A yeast library-hybrid assay to screen maize-Rhizoctonia transcription factors and protein-protein interactions in one experimental pipeline

Q1 Agricultural and Biological Sciences

Agri Gene Pub Date : 2016-08-01 DOI:10.1016/j.aggene.2016.05.002

Xuan Tang , Junwei Shi , Wubei Dong

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引用次数: 1

Abstract

Yeast one- (Y1H) and two-hybrid (Y2H) assays are widely used to study transcription factors (TFs) and protein-protein interactions (PPIs), respectively. Here we combined the Y1H and Y2H into a yeast library-hybrid (YLH) assay, which can systematically screen transcription factors (TFs) and PPIs in one experimental pipeline. In typical TFs, the DNA-binding domain (DBD) and activation domain (AD) evolved separately, but were covalently linked. In the YLH assay, TFs are identified based on functionally conserved ADs, whereas in various Y1H assays TFs are identified based on DBDs. Using the YLH method, we isolated 51 pairs of maize-Rhizoctonia PPIs and 38 novel Rhizoctonia solani TFs. TFs and PPIs related to pathogen virulence and plant resistance responses were isolated by the YLH assay. Our results show that 57.75% of isolated TFs contain typical DBDs such as Zn2Cys6, nucleic acid-binding OB-fold, winged helix repressor DNA-binding, and zinc finger CCHC-type. Key PPI pairs related to major functional categories such as metabolism and cellular signalling were obtained. The percentage of verified PPIs is 69.39%. We proved that common TFs have nonspecific or broad-spectrum activities in the yeast plasmid gene expression system. YLH screening can be conducted on library scales to systematically reveal possible TFs and PPIs at the same time.

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酵母文库-杂交试验筛选玉米-根丝核菌转录因子和蛋白-蛋白相互作用

酵母单杂交(Y1H)法和双杂交(Y2H)法分别被广泛用于研究转录因子(TFs)和蛋白-蛋白相互作用(PPIs)。在这里，我们将Y1H和Y2H结合到酵母文库-杂交(YLH)试验中，该试验可以在一个实验管道中系统地筛选转录因子(TFs)和PPIs。在典型的tf中，dna结合域(DBD)和激活域(AD)是分开进化的，但它们是共价连接的。在YLH检测中，TFs是基于功能保守的ADs识别的，而在各种Y1H检测中，TFs是基于dbd识别的。采用YLH法分离得到51对玉米根丝胞菌PPIs和38对新型番茄根丝胞菌TFs。利用YLH法分离出与病原菌毒力和植物抗性反应相关的TFs和PPIs。结果表明，57.75%的分离TFs含有典型的dbd，如Zn2Cys6、核酸结合OB-fold、翼螺旋抑制因子dna结合和锌指cchc型。获得了与代谢和细胞信号传导等主要功能类别相关的关键PPI对。PPIs验证率为69.39%。我们证明了常见的tf在酵母质粒基因表达系统中具有非特异性或广谱活性。YLH筛选可以在文库尺度上进行，系统地同时发现可能的tf和ppi。

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来源期刊

Agri Gene Agricultural and Biological Sciences-Agricultural and Biological Sciences (miscellaneous)

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期刊介绍： Agri Gene publishes papers that focus on the regulation, expression, function and evolution of genes in crop plants, farm animals, and agriculturally important insects and microorganisms. Agri Gene strives to be a diverse journal and topics in multiple fields will be considered for publication so long as their main focus is on agriculturally important organisms (plants, animals, insects, or microorganisms). Although not limited to the following, some examples of potential topics include: Gene discovery and characterization. Genetic markers to guide traditional breeding. Genetic effects of transposable elements. Evolutionary genetics, molecular evolution, population genetics, and phylogenetics. Profiling of gene expression and genetic variation. Biotechnology and crop or livestock improvement. Genetic improvement of biological control microorganisms. Genetic control of secondary metabolic pathways and metabolic enzymes of crop pathogens. Transcription analysis of beneficial or pest insect developmental stages Agri Gene encourages submission of novel manuscripts that present a reasonable level of analysis, functional relevance and/or mechanistic insight. Agri Gene also welcomes papers that have predominantly a descriptive component but improve the essential basis of knowledge for subsequent functional studies, or which provide important confirmation of recently published discoveries provided that the information is new.