Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes. (26th July 2019)

• Record Type: Journal Article
• Authors: Hu, Zhoubo; Ghosh, Ajit; Stolze, Sara C.; Horváth, Mihály; Bai, Bing; Schaefer, Sabine; Zündorf, Simone; Liu, Shanda; Harzen, Anne; Hajheidari, Mohsen; Sarnowski, Tomasz J.; Nakagami, Hirofumi; Koncz, Zsuzsa; Koncz, Csaba
• Is Part Of: Plant journal. Volume 100:Number 2(2019)
• Page Start: 411
• Page End: 429
• Publication Date: 2019-07-26
• Subjects: recombineering -- site‐directed gene modification -- fluorescent reporters -- affinity purification -- TFIIH protein kinases -- DNA replication‐dependent HISTONE H3.1 -- technical advance; Plant molecular biology -- Periodicals; Plant cells and tissues -- Periodicals; Botany -- Periodicals; 580
• DOI: 10.1111/tpj.14450
• Languages: English
• ISSNs: 0960-7412

Abstract

Summary: To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site‐directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR‐amplified DNA fragments. Application of fast‐track recombineering is illustrated by the simultaneous labelling of CYCLIN‐DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD‐activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 ‐StrepII‐HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T‐DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co‐localization and co‐immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA‐replication‐coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast‐track recombineering is well suited to promote a wide range of protein interaction and proteomics studies. Significance Statement: A PCR‐based BAC‐recombineering protocol accelerating site‐specific modification of plant genes with suitable tags is described and exploited for purification, cellular localization, co‐immunoprecipitation, and mass spectrometry analysis of CDKD;2 and CYCLIN H associated components of TFIIH general transcription factor.