Design and direct assembly of synthesized uracil-containing non-clonal DNA fragments into vectors by USER^TM cloning

This protocol describes how to order and directly assemble uracil-containing non-clonal DNA fragments by uracil excision based cloning (USER cloning). The protocol was generated with the goal of making synthesized non-clonal DNA fragments directly compatible with USER(TM) cloning. The protocol is highly efficient and would be compatible with uracil-containing non-clonal DNA fragments obtained from any synthesizing company. The protocol drastically reduces time and handling between receiving the synthesized DNA fragments and transforming with vector and DNA fragment(s)

Inverse pH Gradient-Assay for Facile Characterization of Proton-Antiporters in <i>Xenopus</i> Oocytes

Xenopus oocytes represent one of the most versatile model systems for characterizing the properties of membrane transporters. However, for studying proton-coupled antiporters, the use of Xenopus oocytes has so far been limited to so-called injection-based transport assays. In such assays, where the compound is injected directly into the oocytes’ cytosol and transport is detected by monitoring substrate efflux, poor control over internal diffusion and concentration are incompatible with mechanistic characterizations. In this study, we present an inverse pH-gradient transport assay. Herein, an outward-facing proton gradient enables the characterization of proton antiporters via facile import-based transport assays. We describe two approaches for establishing sustained outward-facing proton gradients across the oocyte membrane, namely by applying alkaline external conditions or through surprisingly stable carbonyl cyanide m-chlorophenyl-hydrazone (CCCP)-mediated acidification of the cytosol. Previously, genetic evidence has shown that DTX18 from Arabidopsis thaliana is essential for the deposition of the hydroxycinnamic acid amide p-coumaroylagmatine (coumaroylagmatine) defence compound on the leaf surface. However, direct evidence for its ability to transport coumarol-agmatine has not been provided. Here, using Xenopus oocytes as expression hosts, we demonstrate DTX18’s ability to transport coumaroyl-agmatine via both injection-based and inverse pH-gradient transport assays. Notably, by showing that DTX18 is capable of accumulating its substrate against its concentration gradient, we showcase the compatibility of the latter with mechanistic investigations

ABA homeostasis and long-distance translocation are redundantly regulated by ABCG ABA importers

The effects of abscisic acid (ABA) on plant growth, development, and response to the environment depend on local ABA concentrations. Here, we show that in Arabidopsis, ABA homeostasis is regulated by two previously unknown ABA transporters. Adenosine triphosphate–binding cassette subfamily G member 17 (ABCG17) and ABCG18 are localized to the plasma membranes of leaf mesophyll and cortex cells to redundantly promote ABA import, leading to conjugated inactive ABA sinks, thus restricting stomatal closure. ABCG17 and ABCG18 double knockdown revealed that the transporters encoded by these genes not only limit stomatal aperture size, conductance, and transpiration while increasing water use efficiency but also control ABA translocation from the shoot to the root to regulate lateral root emergence. Under abiotic stress conditions, ABCG17 and ABCG18 are transcriptionally repressed, promoting active ABA movement and response. The transport mechanism mediated by ABCG17 and ABCG18 allows plants to maintain ABA homeostasis under normal growth conditions

Multi-Knock—a multi-targeted genome-scale CRISPR toolbox to overcome functional redundancy in plants

International audiencePlant genomes are characterized by large and complex gene families that often result in similar and partially overlapping functions 1. This genetic redundancy severely hampers current efforts to uncover novel phenotypes, delaying basic genetic research and breeding programs 2. Here, we describe the development and validation of Multi-Knock, a genome-scale CRISPR toolbox that overcomes functional redundancy in Arabidopsis by simultaneously targeting multiple gene-family members, thus identifying genetically hidden components. We computationally designed 59,129 optimal single guide RNAs (sgRNAs) that each target 2 to 10 genes within a family at once. Furthermore, partitioning the library into ten sub-libraries directed towards a different functional group allows flexible and targeted genetic screens. From the 5,635 sgRNAs targeting the plant transportome, we generated over 3,500 independent Arabidopsis lines that allowed us to identify and characterize the first known cytokinin tonoplast-localized transporters in plants. With the ability to overcome functional redundancy in plants at the genome-scale level, the developed strategy can be readily deployed by scientists and breeders for basic research and to expedite breeding efforts