transgene immunogenicity. Here, we first validated that AAV capsids with tissue-specific tropism and transgene promoters enabled expression of the immunogenic protein, firefly luciferase, in liver, muscle, or adipose tissue. Cellular immunity was detectable in animals where luciferase was expressed in muscle or adipose, but not liver tissue. With the objective of enhancing tolerance of transduced non-hepatic cells, AAV vectors were engineered to co-express luciferase plus the immune checkpoint protein, PD-L1. In animals where transduced cells expressed luciferase but not PD-L1, there was incremental depletion of transduced cells over time. By contrast, the bioluminescent signal increased incrementally over the study, and was significantly greater, in the muscle and adipose tissue of animals where PD-L1 was co-expressed with luciferase. Our data demonstrate that PD-L1 co-expression facilitates persistent, tissue-targeted expression of immunogenic transgenes without transducing tolerogenic hepatic cells. Our strategy of PD-L1 co-expression may provide a versatile platform for sustained expression of immunogenic transgenes in gene and cell therapies.

Abstract Background Chlamydomonas reinhardtii is gaining recognition as a promising expression system for the production of recombinant proteins. However, its performance as a cellular biofactory remains suboptimal, especially with respect to consistent expression of heterologous genes. Gene silencing mechanisms, position effect, and low nuclear transgene expression are major drawbacks for recombinant protein production in this model system. To unveil the molecular changes following transgene insertion, retention, and expression in this species, we genetically engineered C. reinhardtii wild type strain 137c (strain cc-125 mt+) to express the fluorescent protein mVenus and subsequently analysed its intracellular proteome. Results The obtained transgenic cell lines showed differences in abundance in more than 400 proteins, with multiple pathways altered post-transformation. Proteins involved in chromatin remodelling, translation initiation and elongation, and protein quality control and transport were found in lower abundance. On the other hand, ribosomal proteins showed higher abundance, a signal of ribosomal stress response. Conclusions These results provide new insights into the modifications of C. reinhardtii proteome after transformation, highlighting possible pathways involved in gene silencing. Moreover, this study identifies multiple protein targets for future genetic engineering approaches to improve the prospective use of C. reinhardtii as cell biofactory for industrial applications.

SUMMARY: Female willows exhibit greater drought tolerance and benefit more from exogenous acetic acid (AA)‐improved drought tolerance than males. However, the potential mechanisms driving these sex‐specific responses remain unclear. To comprehensively investigate the sexually dimorphic responsive mechanisms of willows to drought and exogenous AA, here, we performed physiological, proteomic, Lys‐acetylproteomic, and transgenic analyses in female and male Salix myrtillacea exposed to drought and AA‐applicated drought treatments, focusing on protein abundance and lysine acetylation (LysAc) changes. Drought‐tolerant females suffered less drought‐induced photosynthetic and oxidative damage, did not activate AA and acetyl‐CoA biosynthesis, TCA cycle, fatty acid metabolism, and jasmonic acid signaling as strongly as drought‐sensitive males. Exogenous AA caused overaccumulation of endogenous AA and inhibition of acetyl‐CoA biosynthesis and utilization in males. However, exogenous AA greatly enhanced acetyl‐CoA biosynthesis and utilization and further enhanced drought performance of females, possibly determining that AA improved drought tolerance more in females than in males. Interestingly, overexpression of acetyl‐CoA synthetase (ACS) could reprogram fatty acids, increase LysAc levels, and improve drought tolerance, highlighting the involvement of ACS‐derived acetyl‐CoA in drought responses. In addition, drought and exogenous AA induced sexually dimorphic LysAc associated with histones, transcription factors, and metabolic enzymes in willows. Especially, exogenous AA may greatly improve the photosynthetic capacity of S. myrtillacea males by decreasing LysAc levels and increasing the abundances of photosynthetic proteins. While hyperacetylation in glycolysis, TCA cycle, and fatty acid biosynthesis potentially possibly serve as negative feedback to acclimate acetyl‐CoA biosynthesis and utilization in drought‐stressed males and AA‐applicated females. Thus, acetyl‐CoA biosynthesis and utilization determine the sexually dimorphic responses of S. myrtillacea to drought and exogenous AA. [ABSTRACT FROM AUTHOR]

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown. [ABSTRACT FROM AUTHOR]

In order to screen candidate aquaporin genes involved in resisting osmotic stress, we analyzed the physiological responses and the expression levels of aquaporin genes in garlic under drought and salt stress with 'Er Shuizao' as plant material. Different physiological indicators were detected under drought and salt stress treatments. RT-qPCR was used to detect the expression levels of the candidate aquaporin genes in specific tissues. Finally, we screened AsPIP1-3 as a candidate gene and analyzed its function. The results showed that the relative water content and chlorophyll content of leaves decreased, the O2− production rate increased, and H2O2 accumulated in garlic under drought and salt stress. The activities of SOD, POD, and CAT enzymes first increased and then decreased in garlic. The content of soluble sugar and proline increased to maintain cell osmotic balance, and the content of MDA and relative conductivity continued to increase. Most aquaporin gene expression first increased and then decreased in garlic under drought and salt stress. AsPIP1-3 gene expression is up-regulated under drought and salt stress in garlic. The relative expression was the highest on the 6th day of stress, being related to antioxidant enzyme activity and osmotic regulation. The consistent changes in gene expressions and physiological responses indicated that AsPIP1-3 played a role in resisting garlic osmotic stress. AsPIP1-3 was located on the cell membrane, being consistent with the predicted results of subcellular localization. The germination rate and root length of transgenic Arabidopsis under drought stress were significantly different from the wild type. Drought stress reduced the ROS accumulation of transgenic Arabidopsis, and the antioxidant enzyme activity was significantly higher than the wild type. The relative conductivity and MDA content significantly decreased, and the proline content increased under drought stress. The expression level of the genes related to drought stress response (AtRD22, AtP5CS, AtABF3, and AtLEA) significantly increased. The overexpression of AsPIP1-3 genes improved the drought tolerance of transgenic Arabidopsis plants, showing that AsPIP1-3 proteins enhanced drought tolerance. Our study laid a foundation for exploring the regulatory mechanism of garlic to abiotic stress. [ABSTRACT FROM AUTHOR]

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress. [ABSTRACT FROM AUTHOR]

C-repeat binding factor (CBF) transcription factors can activate the expression of a series of cold regulation-related genes, thereby improving the cold resistance of plants. However, no detailed information is known about the biological functions of CBF proteins in yellowhorn (Xanthoceras sorbifolium). In this study, a total of 59 CBF gene family members were identified in five yellowhorn cultivars (WF18, Zhongshi 4, Jinguanxipei 2021, Zhong Guan NO.2, and XsoG11), revealing their intraspecific structural and functional diversity, with 8 core genes present in all cultivars. Phylogenetic and motif analyses highlighted conserved features and species-specific adaptations. Gene duplication events revealed that tandem duplicates are major factors involved in the expansion of this gene family in yellowhorn. Expression profiling under stress conditions demonstrated the involvement of these genes in stress responses. Of particular interest was Xg11_CBF11, which showed strong induction by low-temperature stress. Overexpression of Xg11_CBF11 in Arabidopsis thaliana was performed to validate its cold resistance function. The wild-type and T2 transgenic A. thaliana plants were subjected to low-temperature stress at 4°C for 0, 24, and 48 h, and physiological indexes related to antioxidant enzyme activity, photosynthesis, and cell membrane permeability were determined by comparative test. The results were as follows: the POD and SOD activities of transgenic lines were significantly higher than those of wild-type lines, indicating Xg11_CBF11 improved the adaptability of A. thaliana to low-temperature; The increase of relative conductivity and malondialdehyde, the decrease of chlorophyll content in transgenic lines were smaller than those of wild-type lines, indicating Xg11_CBF11 enhanced the resistance of A. thaliana to low-temperature stress. These results implied that Xg11_CBF11 has a positive regulatory effect on A. thaliana ‘s response to low-temperature stress.

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a crucial role in the viral immune processes of organisms, with STAT being a key transcription factor downstream in this pathway. The STAT gene of Bombyx mori has two splicing forms, named BmSTAT-S and BmSTAT-L. This study compared the effects of the two splicing forms on Bombyx mori nucleopolyhedrovirus (BmNPV) infection through cell-level interference and further explored whether BmSTAT participates in the immune response to BmNPV infection via transgenic intervention at the individual level. Our research results indicated that BmNPV upregulates the expression of BmSTAT-S and BmSTAT-L in Bombyx mori BmE cells and larvae. Furthermore, BmE cells with interference of BmSTAT-S or BmSTAT-L displayed significantly higher expression levels of the viral gene GP41 and increased viral fluorescence compared to the control group after 48 h of infection with BmNPV. Then, we constructed transgenic silkworms with genetic interference, and the results showed that both the transgenic silkworms with systemic interference and midgut-specific interference of the two splice forms of BmSTAT exhibited significantly reduced survival rates and increased viral replication numbers after infection with BmNPV. The above results indicated that the BmSTAT gene is involved in the immune response of Bombyx mori to BmNPV and these findings lay the foundation for further research on the mechanism of JAK/STAT signaling pathway involvement in BmNPV infection.

Abstract Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA3 can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels.

Abstract Plants respond to biotic and abiotic stress by activating and interacting with multiple defense pathways, allowing for an efficient global defense response. RNA silencing is a conserved mechanism of regulation of gene expression directed by small RNAs important in acquired plant immunity and especially virus and transgene repression. Several RNA silencing pathways in plants are crucial to control developmental processes and provide protection against abiotic and biotic stresses as well as invasive nucleic acids such as viruses and transposable elements. Various notable studies have shed light on the genes, small RNAs, and mechanisms involved in plant RNA silencing. However, published research on the potential interactions between RNA silencing and other plant stress responses is limited. In the present study, we tested the hypothesis that spreading and maintenance of systemic post-transcriptional gene silencing (PTGS) of a GFP transgene are associated with transcriptional changes that pertain to non-RNA silencing-based stress responses. To this end, we analyzed the structure and function of the photosynthetic apparatus and conducted whole transcriptome analysis in a transgenic line of Nicotiana benthamiana that spontaneously initiates transgene silencing, at different stages of systemic GFP-PTGS. In vivo analysis of chlorophyll a fluorescence yield and expression levels of key photosynthetic genes indicates that photosynthetic activity remains unaffected by systemic GFP-PTGS. However, transcriptomic analysis reveals that spreading and maintenance of GFP-PTGS are associated with transcriptional reprogramming of genes that are involved in abiotic stress responses and pattern- or effector-triggered immunity-based stress responses. These findings suggest that systemic PTGS may affect non-RNA-silencing-based defense pathways in N. benthamiana, providing new insights into the complex interplay between different plant stress responses.

Abstract The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and β-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular β-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy.

The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems. [ABSTRACT FROM AUTHOR]

Legumin A is a seed storage protein that provides nutrients for seed germination. The purpose of this study was to describe the structure and expression pattern of the EuLEGA gene in Eucommia ulmoides Oliver (E. ulmoides) and to infer its functional role. The 1287 bp coding sequence of the EuLEGA CDS of the EuLEGA gene, encoding a protein containing 428 amino acid residues, was cloned. The structure predicted that the protein belonged to the RmlC (deoxythymidine diphosphates, dTDP)-4-dehydrorhamnose 3,5-epimerase)-like cupin conserved domain family, which contains both RmlC, a key enzyme for the synthesis of rhamnose and legumin A. The overexpression (OE) vector of the EuLEGA gene was constructed and genetically transformed into tobacco and E. ulmoides; the RNA interference (RNAi) vector of the EuLEGA gene was constructed and genetically transformed into E. ulmoides; and the contents of legumin A and rhamnose were detected. The results showed that the EuLEGA gene could significantly increase the content of legumin A in transgenic tobacco leaves and transgenic E. ulmoides regenerative buds, and the OE of this gene in E. ulmoides could promote an increase in rhamnose content. RNAi caused a significant decrease in the legumin A content in the regenerated buds of E. ulmoides. These was a significant increase in legumin A in the transgenic tobacco seeds, and these results indicate that the expression of the EuLEGA gene is closely related to the accumulation of legumin A. Subcellular localization studies revealed that EuLEGA is localized to the cytoplasm with the vacuolar membrane. Analysis of the EuLEGA gene expression data revealed that the expression level of the EuLEGA gene in the samaras was significantly greater than that in the leaves and stems. In addition, the study also demonstrated that GA3 can upregulate the expression levels of the EuLEGA gene, while ABA and MeJA can downregulate its expression levels. [ABSTRACT FROM AUTHOR]

The development of compact CRISPR systems has facilitated delivery but has concurrently reduced gene editing efficiency, thereby limiting the further utilization of CRISPR systems. Enhancing the efficiency of CRISPR systems poses a challenging task and holds significant implications for the advancement of biotechnology. In our work, we report a synthetic dual-antibody system that can stably exist in the intracellular environment, specifically inhibiting the functions of NF-κB and β-catenin. This not only elevates the transgenic expression of the CRISPR system by suppressing the innate immune response within cells to enhance the gene editing efficiency but also demonstrates a notable tumor inhibitory effect. Based on the specific output expression regulation of CRISPR-CasΦ, we constructed a CRISPR-based gene expression platform, which includes sensor modules for detecting intracellular β-catenin and NF-κB, as well as an SDA module to enhance overall efficiency. In vitro experiments revealed that the CRISPR-based gene expression platform exhibited superior CDK5 expression inhibition efficiency and specific cytotoxicity towards tumor cells. In vitro experiments, we found that CRISPR-based gene expression platforms can selectively kill bladder cancer cells through T cell-mediated cytotoxicity. Our design holds significant assistant potential of transgene therapy and may offer the capability to treat other diseases requiring transgene therapy. [ABSTRACT FROM AUTHOR]

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes. [ABSTRACT FROM AUTHOR]

• AAV constructs express transgenes in CNS cells, but microglia present challenges. • Novel, genetically modified AAV capsids show improved microglial transduction. • Myeloid-specific promoters in AAV constructs fail to prevent off-target expression. • Microscopy and PCR controls may minimize false positive AAV transgene detection. Microglia play a central role in the etiology of many neuropathologies. Transgenic tools are a powerful experiment approach to gain reliable and specific control over microglia function. Adeno-associated virus (AAVs) vectors are already an indispensable tool in neuroscience research. Despite ubiquitous use of AAVs and substantial interest in the role of microglia in the study of central nervous system (CNS) function and disease, transduction of microglia using AAVs is seldom reported. This review explores the challenges and advancements made in using AAVs for expressing transgenes in microglia. First, we will examine the functional anatomy of the AAV capsid, which will serve as a basis for subsequent discussions of studies exploring the relationship between capsid mutations and microglia transduction efficacy. After outlining the functional anatomy of AAVs, we will consider the experimental evidence demonstrating AAV-mediated transduction of microglia and microglia-like cell lines followed by an examination of the most promising experimental approaches identified in the literature. Finally, technical limitations will be considered in future applications of AAV experimental approaches. [ABSTRACT FROM AUTHOR]

Sugarcane (Saccharum officinarum) has gained more attention worldwide in recent decades because of its importance as a bioenergy resource and in producing table sugar. However, the production capabilities of conventional varieties are being challenged by the changing climates, which struggle to meet the escalating demands of the growing global population. Genome editing has emerged as a pivotal field that offers groundbreaking solutions in agriculture and beyond. It includes inserting, removing or replacing DNA in an organism's genome. Various approaches are employed to enhance crop yields and resilience in harsh climates. These techniques include zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas). Among these, CRISPR/Cas is one of the most promising and rapidly advancing fields. With the help of these techniques, several crops like rice (Oryza sativa), tomato (Solanum lycopersicum), maize (Zea mays), barley (Hordeum vulgare) and sugarcane have been improved to be resistant to viral diseases. This review describes recent advances in genome editing with a particular focus on sugarcane and focuses on the advantages and limitations of these approaches while also considering the regulatory and ethical implications across different countries. It also offers insights into future prospects and the application of these approaches in agriculture. [ABSTRACT FROM AUTHOR]

[Display omitted] Eosinophilic esophagitis (EoE) is an increasingly common inflammatory condition of the esophagus; however, the underlying immunologic mechanisms remain poorly understood. The epithelium-derived cytokine IL-33 is associated with type 2 immune responses and elevated in esophageal biopsy specimens from patients with EoE. We hypothesized that overexpression of IL-33 by the esophageal epithelium would promote the immunopathology of EoE. We evaluated the functional consequences of esophageal epithelial overexpression of a secreted and active form of IL-33 in a novel transgenic mouse, EoE33. EoE33 mice were analyzed for clinical and immunologic phenotypes. Esophageal contractility was assessed. Epithelial cytokine responses were analyzed in three-dimensional organoids. EoE33 phenotypes were further characterized in ST2−/−, eosinophil-deficient, and IL-13−/− mice. Finally, EoE33 mice were treated with dexamethasone. EoE33 mice displayed ST2-dependent, EoE-like pathology and failed to thrive. Esophageal tissue remodeling and inflammation included basal zone hyperplasia, eosinophilia, mast cells, and T H 2 cells. Marked increases in levels of type 2 cytokines, including IL-13, and molecules associated with immune responses and tissue remodeling were observed. Esophageal organoids suggested reactive epithelial changes. Genetic deletion of IL-13 in EoE33 mice abrogated pathologic changes in vivo. EoE33 mice were responsive to steroids. IL-33 overexpression by the esophageal epithelium generated immunopathology and clinical phenotypes resembling human EoE. IL-33 may play a pivotal role in the etiology of EoE by activating the IL-13 pathway. EoE33 mice are a robust experimental platform for mechanistic investigation and translational discovery. [ABSTRACT FROM AUTHOR]

Most viruses and transposons serve as effective carriers for the introduction of foreign DNA up to 11 kb into vertebrate genomes. However, their activity markedly diminishes with payloads exceeding 11 kb. Expanding the payload capacity of transposons could facilitate more sophisticated cargo designs, improving the regulation of expression and minimizing mutagenic risks associated with molecular therapeutics, metabolic engineering, and transgenic animal production. In this study, we improved the Tol2 transposon by increasing protein expression levels using a translational enhancer (QBI SP163, ST) and enhanced the nuclear targeting ability using the nuclear localization protein H2B (SHT). The modified Tol2 and ST transposon efficiently integrated large DNA cargos into human cell cultures (H1299), comparable to the well-established super PiggyBac system. Furthermore, mRNA from ST and SHT showed a significant increase in transgene delivery efficiency of large DNA payloads (8 kb, 14 kb, and 24 kb) into zebrafish (Danio rerio). This study presents a modified Tol2 transposon as an enhanced nonviral vector for the delivery of large DNA payloads in transgenic applications. [ABSTRACT FROM AUTHOR]

Retroviral-mediated misexpression in chicken embryos has been a powerful research tool for developmental biologists in the last two decades. In the RCASBP retroviral vectors that are widely used for in vivo somatic transgenesis, a coding sequence of interest is under the transcriptional control of a strong viral promoter in the long terminal repeat. While this has proven to be effective for studying secreted signalling proteins, interpretation of the mechanisms of action of nuclear factors is more difficult using this system since it is not clear whether phenotypic effects are cell-autonomous or not, and therefore whether they represent a function of the endogenous protein. Here, we report the consequences of retroviral expression using the RCANBP backbone, in which the transcription factor Dlx5 is expressed under the control of chondrocyte-specific regulatory sequences from the Col2a1 gene. To our knowledge, this is the first demonstration of a tissue-specific phenotype in the chicken embryo. • Tissue-specific transgenesis in chicken embryos. • Targeted misexpression with avian retroviral vectors. • Cell-autonomous effects of Dlx5 on chondrocyte differentiation. [ABSTRACT FROM AUTHOR]

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

Abstract Background Chitinase is an enzyme that hydrolyzes chitin, a major component of the exoskeleton of insects, including plant pests like whiteflies. The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton (Gossypium hirsutum) through Agrobacterium-mediated transformation. Fifty-five putative transgenic cotton plants were obtained, out of which fifteen plants successfully survived and were shifted to the field. Using gene-specific primers, amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation. These five plants were further evaluated for their mRNA expression levels. The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field, were selected to raise their subsequent progenies. Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10 (2) for the ch1 gene and 3.7-fold in P2 (1) for the chi2 gene. Fluorescent in situ hybridization (FISH) confirmed a single copy number of ch1 and chi2 (hemizygous) on chromosome no. 6. Furthermore, the efficacy of transgenes on whitefly was evaluated through an insect bioassay, where after 96 h of infestation, mortality rates of whitefly were calculated to be 78%–80% in transgenic cotton plants. The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant. Conclusion Based on these findings, it can be concluded that the chemically synthesized barley chitinase genes (ch1 and chi2) have the potential to be effective against insects with chitin exoskeletons, including whiteflies. The transgenic cotton plants expressing these genes showed increased resistance to whiteflies, resulting in reduced egg numbers and higher mortality rates.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (r ed pericarp and r ed cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr′ state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr′. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr′ epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr′ sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation. [ABSTRACT FROM AUTHOR]

Nuclear Factor Y (NF-Y) is divided into three different types of subunits, A, B, and C. NF-Ys play crucial roles in plants for controlling gene expression associated with various developmental processes and abiotic stresses, but it is mostly unknown the downstream genes regulated by NF-Ys in plant. One of the potato NF-Y genes, StNF-YA7, increased potato's drought tolerance when overexpressed under the control of constitutive CaMV 35S promoter. Therefore, it was of interest what genes are regulated by the increased expression level of StNF-YA7. To investigate the downstream genes of StNF-YA7, the transcriptome sequencing was carried out for four potato lines, including Solanum tuberosum L ‘Superior’ as wild type (WT), empty vector control (VC), and two StNF-YA7 overexpressor lines (designated to StNF-YA7 #19 & #26). The RNA sequencing data was produced by the Illumina NovaSeq 6000 sequencing system. The number of total raw reads obtained from the RNA sequencing was 36.7 million for WT, 36.2 for VC, 29.3 for StNF-YA7 #19, and 29.5 million for StNF-YA7 #26, respectively. The length of total raw reads for each sample was between 5.92 Gb (StNF-YA7 #19) and 7.42 Gb (WT), and after filtering raw quality reads, the total length was between 5.81 Gb (StNF-YA7 #19) and 7.29 Gb (WT). Each filtered clear read set of four transcriptomes was mapped on the potato reference genome, SolTub_3.0, and the percentage of mapped reads ranged from 89.8 % (VC) to 90.3 % (WT). GC contents range between 43.01 % (StNF-YA7 #19) and 42.44 % (StNF-YA7 #26). Q20 quality score ranges between 98.63 % (StNF-YA7 #26) and 98.74 % (VC).

Abstract Background Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in “carrier” or “pathogenic” states. HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S. aureus infection. However, the contribution of HLA DP to S. aureus infection is unknown yet. Methods In this study, we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes. Neo‐floxed IAβ+/− mice were crossbred with Ella‐Cre and further crossbred with HLA DP401 or HLA‐DRA0101 humanized mice. After several rounds of traditional crossbreeding, we finally obtained HLA DP401‐IAβ−/− and HLA DRA‐IAβ−/− humanized mice, in which human DP401 or DRA0101 molecule was introduced into IAβ−/− mice deficient in endogenous murine MHC class II molecules. A transnasal infection murine model of S. aureus pneumonia was induced in the humanized mice by administering 2 × 108 CFU of S. aureus Newman dropwise into the nasal cavity. The immune responses and histopathology changes were further assessed in lungs in these infected mice. Results We evaluated the local and systemic effects of S. aureus delivered intranasally in HLA DP401‐IAβ−/− and HLA DRA‐IAβ−/− transgenic mice. S. aureus Newman infection significantly increased the mRNA level of IL 12p40 in lungs in humanized mice. An increase in IFN‐γ and IL‐6 protein was observed in HLA DRA‐IAβ−/− mice. We observed a declining trend in the percentage of F4/80+ macrophages in lungs in HLA DP401‐IAβ−/− mice and a decreasing ratio of CD4+ to CD8+ T cells in lungs in IAβ−/− mice and HLA DP401‐IAβ−/− mice. A decreasing ratio of Vβ3+ to Vβ8+ T cells was also found in the lymph node of IAβ−/− mice and HLA DP401‐IAβ−/− mice. S. aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ−/− genetic background mice. Conclusion These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S. aureus pneumonia and study what role DP molecule plays in S. aureus infection.

CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably integrated into the genome of CHO cells using the piggyBac transposon. Each individual transgene contained a unique tag, a DNA barcode, and the resulting polyclonal cell population was cultured for almost a month without any selection. Next, using the high-throughput sequencing, genomic localizations of barcodes, as well as their abundances in the population and transcriptional activities were identified. In total, ~640 transgenes more or less evenly distributed across all chromosomes of CHO cells were characterized. More than half of the transgenes were completely silent. The most active transgenes were identified to be inserted in gene promoters and 5’ UTRs. Transgenes carrying Chinese hamster full-length promoter of the EF-1α gene showed the highest activity. Transgenes with a truncated version of the same promoter and with the mouse PGK gene promoter were on average 10 and 19 times less active, respectively. In total, combinations of genomic loci of CHO cells and transgene promoters that together provide different levels of transcriptional activity of the model reporter construct were described.

In order to screen candidate aquaporin genes involved in resisting osmotic stress, we analyzed the physiological responses and the expression levels of aquaporin genes in garlic under drought and salt stress with ‘Er Shuizao’ as plant material. Different physiological indicators were detected under drought and salt stress treatments. RT-qPCR was used to detect the expression levels of the candidate aquaporin genes in specific tissues. Finally, we screened AsPIP1-3 as a candidate gene and analyzed its function. The results showed that the relative water content and chlorophyll content of leaves decreased, the O2− production rate increased, and H2O2 accumulated in garlic under drought and salt stress. The activities of SOD, POD, and CAT enzymes first increased and then decreased in garlic. The content of soluble sugar and proline increased to maintain cell osmotic balance, and the content of MDA and relative conductivity continued to increase. Most aquaporin gene expression first increased and then decreased in garlic under drought and salt stress. AsPIP1-3 gene expression is up-regulated under drought and salt stress in garlic. The relative expression was the highest on the 6th day of stress, being related to antioxidant enzyme activity and osmotic regulation. The consistent changes in gene expressions and physiological responses indicated that AsPIP1-3 played a role in resisting garlic osmotic stress. AsPIP1-3 was located on the cell membrane, being consistent with the predicted results of subcellular localization. The germination rate and root length of transgenic Arabidopsis under drought stress were significantly different from the wild type. Drought stress reduced the ROS accumulation of transgenic Arabidopsis, and the antioxidant enzyme activity was significantly higher than the wild type. The relative conductivity and MDA content significantly decreased, and the proline content increased under drought stress. The expression level of the genes related to drought stress response (AtRD22, AtP5CS, AtABF3, and AtLEA) significantly increased. The overexpression of AsPIP1-3 genes improved the drought tolerance of transgenic Arabidopsis plants, showing that AsPIP1-3 proteins enhanced drought tolerance. Our study laid a foundation for exploring the regulatory mechanism of garlic to abiotic stress.

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase–treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti–PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

Wheat (Triticum aestivum L.) is an important food crop mainly grown in arid and semiarid regions worldwide, whose productivity is severely limited by drought stress. Although various E3 ubiquitin (Ub) ligases regulate drought stress, only a few SINA‐type E3 Ub ligases are known to participate in such responses. Herein, we identified and cloned 15 TaSINAs from wheat. The transcription level of TaSINA2B was highly induced by drought, osmotic and abscisic acid treatments. Two‐type promoters of TaSINA2B were found in 192 wheat accessions; furthermore wheat accessions with promoter TaSINA2BII showed a considerably higher level of drought tolerance and gene expression levels than those characterizing accessions with promoter TaSINA2BI that was mainly caused by a 64 bp insertion in its promoter. Enhanced drought tolerance of TaSINA2B‐overexpressing (OE) transgenic wheat lines was found to be associated with root growth promotion. Further, an interaction between TaSINA2B and TaSINA1D was detected through yeast two‐hybrid and bimolecular fluorescence complementation assays. And TaSINA1D‐OE transgenic wheat lines showed similar drought tolerance and root growth phenotype to those observed when TaSINA2B was overexpressed. Therefore, the variation of TaSINA2B promoter contributed to the drought stress regulation, while TaSINA2B, interacting with TaSINA1D, positively regulated drought tolerance by promoting root growth. Summary Statement: TaSINA2B is closely associated to drought tolerance, and the 64 bp insertion in promoter drives the enhanced expression of TaSINA2B. TaSINA2B interact with TaSINA1D, both of which positively regulates drought tolerance and root growth in wheat. [ABSTRACT FROM AUTHOR]

SUMMARY: The non‐seed plants (e.g., charophyte algae, bryophytes, and ferns) have multiple human uses, but their contributions to agriculture and research have lagged behind seed plants. While sharing broadly conserved biology with seed plants and the major crops, non‐seed plants sometimes possess alternative molecular and physiological adaptations. These adaptations may guide crop improvements. One such area is the presence of multiple classes of insecticidal proteins found in non‐seed plant genomes which are either absent or widely diverged in seed plants. There are documented uses of non‐seed plants, and ferns for example have been used in human diets. Among the occasional identifiable toxins or antinutritive components present in non‐seed plants, none include these insecticidal proteins. Apart from these discrete risk factors which can be addressed in the safety assessment, there should be no general safety concern about sourcing genes from non‐seed plant species. Significance Statement: Lower plants such as ferns and mosses are underexplored resources for agriculture. Recent investigations have revealed that lower plants contain genes for insecticidal proteins that are now being leveraged for transgenic crop improvement. [ABSTRACT FROM AUTHOR]

In this study, PsMYB2 was successfully cloned using cDNA from Potentilla sericea as a template. It was used to construct the plant overexpression vector pBI121-PsMYB2-GFP, which was transferred into Arabidopsis thaliana wild-type plants. We studied the gene function using real-time quantitative PCR and performed a preliminary characterization and analysis of the function of PsMYB2 under abiotic stresses. This study showed that under cadmium stress, the gene expression of PsMYB2 gene in roots, stems, and leaves was up to 3-6 times higher than the control. The germination rate of transgenic A. thaliana T3 generation seeds reached more than 95%. The O2·-, H2O2, and malondialdehyde contents of the transgenic P. sericea plant lines were increased but lower than those of the wild-type strain. The superoxide dismutase, peroxidase, and hydrogen peroxidase activities were increased in both wild-type and transgenic strains, and the transgenic strains showed higher enzyme activities than the wild-type. We concluded that PsMYB2 could improve plant resistance to cadmium, which provides a theoretical basis for using transgenic plants to remediate cadmium-contaminated soil and for sustainable land use. [ABSTRACT FROM AUTHOR]

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Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1–0.5 (1–2 weeks), > 0.5 cm (3–4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange (Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase (IPT) and an anti-bacterial peptide (PR1aCB). Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained. Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants (deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange (Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.