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Yeast two hybrid

Jayita Chopra, Ramkrishna Sen
Lipid accumulation in oleaginous yeast is generally induced by nitrogen starvation, while oxygen saturation can influence biomass growth. Systematic shake flask studies that help in identifying the right nitrogen source and relate its uptake kinetics to lipid biosynthesis under varying oxygen saturation conditions are very essential for addressing the bioprocessing-related issues, which are envisaged to occur in the fermenter scale production. In the present study, lipid bioaccumulation by P. guilliermondii at varying C:N ratios and oxygen transfer conditions (assessed in terms of kL a) was investigated in shake flasks using a pre-optimized N-source and a two-stage inoculum formulated in a hybrid medium...
April 20, 2018: Bioprocess and Biosystems Engineering
Haidong Ding, Jie He, Yuan Wu, Xiao-Xia Wu, Cailin Ge, Yijun Wang, Silin Zhong, Edgar Peiter, Jian-Sheng Liang, Weifeng Xu
High temperature (HT) stress is a major environmental stress that limits plant growth and development. Mitogen-activated protein kinase (MAPK) cascades play key roles in plant growth and stress signaling, but their involvement in the HT stress response are poorly understood. Here, we describe a 47-kDa MBP-phosphorylated protein (p47-MBPK) activated in tomato (Solanum lycopersicum) leaves under HT, and identify it as SlMPK1 by MS/MS analysis. Silencing of SlMPK1 in transgenic tomato plants resulted in enhanced tolerance to HT, while overexpression resulted in reduced tolerance...
April 20, 2018: Plant Physiology
Haitao Wang, Juan Wang, Yunjie Xie, Zhijun Fu, Taiyun Wei, Xiao-Feng Zhang
BACKGROUND: In China, the rice pathogen Rice yellow stunt virus (RYSV), a member of the genus Nucleorhabdovirus in the family Rhabdoviridae, was a severe threat to rice production during the1960s and1970s. Fundamental aspects of the biology of this virus such as protein localization and formation of the RYSV viroplasm during infection of insect vector cells are largely unexplored. The specific role(s) of the structural proteins nucleoprotein (N) and phosphoprotein (P) in the assembly of the viroplasm during RYSV infection in insect vector is also unclear...
April 20, 2018: Virology Journal
Chun-Lin Chen, Yan Cui, Man Cui, Wen-Juan Zhou, Hui-Lan Wu, Hong-Qing Ling
Fe and Zn are essential micronutrients for plant growth, and the interrelationship regarding their homeostasis is very complicated. In this study, we identified a FIT-binding protein (FBP) using the yeast two-hybrid system. The C-terminus of FBP binds to the bHLH domain of FIT, abolishing the DNA-binding capacity of FIT. Knockout of FBP results in an enhanced expression of NAS genes and a higher NA content, and the fbp mutant exhibits tolerance to excessive Zn. Physiological analyses reveal that the mutant fbp retains a larger amount of Zn in roots and transfers a greater proportion of Fe to shoots than that in wild type under Zn-excessive stress...
April 20, 2018: Plant, Cell & Environment
Hiroyuki Yamazaki, Yoshio Sasagawa, Hideyuki Yamamoto, Haruhiko Bito, Tomoaki Shirao
Drebrin is a major F-actin binding protein in dendritic spines that is critically involved in the regulation of dendritic-spine morphogenesis, pathology, and plasticity. In this study, we aimed to identify a novel drebrin-binding protein involved in spine morphogenesis and synaptic plasticity. We confirmed the beta subunit of Ca2+ /calmodulin-dependent protein kinase II (CaMKIIβ) as a drebrin-binding protein using a yeast two-hybrid system, and investigated the drebrin-CaMKIIβ relationship in dendritic spines using rat hippocampal neurons...
April 20, 2018: Journal of Neurochemistry
Deshui Yu, Libing Liao, Ju Zhang, Yi Zhang, Kedong Xu, Kun Liu, Xiaoli Li, Guangxuan Tan, Ran Chen, Yulu Wang, Xia Liu, Xuan Zhang, Xiaomeng Han, Zhangkun Wei, Chengwei Li
Yeast two-hybrid systems are powerful tools for analyzing interactions between proteins. Vector construction is an essential step in yeast two-hybrid experiments, which require bait and prey plasmids. In this study, we modified the multiple cloning site sequence of the yeast plasmid pGADT7 by site-directed mutagenesis PCR to generate the pGADT7-In vector, which result in an easy and rapid method for constructing yeast two-hybrid vectors using the In-Fusion cloning technique. This method has three key advantages: only one pair of primers and one round of PCR are needed to generate bait and prey plasmids for each gene; it is restriction endonuclease- and ligase-independent and it is fast and easily performed...
April 19, 2018: BioTechniques
Xuli Gao, Ju Zhang, Chaoni Song, Kangyi Yuan, Jianhua Wang, Qiaojun Jin, Jin-Rong Xu
Prp31 is one of the key tri-snRNP components essential for pre-mRNA splicing although its exact molecular function is not well studied. In a previous study, suppressor mutations were identified in the PRP31 ortholog in two spontaneous suppressors of Fgprp4 mutant deleted of the only kinase of the spliceosome in Fusarium graminearum. To further characterize the function of FgPrp31 and its relationship with FgPrp4 kinase, in this study we identified additional suppressor mutations in FgPrp31 and determined the suppressive effects of selected mutations...
April 18, 2018: Current Genetics
Yu Song, Hang Zhang, Hongguang You, Yuanming Liu, Chao Chen, Xu Feng, Xingyu Yu, Shengyang Wu, Libo Wang, Shihua Zhong, Qiang Li, Yanming Zhu, Xiaodong Ding
The plant SnRK1 kinases play the central roles in the processes of energy balance, hormone perception, stress resistance, metabolism, growth and development. However, the functions of these kinases are still elusive. In this study, we used GsSnRK1 of wild soybean as bait to perform library-scale screens by the means of yeast two-hybrid to identify its interacting proteins. The putative interactions were verified by yeast re-transformation and β-galactodase assays, and the selected interactions were further confirmed in planta by BiFC and biochemical Co-IP assays...
April 17, 2018: Plant, Cell & Environment
Yue Gao, Chong Zhang, Xiao Han, Zi Yuan Wang, Lai Ma, De Peng Yuan, Jing Ni Wu, Xiao Feng Zhu, Jing Miao Liu, Dao Pin Li, Yi Bing Hu, Yuan Hu Xuan
Pathogen-host interaction is a complicated process; pathogens mainly infect host plants to acquire nutrients, especially sugars. Rhizoctonia solani, the causative agent of sheath blight disease, is a major pathogen of rice. However, it is not known, as to how this pathogen obtains sugar from rice plants. In this study, we found that the rice sugar transporter, OsSWEET11 is involved in the pathogenesis of sheath blight disease. qRT-PCR and β-d-glucuronidase expression analyses showed that R. solani infection significantly enhanced OsSWEET11 expression in leaves among the clade III SWEET members...
April 16, 2018: Molecular Plant Pathology
Qian Sun, Yuan-Yuan Li, Ying Wang, Hang-Hai Zhao, Tian-Yu Zhao, Zong-Ying Zhang, Da-Wei Li, Jia-Lin Yu, Xian-Bing Wang, Yong-Liang Zhang, Cheng-Gui Han
In polerovirus-host interactions, few cellular proteins have been identified to directly interact with multifunctional P0. To further explore the functions of P0, we identified a Brassica yellows virus genotype A (BrYV-A) P0BrA-interacting protein from Nicotiana benthamiana, Rubisco Assembly Factor 2 (NbRAF2), which localizes in the nucleus, cell periphery, chloroplasts and stromules, and its C-terminal domain (amino acids 183-211) is required for self-interactions. The split ubiquitin membrane-bound yeast two-hybrid system and co-immunoprecipitation assays showed that NbRAF2 interacted with P0BrA, and colocalized in the nucleus and cell periphery...
April 5, 2018: Journal of Experimental Botany
Qianyi Tao, Huanhuan Niu, Zhongyuan Wang, Wenhui Zhang, Hu Wang, Shenhao Wang, Xian Zhang, Zheng Li
In plants, unisexual flowers derived from developmental sex determination form separate stamens and pistils that facilitate cross pollination. In cucumber and melon, ethylene plays a key role in sex determination. Six sex determination-related genes have been identified involving in ethylene biosynthesis in these Cucumis species. The interactions among these genes are thought to involve ethylene signaling; however, the detailed regulatory mechanism remains unknown. In this study, hormone treatment and qPCR assays were used to confirm expression of these sex determination-related genes in cucumber and melon is ethylene sensitive...
April 5, 2018: Journal of Experimental Botany
Haifeng Xu, Guanxian Yang, Jing Zhang, Yicheng Wang, Tianliang Zhang, Nan Wang, Shenghui Jiang, Zongying Zhang, Xuesen Chen
The cold-induced metabolic pathway and anthocyanin biosynthesis play important roles in plant growth. In this study, we identified a bHLH binding motif in the MdMYB15L protein using protein sequence analyses. Yeast two-hybrid and pull-down assays showed that MdMYB15L could interact with MdbHLH33. Overexpressing MdMYB15L in red-fleshed callus inhibited the expression of MdCBF2 and resulted in reduced cold tolerance but did not affect anthocyanin levels. Chip-PCR and EMSA analysis showed that MdMYB15L could bind the type II cis-acting element found in the MdCBF2 promoter...
April 12, 2018: Biochemical and Biophysical Research Communications
Tian Luo, Shubhajit Mitra, Jere W McBride
Ehrlichia chaffeensis is an obligately intracellular bacterium that exhibits tropism for mononuclear phagocytes. The mechanisms involved in E. chaffeensis infection of the host cell and evasion of host defenses are not fully defined, but a subset of type 1 secreted tandem repeat protein (TRP) effectors play important roles. Recently, we determined molecular interactions of TRP120, TRP47, and TRP32 with the eukaryotic host cell. In this investigation, we used yeast two-hybrid analysis to reveal that another E...
April 25, 2018: MSphere
Kelsey D Galimba, Jesús Martínez-Gómez, Verónica S Di Stilio
The floral organ identity gene APETALA3 ( AP3 ) is a MADS-box transcription factor involved in stamen and petal identity that belongs to the B-class of the ABC model of flower development. Thalictrum (Ranunculaceae), an emerging model in the non-core eudicots, has AP3 homologs derived from both ancient and recent gene duplications. Prior work has shown that petals have been lost repeatedly and independently in Ranunculaceae in correlation with the loss of a specific AP3 paralog, and Thalictrum represents one of these instances...
2018: Frontiers in Plant Science
Qianqian Shi, Zhenchuan Mao, Xiaoping Zhang, Jian Ling, Runmao Lin, Xi Zhang, Rui Liu, Yunsheng Wang, Yuhong Yang, Xinyue Cheng, Bingyan Xie
Meloidogyne incognita is highly specialized parasite that interacts with host plants using a range of strategies. The effectors are synthesized in the esophageal glands and secreted into plant cells through a needle-like stylet during parasitism. In this study, based on RNA-seq and bioinformatics analysis, we predicted 110 putative Meloidogyne incognita effectors that contain nuclear localization signals (NLSs). Combining the Burkholderia glumae -pEDV based screening system with subcellular localization, from 20 randomly selected NLS effector candidates, we identified an effector MiISE6 that can effectively suppress B...
2018: Frontiers in Plant Science
Jeremy Forsberg, Xinge Li, Aleksey Zamaraev, Theocharis Panaretakis, Boris Zhivotovsky, Magnus Olsson
Caspase-2 has been implicated in diverse cellular processes, and the identification of factors with which it interacts has steadily increased. In the present study, we report a direct interaction between caspase-2 and factor associated with neutral sphingomyelinase activation (FAN) using yeast two-hybrid screening and co-immunoprecipitation. Further, stable suppression of caspase-2 expression in HEK293T and HeLa cells enabled a systematic investigation of putative novel enzyme functionalities, especially with respect to ceramide production, cell migration, IL-6 production and vesicular homeostasis, all of which have been previously reported to be associated with FAN...
April 2, 2018: Biochemical and Biophysical Research Communications
Hee-Seung Choi, Minji Seo, Hyung-Taeg Cho
Auxin signaling is finalized by activator auxin response factors (aARFs) that are released from Auxin/Indole-3-Acetic Acid (Aux/IAA) repressors and directly activate auxin-responsive genes. However, it remains to be answered how repressor ARFs (rARFs) exert their repression function. In this study, we assessed the molecular and biological functions of two putative co-repressor-binding motifs (EAR and RLFGI) of ARF2 (a rARF) in Arabidopsis thaliana . In the yeast two-hybrid assay, the EAR mutation moderately and the RLFGI mutation, or both motifs, almost completely disrupted the interaction between the co-repressor TOPLESS (TPL) and the repressive motifs-containing middle domain (MD) of ARF2...
2018: Frontiers in Plant Science
Jyoti M Gurung, Ayad A A Amer, Monika K Francis, Tiago R D Costa, Shiyun Chen, Anton V Zavialov, Matthew S Francis
Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family...
2018: Frontiers in Cellular and Infection Microbiology
Aleksandra Bury, Klaas J Hellingwerf
Helical alignment of the α-helical linker of the LOV (light-oxygen-voltage) domain of YtvA from Bacillus subtilis with the α-helical linker of the histidine-protein kinase domain of the Sln1 kinase of the phospho-relay system for osmoregulation of Saccharomyces cerevisiae has been used to construct a light-modulatable histidine protein kinase. In vitro, illumination with blue light inhibits both the ATP-dependent phosphorylation of this hybrid kinase, as well as the phosphoryl transfer to Ypd1, the phosphoryl transfer domain of the Sln1 system...
April 2, 2018: AMB Express
Ruoqi Gao, Nicolas H Piguel, Alexandria E Melendez-Zaidi, Maria Dolores Martin-de-Saavedra, Sehyoun Yoon, Marc P Forrest, Kristoffer Myczek, Gefei Zhang, Theron A Russell, John G Csernansky, D James Surmeier, Peter Penzes
Contactin associated protein-like 2 (CNTNAP2) has emerged as a prominent susceptibility gene implicated in multiple complex neurodevelopmental disorders, including autism spectrum disorders (ASD), intellectual disability (ID), and schizophrenia (SCZ). The presence of seizure comorbidity in many of these cases, as well as inhibitory neuron dysfunction in Cntnap2 knockout (KO) mice, suggests CNTNAP2 may be crucial for proper inhibitory network function. However, underlying cellular mechanisms are unclear. Here we show that cultured Cntnap2 KO mouse neurons exhibit an inhibitory neuron-specific simplification of the dendritic tree...
April 2, 2018: Molecular Psychiatry
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