Recognition Site Research Articles

MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis.

Various signaling pathways are essential for both the innate immune response and the maintenance of cell homeostasis, requiring coordinated interactions among them. In this study, a mutation in the caspase-1 recognition site within MyD88 abolished inflammasome-dependent negative regulation, causing phenotypic changes in mice with some similarities to human NEMO-deficiencies. The MyD88D162E mutation reduced MyD88 protein levels and colon inflammation in DSS-induced colitis mice but did not affect cytokine expression in bone marrow-derived macrophages (BMDMs). However, compared to MyD88wt counterparts, MyD88D162E BMDMs had increased oxidative stress and dysfunctional mitochondria, along with reduced prosurvival Bcl-xL and BTK expression, rendering cells more prone to apoptosis, exacerbated by ibrutinib treatment. NF-κB activation by lipopolysaccharide mitigated this sensitive phenotype. These findings underscore the importance of MyD88wt signaling for NF-κB activation, protecting against macrophage premature apoptosis at resting state. Targeting MyD88 quantity rather than just its signaling could be a promising strategy for MyD88-driven lymphoma treatment.

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Site-selectively Phosphorylated Hsp90 C-terminal Domain Variants Provide Access to Deciphering the Chaperone Code.

The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.

β-d-Ribofuranose as a Core with a Phosphodiester Moiety as the Enzyme Recognition Site for Codrug Development.

An ideal codrug design should be able to control drug release, offer selectivity during drug delivery, and break down into non-toxic fragments after biodegradation. Our design incorporates d-ribofuranose as the core, with carbamate and carbonate groups as linking joints, a phosphodiester moiety as an enzyme recognition site, and lenalidomide and paclitaxel as the constituent drugs. The codrug synthesis involves seven steps with a 33% overall yield. The target codrug increases its water solubility 685 times versus paclitaxel.

Design and Evaluation of Peptide Inhibitors Targeting the Dimerization of SARS-CoV-2 Main Protease.

The severe acute respiratory syndrome virus 2 (SARS-CoV-2) seriously impacted public health. The evolutionarily conserved viral chymotrypsin-like main protease (Mpro) is an important target for anti-SARS-CoV-2 drug development. Previous studies have shown that the eight N-terminal amino acids (N8) of SARS-CoV Mpro are essential for its dimerization, and are used to design inhibitors against SARS-CoV Mpro dimerization. Here, we established a simple readout assay using SDS-PAGE and Coomassie blue staining to measure inhibitory activity of N8 peptide derived from SARS-CoV-2 Mpro. To optimize its inhibitory effect, we then modified the side-chain length, charge, and hydrophilicity of the N8 peptide, and introduced a mutated Mpro recognition sequence. As a result, we obtained a series of potent peptide inhibitors against SARS-CoV-2 Mpro, with N8-A24 being the most efficient with an IC50 value of 1.44 mM. We observed that N8-A24 reduced Mpro dimerization with an IC50 value of 0.86 mM. Molecular docking revealed that N8-A24 formed hydrogen bond interactions with critical dimeric interface residues, thus inhibiting its dimerization and activity. In conclusion, our study not only discovers a series of peptide inhibitors targeting the SARS-CoV-2 Mpro dimerization, but also provides a promising strategy for the rational design of new inhibitors against COVID-19.

Three Years of Google Earth Engine-Based Archaeological Surveys in Iraqi Kurdistan: Results from the Ground

This paper presents the results of a three-year survey (2021–2023), conducted in an area of approximately 356 km2 in Iraqi Kurdistan with the aim of identifying previously undetected archaeological sites. Thanks to the development of a multi-temporal approach based on open multispectral satellite data, greater effectiveness was achieved for the recognition of archaeological sites when compared to the use of single archival or freely accessible satellite images, which are typically employed in archaeological research. In particular, the Google Earth Engine services allowed for the efficient utilization of cloud computing resources to handle hundreds of remote sensing images. Using different datasets, namely Landsat 5, Landsat 7 and Sentinel-2, several products were obtained by processing entire stacks of images acquired at different epochs, thus minimizing the adverse effects on site visibility caused by vegetation, crops and cloud coverage and permitting an effective visual inspection and site recognition. Furthermore, spectral signature analysis of every potential site complemented the method. The developed approach was tested on areas that belong to the Land of Nineveh Archaeological Project (LoNAP) and the Upper Greater Zab Archaeological Reconnaissance (UGZAR) project, which had been intensively surveyed in the recent past. This represented an additional challenge to the method, as the most visible and extensive sites (tells) had already been detected. Three years of direct ground-truthing in the field enabled assessment of the outcomes of the remote sensing-based analysis, discovering more than 60 previously undetected sites and confirming the utility of the method for archaeological research in the area of Northern Mesopotamia.

A Rapidly Inducible DNA Double-Strand Break to Monitor Telomere Formation, DNA Repair, and Checkpoint Activation.

The study of processes that govern genome integrity has been augmented by the ability to create a precise DNA double-strand break (DSB) in a short period of time that allows the kinetics of DNA metabolism and protein recruitment to be followed. Defined DSBs are made by expressing endonucleases with long recognition sites that are rare or absent in the genome, and require that the endonuclease is only active when induced. Research in this area in Schizosaccharomyces pombe was limited because rapidly inducible promoters were not available until around 2005, and several rapidly inducible DSB systems are now available. Here, we describe a system to rapidly induce a modified I-SceI endonuclease that can generate a DSB 20min after induction. I-SceI has no recognition sites in the S. pombe genome, allowing the introduction of complex substrates to monitor the effects of a new DSB in real time. This chapter describes how I-SceI can be most efficiently induced and a simple cell length measurement assay to monitor cell cycle checkpoint activation from a single DSB.

NIR-II Fluorescence/Photoacoustic Dual Ratiometric Probes with Unique Recognition Site for Quantitatively Visualizing H2S2 in Vivo.

Hydrogen persulfide (H2S2) plays a significant role in redox biology and signal transduction; therefore, quantitative visualization of H2S2 in the deep tissue of living organisms is essential for obtaining reliable information about relevant pathophysiological processes directly. However, currently reported H2S2 probes are unsuitable for this purpose because of their poor selectivity for many polysulfide species or their short wavelength, which hinders precise imaging in deep tissues. Herein, for the first time, we report a unique H2S2-mediated dithiole formation reaction. Based on this reaction, we construct the first NIR-II fluorescence (FL) and photoacoustic (PA) dual-ratiometric probe (NIR-II-H2S2) for quantitatively visualizing H2S2 in vivo. This probe shows dual-ratiometric NIR-II fluorescence (I840/I1000, 107-fold) and photoacoustic (PA800/PA900, 6.5-fold) responses towards Na2S2 species with high specificity, excellent sensitivity (1.8nM), improved water solubility, and deep-tissue penetration. More importantly, using NIR-II dual-ratiometric FL/PA imaging, we successfully demonstrated that the probe could be used to accurately quantify the fluctuating H2S2 levels in the liver-injury mouse models induced by lipopolysaccharides or metformin drugs. Overall, this study not only presents a promising tool for H2S2-related pathological research, but also provides a unique recognition site that may be generalized for designing more useful H2S2 imaging agents in the future.

NIR‐II Fluorescence/Photoacoustic Dual Ratiometric Probes with Unique Recognition Site for Quantitatively Visualizing H2S2 in Vivo

Hydrogen persulfide (H2S2) plays a significant role in redox biology and signal transduction; therefore, quantitative visualization of H2S2 in the deep tissue of living organisms is essential for obtaining reliable information about relevant pathophysiological processes directly. However, currently reported H2S2 probes are unsuitable for this purpose because of their poor selectivity for many polysulfide species or their short wavelength, which hinders precise imaging in deep tissues. Herein, for the first time, we report a unique H2S2‐mediated dithiole formation reaction. Based on this reaction, we construct the first NIR‐II fluorescence (FL) and photoacoustic (PA) dual‐ratiometric probe (NIR‐II‐H2S2) for quantitatively visualizing H2S2 in vivo. This probe shows dual‐ratiometric NIR‐II fluorescence (I840/I1000, 107‐fold) and photoacoustic (PA800/PA900, 6.5‐fold) responses towards Na2S2 species with high specificity, excellent sensitivity (1.8 nM), improved water solubility, and deep‐tissue penetration. More importantly, using NIR‐II dual‐ratiometric FL/PA imaging, we successfully demonstrated that the probe could be used to accurately quantify the fluctuating H2S2 levels in the liver‐injury mouse models induced by lipopolysaccharides or metformin drugs. Overall, this study not only presents a promising tool for H2S2‐related pathological research, but also provides a unique recognition site that may be generalized for designing more useful H2S2 imaging agents in the future.

PSATF-6mA: an integrated learning fusion feature-encoded DNA-6 mA methylcytosine modification site recognition model based on attentional mechanisms

DNA methylation is of crucial importance for biological genetic expression, such as biological cell differentiation and cellular tumours. The identification of DNA-6mA sites using traditional biological experimental methods requires more cumbersome steps and a large amount of time. The advent of neural network technology has facilitated the identification of 6 mA sites on cross-species DNA with enhanced efficacy. Nevertheless, the majority of contemporary neural network models for identifying 6 mA sites prioritize the design of the identification model, with comparatively limited research conducted on the statistically significant DNA sequence itself. Consequently, this paper will focus on the statistical strategy of DNA double-stranded features, utilising the multi-head self-attention mechanism in neural networks applied to DNA position probabilistic relationships. Furthermore, a new recognition model, PSATF-6 mA, will be constructed by continually adjusting the attentional tendency of feature fusion through an integrated learning framework. The experimental results, obtained through cross-validation with cross-species data, demonstrate that the PSATF-6 mA model outperforms the baseline model. The in-Matthews correlation coefficient (MCC) for the cross-species dataset of rice and m. musus genomes can reach a score of 0.982. The present model is expected to assist biologists in more accurately identifying 6 mA locus and in formulating new testable biological hypotheses.

Dissecting Immunological Mechanisms Underlying Influenza Viral Nucleoprotein-induced Mucosal Immunity Against Diverse Viral Strains

The nucleoprotein (NP) of type A influenza virus (IAV) is highly conserved across all virus strains, making it an attractive candidate antigen for universal vaccines. While various studies have explored NP-induced mucosal immunity, here we interrogated the mechanistic differences between intramuscular (IM) and intranasal (IN) delivery of a recombinant adenovirus carrying NP fused with a bifunctional CD40 ligand. Despite being less effective than IM delivery in inducing systemic cellular immune responses and antibody-dependent cellular cytotoxicity (ADCC), IN immunization elicited superior antigen-specific recall humoral and cellular response in the nasal associated lymphoid tissue (NALT) of the upper respiratory tract, the initial site of immune recognition and elimination of inhaled pathogens. IN vaccination also induced significantly stronger pulmonary T cell responses in the lower respiratory tract than IM vaccination, in particular the CD8 T cells. Moreover, blocking lymphocyte circulation abrogated IM but not IN immunization induced protection, illustrating the critical role of local memory immune response upon viral infection. Notably, the CD40-targeted nasal delivery not only improved the magnitude but also the breadth of protection, including against lethal challenge with a newly isolated highly pathogenic avian H5N1 strain. These findings are informative for the design of universal mucosal vaccines, where the predominant mode of protection is independent of neutralizing antibodies.

Continuous sign language recognition algorithm based on object detection and variable-length coding sequence.

Currently, continuous sign language recognition faces challenges such as difficulty in acquiring skeletal data, long training time for Three-Dimensional convolutional neural networks, and easy occlusion and blurring of hands. To address these problems, this paper proposes a continuous sign language recognition method based on target detection and coding sequence. The algorithm uses Dual-branch Shuffle Attention Mechanis-You Only Look Once version X (DSA-YOLOX) detection network to detect the head and hands, and encodes the sign language video according to the partition to achieve the transformation from Three-Dimensional to One-Dimensional; and then uses the proposed Bi-directional Long Short-Term Memory (BiLSTM) hand coding sequence classification model with jointly weighted Fast Dynamic Time Warping (FastDTW) to extract hand coding similarity and features while reducing the number of parameters to achieve the classification and recognition of unequal-length hand coding sequences. From the results of ablation experiments and comparison experiments, all parts of the improvement perform well. The word error rate (WER) of this paper's method is reduced by 21.26% compared to Dynamic Time Warping-Hidden Markov Model (DTW-HMM) and 11.53% compared to Long Short-Term Memory-A(LSTM-A); the Giga Floating-point Operations Per Second(GFLOPs) of the algorithm are reduced dramatically, which is about 1/13 of the Visual Alignment Constraint(VAC) model and 1/57 of the Spatial-Temporal Multi-Cue(STMC) model; and the algorithm takes better account of the speed and accuracy of sign language recognition.

Argonaute-Based Nucleic Acid Detection Technology: Advantages, Current Status, Challenges, and Perspectives.

Rapid and accurate detection is a prerequisite for precise clinical diagnostics, ensuring food safety, and facilitating biotechnological applications. The Argonaute system, as a cutting-edge technique, has been successfully repurposed in biosensing beyond the CRISPR/Cas system (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins), which has been extensively researched, but recognition of PAM sequences remains restricted. Argonaute, as a programmable and target-activated nuclease, is repurposed for fabricating novel detection methods due to its unparalleled biological features. In this comprehensive review, we initially elaborate on the current methods for nucleic acid testing and programmable nucleases, followed by delving into the structure and nuclease activity of the Argonaute system. The advantages of Argonaute compared with the CRISPR/Cas system in nucleic acid detection are highlighted and discussed. Furthermore, we summarize the applications of Argonaute-based nucleic acid detection and provide an in-depth analysis of future perspectives and challenges. Recent research has demonstrated that Argonaute-based biosensing is an innovative and rapidly advancing technology that can overcome the limitations of existing methods and potentially replace them. In summary, the implementation of Argonaute and its integration with other technologies hold promise in developing customized and intelligent detection methods for nucleic acid testing across various aspects.

Binding-driven forward tearing protospacer activated CRISPR-Cas12a system and applications for microRNA detection

CRISPR-Cas12a system, characterized by its precise sequence recognition and cleavage activity, has emerged as a powerful and programmable tool for molecular diagnostics. However, current CRISPR-Cas12a-based nucleic acid detection methods, particularly microRNA (miRNA) detection, necessitate additional bio-engineering strategies to exert control over Cas12a activity. Herein, we propose an engineered target-responsive hairpin DNA activator (TRHDA) to mediate forward tearing protospacer activated CRISPR-Cas12a system, which enables direct miRNA detection with high specificity and sensitivity. Target miRNA specifically binding to hairpin DNA can drive forward tearing protospacer in the stem sequence of hairpin structure, facilitating the complementarity between crRNA spacer and protospacer to activate Cas12a. Upon the hairpin DNA as input-responsive activator of Cas12a, a universal biosensing method enables the multiple miRNAs (miR-21, let-7a, miR-30a) detection and also has exceptional capability in identifying single-base mismatches and distinguishing homologous let-7/miR-30 family members. Besides, TRHDA-mediated Cas12a-powered biosensing has realized the evaluation of miR-21 expression levels in diverse cellular contexts by intracellular imaging. Considering the easy programmability of hairpin DNA in responsive region, this strategy could expand for the other target molecules detection (e.g., proteins, micromolecules, peptides, exosomes), which offers significant implications for biomarkers diagnostics utilizing the CRISPR-Cas12a system toolbox.Graphical abstract

Directional Ring Translocation in a pH‐ and Redox‐Driven Tristable [2]Rotaxane

AbstractWe describe the synthesis and characterization of a [2]rotaxane comprising a dibenzo‐24‐crown‐8 (DB24C8) macrocyclic component and a thread containing three recognition sites: ammonium (AmH+), bipyridinium (Bpy2+) and triazolium (Trz+). AmH+ and Bpy2+ are responsive to fully orthogonal stimuli, pH and electrochemical, which allows to precisely control the directional translation of the macrocycle along the axle. A better understanding of the processes driving the operation of the system was obtained thanks to an in‐depth thermodynamic characterization. Orthogonal stimuli responsive tristable rotaxanes represent the starting point for the creation of linear motors and the development of molecular logic gates.

Misincorporations of amino acids in p53 in human cells at artificially constructed termination codons in the presence of the aminoglycoside Gentamicin

Readthrough of a translation termination codon is regulated by ribosomal A site recognition and insertion of near-cognate tRNAs. Small molecules exist that mediate incorporation of amino acids at the stop codon and production of full-length, often functional protein but defining the actual amino acid that is incorporated remains a challenging area. Herein, we report on the development a human cell model that can be used to determine whether rules can be developed using mass spectrometry that define the type of amino acid that is placed at a premature termination codon (PTC) during readthrough mediated by an aminoglycoside. The first PTC we analyzed contained the relatively common cancer-associated termination signal at codon 213 in the p53 gene. Despite of identifying a tryptic peptide with the incorporation of an R at codon 213 in the presence of the aminoglycoside, there were no other tryptic peptides detected across codon 213 that could be recovered; hence we constructed a more robust artificial PTC model. P53 expression plasmids were developed that incorporate a string of single synthetic TGA (opal) stop codons at S127P128A129 within the relatively abundant tryptic p53 peptide 121-SVTCTYSPALNK-132. The treatment of cells stably expressing the p53-TGA129 mutation, treated with Gentamicin, followed by immunoprecipitation and trypsinization of p53, resulted in the identification R, W, or C within the tryptic peptide at codon-TGA129; as expected based on the two-base pairing of the respective anticodons in the tRNA to UGA, with R being the most abundant. By contrast, incorporating the amber or ochre premature stop codons, TAA129 or TAG129 resulted in the incorporation of a Y or Q amino acid, again as expected based on the two base pairings to the anticodons, with Q being the most abundant. A reproducible non-canonical readthrough termination codon-skip event at the extreme C-terminus at codon 436 in the SBP-p53 fusion protein was detected which provided a novel assay for non-canonical readthrough at an extreme C-terminal PTC. The incorporation of amino acids at codons 127, 128, or 129 generally result in a p53 protein that is predicted to be ‘unfolded’ or inactive as defined by molecular dynamic simulations presumably because the production of mixed wild-type p53 and mutant oligomers are known to be inactive through dominant negative effects of the mutation. The data highlight the need to not only produce novel small molecules that can readthrough PTCs or C-terminal termination codons, but also the need to design methods to insert the required amino acid at the position that could result in a ‘wild-type’ functional protein.

A cell-penetrating NS5B-specific nanobody inhibits bovine viral diarrhea virus replication

Bovine viral diarrhea virus (BVDV) causes one of the significant devastating diseases for the cattle industry worldwide. The virus can cross the placenta and result in the persistent infection of the fetus, which has hampered the efficacy and the development of vaccines. Hence, efficient antiviral strategies are urgently needed. In our previous work, a specific nanobody Nb1 against nonstructural protein 5 (NS5B) was successfully isolated, and the replication of BVDV was significantly reduced in the MDBK cell line stably expressing Nb1. Nevertheless, the Nb1 protein itself cannot enter the cells autonomously, which has severely hampered the application of the Nb1. In this work, Nb1fuses with a trans-activating transduction (TAT) peptide to form the TAT-Nb1. The TAT-Nb1 was expressed in Escherichia coli, and then purified by Ni-nitrilotriacetic acid (Ni-NTA) resin. We showed that TAT successfully delivered Nb1 into the MDBK cells, and the TAT-Nb1 efficiently inhibited the replication of BVDV in a dose-and time-dependent manner. Furthermore, the recognition site of Nb1 to NS5B was identified as NS5Baa186-487 by the yeast two-hybrid assay. In summary, this study indicates that the TAT-Nb1 can potentially to be an antiviral drug against BVDV infection, and this research may accelerate the process of Nb1 for clinical use.

Molecularly imprinted electrochemical sensor based on APTES-functionalized indium tin oxide electrode for the determination of sulfadiazine.

Anelectrochemical sensor was developedfor the sensitive and selective detection of sulfadiazine(SDZ), based on a molecularly imprinted polymer (MIP) film formed on an indium tin oxide (ITO) electrode through a self-assembly process. The SDZ-imprinted ITO electrode (SDZ-MIP/APTES-ITO) was prepared through in situ polymerization using sulfadiazine, methacrylic acid (MAA), ethylene glycol dimethacrylate (EGDMA), and 2,2'-azobisisobutyronitrile (AIBN) as the template, functional monomer, cross-linker, and initiator respectively. Before polymerization, the ITO electrode was functionalized with 3-aminopropyltriethoxysilane (APTES) to promote covalent attachment of the polymer to the electrode. After polymerization, the template molecule SDZ was removed to create selective recognition sites, forming the molecularly imprinted polymer electrode (MIP/APTES-ITO), which facilitates sulfadiazine detection. The sensor's performance was evaluated using cyclic and differential pulse voltammetry, demonstrating a linear response in the sulfadiazine concentration range 0.1 to 300μM, with a detection limit of 0.11μM. The MIP-based sensor exhibited goodreproducibility, repeatability, selectivity, and stability in sulfadiazine detection. Its practical applicability was confirmed by the successful quantification of sulfadiazine in spiked milk samples.

Directional Ring Translocation in a pH- and Redox-Driven Tristable [2]Rotaxane.

We describe the synthesis and characterization of a [2]rotaxane comprising a dibenzo-24-crown-8 (DB24C8) macrocyclic component and a thread containing three recognition sites: ammonium (AmH+), bipyridinium (Bpy2+) and triazolium (Trz+). AmH+ and Bpy2+ are responsive to fully orthogonal stimuli, pH and electrochemical, which allows to precisely control the directional translation of the macrocycle along the axle. A better understanding of the processes driving the operation of the system was obtained thanks to an in-depth thermodynamic characterization. Orthogonal stimuli responsive tristable rotaxanes represent the starting point for the creation of linear motors and the development of molecular logic gates.

Wheat TaPYL9-involved signalling pathway impacts plant drought response through regulating distinct osmotic stress-associated physiological indices.

The abscisic acid (ABA) signalling pathway plays a crucial role in plants' response to drought stress. In this study, we aimed to characterize the impact of an ABA signalling module, which consisted of TaPYL9 and its downstream partners in Triticum aestivum, on plant drought adaptation. Our results showed that TaPYL9 protein contains conserved motifs and targets plasma membrane and nucleus after being sorted by the endoplasmic reticulum. In addition, TaPYL9 transcripts in both roots and leaves were significantly upregulated in response to drought stress. We conducted glucuronidase (GUS) histochemical staining analysis for transgenic plants carrying a truncated TaPYL9 promoter, which suggested that cis-elements associate with ABA and drought response, such as ABRE, DRE and recognition sites MYB and MYC, regulating the gene transcription under drought conditions. Using protein interaction assays (i.e., yeast two-hybrid, bimolecular fluorescence complementation (BiFC), co-immunoprecipitation (Co-IP) and in vitro pull-down), we demonstrated interactions between the intermediate segment of TaPYL9, the intermediate segment of TaPP2C6, the N-terminus of TaSnRK2.8 and the C-terminus of the transcription factor TabZIP1 in wheat, indicating the involvement of TaPYL9 in the constitution of an ABA signalling module, namely TaPYL9/TaPP2C6/TaSnRK2.8/TabZIP1. Transgene analysis revealed that TaPYL9, TaSnRK2.8 and TabZIP1 positively regulated drought response, while TaPP2C6 negatively regulated it, and that these genes were closely associated with the regulation of stomata movement, osmolyte accumulation and ROS homeostasis. Electrophoretic mobility shift (EMSA) and transcriptioal activation assays indicated that TabZIP1 interacted promoters of TaP5CS2, TaSLAC1-1 and TaCAT2 and activated transcription of these genes, which regulated proline biosynthesis, stomata movement and ROS scavenging upon drought signalling, respectively. Furthermore, we found that the transcripts of TaPYL9 and stress-responsive genes were positively correlated with yields in wheat cultivars under field drought conditions. Altogether, our findings suggest that the TaPYL9-involved signalling pathway significantly regulates drought response by modulating osmotic stress-associated physiological processes in T. aestivum.