Gateway‐compatible vectors for plant functional genomics and proteomics

TLDR

Gateway cloning uses lambda recombination to enable high‑throughput cloning of target sequences into entry and destination vectors for diverse plant functional genomics applications. The authors review available Gateway destination vectors for plants and provide links and references for further information. They describe pEarleyGate plasmids that fuse FLAG, HA, cMyc, AcV5, or tandem affinity purification tags, with or without fluorescent proteins, to target proteins for Agrobacterium‑mediated plant transformation, enabling affinity purification, immunolocalization, or immunoprecipitation. The pEarleyGate vectors successfully express epitope‑tagged proteins that can be affinity captured or localized by immunofluorescence, and the tags show minimal cross‑reaction with endogenous proteins in both monocots and dicots, indicating broad utility.

Abstract

Summary Gateway cloning technology facilitates high‐throughput cloning of target sequences by making use of the bacteriophage lambda site‐specific recombination system. Target sequences are first captured in a commercially available ‘entry vector’ and are then recombined into various ‘destination vectors’ for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of ‘pEarleyGate’ plasmid vectors for Agrobacterium ‐mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo . We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope‐tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross‐reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.