We present a method that allows simultaneous fusion and cloning of multiple PCR products in a rapid and efficient manner. The procedure is based on the use of PCR primers that contain a single deoxyuridine residue near their 5′ end. Treatment of the PCR products with a commercial deoxyuridine-excision reagent generates long 3′ overhangs designed to specifically complement each other. The combination of this principle with the improved USER cloning technique provides a simple, fast and very efficient method to simultaneously fuse and clone multiple PCR fragments into a vector of interest. Around 90% positive clones were obtained when three different PCR products were fused and cloned into a USER-compatible vector in a simple procedure that, apart from the single PCR amplification step and the bacterial transformation, took approximately one hour. We expect this method to replace overlapping PCR and the use of type IIS restriction enzymes in many of their applications.

White lupin (Lupinus albus L.) is an annual crop cultivated for its protein-rich seeds. It is adapted to poor soils due to the production of cluster roots, which are made of dozens of determinate lateral roots that drastically improve soil exploration and nutrient acquisition (mostly phosphate). Using long-read sequencing technologies, we provide a high-quality genome sequence of a cultivated accession of white lupin (2n = 50, 451 Mb), as well as de novo assemblies of a landrace and a wild relative. We describe a modern accession displaying increased soil exploration capacity through early establishment of lateral and cluster roots. We also show how seed quality may have been impacted by domestication in term of protein profiles and alkaloid content. The availability of a high-quality genome assembly together with companion genomic and transcriptomic resources will enable the development of modern breeding strategies to increase and stabilize white lupin yield.

Plants sequester intermediates of metabolic pathways into different cellular compartments, but the mechanisms by which these molecules are transported remain poorly understood. Monoterpene indole alkaloids, a class of specialized metabolites that includes the anticancer agent vincristine, antimalarial quinine and neurotoxin strychnine, are synthesized in several different cellular locations. However, the transporters that control the movement of these biosynthetic intermediates within cellular compartments have not been discovered. Here we present the discovery of a tonoplast localized nitrate/peptide family (NPF) transporter from Catharanthus roseus, CrNPF2.9, that exports strictosidine, the central intermediate of this pathway, into the cytosol from the vacuole. This discovery highlights the role that intracellular localization plays in specialized metabolism, and sets the stage for understanding and controlling the central branch point of this pharmacologically important group of compounds.