Gene modification by fast‐track recombineering for cellular localization and isolation of components of plant protein complexes

Zhoubo Hu,Hirofumi Nakagami,Zsuzsa Koncz,Sara C Stolze,Ajit Ghosh,Shanda Liu,Sabine Schaefer,Csaba Koncz,Mohsen Hajheidari,Anne Harzen,Simone Zündorf,Tomasz J Sarnowski,Bing Bai,Mihály Horváth

doi:10.1111/tpj.14450

Abstract

SummaryTo 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.

Highlights

The term recombineering refers to cloning technologies that employ phage-encoded recombination enzymes, such as Exo, Beta, and Gam of lambda phage Red system, to achieve in vivo site-specific integration of foreign DNA sequences into genes carried by bacterial chromosomes or plasmids (Thomason et al, 2014)
A positive-negative selectable marker cassette is inserted into the target gene, and this cassette is replaced by a desired tag, or with a DNA fragment carrying a nucleotide exchange or deletion
Selectable markers are inserted into desired positions of genes and replaced with DNA fragments that either code for suitable tags or carry nucleotide exchanges or deletions

Summary

Introduction

The term recombineering refers to cloning technologies that employ phage-encoded recombination enzymes, such as Exo, Beta, and Gam of lambda phage Red system, to achieve in vivo site-specific integration of foreign DNA sequences into genes carried by bacterial chromosomes or plasmids (Thomason et al, 2014). A positive-negative selectable marker cassette is inserted into the target gene, and this cassette is replaced by a desired tag, or with a DNA fragment carrying a nucleotide exchange or deletion. One of the most popular positiveÀnegative selectable markers is the galactokinase (galK) gene, the integration of which into the target site is selected for on minimal medium in a galKÀ host. The exchange of galK marker with desired sequences is achieved by counter-selection on deoxygalactose-containing medium

Results

Discussion

Conclusion