Development of acetosyringone-inducible Gateway ® and Golden Gate expression vectors for heterologous gene expression in Agrobacterium tumefaciens

As a plant genetic engineer, Agrobacterium tumefaciens utilizes phenolic compounds, such as acetosyringone, to activate its virulence genes during the infection and transformation process. In this study, two novel broad-host-range (BHR) acetosyringone-inducible expression vectors were constructed. U...

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Published in:In vitro cellular & developmental biology. Plant 2020-10, Vol.56 (5), p.578-587
Main Authors: Toh, Wai Keat, Loo, Eliza Po-lian, Tee, Chong Siang, Loh, Pek Chin, Wong, Hann Ling
Format: Article
Language:English
Subjects:
Biomedical and Life Sciences
BIOTECHNOLOGY
Cell Biology
Developmental Biology
Life Sciences
Plant Breeding/Biotechnology
Plant Genetics and Genomics
Plant Sciences
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Summary:As a plant genetic engineer, Agrobacterium tumefaciens utilizes phenolic compounds, such as acetosyringone, to activate its virulence genes during the infection and transformation process. In this study, two novel broad-host-range (BHR) acetosyringone-inducible expression vectors were constructed. Unlike Agrobacterium binary vectors which are developed for plant transformation, these vectors were designed for heterologous gene expression in A. tumefaciens. To this end, an acetosyringone-inducible virB promoter (PvirB) was placed upstream of a Gateway® cassette for plasmid vector pASE1 and Type IIS recognition sites, BpiI for plasmid vector pASE2, thus allowing the PvirB to drive the expression of the insert, which may be cloned into pASE1 and pASE2 via Gateway® LR Cloning and Golden Gate Cloning approaches, respectively. Here, we tested the functionality and inducibility of pASE1 and pASE2 vectors by expressing the reporter gene superfolder green fluorescent protein (sfGFP) in the presence of acetosyringone. The expression of the reporter gene sfGFP after induction by acetosyringone was verified by Western blotting. Interestingly, the presence of the attB1 site in pASE1 imposed a tight regulation on the inducibility of the PvirB, This is the first report that indicates that an attB1 site may hinder downstream gene expression in a bacterium. We envision that pASE2 can be used to express key virulence genes to enhance plant transformation and advance the genetic studies of Agrobacterium.
ISSN:1054-5476
1475-2689
DOI:10.1007/s11627-020-10095-8