Journal Article

Global Protein Interactome Mapping in Rice Using Barcode‐Indexed PCR Coupled with HiFi Long‐Read Sequencing

Tong, Xiaohong, Ying, Jiezheng, Luo, Jinjin, Cheng, Yu, Liu, Wanning, Zhang, Jian, Zhang, Sanqiang, Liu, Xixi, Zhang, Fengyong, Zhang, Jianwei, Wang, Yifeng, Wang, Shiyi, Ergashev, Mukhammadjon Arabboevich, Chen, Lijuan, Xia, Dandan, Chen, Yiting, Liu, Zhichao, Yuan, Wenya, Huang, Jie, Li, Mengyuan, Yuan, Yang, Li, Yanan, Li, Zhiyong, Li, Guanghao, Xu, Huayu, Xue, Dawei, ugli, Abdullaev Mirtemir Baxodir, Liu, Xinyong, Hou, Yuxuan

2025

Overview

Establishing the protein–protein interaction network sheds light on functional genomics studies by providing insights from known counterparts. However, the rice interactome has barely been studied due to the lack of massive, reliable, and cost‐effective methodologies. Here, the development of a barcode‐indexed PCR coupled with HiFi long‐read sequencing pipeline (BIP‐seq) is reported for high throughput Protein Protein Interaction (PPI)identification. BIP‐seq is essentially built on the integration of library versus library Y2H mating strategy to facilitate the efficient acquisition of random PPI colonies, semi‐mechanized dual barcode‐indexed yeast colony PCR for the large‐scale indexed amplification of bait and prey cDNAs, and massive pac‐bio sequencing of PCR amplicon pools. It is demonstrated that BIP‐seq could map over 15 000 high‐confidence (≈62.5% could be verified by Bimolecular fluorescence Complementation (BiFC)) rice PPIs within 2 months, outperforming the other reported methods. In addition, the obtained 23 032 rice PPIs, including 22,665 newly identified PPIs, greatly expanded the current rice PPI dataset, provided a comprehensive overview of the rice PPIs networks, and could be a valuable asset in facilitating functional genomics research in rice. BIP‐seq is developed, a cost‐effective pipeline for high‐throughput protein–protein interaction (PPI) identification using yeast two‐hybrid system. BIP‐seq successfully map over 15 000 high‐confidence rice PPIs (≈62.5% could be verified by BiFC) within 2 months. A rice PPI database (RiPPID) containing 23032 PPIs is established, which greatly enhances the understanding of the interactome in rice.

• Publisher: John Wiley & Sons, Inc,John Wiley and Sons Inc,Wiley
• Subject: Bar codes / barcode‐indexed PCR / HiFi sequencing / High-Throughput Nucleotide Sequencing - methods / Oryza - genetics / Oryza - metabolism / Plant Proteins - genetics / Plant Proteins - metabolism / Polymerase Chain Reaction - methods / Protein Interaction Mapping - methods / Protein Interaction Maps - genetics / Proteins / protein–protein interactome / rice / Robots / Thermal cycling / Yeast