Neuron-specific gene NSG1 binds to and positively regulates sortilin ectodomain shedding via a metalloproteinase-dependent mechanism.

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-ß pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here, we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation, we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin, a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or progranulin uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A disintegrin and metalloproteinase 10 KO cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by A disintegrin and metalloproteinase 10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Insights into Phakopsora pachyrhizi Effector-Effector Interactions.

The multifaceted role of pathogen-encoded effectors in plant-pathogen interactions is complex and not fully understood. Effectors operate within intricate host environments, interacting with host proteins and other effectors to modulate virulence. The complex interplay between effectors raises the concept of metaeffectors, wherein some effectors regulate the activity of others. While previous research has demonstrated the importance of effector repertoires in pathogen virulence, only a limited number of studies have investigated the interactions between these effectors. This study explores the interactions among Phakopsora pachyrhizi effector candidates (PpECs). P. pachyrhizi haustorial transcriptome analysis identified a collection of predicted PpECs. Among these, PpEC23 was found to interact with PpEC48, prompting further exploration into their potential interaction with other effectors. Here, we utilized a yeast two-hybrid screen to explore protein-protein interactions between PpECs. A split-luciferase complementation assay also demonstrated that these interactions could occur within soybean cells. Interestingly, PpEC48 displayed the ability to interact with several small cysteine-rich proteins (SCRPs), suggesting its affinity for this specific class of effectors. We show that these interactions involve a histidine-rich domain within PpEC48, emphasizing the significance of structural motifs in mediating effector interactions. The unique nature of PpEC48, showing no sequence matches in other organisms, suggests its relatively recent evolution and potential orphan gene status. Our work reveals insights into the intricate network of interactions among P. pachyrhizi effector-effector interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

The mitochondrial carrier homolog 2 is involved in down-regulation of influenza A virus replication.

BACKGROUND: The mitochondrial carrier homolog 2 (MTCH2) is a mitochondrial outer membrane protein regulating mitochondrial metabolism and functions in lipid homeostasis and apoptosis. Experimental data on the interaction of MTCH2 with viral proteins in virus-infected cells are very limited. Here, the interaction of MTCH2 with PA subunit of influenza A virus RdRp and its effects on viral replication was investigated. METHODS: The human MTCH2 protein was identified as the influenza A virus PA-related cellular factor with the Y2H assay. The interaction between GST.MTCH2 and PA protein co-expressed in transfected HEK293 cells was evaluated by GST-pull down. The effect of MTCH2 on virus replication was determined by quantification of viral transcript and/or viral proteins in the cells transfected with MTCH2-encoding plasmid or MTCH2-siRNA. An interaction model of MTCH2 and PA was predicted with protein modeling/docking algorithms. RESULTS: It was observed that PA and GST.MTCH2 proteins expressed in HEK293 cells were co-precipitated by glutathione-agarose beads. The influenza A virus replication was stimulated in HeLa cells whose MTCH2 expression was suppressed with specific siRNA, whereas the increase of MTCH2 in transiently transfected HEK293 cells inhibited viral RdRp activity. The results of a Y2H assay and protein-protein docking analysis suggested that the amino terminal part of the viral PA (nPA) can bind to the cytoplasmic domain comprising amino acid residues 253 to 282 of the MTCH2. CONCLUSION: It is suggested that the host mitochondrial MTCH2 protein is probably involved in the interaction with the viral polymerase protein PA to cause negative regulatory effect on influenza A virus replication in infected cells.

Polyubiquitin protein of Aedes aegypti as an interacting partner of dengue virus envelope protein.

Dengue virus (DENV) is an arbovirus that comprises four antigenically different serotypes. Aedes aegypti (Diptera: Culicidae) acts as the principal vector for DENV transmission, and vector control is crucial for dengue fever epidemic management. To design effective vector control strategies, a comprehensive understanding of the insect vector and virus interaction is required. Female Ae. aegypti ingests DENV during the acquisition of a blood meal from an infected human. DENV enters the insect midgut, replicates inside it and reaches the salivary gland for transmitting DENV to healthy humans during the subsequent feeding cycles. DENV must interact with the proteins present in the midgut and salivary glands to gain entry and accomplish successful replication and transmission. Ae. aegypti midgut cDNA library was prepared, and yeast two-hybrid screening was performed against the envelope protein domain III (EDIII) protein of DENV-2. The polyubiquitin protein was selected from the various candidate proteins for subsequent analysis. Polyubiquitin gene was amplified, and the protein was purified in a heterologous expression system for in vitro interaction studies. In vitro pull-down assay presented a clear interaction between polyubiquitin protein and EDIII. To further confirm this interaction, a dot blot assay was employed, and polyubiquitin protein was found to interact with DENV particles. Our results enable us to suggest that polyubiquitin plays an important role in DENV infection within mosquitoes.

Chaetocin disrupts the SUV39H1-HP1 interaction independent of SUV39H1 methyltransferase activity.

Chemical tools to control the activities and interactions of chromatin components have broad impact on our understanding of cellular and disease processes. It is important to accurately identify their molecular effects to inform clinical efforts and interpretations of scientific studies. Chaetocin is a widely used chemical that decreases H3K9 methylation in cells. It is frequently attributed as a specific inhibitor of the histone methyltransferase activities of SUV39H1/SU(VAR)3-9, although prior observations showed chaetocin likely inhibits methyltransferase activity through covalent mechanisms involving its epipolythiodixopiperazine disulfide 'warhead' functionality. The continued use of chaetocin in scientific studies may derive from the net effect of reduced H3K9 methylation, irrespective of a direct or indirect mechanism. However, there may be other molecular impacts of chaetocin on SUV39H1 besides inhibition of H3K9 methylation levels that could confound the interpretation of past and future experimental studies. Here, we test a new hypothesis that chaetocin may have an additional downstream impact aside from inhibition of methyltransferase activity. Using a combination of truncation mutants, a yeast two-hybrid system, and direct in vitro binding assays, we show that the human SUV39H1 chromodomain (CD) and HP1 chromoshadow domain (CSD) directly interact. Chaetocin inhibits this binding interaction through its disulfide functionality with some specificity by covalently binding with the CD of SUV39H1, whereas the histone H3-HP1 interaction is not inhibited. Given the key role of HP1 dimers in driving a feedback cascade to recruit SUV39H1 and to establish and stabilize constitutive heterochromatin, this additional molecular consequence of chaetocin should be broadly considered.

Technologies to Study Genetics and Molecular Pathways.

Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.

Grape SnRK2.7 Positively Regulates Drought Tolerance in Transgenic Arabidopsis.

In this study, we obtained and cloned VvSnRK2.7 by screening transcriptomic data to investigate the function of the grape sucrose non-fermenting kinase 2 (SnRK2) gene under stress conditions. A yeast two-hybrid (Y2H) assay was used to further screen for interaction proteins of VvSnRK2.7. Ultimately, VvSnRK2.7 was heterologously expressed in Arabidopsis thaliana, and the relative conductivity, MDA content, antioxidant enzyme activity, and sugar content of the transgenic plants were determined under drought treatment. In addition, the expression levels of VvSnRK2.7 in Arabidopsis were analyzed. The results showed that the VvSnRK2.7-EGFP fusion protein was mainly located in the cell membrane and nucleus of tobacco leaves. In addition, the VvSnRK2.7 protein had an interactive relationship with the VvbZIP protein during the Y2H assay. The expression levels of VvSnRK2.7 and the antioxidant enzyme activities and sugar contents of the transgenic lines were higher than those of the wild type under drought treatment. Moreover, the relative conductivity and MDA content were lower than those of the wild type. The results indicate that VvSnRK2.7 may activate the enzyme activity of the antioxidant enzyme system, maintain normal cellular physiological metabolism, stabilize the berry sugar metabolism pathway under drought stress, and promote sugar accumulation to improve plant resistance.

VvJAZ13 Positively Regulates Cold Tolerance in Arabidopsis and Grape.

Cold stress adversely impacts grape growth, development, and yield. Therefore, improving the cold tolerance of grape is an urgent task of grape breeding. The Jasmonic acid (JA) pathway responsive gene JAZ plays a key role in plant response to cold stress. However, the role of JAZ in response to low temperatures in grape is unclear. In this study, VvJAZ13 was cloned from the 'Pinot Noir' (Vitis vinefera cv. 'Pinot Noir') grape, and the potential interacting protein of VvJAZ13 was screened by yeast two-hybrid (Y2H). The function of VvJAZ13 under low temperature stress was verified by genetic transformation. Subcellular localization showed that the gene was mainly expressed in cytoplasm and the nucleus. Y2H indicated that VvF-box, VvTIFY5A, VvTIFY9, Vvbch1, and VvAGD13 may be potential interacting proteins of VvJAZ13. The results of transient transformation of grape leaves showed that VvJAZ13 improved photosynthetic capacity and reduced cell damage by increasing maximum photosynthetic efficiency of photosystem II (Fv/Fm), reducing relative electrolyte leakage (REL) and malondialdehyde (MDA), and increasing proline content in overexpressed lines (OEs), which played an active role in cold resistance. Through the overexpression of VvJAZ13 in Arabidopsis thaliana and grape calli, the results showed that compared with wild type (WT), transgenic lines had higher antioxidant enzyme activity and proline content, lower REL, MDA, and hydrogen peroxide (H2O2) content, and an improved ability of scavenging reactive oxygen species. In addition, the expression levels of CBF1-2 and ICE1 genes related to cold response were up-regulated in transgenic lines. To sum up, VvJAZ13 is actively involved in the cold tolerance of Arabidopsis and grape, and has the potential to be a candidate gene for improving plant cold tolerance.

Studies on the PII-PipX-NtcA Regulatory Axis of Cyanobacteria Provide Novel Insights into the Advantages and Limitations of Two-Hybrid Systems for Protein Interactions.

Yeast two-hybrid approaches, which are based on fusion proteins that must co-localise to the nucleus to reconstitute the transcriptional activity of GAL4, have greatly contributed to our understanding of the nitrogen interaction network of cyanobacteria, the main hubs of which are the trimeric PII and the monomeric PipX regulators. The bacterial two-hybrid system, based on the reconstitution in the E. coli cytoplasm of the adenylate cyclase of Bordetella pertussis, should provide a relatively faster and presumably more physiological assay for cyanobacterial proteins than the yeast system. Here, we used the bacterial two-hybrid system to gain additional insights into the cyanobacterial PipX interaction network while simultaneously assessing the advantages and limitations of the two most popular two-hybrid systems. A comprehensive mutational analysis of PipX and bacterial two-hybrid assays were performed to compare the outcomes between yeast and bacterial systems. We detected interactions that were previously recorded in the yeast two-hybrid system as negative, as well as a "false positive", the self-interaction of PipX, which is rather an indirect interaction that is dependent on PII homologues from the E. coli host, a result confirmed by Western blot analysis with relevant PipX variants. This is, to our knowledge, the first report of the molecular basis of a false positive in the bacterial two-hybrid system.

An in silico prediction of interaction models of influenza A virus PA and human C14orf166 protein from yeast-two-hybrid screening data.

The human C14orf166 protein, also known as RNA transcription, translation, and transport factor, shows positive modulatory activity on the cellular RNA polymerase II enzyme. This protein is a component of the tRNA-splicing ligase complex and is involved in RNA metabolism. It also functions in the nucleo-cytoplasmic transport of RNA molecules. The C14orf166 protein has been reported to be associated with some types of cancer. It has been shown that the C14orf166 protein binds to the influenza A virus RNA polymerase PA subunit and has a stimulating effect on viral replication. In this study, candidate interactor proteins for influenza A virus PA protein were screened with a Y2H assay using HEK293 Matchmaker cDNA. The C14orf166 protein fragments in different sizes were found to interact with the PA. The three-dimensional structures of the viral PA and C14orf166 proteins interacting with the PA were generated using the I-TASSER algorithm. The interaction models between these proteins were predicted with the ClusPro protein docking algorithm and analyzed with PyMol software. The results revealed that the carboxy-terminal end of the C14orf166 protein is involved in this interaction, and it is highly possible that it binds to the carboxy-terminal of the PA protein. Although amino acid residues in the interaction area of the PA protein with the C14orf166 showed distribution from 450th to 700th position, the intense interaction region was revealed to be at amino acid positions 610-630.

Anthocyanin accumulation and transcriptional regulation in purple flowering stalk (Brassica campestris L. var. purpurea Bailey).

KEY MESSAGE: 1. Purple flowering stalk (Brassica campestris L. ssp. chinensis L. var. purpurea Bailey) is a crop with the high-level anthocyanin. 2. Increased abundance of LBGs promoted the synthesis of anthocyanin. 3. TTG2 (WRKY) interacted with TTG1 (WD40), probably regulating anthocyanin accumulation by shaping a MBWW complex. Brassica crops are a class of nutrient-rich vegetables. Here, two Brassica Crops-Flowering Stalk cultivars, purple flowering stalk (Brassica campestris L. var. purpurea Bailey) and pakchoi (Brassica campestris ssp. chinensis var. communis) were investigated. HPLC-ESI-MS/MS analysis demonstrated that Cy 3-p-coumaroylsophoroside-5-malonylglucoside and Cy 3-diferuloylsophoroside-5-malonylglucoside were identified as the major anthocyanin in peel of purple flowering stalk. The transcript level of structural genes including C4H, CHS, F3H, DFR, ANS and UFGT, and regulatory genes such as TT8, TTG1, Bra004162, Bra001917 and TTG2 in peel of purple flowering stalk were significantly higher than that in peel of pakchoi. In addition, the TTG2(WRKY) interacted only with TTG1(WD40) and the interaction between TT8 (bHLH) and TTG1/Bra004162(MYB)/Bra001917(MYB) were identified. Else, the WD40-WRKY complex (TTG1-TTG2) could activate the transcript of TT12. Our study laid a foundation for the research on the anthocyanin accumulation in Brassica crops.

Screening of Tnfaip1-Interacting Proteins in Zebrafish Embryonic cDNA Libraries Using a Yeast Two-Hybrid System.

TNFAIP1 regulates cellular biological functions, including DNA replication, DNA repair, and cell cycle, by binding to target proteins. Identification of Tnfaip1-interacting proteins contributes to the understanding of the molecular regulatory mechanisms of their biological functions. In this study, 48 hpf, 72 hpf, and 96 hpf wild-type zebrafish embryo mRNAs were used to construct yeast cDNA library. The library titer was 1.12 × 107 CFU/mL, the recombination rate was 100%, and the average length of the inserted fragments was greater than 1000 bp. A total of 43 potential interacting proteins of Tnfaip1 were identified using zebrafish Tnfaip1 as a bait protein. Utilizing GO functional annotation and KEGG signaling pathway analysis, we found that these interacting proteins are mainly involved in translation, protein catabolic process, ribosome assembly, cytoskeleton formation, amino acid metabolism, and PPAR signaling pathway. Further yeast spotting analyses identified four interacting proteins of Tnfaip1, namely, Ubxn7, Tubb4b, Rpl10, and Ybx1. The Tnfaip1-interacting proteins, screened from zebrafish embryo cDNA in this study, increased our understanding of the network of Tnfaip1-interacting proteins during the earliest embryo development and provided a molecular foundation for the future exploration of tnfaip1's biological functions.

Uncovering the involvement of DoDELLA1-interacting proteins in development by characterizing the DoDELLA gene family in Dendrobium officinale.

BACKGROUND: Gibberellins (GAs) are widely involved in plant growth and development. DELLA proteins are key regulators of plant development and a negative regulatory factor of GA. Dendrobium officinale is a valuable traditional Chinese medicine, but little is known about D. officinale DELLA proteins. Assessing the function of D. officinale DELLA proteins would provide an understanding of their roles in this orchid's development. RESULTS: In this study, the D. officinale DELLA gene family was identified. The function of DoDELLA1 was analyzed in detail. qRT-PCR analysis showed that the expression levels of all DoDELLA genes were significantly up-regulated in multiple shoots and GA3-treated leaves. DoDELLA1 and DoDELLA3 were significantly up-regulated in response to salt stress but were significantly down-regulated under drought stress. DoDELLA1 was localized in the nucleus. A strong interaction was observed between DoDELLA1 and DoMYB39 or DoMYB308, but a weak interaction with DoWAT1. CONCLUSIONS: In D. officinale, a developmental regulatory network involves a close link between DELLA and other key proteins in this orchid's life cycle. DELLA plays a crucial role in D. officinale development.

NGPINT: a next-generation protein-protein interaction software.

Mapping protein-protein interactions at a proteome scale is critical to understanding how cellular signaling networks respond to stimuli. Since eukaryotic genomes encode thousands of proteins, testing their interactions one-by-one is a challenging prospect. High-throughput yeast-two hybrid (Y2H) assays that employ next-generation sequencing to interrogate complementary DNA (cDNA) libraries represent an alternative approach that optimizes scale, cost and effort. We present NGPINT, a robust and scalable software to identify all putative interactors of a protein using Y2H in batch culture. NGPINT combines diverse tools to align sequence reads to target genomes, reconstruct prey fragments and compute gene enrichment under reporter selection. Central to this pipeline is the identification of fusion reads containing sequences derived from both the Y2H expression plasmid and the cDNA of interest. To reduce false positives, these fusion reads are evaluated as to whether the cDNA fragment forms an in-frame translational fusion with the Y2H transcription factor. NGPINT successfully recognized 95% of interactions in simulated test runs. As proof of concept, NGPINT was tested using published data sets and it recognized all validated interactions. NGPINT can process interaction data from any biosystem with an available genome or transcriptome reference, thus facilitating the discovery of protein-protein interactions in model and non-model organisms.

A human kinase yeast array for the identification of kinases modulating phosphorylation-dependent protein-protein interactions.

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.

ENHANCED GRAVITROPISM 2 coordinates molecular adaptations to gravistimulation in the elongation zone of barley roots.

Root gravitropism includes gravity perception in the root cap, signal transduction between root cap and elongation zone, and curvature response in the elongation zone. The barley (Hordeum vulgare) mutant enhanced gravitropism 2 (egt2) displays a hypergravitropic root phenotype. We compared the transcriptomic reprogramming of the root cap, the meristem, and the elongation zone of wild-type (WT) and egt2 seminal roots upon gravistimulation in a time-course experiment and identified direct interaction partners of EGT2 by yeast-two-hybrid screening and bimolecular fluorescence complementation validation. We demonstrated that the elongation zone is subjected to most transcriptomic changes after gravistimulation. Here, 33% of graviregulated genes are also transcriptionally controlled by EGT2, suggesting a central role of this gene in controlling the molecular networks associated with gravitropic bending. Gene co-expression analyses suggested a role of EGT2 in cell wall and reactive oxygen species-related processes, in which direct interaction partners of EGT2 regulated by EGT2 and gravity might be involved. Taken together, this study demonstrated the central role of EGT2 and its interaction partners in the networks controlling root zone-specific transcriptomic reprogramming of barley roots upon gravistimulation. These findings can contribute to the development of novel root idiotypes leading to improved crop performance.

The Bax inhibitor GmBI-1α interacts with a Nod factor receptor and plays a dual role in the legume-rhizobia symbiosis.

The gene networks surrounding Nod factor receptors that govern the symbiotic process between legumes and rhizobia remain largely unexplored. Here, we identify 13 novel GmNFR1α-associated proteins by yeast two-hybrid screening, and describe a potential interacting protein, GmBI-1α. GmBI-1α had the highest positive correlation with GmNFR1α in a co-expression network analysis, and its expression at the mRNA level in roots was enhanced by rhizobial infection. Moreover, GmBI-1α-GmNFR1α interaction was shown to occur in vitro and in vivo. The GmBI-1α protein was localized to multiple subcellular locations, including the endoplasmic reticulum and plasma membrane. Overexpression of GmBI-1α increased the nodule number in transgenic hairy roots or transgenic soybean, whereas down-regulation of GmBI-1α transcripts by RNA interference reduced the nodule number. In addition, the nodules in GmBI-1α-overexpressing plants became smaller in size and infected area with reduced nitrogenase activity. In GmBI-1α-overexpressing transgenic soybean, the elevated GmBI-1α also promoted plant growth and suppressed the expression of defense signaling-related genes. Infection thread analysis of GmBI-1α-overexpressing plants showed that GmBI-1α promoted rhizobial infection. Collectively, our findings support a GmNFR1α-associated protein in the Nod factor signaling pathway and shed new light on the regulatory mechanism of GmNFR1α in rhizobial symbiosis.

Screening of genes encoding proteins that interact with Nrf2: Probing a cDNA library from Mytilus coruscus using a yeast two-hybrid system.

The Nuclear factor Erythroid 2-related factor 2 (Nrf2) is the most important endogenous antioxidant factor in organisms, and it has been demonstrated that it exerts extensive control over the immune response by interacting with crucial innate immunity components directly or indirectly. Although Nrf2 has been widely confirmed to be involved in stress resistance in mammals and some fish, its contribution to mollusks oxidative stress resistance has not frequently been documented. In this investigation, total RNA was taken from the digestive gland of M. coruscus, and a cDNA library was constructed and screened using the GATEWAY recombination technology. The Nrf2 cDNA sequence of M. coruscus was cloned into the pGBKT7 vector to prepare the bait plasmid. Using yeast two-hybrid system, after auxotrophic medium screening, sequencing, and bioinformatics analysis, 13 binding proteins that interacted with Nrf2 were finally identified. They were QM-like protein, 40S ribosomal protein S4 (RPS4), ribosomal protein S2 (RPS2), ribosomal protein L12 (RPL12), EF1-alpha mRNA for elongation factor 1 alpha (eEF1-alpha), ferritin, alpha-amylase, trypsin, vdg3, period clock protein, cyclophilin A isoform 1 (CYP A), serine protease CFSP2, histone variant H2A.Z (H2A.Z). For a better understanding the physiological function of Nrf2 in animals and as a potential target for future research on protein roles in Nrf2 interactions, it is crucial to clarify these protein interactions.

Investigating Protein-Protein Interactions Between Grapevine Leafroll-Associated Virus 3 and Vitis vinifera.

Grapevine leafroll disease (GLD) is a globally important disease that affects the metabolic composition and biomass of grapes, leading to a reduction in grape yield and quality of wine produced. Grapevine leafroll-associated virus 3 (GLRaV-3) is the main causal agent for GLD. This study aimed to identify protein-protein interactions between GLRaV-3 and its host. A yeast two-hybrid (Y2H) library was constructed from Vitis vinifera mRNA and screened against GLRaV-3 open reading frames encoding structural proteins and those potentially involved in systemic spread and silencing of host defense mechanisms. Five interacting protein pairs were identified, three of which were demonstrated in planta. The minor coat protein of GLRaV-3 was shown to interact with 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase 02, a protein involved in primary carbohydrate metabolism and the biosynthesis of aromatic amino acids. Interactions were also identified between GLRaV-3 p20A and an 18.1-kDa class I small heat shock protein, as well as MAP3K epsilon protein kinase 1. Both proteins are involved in the response of plants to various stressors, including pathogen infections. Two additional proteins, chlorophyll a-b binding protein CP26 and a SMAX1-LIKE 6 protein, were identified as interacting with p20A in yeast but these interactions could not be demonstrated in planta. The findings of this study advance our understanding of the functions of GLRaV-3-encoded proteins and how the interaction between these proteins and those of V. vinifera could lead to GLD.

Evolution of the 14-3-3 gene family in monocotyledons and dicotyledons and validation of MdGRF13 function in transgenic Arabidopsis thaliana.

KEY MESSAGE: The 14-3-3 family is more highly conserved among monocotyledons, and overexpression of MdGRF13 improved drought and salt tolerance in transgenic Arabidopsis thaliana. The 14-3-3 are highly conserved regulatory proteins found in eukaryotes and play an essential role in plant growth, development and stress response. However, the 14-3-3 gene family evolution in monocotyledons and dicotyledons and the biological functions of the MdGRF13 under abiotic stress remain unknown. In our study, 195 members of the 14-3-3 family were identified from 12 species and divided into ε group and the Non-ε group. Synteny analysis within the 14-3-3 family indicated that segmental duplication events contributed to the expansion of the family. Selective pressure analysis indicated that purifying selection was a vital force in the 14-3-3 genes evolution, and monocotyledons had a lower million years ago (Mya) mean values than dicotyledons. Meanwhile, the codon adaptation index (CAI) and frequency of optical codons (FOP) are higher and the effective number of codons (Nc) is lower in monocotyledons 14-3-3 genes compared to dicotyledons. Moreover, the yeast two-hybrid (Y2H) demonstrated that MdGRF13 interacts with MdRD22, MdLHP1a and MdMORF1. Significantly, the malondialdehyde (MDA) content and relative conductivity were decreased, while the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were increased in transgenic Arabidopsis compared to the wild type (WT) under drought and salt stress. These results suggest that overexpression of MdGRF13 significantly improved the tolerance to drought and salt stress in transgenic Arabidopsis. Thus, our results provide a theoretical basis for exploring the evolution and function of the 14-3-3 gene family in monocotyledons and dicotyledons.