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A LexA-based yeast two-hybrid system for studying light- switchable interactions of phytochromes with their interacting partners

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aBIOTECH 2021年第2期2卷 105-116页

作者： Hong Li Xinyan Qin Pengyu Song Run Han Jigang Li State Key Laboratory of Plant Physiology and Biochemistry College of Biological SciencesChina Agricultural UniversityBeijing 100193China

Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light *** exist in vivo in two forms,the inactive Pr form and the active Pfr form,that are interconvertible by treatments with R or FR *** is believed that phytochromes transduce light signals by interacting with their signaling partners.A GAL4-based lightswitchable yeast two-hybrid(Y2H)system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling ***,several pairs of interactions between phytochromes and their interactors,such as the phyACOP1 and phyA-TZP interactions,were demonstrated by other assay systems but were not detected by this GAL4 Y2H ***,we report a modified LexA Y2H system,in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome *** conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin(PCB)extracted from *** well-defined interaction pairs,including phyA-FHY1 and phyB-PIFs,are well reproducible in this ***,we show that our system is successful in detecting the phyA-COP1 and phyA-TZP ***,our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.

关键词： Phytochrome Interaction yeast two-hybrid LexA Light-switchable

Identification of FANCA as a protein interacting with Centromere-associated protein E Using a yeast two-hybrid

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Identification of FANCA as a protein interacting with Centro...

Construction of a yeast two-hybrid cDNA library form rice spikelets infected with Ustilaginoidea virens

华东六省一市生物化学与分子生物学会2009年学术交流会

作者： Jian Du~(1,5) Li Jian Chen~(2,5) Ji Long Shen~3 Sha Sha Hua~4 Xin Cai~4 Xue biao Yao~4 (1 Department of Biochemistry and Molecular Biology,Anhui Medical University,Hefei 230032, China 2 Department of Anaesthesiology,The First Affiliated Hospital of Anhui Medical University,Hefei 230032,China 3 Department of Microbiology and Parasitology,Anhui Medical University,Provincial Key Laboratory of Genomic Research,Anhui,China.4 Laboratory of Cellular Dynamics,University of Science and Technology of China,and National Laboratory for Physical Sciences,Hefei 230027,China 5 These authors contributed equally to this work.)

＜正＞This study sought to isolate and identify proteins that interact with CENP-E, provide new clues for exploring the function of CENP-E in cell cycle control and the pathogenesisof *** two hybrid screen（MATCH-MAKE... 详细信息

关键词： FANCA Centromere-associated protein E yeast two-hybrid interaction

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Construction of a yeast two-hybrid cDNA library form rice sp...

Screening and Identifying of Interaction Protein AtL5 in Arabidopsis thaliana

中国植物病理学会2015年学术年会

作者： WANG Yu-qiu FAN Jing YANG Juan LI Guo-bang LI Liang ZHAO Zhi-xue WANG Wen-ming Rice Research Institute Sichuan Agricultural University

Occurrence of rice false smut disease is increasing worldwide,but the underlying mechanism of rice-Ustilaginoidea virens interaction is still *** isolated the total RNA from ***-infected spikelets of an indica rice Chuannong HIS at 13 days post inoculation(dpi),when pathogen hyphae extended into inner floral organs of rice *** RNA was used for synthesizing the first-strand cDNA by Oligo(dT) primer,ds cDNA was amplified and mixed with vector pGADT7-Rec after *** yeast two-hybrid cDNA library was constructed by homologous recombination technology in yeast line *** cDNA library titer was 5.7×10cfu/ml,and the average length of the insertions was 750 *** is indicated that the cDNA library has good quality and can provide a valuable resource for studying molecular interactions between *** and rice.

关键词： Ustilaginoidea virens rice cDNA library yeast two-hybrid

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Journal of Biosciences and Medicines 2024年第7期12卷 184-193页

作者： Jianzhong Huang Xiaoju Zhong Xiuying Guan Peng Jia Hongbin Zhang Kai Chen Zhuojun Li Guangyu Chen Chuitang Zeng Key Laboratory of Chronic Diseases Fuzhou Medical University Fuzhou China

Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.

关键词： Cell Death NBS-LRR Nicotiana benthamiana yeast two-hybrid

Interaction between MAPKs and MKPs in hexaploid chrysanthemum illuminates functional paralogue diversification in polyploids

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Horticultural Plant Journal 2023年第1期9卷 158-168页

作者： Qi Yu Luyao Zhang Daojin Sun Yueheng Hu Peiling Li Xue Zhang Lian Ding Lijie Zhou Zhiyong Guan Weimin Fang Fadi Chen Aiping Song State Key Laboratory of Crop Genetics and Germplasm Enhancement Key Laboratory of LandscapingMinistry of Agriculture and Rural AffairsCollege of HorticultureNanjing Agricultural UniversityNanjingJiangsu 210095China Henan Key Laboratory of Tea Comprehensive Utilization in South Henan Xinyang Agriculture and Forestry UniversityXinyangHenan 464000China

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 *** to lacking of overall understanding for the regulatory networks between Chrysanthemum morifolium MKPs(CmMKPs)and *** 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 ***,the expression of CmMKP1 and CmMKP1-LIKE1showed 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 *** 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.

关键词： Chrysanthemum Evolution MAPK MKP Protein structure yeast two-hybrid

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Identification of anti-Mycobacterium tuberculosis agents targeting the interaction of bacterial division proteins FtsZ and SepFe

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Acta Pharmaceutica Sinica B 2023年第5期13卷 2056-2070页

作者： Hongjuan Zhang Ying Chen Yu Zhang Luyao Qiao Xiangyin Chi Yanxing Han Yuan Lin Shuyi Si Jiandong Jiang State Key Laboratory of Bioactive Substance and Function of Natural Medicines Institute of Materia MedicaChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100050China Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100050China

Tuberculosis(TB)is one of the deadly diseases caused by Mycobacterium tuberculosis(Mtb),which presents a significant public health *** of TB relies on the combination of several anti-TB drugs to create shorter and safer ***,new anti-TB agents working by different mechanisms are urgently ***,a tubulin-like protein with GTPase activity,forms a dynamic Z-ring in cell *** of FtsZ inhibitors are designed to inhibit GTPase *** Mtb,the function of Z-ring is modulated by SepF,a FtsZ binding *** FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of ***,we established a yeast twohybrid based screening system to identify inhibitors of FtsZ/SepF interaction in *** this system,we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and ***,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 ***,T0349 can inhibit bacterial cell division by inducing filamentation and abnormal *** data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.

关键词： Anti-Mycobacterium tuberculosis FtsZ SepF Bacterial division yeast two-hybrid CRISPRi Protein—protein interaction Inhibitor

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Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by yeasttwo-hybrid System

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Animal Husbandry and Feed Science 2018年第4期10卷 267-271页

作者： Wang Zhongze Qin Cuili Kong Ning Zuo Yewen Wang Meng Zheng Hao Tong Wu Li Liwei Yu Hai Li Zhili Shan Tongling Tong Guangzhi Li Xue Henan University of Science and Technology Luoyang 471023 China Shanghai Institute of Veterinary Medicine Chinese Academy of Agricultural Sciences Shanghai 200241 China 不详

[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus （PEDV） N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage （PAM） by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins （FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598） interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation（CoIP） test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.

关键词： Porcine epidemic diarrhea virus (PEDV) yeast two-hybrid N protein Protein interaction

OsFY,a Homolog of AtFY,Encodes a Protein that Can Interact with OsFCA-γ in Rice(Oryza sativa L.)

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Acta Biochimica et Biophysica Sinica 2006年第7期38卷 492-499页

作者： Qi LU Zheng-Kai XU Ren-Tao SONG Shanghai Key Laboratory of Bio-energy Crop,School of Life Sciences,Shanghai University Shanghai 200444,China Shanghai Key Laboratory of Bio-energy Crop School of Life Sciences Shanghai UniversityShanghai 200444 China

FCA and FY are flowering time related genes involved in the autonomous flowering pathwayin *** interacts with FY to regulate the alternative processing of FCA *** FCA/FY interaction is also required for the regulation of FLC expression,a major floral repressor in ***,it is not clear if the regulation of this autonomous flowering pathway is also present in monocotplants,such as ***,alternative RNA processing of OsFCA was observed in rice,which stronglysuggested the existence of an autonomous flowering pathway in *** this work,we cloned the cDNA ofthe autonomous flowering pathway gene OsFY from *** predicted OsFY protein contained a conserved7 WD-repeat region and at least two Pro-Pro-Leu-Pro motifs compared to Arabidopsis *** protein-protein interaction between OsFY and OsFCA-γ,the key feature of their gene function,was also demon-strated using the yeast two-hybrid *** GenBank database search provided evidence of expressionfor other autonomous pathway gene homologs in *** results indicate that the autonomous floweringpathway is present in monocots,and the regulation through FY and FCA interaction is conserved betweenmonocots and dicots.

关键词： OsFY autonomous flowering pathway OsFCA protein interaction yeast two-hybrid

Identification of karyopherin-alpha 2 as an Oct4 associated protein

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Journal of Genetics and Genomics 2008年第12期35卷 723-728页

作者： Xiangqun Li Lei Sun Ying Jin Institute of Genetics and Developmental Biology Chinese Academy of Sciences Beijing 100101 China Shanghai Stem Cell Institute Institutes of Medical Sciences School of Medicine Shanghai Jiao Tong University Shanghai 200025 China Key Laboratory of Stem Cell Biology Institute of Health Sciences Shanghai Institutes for Biological Sciences Chinese Academy of Sciences/School of Medicine Shanghai Jiao Tong University Shanghai 200025 China

The POU domain transcription factor Oct4 is a master regulator in maintaining self-renewal and pluripotency of embryonic stem （ES） cells. To further explore the functional network of Oct4, the yeast two-hybrid system was used to search for Oct4 interacting proteins. PH domain （containing POU domain and homeodomain） of human OCT4 was used as a bait. From the human testis cDNA library, we identified a strong interaction between OCT4 and karyopberin-alpha 2 （KPNA-2）. KPNA2 is involved in active nuclear import of proteins. This finding was confirmed by glutathione S-transferase pull-down and co-immunoprecipitation assays. The interaction between OCT4 and KPNA-2 was further mapped to multiple regions of the two proteins. In addition, we studied nuclear localization signal （NLS） of mouse Oct4 and demonstrated that it is essential for Oct4 nuclear localization. Thus, our data suggest that Oct4 nuclear localization may be mediated by its interaction with KPNA-2.

关键词： Oct4 karyopherin-alpha 2 yeast two-hybrid nuclear localization signal

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