Search Results (911)

AT-hook motif nuclear-localized (AHL) proteins are pivotal in plant growth, development, and stress responses. Nevertheless, there is limited research on AHL proteins in Malus sieversii. Our study identified 25 AHL genes from the M. sieversii genome, named MsAHL1–MsAHL25. The encoded protein sequences had lengths ranging from 195 to 554 amino acids, molecular weights from 19.17 to 58.53 kDa, and isoelectric points from 4.67 to 10.09. Chromosomal mapping revealed that these 25 genes were unevenly distributed across 10 chromosomes. Collinearity analysis of AHL genes in M. sieversii implied that gene loss might have occurred during its evolution. The phylogenetic tree classified the AHL proteins of M. sieversii into two subfamilies, showing a close relationship with multiple proteins of M. domestica. Promoter analysis indicated that the AHL genes in M. sieversii harbored numerous stress- and hormone-responsive elements, suggesting their potential role in various stress responses. qRT-PCR analysis of six representative MsAHLs under biotic and abiotic stresses demonstrated that the expression of MsAHL13, MsAHL15, and MsAHL17 was significantly upregulated under salt, drought, and cold stresses, while MsAHL01 expression was inhibited under low-temperature stress. All six MsAHLs were induced by the pathogen Valsa mali. Subcellular localization analysis of the specifically expressed protein MsAHL13 showed its nuclear location. Furthermore, luciferase and yeast two-hybrid assays confirmed the in vitro physical interaction between the MsAHL13 and MsMYB1 proteins. This research offers an important theoretical basis for further exploration of the functional mechanisms of this gene family in responding to environmental stresses. Full article

Superoxide dismutase (SOD) is a key enzyme in the plant antioxidant system. It plays an essential role in plant adversity stress by scavenging excess reactive oxygen species to protect cells from oxidative damage. Isatis indigotica, being a mildly saline-tolerant plant, can be grown in soils containing a certain amount of saline–alkaline content. In order to reveal the SOD gene family members and their potential roles under saline and alkaline stress, the present study used a bioinformatics approach to identify 9 potential IiSOD genes in the I. indigotica genome. It analyzed the expression patterns of SOD family genes (IiSODs) in response to alkaline stress. According to the results of quantitative real-time PCR (qRT-PCR), the expression levels of the IiSOD7 gene significantly increased within 120 h of alkaline stress treatment, while the expression level of the IiSOD8 gene was the highest among all detected genes at 120 h of alkaline stress. The rest of the genes showed different degrees of expression. Alkaline stress showed significant and dynamic changes in the content of indigo and indirubin in leaves of I. indigotica. Finally, the yeast one-hybrid assay confirmed that IiWRKY54 was able to activate the expression of IiSOD2 and IiSOD7. Combined with qRT-PCR analysis, it was further hypothesized that IiWRKY54 might enhance the alkaline tolerance of I. indigotica by regulating the expression of IiSOD2 and IiSOD7. Taken together, this study lays the foundation for elucidating the function of the IiSOD gene in salinity stress tolerance of I. indigotica as well as promoting the genetic breeding of alkaline-tolerant varieties of I. indigotica. Full article

In response to abiotic stress, plants utilize hub protein-mediated signaling networks, with members of the SIMILAR TO RCD ONE (SRO) protein family playing a pivotal role in regulating stress resistance pathways. This study investigates the functional role of the soybean GmRCD1 protein and its interaction mechanisms to elucidate its molecular regulatory network in stress resistance responses. By employing yeast two-hybrid technology to screen a soybean cDNA library under high-salt stress conditions, 17 potential interacting proteins were identified, which include NAC transcription factors (e.g., GmNAC058), ubiquitin–proteasome proteins, and ribosomal proteins. Subsequent validation using GST pull-down and bimolecular fluorescence complementation assays confirmed the direct interaction between GmRCD1 and GmNAC058, which is mediated by the RST domain of GmRCD1 and the C-terminal disordered region (amino acids 288–323) of GmNAC058. Subcellular localization studies revealed that both proteins are nuclear-localized, aligning with their roles in transcriptional regulation. Furthermore, PAR binding assays demonstrated that both GmRCD1 and AtRCD1 can bind to PAR polymers; however, PARP activity analysis revealed that neither protein exhibits catalytic activity, indicating their participation in stress responses via non-enzymatic mechanisms. This study represents the first to elucidate the interaction network and structural basis between soybean GmRCD1 and GmNAC058, providing crucial theoretical support for understanding the multifunctional roles of plant hub proteins in stress resistance regulation and for molecular breeding in soybean. Full article

Dirigent proteins (DIR) are involved in lignan biosynthesis, stress responses, and disease resistance in plants. However, systematic characterization of the DIR gene family in watermelon (Citrullus lanatus) remains limited. Here, we identified 22 ClDIR genes in watermelon using bioinformatics methods, designated ClDIR1 to ClDIR22, which were unevenly distributed across eight chromosomes and classified into three subfamilies (DIR-a, DIR-b/d, DIR-e) based on phylogenetic analysis, with DIR-b/d being the largest. Synteny analysis revealed that tandem duplication primarily drove ClDIR family expansion, and collinear relationships with Arabidopsis, rice, and cucurbit species indicated evolutionary conservation. Cis-acting element analysis showed abundant stress- and hormone-responsive elements in ClDIR promoters, suggesting roles in stress regulation. Tissue-specific expression analysis demonstrated distinct patterns, with most genes highly expressed in roots. Expression profiling under 16 abiotic and biotic stresses showed 18 ClDIR genes responded to stress, with ClDIR8 differentially expressed across all conditions. qRT-PCR validation of six key genes (ClDIR5, ClDIR8, ClDIR9, ClDIR12, ClDIR16, ClDIR22) confirmed their expression patterns under high-temperature, drought, salt, and low-temperature stresses, showing a high degree of consistency with transcriptome data. Subcellular localization indicated ClDIR8 is peroxisome-localized. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays validated two ClDIR8-interacting proteins, Cla97C02G049920 (encoding peroxidase) and Cla97C08G152180 (encoding catalase). These findings provide insights into ClDIR genes in watermelon, highlighting ClDIR8 as a key stress-responsive candidate for further functional studies and breeding. Full article

The apoplast is often the first point of contact between plant cells and invading pathogens, serving as an important site for defense signaling. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein from Phytolacca americana (pokeweed), is localized to the apoplast and is hypothesized to accompany a pathogen to the cytosol, where it would inactivate host ribosomes to prevent pathogen spread. However, it is not known whether PAP interacts with other proteins in the apoplast. In this study, we identified Phytolacca americana cysteine protease 1 (PaCP1), an extracellular cysteine protease, as a novel PAP interactor. Sequence and structural analyses classified PaCP1 as a member of the C1A subfamily of papain-like cysteine proteases. Immunoprecipitation, mass spectrometry, and yeast two-hybrid analysis showed that PAP specifically binds the mature, active form of PaCP1. Curiously, PaCP1 cleaves PAP at its N- and C-termini, generating peptides that enhance MAPK phosphorylation in pokeweed leaves, indicating their potential role in stress signaling. PaCP1 processing of PAP to generate bioactive peptides diversifies the function of a ribosome-inactivating protein beyond its canonical inhibition of translation. Our findings present a novel extracellular role for PAP and advance our understanding of how protein interactions in the apoplast contribute to plant immune responses. Full article

Late-spring frost events severely damage low-chill peach blossoms, causing significant yield losses. Although 5-aminolevulinic acid (ALA) enhances cold tolerance through the PpC3H37-PpWRKY18 module, the regulatory mechanism of ALA on PpC3H37 remains to be elucidated. Using yeast one-hybrid screening with the PpC3H37 promoter as bait, we identified PpDof9 as a key interacting transcription factor. A genome-wide analysis revealed 25 PpDof genes in peaches (Prunus persica). These genes exhibited variable physicochemical properties, with most proteins predicted as nuclear-localized. Subcellular localization experiments in tobacco revealed that PpDof9 was localized to the nucleus, consistent with predictions. A synteny analysis indicated nine segmental duplication pairs and tandem duplications on chromosomes 5 and 6, suggesting duplication events drove family expansion. A conserved motif analysis confirmed universal presence of the Dof domain (Motif 1). Promoter cis-element screening identified low-temperature responsive (LTR) elements in 12 PpDofs, including PpDof1, PpDof8, PpDof9, and PpDof25. The quantitative real-time PCR (qRT-PCR) results showed that PpDof1, PpDof8, PpDof9, PpDof15, PpDof16, and PpDof25 were significantly upregulated under low-temperature stress, and this upregulation was further enhanced by ALA pretreatment. Our findings demonstrate ALA-mediated modulation of specific PpDof TFs in cold response and provide candidates (PpDof1, PpDof9, PpDof8, PpDof25) for enhancing floral frost tolerance in peaches. Full article

The innate immune response is an important defense against invading pathogens. Stimulator of interferon gene (STING) plays an important role in the cyclic GMP-AMP synthase (cGAS)-mediated activation of type I IFN responses. However, some viruses have evolved the ability to inhibit the function of STING and evade the host antiviral defenses. Understanding both the mechanism of action and the viruses targets of STING effector is important because of their importance to evade the host antiviral defenses. In this study, the STING (IpSTING) of Ictalurus punctatus was first identified and characterized. Subsequently, the yeast two-hybrid system (Y2HS) was used to screen for proteins from channel catfish virus (CCV, Ictalurid herpesvirus 1) that interact with IpSTING. The ORFs of the CCV were cloned into the pGBKT7 vector and expressed in the AH109 yeast strain. The bait protein expression was validated by autoactivation, and toxicity investigation compared with control (AH109 yeast strain transformed with empty pGBKT7 and pGADT7 vector). Two positive candidate proteins, ORF41 and ORF65, were identified through Y2HS screening as interacting with IpSTING. Their interactions were further validated using co-immunoprecipitation (Co-IP). This represented the first identification of interactions between IpSTING and the CCV proteins ORF41 and ORF65. The data advanced our understanding of the functions of ORF41 and ORF65 and suggested that they might contribute to the evasion of host antiviral defenses. However, the interaction mechanism between IpSTING, and CCV proteins ORF41 and ORF65 still needs to be further explored. Full article

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

In order to mitigate the reduction in soybean yield caused by soil salinization, a soybean gene, GmSNF4, which promotes plant tolerance to salt–alkali stress, was identified in this study. The STRING database was used to predict the interaction between GmSNF4 and GmPKS4. The GmPKS4 gene was experimentally shown to be involved in salt–alkali stress tolerance. Firstly, the yeast two-hybrid technique and bimolecular fluorescence complementation (BiFC) technique were used to confirm the interaction between GmSNF4 and GmPKS4: the AMPK-CBM-CBS1 conserved domain was thereby determined to be the region of the GmSNF4 protein involved in the interaction. Secondly, the GmSNF4 gene was induced by salt–alkali stress according to qRT-PCR analysis, and the GmSNF4 protein was localized in the nucleus and cytoplasm. Finally, analysis of GmSNF4’s role in resistance to salt–alkali stress in transgenic soybean plants showed that transgenic lines had better phenotypic, physiological, and stress-related gene expression than non-transgenic soybeans. Thus, GmSNF4 may play a significant role in plant salt–alkali stress tolerance. Full article

Drought stress poses a major constraint on global crop productivity. Although aspartic proteases (APs) are primarily characterized in plant disease resistance, their roles in abiotic stress adaptation remain largely unexplored. Here, we demonstrate that rice (Oryza sativa) OsAP4 critically regulates drought stress tolerance at the seedling stage. Genetic manipulation through overexpression (OsAP4-OE) or CRISPR knockout (OsAP4-KO) resulted in significantly reduced or enhanced stress tolerance compared to wild-type plants, respectively. Through integrated approaches including yeast two-hybrid, bimolecular fluorescence complementation, pull-down, co-immunoprecipitation, and protein degradation assays, we established that OsAP4 physically interacts with and destabilizes OsCATA/OsCATC, two catalase enzymes responsible for reactive oxygen species (ROS) scavenging. Importantly, OsAP4 modulates ROS production under drought stress treatment conditions. Together, these findings reveal a novel OsAP4-OsCATA/OsCATC regulatory module governing rice drought stress responses. Full article

Background/Objectives: The potential of DNA as an information-dense storage medium has inspired a broad spectrum of creative systems. In particular, hybrid biomolecular systems that integrate new materials and chemistries with DNA could drive novel functions. In this work, we explore the potential for proteins to serve as molecular file addresses. We stored DNA-encoded data in yeast and leveraged yeast surface display to readily produce the protein addresses and make them easy to access on the cell surface. Methods: We generated yeast populations that each displayed a distinct protein on their cell surfaces. These proteins included binding partners for cognate antibodies as well as chromatin-associated proteins that bind post-translationally modified histone peptides. For each specific yeast population, we transformed a library of hundreds of DNA sequences collectively encoding a specific image file. Results: We first demonstrated that the yeast retained file-encoded DNA through multiple cell divisions without a noticeable skew in their distribution or a loss in file integrity. Second, we showed that the physical act of sorting yeast displaying a specific file address was able to recover the desired data without a loss in file fidelity. Finally, we showed that analog addresses can be achieved by using addresses that have overlapping binding specificities for target peptides. Conclusions: These results motivate further exploration into the advantages proteins may confer in molecular information storage. Full article

Grapevine leafroll disease is one of the most devastating diseases in the global viticulture industry. Grapevine leafroll-associated virus 4 is one of the main pathogens causing this disease. In this study, we obtained the complete genome sequences of two Chinese isolates of GLRaV-4 from ‘Baisainie’ and ‘Fantasy Seedless’ grapevines through high-throughput sequencing and overlapping RT-PCR combined with RACE technology. The sequences contain 13,814 and 13,824 nucleotides and code six open reading frames, respectively. Phylogenetic trees based on the coat protein (CP) and heat shock protein 70 (HSP70) genes show that in addition to other GLRaV-4 strains (strains 5, 6, 9, Pr, and Car), the GLRaV-4 strains were divided into two distinct groups. The two isolates obtained in this study were classified into separate branches within GLRaV-4 Group 1. Additionally, we systematically investigated the interactions between the proteins encoded by GLRaV-4 using the yeast two-hybrid and bimolecular fluorescence complementation techniques. We found significant interactions between the GLRaV-4-encoded p23 and HSP70 and CP. This study first reports the complete genomes of two different GLRaV-4 isolates from China and suggests that p23 protein encoded by GLRaV-4 may play an important role in viral pathogenicity due to its interactions with the other two proteins. Full article

The investigation of phosphorus metabolism and regulatory mechanisms is conducive to maintaining stable production of crops within a low-phosphorus environment. In phosphorus signal transduction, a few phosphorus starvation response (PHR) transcription factors were identified to bind to the characteristic cis-element, namely the PHR1 binding sequence (P1BS). While the molecular function of the PHR transcription factor has been intensively elucidated, here, we explore a novel transcription factor, BnaA01.KAN3, that undergoes specific binding to the P1BS by yeast one-hybrid and electrophoretic mobility shift assays, and its expression is induced with low-phosphorus stress. BnaA01.KAN3 possessed transcriptional activation and was located in the nucleus. The spatiotemporal expression pattern of BnaA01.KAN3 exhibited tissue specificity in developmental seed, and its expression level was especially high 25–30 days after pollination. Regarding the phenotype analysis, the independent heterologous overexpression lines of BnaA01.KAN3 in Arabidopsis thaliana exhibited not only significantly longer taproots but also an increased number of lateral roots compared to that of the wild type undergoing low-phosphorus treatment, while no differences were seen under normal phosphorus conditions. Furthermore, these lines showed higher anthocyanin and inorganic phosphorus contents with normal and low-phosphorus treatment, suggesting that BnaA01.KAN3 could enhance phosphorus uptake or remobilization to cope with low-phosphorus stress. In summary, this study characterized the transcription factor BnaA01.KAN3 that modulates low-phosphate adaptation and seed development, providing insights for improving phosphorus use efficiency and yield traits in Brassica napus. Full article

Hemerocallis spp. exhibit distinct flower opening times, categorized into nocturnal and diurnal types. Previous studies have demonstrated that the circadian clock and CONSTANS (CO) genes play crucial roles in regulating flowering in Hemerocallis. However, the key genes that integrate flowering pathways remain largely unknown. To address this gap, we identified potential homologs of the FLOWERING LOCUS T (FT) gene in Hemerocallis. A yeast one-hybrid assay revealed that HfCOL2 and HfLHY directly bind to the HfFT1 and HfFT2 promoters, thereby activating FT transcription. The expression analysis reveals that HfCOL2 expression rhythms not only display opposing patterns between nocturnal and diurnal opening types of Hemerocallis but also between leaf and flower tissues. The peak expression of HfCOL2 in flowers aligns closely with the respective opening times of diurnally and nocturnally flowering Hemerocallis. The overexpression of HfCOL2 in tobacco plants led to early flowering and prolonged flower longevity. In Hemerocallis, the HfCOL2 gene plays a pivotal role not only in photoperiod-induced flowering but also in the circadian rhythm-mediated regulation of flower opening time. Due to the limited availability of plant materials exhibiting distinct flower opening rhythms, research in this area has been constrained. Identifying the key genes in the flowering pathway of Hemerocallis can facilitate a better understanding of the mechanisms by which plants respond to circadian rhythms. Full article

Chaperonin 60 proteins plays an important role in plant growth and development as well as the response to abiotic stress. As part of the protein homeostasis system, molecular chaperones have attracted increasing attention in recent years due to their involvement in the folding and assembly of key proteins in photosynthesis. However, little is known about the function of maize chaperonin 60 protein. In the study, a gene encoding the chaperonin 60 proteins was cloned from the maize inbred line B73, and named ZmCpn60-3. The gene was 1, 818 bp in length and encoded a protein consisting of 605 amino acids. Phylogenetic analysis showed that ZmCpn60-3 had high similarity with OsCPN60-1, belonging to the β subunits of the chloroplast chaperonin 60 protein family, and it was predicted to be localized in chloroplasts. The ZmCpn60-3 was highly expressed in the stems and tassels of maize, and could be induced by exogenous plant hormones, mycotoxins, and pathogens; Overexpression of ZmCpn60-3 in Arabidopsis improved the resistance to Pst DC3000 by inducing the hypersensitive response and the expression of SA signaling-related genes, and the H₂O₂ and the SA contents of ZmCpn60-3-overexpressing Arabidopsis infected with Pst DC3000 accumulated significantly when compared to the wild-type controls. Experimental data demonstrate that flg22 treatment significantly upregulated transcriptional levels of the PR1 defense gene in ZmCpn60-3-transfected maize protoplasts. Notably, the enhanced resistance phenotype against Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) in ZmCpn60-3-overexpressing transgenic lines was specifically abolished by pretreatment with ABT, a salicylic acid (SA) biosynthetic inhibitor. Our integrated findings reveal that this chaperonin protein orchestrates plant immune responses through a dual mechanism: triggering a reactive oxygen species (ROS) burst while simultaneously activating SA-mediated signaling cascades, thereby synergistically enhancing host disease resistance. Additionally, yeast two-hybrid assay preliminary data indicated that ZmCpn60-3 might bind to ZmbHLH118 and ZmBURP7, indicating ZmCpn60-3 might be involved in plant abiotic responses. The results provided a reference for comprehensively understanding the resistance mechanism of ZmCpn60-3 in plant responses to abiotic or biotic stress. Full article