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Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes
Author(s) -
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
Publication year - 2019
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.14450
Subject(s) - recombineering , biology , transcription factor ii h , genetics , gene , microbiology and biotechnology , homologous recombination , dna repair , nucleotide excision repair
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 ( BAC s). 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 mC herry (green and red fluorescent protein) tags, and a PIPL (His 18 ‐Strep II ‐ 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.