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The house mouse X and Y chromosomes have recently acquired multicopy, rapidly evolving gene families representing an evolutionary arms race. This arms race between proteins encoded by X-linked Slxl1/Slx and Y-linked Sly gene families can distort offspring sex ratio, but how these proteins compete remains unknown. Here, we report how Slxl1/Slx and Sly encoded proteins compete in a protein family--specific and dose-dependent manner using yeast. Specifically, SLXL1 competes with SLY1 and SLY2 for binding to the Spindlin SPIN1. Similarly, SLX competes with SLY2 for binding the Spindlin SSTY2. These competitions are driven by the N termini of SLXL1, SLX, SLY1, and SLY2 binding to the third Tudor domains of SPIN1 and SSTY2. SLY1 and SLY2 form homo- and heterodimers, suggesting that the competition is between complex multimers. Residues under positive selection mapping to the interaction domains and rapid exon gain/loss are consistent with competition between the X- and Y-linked gene families. Our findings support a model in which dose-dependent competition of these X- and Y-linked encoded proteins to bind Spindlins occurs in haploid X- and Y-spermatids to influence X- versus Y-sperm fitness and thus sex ratio. [ABSTRACT FROM AUTHOR]

MADS-box genes are classified into five categories: ABCDE, including SEP1 , SEP2 , SEP3 , SEP4 , and other homologous genes, which play important roles in floral organ development. In this study, the cDNA sequence of the HrSEP1 gene was cloned by RT-PCR and confirmed that this gene belongs to the MADS-box gene family. In addition, subcellular localization experiments showed that the HrSEP1 protein was localized in the nucleus. We verified the interaction of HrSEP1 with HrSOC1 , HrSVP , and HrAP1 using yeast two-hybrid and bimolecular fluorescence complementation assays. These genes jointly regulate the growth and development of floral organs. We also found a strong synergy between HrSEP1 and AP1 genes in sepals, petals, and stamens by transgenic methods and fluorescence quantitative PCR, suggesting that HrSEP1 and AP1 may co-regulate the development of these structures. In conclusion, the expression of HrSEP1 has a certain effect on the development of floral organs, and these findings lay the foundation for further research on the biological functions of MADS transcription factors in Hippophae rhamnoides. [ABSTRACT FROM AUTHOR]

A yeast two-hybrid cDNA library of cucumber was constructed by Gateway technology under the stress of Pseudoperonospora cubensis and Corynespora cassiicola using the disease resistant inbred line D9320. The results showed that the number of yeast library clones was 350, the titer of library was 3.5×107 CFU/mL and the rate of recombination was 100%. The length of all the fragments was 750-2 000 bp. The average length was more than 1 000 bp. The yeast two hybrid cDNA library of cucumber under the stress of Pseudoperonospora cubensis and Corynespora cassiicola was constructed successfully, which could be used for screening interaction proteins of the double-disease resistance mechanism in cucumber. [ABSTRACT FROM AUTHOR]

【Objective】This study is aimed to construct a konjac cDNA library and screen for interacting proteins of the transcription factor WRKY72 in order to elucidate the molecular mechanism underlying WRKY72-mediated konjac resistance against diseases.【Method】Konjac plants at different stages of bacterial soft rot infection were used as experimental materials. Total RNA was extracted, pooled in equal amounts, and used to construct a konjac cDNA library. The bait vector containing the transcription factor WRKY72 was constructed using the homologous recombination method, and a yeast two-hybrid screen was performed to identify candidate target proteins, which were further verified individually.【Result】The yeast library titer was 1.6×107 CFU/mL, and the average length of the inserted fragments was around 1 000 bp. The bait vector was verified to have no self-activation in yeast. The bait vector pGBKT7-WRKY72 was used to screen library and 41 positive interacting proteins were obtained, including transport proteins, channel proteins, heat shock proteins, chlorophyll-binding proteins, and peroxidases. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analyses were performed on these interacting proteins, and 12 of them were further verified by one-on-one yeast two-hybrid assays.【Conclusion】A high-quality konjac cDNA library was successfully constructed, and the interacting proteins of the transcription factor WRKY72 were screened, providing a theoretical basis for elucidating the mechanism of resistance against soft rot disease in konjac. [ABSTRACT FROM AUTHOR]

Simple Summary: Infectious bronchitis (IB) is a highly contagious viral disease that affects poultry and leads to significant economic losses. Previous studies have demonstrated that Nsp2 functions as a potential virulence factor for infectious bronchitis virus (IBV). However, the host proteins that interact with Nsp2 and their roles in IBV pathogenesis remain largely unidentified. In this study, we identified ten host proteins that interact with IBV Nsp2 through yeast two-hybrid assays and molecular docking simulations. The intracellular interactions of Nsp2 with the proteins ATP1B3, DNAJA1, and ISCA1 were further validated using co-immunoprecipitation and confocal microscopy. Analyses incorporating Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and the Protein-Protein Interaction (PPI) database revealed that these host proteins are involved in ATPase activation, iron-sulfur (Fe-S) cluster binding, ion homeostasis, and the regulation of innate immunity. Furthermore, we observed significant changes in the expression levels of these Nsp2-interacting host proteins during IBV infection. This study establishes a foundation for the further exploration of the role and mechanisms of Nsp2 in IBV proliferation and provides valuable insights for developing novel antiviral strategies. Infectious bronchitis (IB) is a highly contagious acute viral disease that leads to substantial economic losses in the poultry industry. Previous research conducted in our laboratory has indicated that Nsp2 may serve as a key virulence factor within the IBV genome, as evidenced by its pronounced divergence between the field strain and its attenuated counterpart. Understanding the interaction between Nsp2 and host proteins is crucial to elucidating the role of the Nsp2 protein in the pathogenesis and proliferation of IBV. Currently, much remains to be uncovered regarding the host proteins that interact with the IBV Nsp2 protein. In this study, 10 host proteins, including COX1, COX3, NFIA, ITGA1, ATP1B1, ATP1B3, ABCB1, ISCA1, DNAJA1, and IREB2, were screened to interact with IBV Nsp2 through yeast two-hybrid experiments and molecular docking simulations. Furthermore, the interaction of Nsp2 with ATP1B3, DNAJA1, and ISCA1 proteins was further validated through co-immunoprecipitation and confocal experiments. The GO, KEGG, and PPI databases revealed that the host proteins interacting with Nsp2 are primarily associated with ATPase activation, Fe-S cluster binding, ion homeostasis, and innate immune regulation. The examination of the expression levels of these Nsp2-interacting host proteins during IBV infection demonstrated the significant downregulation of COX3, COX1, ATP1B1, and ATP1B3, while NFIA, DNAJA1, and IREB2 showed significant upregulation. Moreover, our study identified that IBV enhances viral replication by upregulating DNAJA1 expression, although the underlying mechanism requires further investigation. These findings provide valuable insights into the potential role of the Nsp2 protein in the pathogenesis of IBV. [ABSTRACT FROM AUTHOR]

MADS-box genes are classified into five categories: ABCDE, including SEP1, SEP2, SEP3, SEP4, and other homologous genes, which play important roles in floral organ development. In this study, the cDNA sequence of the HrSEP1 gene was cloned by RT-PCR and confirmed that this gene belongs to the MADS-box gene family. In addition, subcellular localization experiments showed that the HrSEP1 protein was localized in the nucleus. We verified the interaction of HrSEP1 with HrSOC1, HrSVP, and HrAP1 using yeast two-hybrid and bimolecular fluorescence complementation assays. These genes jointly regulate the growth and development of floral organs. We also found a strong synergy between HrSEP1 and AP1 genes in sepals, petals, and stamens by transgenic methods and fluorescence quantitative PCR, suggesting that HrSEP1 and AP1 may co-regulate the development of these structures. In conclusion, the expression of HrSEP1 has a certain effect on the development of floral organs, and these findings lay the foundation for further research on the biological functions of MADS transcription factors in Hippophae rhamnoides.

Abstract Background The Flavonoid 3’-hydroxylase gene(F3’H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. Results In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3’H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3’H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. Conclusions The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke.

Background: The Flavonoid 3'-hydroxylase gene(F3'H) is an important structural gene in the anthocyanin synthesis pathway of plants, which has been proven to be involved in the color formation of organs such as leaves, flowers, and fruits in many plants. However, the mechanism and function in barley are still unclear. Results: In order to explore the molecular mechanism of the grain color formation of purple qingke, we used the cultivated qingke variety Nierumzha (purple grain) and the selected qingke variety Kunlun 10 (white grain) to conduct transcriptomic sequencing at the early milk, late milk and soft dough stage. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct weighted gene co-expression network related to grain color formation, and three key modules (brown, yellow, and turquoise modules) related to purple grain of qingke were selected. F3'H (HORVU1Hr1G094880) was selected from the hub gene of the module for the yeast library, yeast two-hybrid (Y2H), subcellular localization and other studies. It was found that in purple qingke, HvnF3'H mainly distributed in the cytoplasm and cell membrane and interacted with several stress proteins such as methyltransferase protein and zinc finger protein. Conclusions: The results of this study provide reference for the regulation mechanism of anthocyanin-related genes in purple grain qingke. [ABSTRACT FROM AUTHOR]

Copyright of Chinese Journal of Preventive Veterinary Medicine / Zhongguo Yufang Shouyi Xuebao is the property of Chinese Journal of Preventive Veterinary Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

VQ1 and VQ10 are largely unstructured homologous proteins with a significant potential for protein–protein interactions. Yeast two-hybrid (Y2H) analysis confirmed that both proteins interact not only with themselves and each other but also with other VQ and WRKY proteins. Screening an Arabidopsis Y2H library with VQ1 as bait identified 287 interacting proteins. Validation of the screening confirmed that interactions with VQ1 also occurred with VQ10, supporting their functional homology. Although VQ1 or VQ10 proteins do not localize in plastids, 47 VQ1-targets were found to be plastidial proteins. In planta interaction with the isoprenoid biosynthetic enzyme 1-deoxy-D-xylulose-5-phosphate synthase (DXS) was confirmed by co-immunoprecipitation. DXS oligomerizes through redox-regulated intermolecular disulfide bond formation, and the interaction with VQ1 or VQ10 do not involve their unique C residues. The VQ-DXS protein interaction did not alter plastid DXS localization or its oligomerization state. Although plants with enhanced or reduced VQ1 and VQ10 expression did not exhibit significantly altered levels of isoprenoids compared to wild-type plants, they did display significantly improved or diminished photosynthesis efficiency, respectively. [ABSTRACT FROM AUTHOR]

[Objective] WRKY, as a plant idiosyncratic transcription factor, is involved in plant growth and development. DoWRKY40 gene in yam (Dioscorea opposita Thunb.) was cloned and its expression pattern was analyzed. The protein interacting with DoWRKY40 was screened by yeast two-hybrid technique to explore the regulatory mechanism of WRKY40 in the process of yam expansion. [Method] DoWRKY40 gene was cloned from ‘Dahechangyu’ yam, and bioinformatics analysis, expression pattern analysis and subcellular localization were performed. Yeast libraries of yam tuber at different developmental stages were constructed and DoWRKY40 interacting proteins were screened. [Result] The length of open reading frame of DoWRKY40 was 927 bp; DoWRKY40 belonged to group II of the WRKY family and contained a typical WRKY transcription factor conserved domain, which was localized in the nucleus. It was closely related to Dioscorea cayenensis subsp. The expression of DoWRKY40 gene was the highest at 120 d after the growth and development of yam tuber, and it was expressed in the leaves, stems and tubers with tissue specificity. The library capacity of yam cDNA library was 1.484×108; pGBKT7-WRKY40 decoy vector had no self-activation activity. Four kinds of proteins interacting with DoWRKY40 were screened from yam library by yeast two-hybridization, which were involved in plant growth and development, cell cycle regulation and cell expansion, signal transduction and environmental stress response, respectively. [Conclusion] The DoWRKY40 gene is cloned, which responds to the growth and development process of yam. The four interacting proteins are screened, suggesting that it plays a regulatory role in the expansion and signal transduction of yam cells. [ABSTRACT FROM AUTHOR]

Copyright of Acta Prataculturae Sinica is the property of Acta Prataculturae Sinica Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Photoperiod is a pivotal regulatory factor in the flowering of Camellia oleifera Abel. (C. oleifera). GIGANTEA (GI) serves as a pivotal regulator, not only orchestrating the intricate circadian rhythm but also governing photoperiod-dependent flowering. In order to explore the function of GI in C. oleifera (CoGI), we obtained a CoGI gene-coding sequence and analyzed a CoGI protein sequence using bioinformatics. Furthermore, we conducted a spatiotemporal expression analysis of CoGI. And a yeast two-hybridization assay was used to screen the interacting proteins of CoGI. Evolutionary analysis revealed high conservation of the CoGI protein, which clustered with the GI protein from Camellia sinensis (CsGI) on a common evolutionary branch. The expression of CoGI was different in each part, and a tissue expression analysis revealed that the relative expression level of the CoGI gene is highest in the leaves of C. oleifera, while it is at its lowest in the seed coats. Transgenic Arabidopsis thaliana (Arabidopsis) overexpressing CoGI exhibited early flowering under long-day conditions. In addition, the yeast two-hybrid library screening revealed interactions between seven C. oleifera proteins and CoGI: CoACR9, CoLAO, CoDExH12-like, CoIT1K-like, CoUPF0481, CoIDM3, and CoAt4g27190-like. The findings demonstrated that CoGI is crucial to C. oleifera's flowering. [ABSTRACT FROM AUTHOR]

ABSTRACT Legionella pneumophila uses over 300 translocated effector proteins to rewire host cells during infection and create a replicative niche for intracellular growth. To date, several studies have identified L. pneumophila effectors that indirectly and directly regulate the activity of other effectors, providing an additional layer of regulatory complexity. Among these are “metaeffectors,” a special class of effectors that regulate the activity of other effectors once inside the host. A defining feature of metaeffectors is direct, physical interaction with a target effector. Metaeffector identification, to date, has depended on phenotypes in heterologous systems and experimental serendipity. Using a multiplexed, recombinant barcode-based yeast two-hybrid technology we screened for protein–protein interactions among all L. pneumophila effectors and 28 components of the Dot/Icm type IV secretion system (>167,000 protein combinations). Of the 52 protein interactions identified by this approach, 44 are novel protein interactions, including 10 novel effector–effector interactions (doubling the number of known effector–effector interactions).IMPORTANCESecreted bacterial effector proteins are typically viewed as modulators of host activity, entering the host cytosol to physically interact with and modify the activity of one or more host proteins in support of infection. A growing body of evidence suggests that a subset of effectors primarily function to modify the activities of other effectors inside the host. These “effectors of effectors” or metaeffectors are often identified through experimental serendipity during the study of canonical effector function against the host. We previously performed the first global effector-wide genetic interaction screen for metaeffectors within the arsenal of Legionella pneumophila, an intracellular bacterial pathogen with over 300 effectors. Here, using a high-throughput, scalable methodology, we present the first global interaction network of physical interactions between L. pneumophila effectors. This data set serves as a complementary resource to identify and understand both the scope and nature of non-canonical effector activity within this important human pathogen.

The utilization of human cDNA libraries in yeast genetic screens is an approach that has been used to identify novel gene functions and/or genetic and physical interaction partners through forward genetics using yeast two-hybrid (Y2H) and classical cDNA library screens. Here, we summarize several challenges that have been observed during the implementation of human cDNA library screens in Saccharomyces cerevisiae (budding yeast). Upon the utilization of DNA repair deficient-yeast strains to identify novel genes that rescue the toxic effect of DNA-damage inducing drugs, we have observed a wide range of transcripts that could rescue the strains. However, after several rounds of screening, most of these hits turned out to be false positives, most likely due to spontaneous mutations in the yeast strains that arise as a rescue mechanism due to exposure to toxic DNA damage inducing-drugs.The observed transcripts included mitochondrial hits, non-coding RNAs, truncated cDNAs, and transcription products that resulted from the internal priming of genomic regions. We have also noticed that most cDNA transcripts are not fused with the GAL4 activation domain (GAL4AD), rendering them unsuitable for Y2H screening. Consequently, we utilized Sanger sequencing to screen 282 transcripts obtained from either four different yeast screens or through direct fishing from a human kidney cDNA library. The aim was to gain insights into the different transcription products and to highlight the challenges of cDNA screening approaches in the presence of a significant number of undesired transcription products. In summary, this study describes the challenges encountering human cDNA library screening in yeast as a valuable technique that led to the identification of important molecular mechanisms. The results open research venues to further optimize the process and increase its efficiency.

【Objective】Pogostemon cablin (Blanco) Benth. is a frequently-used aromatic herb eliminating dampness in clinical settings, with its primary bioactive compound, patchouli alcohol, demonstrating potent antiviral and anti-inflammatory and other beneficial properties. Patchoulialcohol synthase (PcPTS) plays an essential role in the biosynthesis pathway of patchouli alcohol. However, the precise mechanisms by which PcPTS modulates patchouli alcohol production at the level of protein-protein interactions is still unclear. This study aims to identify interacting proteins of PcPTS in order to elucidate its regulatory mechanisms and functions in the biosynthesis pathway of patchouli alcohol.【Method】The yeast two-hybrid technique and GST pull-down combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed to identify potential interacting proteins of PcPTS. The annotation of these interacting proteins was conducted by the MASCOT software and BLAST analysis. Proteins known to be involved in protein translocation, modification, degradation and regulation of secondary metabolism were selected for further analysis. Their interactions with PcPTS proteins were initially confirmed through yeast two-hybrid technology, and subsequently analyzed by using bioinformatics tools.【Result】The cDNA sequence of PcPTS was successfully cloned by PCR, resulting in an open reading frame (ORF) of 1 659 bp, encoding 552 amino acids. The primary cDNA library and the yeast hybrid cDNA library were constructed and obtained, with capacities of 7.6×106 and 8.6×106 colony-forming units (CFU), respectively. Both libraries achieved a 100% recombination rate, with an average length of the inserted fragment greater than 800 bp. The constructed bait vector pGBKT7-PcPTS exhibited no toxicity towards yeast strains and did not exhibit any self-activating activity in yeast cells. The positive clones identified through yeast two-hybrid screening were subjected to sequencing and BLAST analysis, resulting in the identification of 17 candidate proteins. Subsequently, the GST-PcPTS expression vector was effectively constructed, and the GST-PcPTS bait protein was obtained through induced expression purification. This bait protein was utilized to capture interacting proteins from the total protein of Pogostemon cablin, and 98 candidate interacting proteins were identified. Among these candidates, 14 proteins potentially interacting with PcPTS were selected and confirmed through point-to-point verification with yeast two-hybridization. The results indicated that PcC3H6, PcENO3, PcACR11 and PcHAD interacted with PcPTS. Bioinformatics analysis indicated that they belonged to the CCCH zinc finger protein family, enolase proteins, ACR subfamily and HAD-like superfamily, respectively.【Conclusion】Four proteins (PcC3H6, PcENO3, PcACR11, and PcHAD) were identified as interacting with PcPTS through initial screening and validation. This finding aids in elucidating the role of PcPTS in the biosynthesis pathway of patchouli alcohol and provides a robust basis for future investigations into the regulatory mechanisms of patchouli alcohol synthesis.

【Objective】To screen the interaction proteins of transcription factors of Setosphaeria turcica, and to analyze the molecular mechanism of melanin-regulated transcription factor StMR1 in regulating the pathogenicity of S. turcica. This study provides a reference for elucidating the regulatory network of transcription factors in the infection process of S. turcica and elucidating the pathogenic mechanism of the pathogen.【Method】Different germination stages of hyphae and spores of S. turcica were collected as test materials, and the cDNA library of S. turcica was constructed by gateway method, The bait vector of transcription factor StMR1 was constructed by homologous recombination, and its interacting proteins were screened by yeast two-hybrid technology and verified one-to-one.【Result】The average fragment length of the constructed library was greater than 1 000 bp, the library capacity of the primary library and the secondary library were 1.2×107 and 1.04×107 CFU, respectively, and the recombination rate was 100%, which could be used for yeast two-hybrid screening. The bait vector pGBKT7-StMR1 that could be used for screen library was successfully constructed, and three interacting proteins were obtained through primary screening and rescreening, and one-to-one verification of the interaction between short-chain dehydrogenase, glycosyltransferase and leucine-rich repeat protein and transcription factor StMR1 were verified.【Conclusion】A high quality and abundant cDNA library was successfully constructed and the protein interacting with StMR1 was screened [ABSTRACT FROM AUTHOR]

Voltage-dependent anion channels (VDACs) are known to be involved in the transport of molecules across the outer mitochondrial membrane. S-adenosylmethionine synthetase (SAMS) serves as a pivotal enzyme in synthesizing S-adenosylmethionine from ATP and L-methionine. Previously, we identified a high amount of accumulation of barley SAMS1 transcripts in grains. In this study, we conducted a yeast two-hybrid assay to identify proteins interacting with HvSAMS1. HvVDAC1 was identified from clones screened by a yeast two-hybrid assay and was then characterized. The interaction between HvSAMS1 and HvVDAC1 was cross-validated through a BiFC assay in barley protoplasts. HvVDAC1 was primarily localized in small vesicles near the plasma membrane in barley protoplasts. A β-galactosidase assay, using HvVDAC1 and HvSAMS1 deletion derivatives, was conducted to investigate which part(s) of HvSAMS1 is (are) involved in the interaction with HvVDAC1 in yeast. The β-galactosidase activities of HvSAMS1 Fragment II (1st to 116th amino acids) and Fragment III (114th to 194th amino acids) were as high as the positive control, implying that HvSAMS1 Fragment II and Fragment III play important roles in the binding of HvSAMS1 to HvVDAC1. HvVDAC1 was expressed in dry seeds, imbibed seeds, and germinating seeds, exhibiting a gradual increase in that order. HvVDAC1 expression was regulated by exogenous plant hormones, in this case GA and ABA, in germinating seeds. HvVDAC1 was also expressed during the grain development stage, exhibiting high levels of transcript accumulation before and after fertilization in grains. These results contribute to advancing our fundamental understanding of HvVDAC1, providing insights into barley grain development and germination, with potential applications in further functional studies. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Guangdong Agricultural Sciences is the property of South China Agricultural University, Guangdong Academy of Agricultural Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

Plant acyl-CoA-binding proteins (ACBPs) function in plant development and stress responses, with some ACBPs interacting with protein partners. This study tested the interaction between two Class II GmACBPs (Glycine max ACBPs) and seven kinases, using yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC). The results revealed that both GmACBP3.1 and GmACBP4.1 interact with two soybean kinases, a mitogen-activated protein kinase MPK2, and a serine/threonine-protein kinase SAPK2, highlighting the significance of the ankyrin-repeat (ANK) domain in facilitating protein–protein interactions. Moreover, an in vitro kinase assay and subsequent Phos-tag SDS-PAGE determined that GmMPK2 and GmSAPK2 possess the ability to phosphorylate Class II GmACBPs. Additionally, the kinase-specific phosphosites for Class II GmACBPs were predicted using databases. The HDOCK server was also utilized to predict the binding models of Class II GmACBPs with these two kinases, and the results indicated that the affected residues were located in the ANK region of Class II GmACBPs in both docking models, aligning with the findings of the Y2H and BiFC experiments. This is the first report describing the interaction between Class II GmACBPs and kinases, suggesting that Class II GmACBPs have potential as phospho-proteins that impact signaling pathways. [ABSTRACT FROM AUTHOR]

The secondary laticifer is the position for synthesis and storage of natural rubber (NR), which is differentiated from the vascular cambium cells of bark in stem of rubber trees (Hevea brasiliensis). The quantity of secondary laticifer is depended on the frequency of the secondary laticifer differentiation from cambia, which is the main index of yield breeding of rubber tree. In previous studies, we found trichostatin A (TSA), an inhibitor of histone deacetylase (HDA), can also induce laticifer differentiation, and the histone deacetylase gene (HbHDA6) is a participator in laticifer differentiation. Because of the molecular mechanism of secondary laticifer differentiation regulated by histone acetylation has not been clarified. Therefore, we construct a yeast two-hybrid cDNA library used the vascular cambium tissues treatment by coronatine (COR), and screening the yeast two-hybrid library by HbHDA6 gene as the bait, for determining the proteins interacting with HbHDA6. The results were as follows: (1) The homogenized yeast two-hybrid cDNA library of vascular cambium was constructed by the technology of Gateway. The capacity of the primary library was 6.34 × 106 CFU·mL-1, the total number of clones was 1.27 × 107, and the capacity of secondary library was 7.72 × 106 CFU·mL-1, the total number of clones was 1.54 × 107, and the recombination rates of two libraries were 100%. The average length of inserted fragments was 1.1 kb and 1.2 kb in primary and secondary library, respectively. (2) The bait vector of pGBKT7-HbHDA6 for screening the proteins interacting with HbHDA6 was successfully constructed and confirmed no self-activation activity. (3) The pGBKT7-HbHDA6 bait vector was used to screen the constructed yeast two-hybrid cDNA library, and 22 proteins interacting with HbHDA6 were obtained by NCBI_BLAST comparison and removing duplicates, including CLP1, ERF3, ERF4, HSP82, LARP6a, APT5, PP2A, APT5, FBA6, etc. The results provide a theoretical basis for analyzing the molecular regulatory network of the secondary laticifer differentiation of rubber tree, and provide candidate genes for the rubber production potential of genetically modified and a new clue for the genetic improvement and breeding of high-performance NR.

SnRK (sucrose non-fermenting protein kinase family), a class of serine/threonine (Ser/Thr) protein kinases, is widely distributed in plants and is divided into three subfamilies: SnRK1, SnRK2, and SnRK3. The SnRK2 family, in particular, plays a significant role in osmotic stress resistance and ABA signaling pathways. However, research on the relationship between the SnRK2 gene and iron (Fe) deficiency in apples is limited. Studies have shown that the SnRK2.4 gene exhibited higher regulation of expression under Fe-deficient conditions compared to other genes. Yet, the mode of action of this gene in regulating Fe-deficiency stress remains unexplained. Therefore, the MhSnRK2.4 gene (Genbank ID:103411041) was cloned from Malus halliana in the present study. It was observed that transgenic Arabidopsis plants exhibited more robust growth and reduced chlorosis under Fe-deficiency stress when compared to the wild-type control. The overexpressed apple calli also exhibited enhanced growth Fe-deficiency stress. In addition, measurements of physiological indices showed that overexpression of the MhSnRK2.4 gene increased the tolerance of Arabidopsis thaliana to Fe-deficiency damage by promoting chlorophyll synthesis, Fe-deficiency damage by promoting chlorophyll synthesis, increasing the activity of antioxidant end enzymes, and promoting Fe reduction, and apple calli to Fe-deficiency damage by decreasing pH, increasing the activity of antioxidant end enzymes, and promoting Fe uptake. In conclusion, the overexpression of the MhSnRK2.4 gene enhanced the resistance to Fe-deficiency stress in Arabidopsis and apple calli, increasing antioxidant enzyme activity, and promoting Fe reduction.

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

Infectious bronchitis (IB) is a highly contagious acute viral disease that leads to substantial economic losses in the poultry industry. Previous research conducted in our laboratory has indicated that Nsp2 may serve as a key virulence factor within the IBV genome, as evidenced by its pronounced divergence between the field strain and its attenuated counterpart. Understanding the interaction between Nsp2 and host proteins is crucial to elucidating the role of the Nsp2 protein in the pathogenesis and proliferation of IBV. Currently, much remains to be uncovered regarding the host proteins that interact with the IBV Nsp2 protein. In this study, 10 host proteins, including COX1, COX3, NFIA, ITGA1, ATP1B1, ATP1B3, ABCB1, ISCA1, DNAJA1, and IREB2, were screened to interact with IBV Nsp2 through yeast two-hybrid experiments and molecular docking simulations. Furthermore, the interaction of Nsp2 with ATP1B3, DNAJA1, and ISCA1 proteins was further validated through co-immunoprecipitation and confocal experiments. The GO, KEGG, and PPI databases revealed that the host proteins interacting with Nsp2 are primarily associated with ATPase activation, Fe-S cluster binding, ion homeostasis, and innate immune regulation. The examination of the expression levels of these Nsp2-interacting host proteins during IBV infection demonstrated the significant downregulation of COX3, COX1, ATP1B1, and ATP1B3, while NFIA, DNAJA1, and IREB2 showed significant upregulation. Moreover, our study identified that IBV enhances viral replication by upregulating DNAJA1 expression, although the underlying mechanism requires further investigation. These findings provide valuable insights into the potential role of the Nsp2 protein in the pathogenesis of IBV.

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[Objective] Sugarcane is an important sugar crop, and temperature, salinity, water and other factors are the key environmental factors restricting its growth and development. Calcineurin B-Like protein (CBL) family represents a group of Ca2+ binding proteins and plays a key role in decoding calcium signals by specifically interacting with and regulating a family of protein kinases CIPKs (for CBL-interacting protein kinases) in abiotic stress signal transduction pathway. At present, the whole genome of sugarcane has been sequenced, but its CPL-CIPK gene family members have not been identified, and the interaction regulation mechanism is still unknown. Here, the members of CBL and CIPK in sugarcane were identified and the CPL-CIPK interaction relationship was revealed, which may provide gene resources and theoretical basis for studying the mechanism of CPL-CIPK interaction in sugarcane. [Method] The expression levels of CBLs and CIPKs under low temperature, high temperature, NaHCO3 and PEG were analyzed by RT-qPCR from sugarcane GT58. The interactions between SsCBLs and SsCIPKs were analyzed by yeast two-hybrid assay. [Result] There were 19 CBL genes and 82 CIPK genes in the whole genome of sugarcane, which distributed in different evolutionary branches with gene replication. The physicochemical properties of gene family members were different, the structural domains and protein motifs were highly conserved, and the distribution of cis-acting elements was diverse. SsCBL7/SsCBL12/SsCIPK1/SsCIPK5 were specifically expressed under low temperature, high temperature, drought and high salt and other abiotic stress regulation. Meanwhile, SsCBL1 interacted with SsCIPK47 and SsCIPK81, and SsCBL8 interacted with SsCIPK47 and SsCIPK81. [Conclusion] The CBL-CIPK signaling system may play an important role in response to abiotic stress during the growth and development of sugarcane. [ABSTRACT FROM AUTHOR]

[Objective] Screening the interacting protein of WDR40 protein TRANSPARENT TESTA GLABRA 1（TTG1）by constructing the cDNA library of Chinese cabbage seeds, the molecular mechanism of TTG1 in the regulation of the formation of MBW ternary complex will be explored. [Method] Seeds of brown-seeded Chinese cabbage ‘B147’ were used as materials for RNA extraction and cDNA library construction. The bait vector pGBKT7-TTG1 was constructed by gateway technology and then yeast two-hybrid screening were performed. [Result] The library capacity was 1.2×107 CFU and library titer was 5.0×107 CFU/mL with average length of the insert greater than 1 000 bp. The bait vector had no self-activating activity in yeast. A total of 38 positive interacting proteins were obtained by hybridizing the bait vector pGBKT7-TTG1 with the cDNA library. Function prediction revealed one of the proteins annotated as a MYB73 transcription factor. Sequence analysis results showed that this gene contained R2R3-MYB type suppressor conserved motifs C1 and C2, speculated that this gene may be the R2R3-MYB suppressor involving in the seed coat color formation in Chinese cabbage, suggesting that there may be different MYB transcription factors in the regulation network affecting the formation of proanthocyanidins. [Conclusion] In this study, a yeast two-hybrid cDNA library of Chinese cabbage seed-specific tissue is constructed and a total of 38 TTG1-positive interacting proteins are obtained. We found the R2R3-MYB type suppressor MYB73 that may affect the formation of proanthocyanidin color in cabbage seed coat for the first time. This study lays a good foundation for exploring the regulatory network of proanthocyanindins formation. [ABSTRACT FROM AUTHOR]

Plants absorb and transport phosphorus through phosphorus transporters, and sufficient phosphorus can improve crop yield, quality, and stress resistance. High temperature is an important environmental factor affecting the growth and development of potato. In this study, we overexpressed the phosphorus transporter StPHO1.2 in potato mediated by Agrobacterium tumefaciens, and then compared the growth of transgenic strains and wild-type strains at high temperature (35°C) and normal temperature (22°±1°C. The results showed that overexpression of StPHO1.2 could improve the heat resistance and promote the growth of potato. And the higher the phosphorus concentration, the stronger the resistance, the better the growth. Subcellular localization in tobacco showed that StPHO1.2 was expressed on cell membrane. Therefore, the membrane system library plasmid was selected to screen the interacting proteins of StPHO1.2. This study demonstrated that the phosphorus transporter StPHO1.2 interacted with both the calcium ion transporter associated protein (StCAX1) and the photosynthetic system II protein subunit (StPsbR) by the yeast two-hybrid and BiFC. In conclusion, overexpression of StPHO1.2 may improve the heat tolerance and promote the growth of potato by affecting photosynthesis and signal transduction in potato. These results provide theoretical basis and reference for further understanding of the function of phosphorus transporters and promote the breeding of new potato varieties. [ABSTRACT FROM AUTHOR]

Copyright of Guihaia is the property of Guihaia Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Influenza A viruses are enveloped viruses with a genome of eight singlestranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxyterminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription. [ABSTRACT FROM AUTHOR]

Copyright of Acta Agriculturae Zhejiangensis is the property of Acta Agriculturae Zhejiangensis Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Photoperiod is a pivotal regulatory factor in the flowering of Camellia oleifera Abel. (C. oleifera). GIGANTEA (GI) serves as a pivotal regulator, not only orchestrating the intricate circadian rhythm but also governing photoperiod-dependent flowering. In order to explore the function of GI in C. oleifera (CoGI), we obtained a CoGI gene-coding sequence and analyzed a CoGI protein sequence using bioinformatics. Furthermore, we conducted a spatiotemporal expression analysis of CoGI. And a yeast two-hybridization assay was used to screen the interacting proteins of CoGI. Evolutionary analysis revealed high conservation of the CoGI protein, which clustered with the GI protein from Camellia sinensis (CsGI) on a common evolutionary branch. The expression of CoGI was different in each part, and a tissue expression analysis revealed that the relative expression level of the CoGI gene is highest in the leaves of C. oleifera, while it is at its lowest in the seed coats. Transgenic Arabidopsis thaliana (Arabidopsis) overexpressing CoGI exhibited early flowering under long-day conditions. In addition, the yeast two-hybrid library screening revealed interactions between seven C. oleifera proteins and CoGI: CoACR9, CoLAO, CoDExH12-like, CoIT1K-like, CoUPF0481, CoIDM3, and CoAt4g27190-like. The findings demonstrated that CoGI is crucial to C. oleifera’s flowering.

Influenza A viruses are enveloped viruses with a genome of eight single-stranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxy-terminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription.

Interactions between proteins are fundamental for every biological process and especially important in cell signaling pathways. Biochemical techniques that evaluate these protein-protein interactions (PPIs), such as in vitro pull downs and coimmunoprecipitations, have become popular in most laboratories and are essential to identify and validate novel protein binding partners. Most PPIs occur through small domains or motifs, which are challenging and laborious to map by using standard biochemical approaches because they generally require the cloning of several truncation mutants. Moreover, these classical methodologies provide limited resolution of the interacting interface. Here, we describe the development of an alternative technique to overcome these limitations termed "Protein Domain mapping using Yeast 2 Hybrid-Next Generation Sequencing" (DoMY-Seq), which leverages both yeast two-hybrid and next-generation sequencing techniques. In brief, our approach involves creating a library of fragments derived from an open reading frame of interest and enriching for the interacting fragments using a yeast two-hybrid reporter system. Next-generation sequencing is then subsequently employed to read and map the sequence of the interacting fragment, yielding a high-resolution plot of the binding interface. We optimized DoMY-Seq by taking advantage of the well-described and high-affinity interaction between KRAS and CRAF, and we provide high-resolution domain mapping on this and other protein-interacting pairs, including CRAF-MEK1, RIT1-RGL3, and p53-MDM2. Thus, DoMY-Seq provides an unbiased alternative method to rapidly identify the domains involved in PPIs by advancing the use of yeast two-hybrid technology.

In order to verify whether the cryptochrome protein IgCRYl of Isochrysis galbana interacted with the downstream protein IgCOPl, the total RNA of Isochrysis galbana was extracted and reverse transcribed into cDNA as a template to amplify the target fragment. The BE)-IgCRYl and AD-IgCOPl vectors were constructed and co-transfected into the yeast, strain AH109 for the yeast two-hybrid verification. And the bioinformatics analysis of IgCOPl protein was performed. The results showed that the CDS sequences of IgCRYl and IgCOPl cloned from Isochrysis galbana were 1 560 bp and 1185 bp, respectively. The mutual interaction between IgCRYl protein and IgCOPl protein was verified by the yeast two--hybrid system. The bioinformatics analysis of the interacting protein IgCOPl revealed that the molecular size of the protein was 43. 7 kDa, and the number of encoded amino acids was 394, which was a typical hydrophilic protein with 62. 69% of its secondary structure irregularly coiled. The phylogenetic tree analysis showed that IgCOPl protein was clustered at the bottom of the branch, and had a distant relationship with the other branches. The verification of the interactions between IgCRYl protein and IgCOPl protein provided an important theoretical basis for the biological process of light signal regulation in Isochrysis galbana. [ABSTRACT FROM AUTHOR]

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 litis 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. [ABSTRACT FROM AUTHOR]

Nanobodies are a novel type of engineered antibodies with small size, high stability, and strong affinity, offering new possibilities for scientific research. The FLAG tag is a short peptide tag widely used in biological research, playing a significant role in various studies. In order to prepare nanobodies against the FLAG tag, high‑affinity nanobodies were screened using yeast two‑hybrid technology and the prepared FLAG nanobodies were subsequently tested for performance. Through DNA recombination technology, a bait vector containing the FLAG tag was constructed. Using yeast two‑hybrid technology, nanobodies against the FLAG tag were screened from a camel‑derived nanobody yeast library. Five single candidate antibody DNA sequences were screened from the yeast library. To exclude interference from the expression of protein sequences in the carrier itself on antibody screening, the possibility of non‑specific hybridization was ruled out through a‘point‑to‑ point’verification method. This operation confirmed that all five screened nanobodies specifically reacted with the FLAG tag. For the production of nanobodies, five nanobody prokaryotic expression vectors were constructed and expressed using the Escherichia coli system. SDS‑PAGE and Western blot(WB)analysis showed that two soluble anti‑FLAG tagged protein nanobodies were successfully obtained. The effects of these two nanobodies were compared with commercially available conventional FLAG tag antibodies. The results showed that both the prepared nanobodies and the commercial antibodies recognized FLAG peptides and fusion proteins with FLAG tags, and there were no significant differences in specificity, indicating that the prepared FLAG nanobodies had good application prospects. Based on yeast two‑hybrid technology, FLAG tag nanobodies were successfully screened and prepared. This achievement not only enriches the types of nanobodies but also provides a new approach for the development and application of antibodies. This study provides strong support for further research on the application of FLAG tags in biological research, as well as the application of nanobodies in bioengineering. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is one of the deadly diseases caused by Mycobacterium tuberculosis (Mtb), which presents a significant public health challenge. Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens. Therefore, new anti-TB agents working by different mechanisms are urgently needed. FtsZ, a tubulin-like protein with GTPase activity, forms a dynamic Z-ring in cell division. Most of FtsZ inhibitors are designed to inhibit GTPase activity. In Mtb, the function of Z-ring is modulated by SepF, a FtsZ binding protein. The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division. Here, we established a yeast two-hybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M. tuberculosis. Using this system, we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice. Moreover, we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down, fluorescence polarization (FP), surface plasmon resonance (SPR) and CRISPRi knockdown assays. Furthermore, T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum. Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.

Mitogen-activated protein kinases (MAPKs, also known as MPKs) regulate diverse cellular and physiological functions, and dual-specificity MAPK phosphatases (MKPs) modulate MAPK signalling through MAPK dephosphorylation and inactivation. Due to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs (CmMKPs) and C. morifolium MAPKs (CmMPKs), we systematically studied the interactions between four groups of CmMPKs and eight identified CmMKPs in chrysanthemum and found that the interaction between the specific CmMKP and the specific CmMPK differed from those in other plants. Furthermore, the expression of CmMKP1 and CmMKP1-LIKE1 showed opposite trends during the development of chrysanthemum flower buds under salt treatment and Alternaria alternata inoculation, but these genes could interact with the same CmMPKs, providing insight into the subfunctionalization of paralogues. Amino acid variations (M87V, T277P and V6L) in dual-specificity protein phosphatases (DsPTP1)-LIKE1/2/3 changed the interactions of these proteins with the four CmMPK groups in chrysanthemum, providing evidence for the de/neofunctionalization of paralogues in polyploids, suggesting that we can identify the key functional sites of proteins by studying polyploid paralogues.

In order to screen the target proteins interacting with effector proteins of the wheat leaf rust and to lay a foundation for dissecting the defense response mechanism of the effector proteins interfering with hosts and disease resistance breeding, the samples of interaction between the isolate13-5-28-1 (JHKT) and susceptible wheat variety Thatcher at 0-12 d were collected. The three-frame homogenization was constructed by SMART method to screen the wheat cDNA library interacting with the wheat leaf rust. The homologous recombination method was used to construct the recombinant bait vector pGBKT7-Pt34084 of the effector factor Pt34084, and it was used as the bait protein to screen the interaction target protein by co-transformation method. The results showed that the cDNA library structure of the interaction between the wheat leaf rust and wheat was successfully constructed. The library capacity was about 1.05×107 CFU/mL, the titer was 1.2×109 CFU/mL, the average insertion fragment was more than 1 kb, and the recombination positive rate was 100%. The cDNA library met the requirement. The constructed bait recombinant vector pGBKT7-Pt34084 inhibited its self-activation after the addition of 20 mmol/L 3-AT and 400 ng/mL ABA to SD/-Trp-His-Ade medium. By this library a total of 16 different candidate interacting proteins from the wheat and the leaf rust were screened. These proteins were involved in plant metabolism, hormone signal transduction, plant disease resistance and other aspects. It indicated that Pt34084 could interfere with the host defense response and promote leaf rust infection through multiple pathways. The results are conducive to analyzing the pathogenic mechanism of wheat leaf rust and lay a foundation for creating new effective strategies to prevent and control wheat leaf rust. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Chinese Institute of Food Science & Technology / Zhongguo Shipin Xuebao is the property of Journal of Chinese Institute of Food Science & Technology Periodical Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. [ABSTRACT FROM AUTHOR]

Simple Summary: In insects, sex determination is generated using intricate and variegate biological processes that may be effectively described as variations on a common theme. In the model system Drosophila melanogaster, genetic and biochemical studies have shown that the female-specific Transformer (TRA) and the non-sex-specific Transformer2 (TRA2) are RNA-binding proteins that physically interact to promote female differentiation by female-specific alternative splicing of downstream genes. This tra gene responds and transduces different primary sex-determining signals, and its master function is widely conserved in Diptera, Coleoptera, and Hymenoptera. Here, combining yeast two-hybrid and computational methodologies, we demonstrate that the TRA and TRA2 orthologs of the agricultural pest Ceratitis capitata physically interact through a molecular mechanism that could be evolutionarily conserved in other species. These technical approaches can be helpful to verify or to identify other proteins interacting with TRA and TRA2, for example, those promoting male sex determination in this and other species, as well as to design new compounds that could induce masculinization of XX individuals in applications of the Sterile Insect Technique. In the model system for genetics, Drosophila melanogaster, sexual differentiation and male courtship behavior are controlled by sex-specific splicing of doublesex (dsx) and fruitless (fru). In vitro and in vivo studies showed that female-specific Transformer (TRA) and the non-sex-specific Transformer 2 (TRA2) splicing factors interact, forming a complex promoting dsx and fru female-specific splicing. TRA/TRA2 complex binds to 13 nt long sequence repeats in their pre-mRNAs. In the Mediterranean fruitfly Ceratitis capitata (Medfly), a major agricultural pest, which shares with Drosophila a ~120 million years old ancestor, Cctra and Cctra2 genes seem to promote female-specific splicing of Ccdsx and Ccfru, which contain conserved TRA/TRA2 binding repeats. Unlike Drosophila tra, Cctra autoregulates its female-specific splicing through these putative regulatory repeats. Here, a yeast two-hybrid assay shows that CcTRA interacts with CcTRA2, despite its high amino acid divergence compared to Drosophila TRA. Interestingly, CcTRA2 interacts with itself, as also observed for Drosophila TRA2. We also generated a three-dimensional model of the complex formed by CcTRA and CcTRA2 using predictive approaches based on Artificial Intelligence. This structure also identified an evolutionary and highly conserved putative TRA2 recognition motif in the TRA sequence. The Y2H approach, combined with powerful predictive tools of three-dimensional protein structures, could use helpful also in this and other insect species to understand the potential links between different upstream proteins acting as primary sex-determining signals and the conserved TRA and TRA2 transducers. [ABSTRACT FROM AUTHOR]

Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus in the family Luteoviridae, causes severe damage and represents a great threat to sugarcane cultivation and sugar industry development. In this study, inoculation of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the SCYLV P0 gene induced typical mosaic, leaf rolling symptoms and was associated with a hypersensitive-like response (HLR) necrosis symptom, which is accompanied with a systemic burst of H2O2 and also leads to higher PVX viral genome accumulation levels. Our results demonstrate that SCYLV P0 is a pathogenicity determinant and plays important roles in disease development. To further explore its function in pathogenic processes, a yeast two-hybrid assay was performed to screen the putative P0-interacting host factors. The recombinant plasmid pGBKT7-P0 was constructed as a bait and transformed into the yeast strain Y2HGold. The ROC22 cultivar (an important parental resource of the main cultivar in China) cDNA prey library was constructed and screened by co-transformation with the P0 bait. We identified 28 potential interacting partners including those involved in the optical signal path, plant growth and development, transcriptional regulation, host defense response, and viral replication. To our knowledge, this is the first time we have reported the host proteins interacting with the P0 virulence factor encoded by sugarcane yellow leaf virus. This study not only provides valuable insights into elucidating the molecular mechanism of the pathogenicity of SCYLV, but also sheds light on revealing the probable new pathogenesis of Polerovirus in the future. [ABSTRACT FROM AUTHOR]