A Gateway Cloning Vector Set for High-Throughput Functional Analysis of Genes in Planta

TLDR

With two plant genomes sequenced, assigning functions to the growing number of predicted genes remains a challenge, as only about 55 % of Arabidopsis genes have putative functions and fewer than 8 % have been experimentally validated, and conventional cloning methods hinder transgene production. The authors aim to facilitate high‑throughput functional analysis by producing a Gateway‑compatible Agrobacterium system that generates diverse chimeric transgenes for expression, localization, promoter, and complementation studies. Derived from pCambia T‑DNA vectors (except a chemically inducible variant), the system supports GFP and GUS fusion expression for subcellular localization, promoter‑reporter construction, and efficient cloning of genomic fragments for complementation in diverse plant species. The resulting Gateway‑compatible binary vector set enables rapid, reliable cloning and is freely available for noncommercial use, allowing constitutive or inducible ectopic gene expression.

Abstract

The current challenge, now that two plant genomes have been sequenced, is to assign a function to the increasing number of predicted genes. In Arabidopsis, approximately 55% of genes can be assigned a putative function, however, less than 8% of these have been assigned a function by direct experimental evidence. To identify these functions, many genes will have to undergo comprehensive analyses, which will include the production of chimeric transgenes for constitutive or inducible ectopic expression, for antisense or dominant negative expression, for subcellular localization studies, for promoter analysis, and for gene complementation studies. The production of such transgenes is often hampered by laborious conventional cloning technology that relies on restriction digestion and ligation. With the aim of providing tools for high throughput gene analysis, we have produced a Gateway-compatible Agrobacterium sp. binary vector system that facilitates fast and reliable DNA cloning. This collection of vectors is freely available, for noncommercial purposes, and can be used for the ectopic expression of genes either constitutively or inducibly. The vectors can be used for the expression of protein fusions to the Aequorea victoria green fluorescent protein and to the beta-glucuronidase protein so that the subcellular localization of a protein can be identified. They can also be used to generate promoter-reporter constructs and to facilitate efficient cloning of genomic DNA fragments for complementation experiments. All vectors were derived from pCambia T-DNA cloning vectors, with the exception of a chemically inducible vector, for Agrobacterium sp.-mediated transformation of a wide range of plant species.