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

Abstract

Abstract 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.

Keywords

• barcode‐indexed PCR
• HiFi sequencing
• protein–protein interactome
• rice