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Strawberry (Fragaria × ananassa) fruits contain high concentrations of flavonoids. In unripe strawberries, the flavonoids are mainly represented by proanthocyanidins (PAs), while in ripe fruits the red-coloured anthocyanins also accumulate. Most of the structural genes leading to PA biosynthesis in strawberry have been characterized, but no information is available on their transcriptional regulation. In Arabidopsis thaliana the expression of the PA biosynthetic genes is specifically induced by a ternary protein complex, composed of AtTT2 (AtMYB123), AtTT8 (AtbHLH042) and AtTTG1 (WD40-repeat protein). A strategy combining yeast-two-hybrid screening and agglomerative hierarchical clustering of transcriptomic and metabolomic data was undertaken to identify strawberry PA regulators. Among the candidate genes isolated, four were similar to AtTT2, AtTT8 and AtTTG1 (FaMYB9/FaMYB11, FabHLH3 and FaTTG1, respectively) and two encode putative negative regulators (FaMYB5 and FabHLH3Δ). Interestingly, FaMYB9/FaMYB11, FabHLH3 and FaTTG1 were found to complement the tt2-1, tt8-3 and ttg1-1 transparent testa mutants, respectively. In addition, they interacted in yeast and activated the Arabidopsis BANYULS (anthocyanidin reductase) gene promoter when coexpressed in Physcomitrella patens protoplasts. Taken together, these results demonstrated that FaMYB9/FaMYB11, FabHLH3 and FaTTG1 are the respective functional homologues of AtTT2, AtTT8 and AtTTG1, providing new tools for modifying PA content and strawberry fruit quality.

High‐throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome‐scale interaction mapping. Here, we report Barcode Fusion Genetics‐Yeast Two‐Hybrid (BFG‐Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG‐Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein‐pair barcodes that can be quantified via next‐generation sequencing. We applied BFG‐Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG‐Y2H increases the efficiency of protein matrix screening, with quality that is on par with state‐of‐the‐art Y2H methods. Synopsis Barcode Fusion Genetics‐Yeast Two‐Hybrid, a new technology allowing many‐by‐many screening of protein interactions in a single pooled assay, is presented. High‐quality interactions are identified in search matrices up to ~2.5 million protein pairs in scale. Barcode Fusion Genetics (BFG) enables phenotypic analysis of millions of strains, each carrying two engineered loci. BFG is applied to perform highly multiplexed yeast two‐hybrid protein interaction assays (BFG‐Y2H). Protein interactions for ˜2.5 million protein pairs are tested in a single BFG‐Y2H run. The quality of BFG‐Y2H results is on par with current state‐of‐the‐art Y2H methods. Graphical Abstract Barcode Fusion Genetics‐Yeast Two‐Hybrid, a new technology allowing many‐by‐many screening of protein interactions in a single pooled assay, is presented. High‐quality interactions are identified in search matrices up to ~2.5 million protein pairs in scale.

The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide‐binding leucine‐rich‐repeat (NLR) receptor MLA1 and therefore is designated AVRA1. Here, we show that AVRA1 and the sequence‐unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two‐hybrid next‐generation interaction screens (Y2H‐NGIS), followed by binary Y2H and in planta protein–protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)‐localized J‐domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER signal peptide‐independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J‐domain protein HvERdj3B appears to be essential and can be regulated by AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J‐domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition. HvERdj3B, identified as a host target of AVRA1 and BEC1016, is an immunity‐associated chaperone in the endoplasmic reticulum, into which these effectors enter signal peptide‐independently.

G‐protein‐coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR‐mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two‐hybrid (MYTH) approach and identified interacting partners for 48 selected full‐length human ligand‐unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5‐HT4d, and adenosine ADORA2A receptors. Our data represent the first large‐scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins. Synopsis The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology. MYTH is used to identify interacting partners for 48 human GPCRS, resulting in an interactome containing 686 proteins and 987 unique interactions. Bioinformatics analyses of the GPCR interactome indicate enrichment for diverse pathways, diseases, molecular functions, biological processes, domains, and drug targets. Orthogonal analyses using co‐immunoprecipitation and BRET validate a subset of the identified interactions. Functional characterization of novel GPCR interactions identifies potential roles in neurobiological processes. Graphical Abstract The complete interactome of 48 disease‐associated, full‐length human G‐coupled protein receptors (GPCRs) is mapped in the native environment of the cellular membrane, using the membrane yeast two‐hybrid (MYTH) technology.

Post‐translational protein modifications, such as tyrosine phosphorylation, regulate protein–protein interactions (PPIs) critical for signal processing and cellular phenotypes. We extended an established yeast two‐hybrid system employing human protein kinases for the analyses of phospho‐tyrosine (pY)‐dependent PPIs in a direct experimental, large‐scale approach. We identified 292 mostly novel pY‐dependent PPIs which showed high specificity with respect to kinases and interacting proteins and validated a large fraction in co‐immunoprecipitation experiments from mammalian cells. About one‐sixth of the interactions are mediated by known linear sequence binding motifs while the majority of pY‐PPIs are mediated by other linear epitopes or governed by alternative recognition modes. Network analysis revealed that pY‐mediated recognition events are tied to a highly connected protein module dedicated to signaling and cell growth pathways related to cancer. Using binding assays, protein complementation and phenotypic readouts to characterize the pY‐dependent interactions of TSPAN2 (tetraspanin 2) and GRB2 or PIK3R3 (p55γ), we exemplarily provide evidence that the two pY‐dependent PPIs dictate cellular cancer phenotypes. Synopsis A modified yeast two‐hybrid approach employed on a large scale generates a network of 292 human phospho‐tyrosine (pY)‐dependent protein–protein interactions. Conditional interactions are validated, and pY‐dependent interaction specificity and network features are assessed. A pY‐dependent protein interaction data set is generated using a modified yeast two‐hybrid approach. Network analyses assess the extent of known linear motif‐based pY recognition, pointing toward the importance of context for interaction specificity, and reveal a highly connected pY‐recognition module in the human proteome. A large fraction of PPIs is validated by co‐immunoprecipitation with good success rate. pY‐dependent TSPAN2 interactions are related to cancer phenotypes. Graphical Abstract A modified yeast two‐hybrid approach employed on a large scale generates a network of 292 human phospho‐tyrosine (pY)‐dependent protein–protein interactions. Conditional interactions are validated, and pY‐dependent interaction specificity and network features are assessed.

Protein kinases play an important role in cellular signaling pathways and their dysregulation leads to multiple diseases, making kinases prime drug targets. While more than 500 human protein kinases are known to collectively mediate phosphorylation of over 290,000 S/T/Y sites, the activities have been characterized only for a minor, intensively studied subset. To systematically address this discrepancy, we developed a human kinase array in Saccharomyces cerevisiae as a simple readout tool to systematically assess kinase activities. For this array, we expressed 266 human kinases in four different S. cerevisiae strains and profiled ectopic growth as a proxy for kinase activity across 33 conditions. More than half of the kinases showed an activity‐dependent phenotype across many conditions and in more than one strain. We then employed the kinase array to identify the kinase(s) that can modulate protein–protein interactions (PPIs). Two characterized, phosphorylation‐dependent PPIs with unknown kinase–substrate relationships were analyzed in a phospho‐yeast two‐hybrid assay. CK2α1 and SGK2 kinases can abrogate the interaction between the spliceosomal proteins AAR2 and PRPF8, and NEK6 kinase was found to mediate the estrogen receptor (ERα) interaction with 14‐3‐3 proteins. The human kinase yeast array can thus be used for a variety of kinase activity‐dependent readouts. Synopsis A large array of human kinases is developed in yeast, covering the activities of more than 50% of the known human kinome and including many difficult‐to‐study kinases. The array is used to identify human kinases modulating phosphorylation‐dependent protein interactions. Growth phenotypes of 266 human protein kinases expressed in yeast are systematically assayed across four laboratory strains and 73 growth conditions. The human kinase yeast array contains 150 highly active human kinases and is a versatile tool for studying human kinase activities, including low abundant, tissue‐specific and “dark” kinases with largely unknown functions. The array is used in a phospho‐yeast two‐hybrid assay to identify kinases regulating phosphorylation‐dependent protein–protein interactions. SGK2 and CK2α1 are shown to modulate phospho‐dependent interactions between spliceosomal proteins AAR2 and PRPF8, while NEK6 and PAK5 modulate the association between ERα and 14‐3‐3. Graphical Abstract A large array of human kinases is developed in yeast, covering the activities of more than 50% of the known human kinome and including many difficult‐to‐study kinases. The array is used to identify human kinases modulating phosphorylation‐dependent protein interactions.

Background: The responsible genes have not yet been identified for many genetically mapped disease loci. Physically interacting proteins tend to be involved in the same cellular process, and mutations in their genes may lead to similar disease phenotypes. Objective: To investigate whether protein–protein interactions can predict genes for genetically heterogeneous diseases. Methods: 72 940 protein–protein interactions between 10 894 human proteins were used to search 432 loci for candidate disease genes representing 383 genetically heterogeneous hereditary diseases. For each disease, the protein interaction partners of its known causative genes were compared with the disease associated loci lacking identified causative genes. Interaction partners located within such loci were considered candidate disease gene predictions. Prediction accuracy was tested using a benchmark set of known disease genes. Results: Almost 300 candidate disease gene predictions were made. Some of these have since been confirmed. On average, 10% or more are expected to be genuine disease genes, representing a 10-fold enrichment compared with positional information only. Examples of interesting candidates are AKAP6 for arrythmogenic right ventricular dysplasia 3 and SYN3 for familial partial epilepsy with variable foci. Conclusions: Exploiting protein–protein interactions can greatly increase the likelihood of finding positional candidate disease genes. When applied on a large scale they can lead to novel candidate gene predictions.

Summary •  Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH‐type zinc finger proteins involved in plant stress tolerance are poorly understood. •  A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH‐type zinc finger protein, was isolated from a salt‐induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two‐hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis‐related protein 5), which interacted with GhZFP1, were isolated. •  GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. •  Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

DNA damage‐induced apoptosis suppressor (DDIAS) facilitates the survival of lung cancer by suppressing apoptosis. Moreover, DDIAS promotes tyrosine phosphorylation of signal transducer and activator of transcription 3 (STAT3) via their interaction. Here, we identified miconazole as an inhibitor of DDIAS/STAT3 interaction by screening a chemical library using a yeast two‐hybrid assay. Miconazole inhibited growth, migration and invasion of lung cancer cells. Furthermore, miconazole suppressed STAT3 tyrosine Y705 phosphorylation and the expression of its target genes, such as cyclin D1, survivin and snail but had no suppressive effect on the activation of ERK1/2 or AKT, which is involved in the survival of lung cancer. As expected, no interaction between DDIAS and STAT3 occurred in the presence of miconazole, as confirmed by immunoprecipitation assays. Mouse xenograft experiments showed that miconazole significantly suppressed both tumor size and weight in an NCI‐H1703 mouse model. Tyrosine phosphorylation of STAT3 at Y705 and expression of its targets, such as cyclin D1, survivin and snail, were decreased in miconazole‐treated tumor tissues, as compared with those in vehicle‐treated tumor tissues. These data suggest that miconazole exerts an anti–cancer effect by suppressing STAT3 activation through inhibiting DDIAS/STAT3 binding. DNA damage‐induced apoptosis suppressor promotes activation of tyrosine phosphorylation of STAT3 through their interaction. Miconazole exerts an anti–cancer effect by suppressing STAT3 activation through inhibition of DDIAS/STAT3 binding.

MST2 (STK3) is a major upstream kinase in the Hippo signalling pathway, an evolutionary conserved pathway in regulation of organ size, self‐renewal and tissue homeostasis. Its downstream effectors are the transcriptional regulators YAP and TAZ. This pathway is regulated by a variety of factors, such as substrate stiffness or cell–cell contacts. Using a yeast two‐hybrid screen, we detected a novel interaction between the kinases MST2 and CDK5, which we further confirmed by co‐immunoprecipitation experiments. Cyclin‐dependent kinase 5 (CDK5) is an unusual member of the family of cyclin‐dependent kinases, involved in tumour growth and angiogenesis. Although a link between CDK5 and Hippo has been previously postulated, the mode of action is still elusive. Here, we show that knockdown of CDK5 causes reduced transcriptional activity of YAP and that CDK5 influences the phosphorylation levels of the Hippo upstream kinase LATS1. Moreover, a phosphoproteomics approach revealed that CDK5 interferes with the phosphorylation of DLG5, another upstream kinase, which regulates the Hippo pathway. Hence, CDK5 seems to act as a signalling hub for integrating the Hippo pathway and other signalling cascades. These interactions might have important implications for the use of CDK5 inhibitors, which are already in clinical use for tumour diseases. By using a yeast‐two‐hybrid system, immune precipitation and a proteomics approach, we identify a novel interaction between CDK5 and MST2, which modulates the mechano‐sensitive Hippo signalling pathway in a liver tumour cell line.