The article reviews the use of Gateway cloning technology in plant functional genomics and proteomics. Gateway technology facilitates high-throughput cloning by utilizing the bacteriophage lambda site-specific recombination system. Target sequences are captured in an entry vector and then recombined into various destination vectors for expression in different organisms. The authors describe a set of pEarleyGate plasmid vectors designed for Agrobacterium-mediated plant transformation, which can translateally fuse epitope tags or fluorescent proteins to target proteins. These vectors are useful for affinity purification, immunolocalization, and protein localization studies. The article also provides detailed protocols for using the pEarleyGate vectors and discusses their applications in plant research.The article reviews the use of Gateway cloning technology in plant functional genomics and proteomics. Gateway technology facilitates high-throughput cloning by utilizing the bacteriophage lambda site-specific recombination system. Target sequences are captured in an entry vector and then recombined into various destination vectors for expression in different organisms. The authors describe a set of pEarleyGate plasmid vectors designed for Agrobacterium-mediated plant transformation, which can translateally fuse epitope tags or fluorescent proteins to target proteins. These vectors are useful for affinity purification, immunolocalization, and protein localization studies. The article also provides detailed protocols for using the pEarleyGate vectors and discusses their applications in plant research.