Search Results (134)

As a pivotal model organism in Lepidoptera research, the silkworm (Bombyx mori) holds significant importance in life science due to its economic value and biotechnological applications. Advancements in proteomics and bioinformatics have enabled substantial progress in characterizing the B. mori proteome. Systematic screening and identification of protein–protein interactions (PPIs) have progressively elucidated the molecular mechanisms governing key biological processes, including viral infection, immune regulation, and growth development. This review comprehensively summarizes traditional PPI detection techniques, such as yeast two-hybrid (Y2H) and immunoprecipitation (IP), alongside emerging methodologies such as mass spectrometry-based interactomics and artificial intelligence (AI)-driven PPI prediction. We critically analyze the strengths, limitations, and technological integration strategies for each approach, highlighting current field challenges. Furthermore, we elaborate on the molecular regulatory networks of Bombyx mori nucleopolyhedrovirus (BmNPV) from multiple perspectives: apoptosis and cell cycle regulation; viral protein invasion and trafficking; non-coding RNA-mediated modulation; metabolic reprogramming; and host immune evasion. These insights reveal the dynamic interplay between viral replication and host defense mechanisms. Collectively, this synthesis aims to provide a robust theoretical foundation and technical guidance for silkworm genetic improvement, infectious disease management, and the advancement of related biotechnological applications. Full article

The 14-3-3 proteins play crucial roles in regulating plant growth, development, signal transduction and abiotic stress responses. However, there exists a scarcity of research on the role of 14-3-3 proteins in responding to abiotic stress in apples. In this study, we isolated the MdGRF22 gene from the apple 14-3-3 family. Through the screening of interacting proteins and genetic transformation of Arabidopsis thaliana and apple callus tissues, the function of the MdGRF22 gene under drought stress was verified. The coding sequence (CDS) of MdGRF22 consists of 786 bp and encodes for 261 amino acids. Through sequence alignment, the conserved 14-3-3 domain was identified in MdGRF22 and its homologous genes, which also share similar gene structures and conserved motifs. Subcellular localization revealed that the MdGRF22 protein was predominantly located in the cytoplasm and cell membrane. The yeast two-hybrid (Y2H) analysis demonstrated a possible interaction between MdGRF22 and MdSK. In addition, MdGRF22 transgenic plants generally exhibited lower superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities, higher malondialdehyde (MDA) levels and relative electrolyte leakage under drought conditions compared with wild-type (WT) plants. Our study suggests that MdGRF22 may reduce the drought resistance of transgenic A. thaliana and callus tissues by interacting with MdSK. This study provides a theoretical basis for further exploring the function of 14-3-3 family genes. Full article

Dendrobium officinale is a traditional and valuable medicinal herb, with extensive research conducted on its polysaccharides, alkaloids, and other components, yet studies on anthocyanins remain limited. In this study, we analyzed the expression levels of GST family genes in green and purplish D. officinale and found that DoGSTF11 is highly expressed in the purplish variety. DoGSTF11 is localized to the nucleus and cell membrane but lacks transcriptional activation activity. Overexpression of DoGSTF11 in tomato enhances anthocyanin accumulation, suggesting a role in anthocyanin sequestration or transport. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays further revealed that DoGSTF11 interacts with DoGST31, while DoIDD12 and DoNF-YB3 are potential transcriptional regulators based on promoter-binding assays and expression correlation. In conclusion, our study demonstrates that DoGST11 positively regulates anthocyanin accumulation in D. officinale. These findings provide valuable insights into the metabolic engineering of flavonoids in D. officinale. Full article

BEL1-like homeodomain protein 3 (BLH3) plays a crucial role in plant development. However, its involvement in the salt stress response has not been studied. In this study, we investigated the molecular mechanism underlying the response of LpBLH3 to salt stress in Lilium pumilum (L. pumilum) using various techniques, including quantitative PCR (RT-qPCR), determination of physiological indices of plant after Saline-Alkali stress, yeast two-hybrid screening, luciferase complementation imaging (LCI), and chromosome walking to obtain the promoter sequence, analyzed by PlantCARE, electrophoretic mobility shift assay (EMSA), and then dual-luciferase reporter assay(LUC). RT-qPCR analysis revealed that LpBLH3 is most highly expressed in the leaves of L. pumilum. The expression of LpBLH3 peaks at 24 or 36 h in the leaves under different saline stress. Under various treatments, compared to the wild type (WT), the LpBLH3 overexpression lines exhibited less chlorosis and leaf curling and stronger photosynthesis. The overexpression of LpBLH3 can enhance lignin accumulation in root and stem by positively modulating the expression of crucial genes within the lignin biosynthesis pathway. Y2H and LCI analyses demonstrated that LpBLH3 interacts with LpKNAT3. Additionally, EMSA and LUC analyses confirmed that LpBLH3 can bind to the promoter of LpABI5 and upregulate the expression of ABI5 downstream genes (LpCAT1/LpATEM/LpRD29B). In summary, LpBLH3 enhances the plant’s salt tolerance through the ABA pathway and lignin synthesis. This study can enrich the functional network of the BLH transcription factor family, obtain Lilium pumilum lines with good saline-alkali resistance, expand the planting area of Lilium pumilum, and improve its medicinal and ornamental values. Additionally, the functional analysis of the BLH transcription factor family provides new insights into how crops adapt to the extreme growth environment of saline-alkali soils. Full article

Spinach (Spinacia oleracea L.), a globally consumed, nutrient-dense vegetable, contains diverse vitamins and minerals. However, elevated temperatures can constrain yield by interrupting leaf development and photosynthetic efficiency. The mitochondrial transcription termination factor (mTERF) family, which regulates organellar gene expression, plays crucial roles in plant growth and photosynthetic regulation. Thus, characterization of the spinach mTERF (SomTERF) family is critical for elucidating thermotolerance mechanisms in this crop. In this study, we systematically identified 31 SomTERF genes from the spinach genome, which are distributed across five chromosomes and nine unassembled genomic scaffolds. Subcellular localization predictions indicated that these proteins predominantly target chloroplasts and mitochondria. Conserved domain analyses confirmed that all SomTERF proteins possess canonical mTERF domains and ten conserved motifs. Phylogenetic clustering segregated these proteins into nine distinct subgroups (I–IX), with significant divergence observed in gene copy numbers among subgroups. Cis-element screening identified an abundance of heat-, cold-, and hormone-responsive motifs within SomTERF promoter regions. Notably, seven members (including SomTERF5) exhibited pronounced enrichment of heat shock elements (HSEs). Organ-specific expression profiling revealed preferential leaf expression of these seven genes. Comparative RT-qPCR in heat-sensitive (Sp73) and heat-tolerant (Sp75) cultivars under thermal stress demonstrated genotype-dependent expression dynamics. Functional validation of SomTERF5 was achieved through cloning, and transgenic Arabidopsis overexpressing SomTERF5 showed significantly enhanced thermotolerance, as evidenced by improved survival rates following heat treatment. Yeast two-hybrid (Y2H) assays further revealed physical interaction between SomTERF5 and SopTAC2. This study provides a comprehensive foundation for understanding mTERF-mediated developmental regulation and advanced molecular breeding strategies for developing heat-resilient spinach varieties. Full article

The Rare Cold-Inducible 2 (RCI2) gene encodes a conserved hydrophobic peptide that plays a crucial role in ion homeostasis, membrane stability, and responses to abiotic stress. In this study, six members of the MsRCI2 gene family were identified in Medicago sativa L., all of which contain highly conserved PMP₃ domains. Comparative collinearity analysis revealed syntenic relationships between M. sativa and M. truncatula, with each gene displaying distinct expression profiles under various stress conditions. Among them, MsRCI2B was significantly upregulated in response to salt stress. Alfalfa plants overexpressing MsRCI2B exhibited enhanced salt tolerance, as evidenced by increased antioxidant enzyme activities and reduced accumulation of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and superoxide anion (O₂⁻) compared to wild-type plants. Furthermore, the transgenic lines maintained better Na⁺/K⁺ homeostasis under salt stress, reflected by a lower Na⁺/K⁺ ratio and significantly elevated expression of key ion transport genes, including MsSOS1, MsAKT1, and MsNHX1. To elucidate the molecular mechanisms underlying MsRCI2B function, a yeast two-hybrid (Y2H) screen identified 151 potential interacting proteins. Gene Ontology (GO) enrichment analysis revealed that these interactors are mainly involved in antioxidant defense and ion transport. Further validation confirmed direct interactions between MsRCI2B and both calmodulin (CaM) and vacuola H⁺-ATPase (V-H⁺-ATPase), suggesting that MsRCI2B contributes to ion homeostasis through interactions with CaM and V-H⁺-ATPase, thereby promoting Na⁺/K⁺ balance and enhancing salt tolerance. These findings provide new insights into the role of MsRCI2B in salt stress responses and underscore its potential as a genetic target for enhancing salinity tolerance in forage crops. Full article

Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with most land plants, facilitating mineral nutrient uptake in exchange for photosynthates. As one of the most commercially used rootstocks in citrus, Poncirus trifoliata heavily depends on AM fungi for nutrient absorption. The GRAS gene family plays essential roles in plant growth and development, signaling transduction, and responses to biotic and abiotic stresses. However, the identification and functional characterization of GRAS family genes in P. trifoliata remains largely unexplored. In this study, a comprehensive genome-wide analysis of PtGRAS family genes was conducted, including their identification, physicochemical properties, phylogenetic relationships, gene structures, conserved domains, chromosome localization, and collinear relationships. Additionally, the expression profiles and protein interaction of these genes under AM symbiosis were systematically investigated. As a result, 41 GRAS genes were identified in the P. trifoliata genome, and classified into nine distinct clades. Collinearity analysis revealed seven segmental duplications but no tandem duplications, suggesting that segmental duplication played a more important role in the expansion of the PtGRAS gene family compared to tandem duplication. Additionally, 18 PtGRAS genes were differentially expressed in response to AM symbiosis, including orthologs of RAD1, RAM1, and DELLA3 in P. trifoliata. Yeast two-hybrid (Y2H) screening further revealed that PtGRAS6 and PtGRAS20 interacted with both PtGRAS12 and PtGRAS18, respectively. The interactions were subsequently validated through bimolecular fluorescence complementation (BiFC) assays. These findings underscored the crucial role of GRAS genes in AM symbiosis in P. trifoliata, and provided valuable candidate genes for improving nutrient uptake and stress resistance in citrus rootstocks through molecular breeding approaches. Full article

Background: The attractive inflorescence of Chrysanthemum morifolium, its capitulum, is always composed of ray (female, zygomorphy) and disc (bisexual, actinomorphy) florets, but the formation mechanism remains elusive. The gene diversification pattern of the ECE (CYC/TB1) clade has been speculated to correlate with the capitulum. Within the three subclades of ECE, the involvement of CYC2 in defining floret identity and regulating flower symmetry has been demonstrated in many species of Asteraceae, including C. morifolium. Differential expression of the other two subclade genes, CYC1 and CYC3, in different florets has been reported in other Asteraceae groups, yet their functions in flower development have not been investigated. Methods: Here, a CYC1 gene, CmCYC1a, was isolated and its expression pattern was studied in C. morifolium. The function of CmCYC1a was identified with gene transformation in Arabidopsis thaliana and yeast two-hybrid (Y2H) assays were performed to explore the interaction between CmCYC1 and CmCYC2. Results: CmCYC1a was expressed at higher levels in disc florets than in ray florets and the expression of CmCYC1a was increased in both florets during the flowering process. Overexpression of CmCYC1a in A. thaliana changed flower symmetry from actinomorphic to zygomorphic, with fewer stamens. Furthermore, CmCYC1a could interact with CmCYC2b, CmCYC2d, and CmCYC2f in Y2H assays. Conclusions: The results provide evidence for the involvement of CmCYC1a in regulating flower symmetry and stamen development in C. morifolium and deepen our comprehension of the contributions of ECE genes in capitulum formation. Full article

Thioredoxin z (TRX z) plays a significant role in chloroplast development by regulating the transcription of chloroplast genes. In this study, we identified a pentatricopeptide repeat (PPR) protein, rice albino seedling-lethal (RAS), that interacts with OsTRX z. This interaction was initially discovered by using a yeast two-hybrid (Y2H) screening technique and was further validated through Y2H and bimolecular fluorescence complementation (BiFC) experiments. RAS contains 16 PPR motifs and features a small MutS-related (SMR) domain at its C-terminus. CRISPR/Cas9-generated ras mutants exhibited an albino seedling-lethal phenotype characterized by abnormal chloroplast structures and a significantly reduced chlorophyll content. RAS localizes to the chloroplast and is predominantly expressed in young leaves. Mutations in RAS affect RNA editing at the rpl2, rps14, and ndhA sites, as well as RNA splicing at the rpl2, atpF, and ndhA transcripts within the chloroplast. Furthermore, the expression levels of genes associated with chloroplast formation are altered in the ras mutant. Both OsTRX z and RAS were found to interact with chloroplast signal recognition particle (cpSRP) proteins, indicating that their proper localization within the chloroplast may be dependent on the SRP pathway. Collectively, our findings highlight the critical role of RAS in chloroplast development, as it is involved in RNA processing and the regulation of chloroplast gene expression. Full article

The recent coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has exerted considerable impact on global health. To prepare for rapidly mutating viruses and for the forthcoming pandemic, effective therapies targeting the critical stages of the viral life cycle need to be developed. Viruses are dependent on the interaction between the receptor-binding domain (RBD) of the viral Spike (S) protein (S-RBD) and the angiotensin-converting enzyme 2 (ACE2) receptor to efficiently establish infection and the following replicate. Targeting this interaction provides a promising strategy to inhibit the entry process of the virus, which in turn has both preventive and therapeutic effects. In this study, we developed a robust and straightforward assay based on the Yeast-Two Hybrid system (Y2H) for identifying inhibitors targeting the S-RBD-ACE2 interaction of SARS-CoV-2. Through high-throughput screening, two compounds were identified as potential entry inhibitors. Among them, IMB-1C was superior in terms of pseudovirus entry inhibition and toxicity. It could bind to both ACE2 and S-RBD and induce conformational change in the S-RBD+ACE2 complex. This is the first study to verify the feasibility of utilizing the Y2H system to discover potent SARS-CoV-2 inhibitors targeting the receptor recognition stage. This approach may also be applied in the discovery of other virus receptor recognition inhibitors. Full article

The determinate inflorescence trait of Brassica napus L. is associated with various desirable agricultural characteristics. BnTFL1s (BnaA10.TFL1 and BnaC09.TFL1), which encode the transcription factor TERMINAL FLOWER 1 (TFL1), have previously been identified as candidate genes controlling this trait through map-based cloning. However, the mechanism underlying the effects of the BnTFL1 proteins remains unclear. Further, proteins generally interact with each other to fulfill their biological functions. The objective of this study was to construct a cDNA library of the shoot apical meristem (SAM) of B. napus and screen for proteins that interact with BnTFL1s, to better understand its mechanism of action. The recombination efficiency of the yeast two-hybrid (Y2H) library that we constructed was 100%, with insertion fragment lengths ranging from 750 to 2000 bp and a capacity of approximately 1.44 × 10⁷ CFUs (colony-forming units), sufficient for screening protein interactions. Additionally, the bait vector pGBKT7-BnTFL1s was transformed into yeast cells alongside positive and negative controls, demonstrating no toxicity to the yeast cells and no self-activation. This bait was used to screen the SAM cDNA library of B. napus, ultimately identifying two BnTFL1s-interacting proteins: 14-3-3-like protein GF14 omega GRF2. These interactions were verified through one-to-one interaction experiments. This study provides a foundation for further research on the biological functions of the BnTFL1s genes and their regulatory role in inflorescence formation in B. napus, while providing a reference for studying similar mechanisms in other plants. Full article

The bHLH (basic Helix–Loop–Helix) transcription factor serves as pivotal controller in plant growth and development. In a previous study, the overexpression of SlUPA-like in Solanum lycopersicum L. Ailsa Craig (AC⁺⁺) altered the JA (Jasmonic acid) response and endogenous GA (Gibberellic acid) content. However, the detailed regulation mechanism was not fully explored. In the present research, we found that the overexpression of SlUPA-like influenced the accumulation of GA, JA and BR (Brassinolide). RNA-Seq data illustrated that the expression levels of genes related to these plant hormones were significantly affected. Additionally, the interaction of SlUPA-like with SlMYB21, SlMYC2 and SlDELLA was characterized by employing Y2H (Yeast Two-Hybrid) and BiFC (Bimolecular Fluorescence Complementation) assay. Furthermore, Dual-LUC (Dual-Luciferase) assay and EMSA (Electrophoretic Mobility Shift Assay) identified that SlUPA-like directly targeted the E-box motif in the promoter of SlGID2 and activated the transcription of SlGID2. These results shed light on the potential role of SlUPA-like in mediating crosstalk among multiple plant hormones and established a robust theoretical framework for further unraveling the functions of SlUPA-like transcription factors in the context of plant growth and hormone signal transduction. Full article

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation. Full article

The IV subfamily of receptor-like cytoplasmic kinase (RLCK-IV), known as calcium-binding receptor-like cytoplasmic kinases (CRCKs), plays a vital role in plant signal transduction, particularly in coordinating growth and responses to abiotic stresses. However, our comprehension of CRCK genes in Populus deltoides, a species characterized as fast-growing and pest-resistant but with drought intolerance, is limited. Here, we identify 6 members of the CRCK subfamily on a genome-wide scale in P. deltoides, denoted as PdeCRCK1–PdeCRCK6. An evolutionary and structural analysis revealed highly conserved kinase catalytic domains across all PdeCRCKs, characterized by calmodulin (CaM)-binding sites and serine (Ser)/threonine (Thr) phosphorylation sites. The cis-acting elements of promoters indicated the presence of responsive elements for plant hormones, abiotic stresses, and transcription factor binding sites, which is supported by the distinct transcriptional expression patterns of PdeCRCKs under abscisic acid (ABA), polyethylene glycol (PEG), and mannitol treatments. A transient overexpression of PdeCRCK3/5/6 in tobacco (Nicotiana benthamiana) leaves indicated their involvement in reactive oxygen species (ROS) scavenging, polyamine gene synthesis, and ABA signaling pathway modulation. Immunoprecipitation–Mass Spectrometry (IP–MS) and a yeast two-hybrid (Y2H) assay showed that PdeCRCK6 interacted with AAA-type ATPase proteins and ubiquitin, suggesting its potential function in being involved in chloroplast homeostasis and the 26S ubiquitin protease system. Taken together, these findings offer a comprehensive analysis of the RLCK-IV subfamily members in P. deltoides, especially laying a foundation for revealing the potential mechanism of PdeCRCK6 in response to osmotic stresses and accelerating the molecular design breeding of drought tolerance in poplar. Full article

Brassinazole-resistant (BZR) transcription factors are important transcription factors in Brassinosteroid (BR)-responsive gene expression. However, limited knowledge exists regarding the BZR genes in wheat and a limited number of BZR family genes have been previously reported in wheat. In this study, the synteny analyses of the TaBZR genes suggested that gene duplication events have played an essential role in the TaBZR family during evolution. The results of RT-qPCR and transcriptome data analyses exhibited remarkable expression patterns in the BZR genes in different tissues and under different treatments. The yeast two-hybrid (Y2H) screen result showed that the TaBZR2.1 protein interacts with Argonaute 4 (AGO4). Taken together, our results not only provide us a basis for understanding the molecular characteristics and expression patterns of the TaBZR family genes but also offered the functional characterization of TaBZR2.1 in wheat. Full article