Gateway cloning technology enables high-throughput cloning of target sequences using the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available 'entry vector' and then recombined into various 'destination vectors' for expression in different experimental organisms. This technology has been adopted by many plant laboratories 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. The authors review various types of Gateway destination vectors available to the plant research community and provide links and references for further information. They 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. These epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. The utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy is demonstrated. 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. The authors also describe a series of pEarleyGate vectors designed for transient or stable expression of proteins fused to various oligopeptide epitope tags and/or GFP, YFP or CFP. Representative immunoblotting, affinity purification and protein localization data are provided to illustrate the usefulness of pEarleyGate vectors. The Gateway cloning system exploits the accurate, site-specific recombination system of bacteriophage lambda to shuttle sequences between plasmids bearing compatible recombination sites. The preferred method for initially capturing sequences of interest is topoisomerase-mediated cloning, which eliminates the need for conventional DNA ligase-mediated molecular cloning. The resulting recombinant plasmid has the target DNA sequences flanked by attL recombination sequences. Once flanked by attL recombination sites, the sequence can be recombined with attR sites using the LR clonase reaction mix. This reaction transfers the target sequence into a desired destination vector. Destination vectors contain a gene (ccdB) that is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction. This negative selection, combined with positive selection for an antibiotic resistance marker, ensures that resulting colonies contain plasmids that have undergone recombination. The ease and speed with which a captured target sequence can be shuttled simultaneously into a variety of destination vectors are great advantages for high-throughput functional genomics/proteomics investigations. Several laboratories have developed Gateway-compatibleGateway cloning technology enables high-throughput cloning of target sequences using the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available 'entry vector' and then recombined into various 'destination vectors' for expression in different experimental organisms. This technology has been adopted by many plant laboratories 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. The authors review various types of Gateway destination vectors available to the plant research community and provide links and references for further information. They 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. These epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. The utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy is demonstrated. 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. The authors also describe a series of pEarleyGate vectors designed for transient or stable expression of proteins fused to various oligopeptide epitope tags and/or GFP, YFP or CFP. Representative immunoblotting, affinity purification and protein localization data are provided to illustrate the usefulness of pEarleyGate vectors. The Gateway cloning system exploits the accurate, site-specific recombination system of bacteriophage lambda to shuttle sequences between plasmids bearing compatible recombination sites. The preferred method for initially capturing sequences of interest is topoisomerase-mediated cloning, which eliminates the need for conventional DNA ligase-mediated molecular cloning. The resulting recombinant plasmid has the target DNA sequences flanked by attL recombination sequences. Once flanked by attL recombination sites, the sequence can be recombined with attR sites using the LR clonase reaction mix. This reaction transfers the target sequence into a desired destination vector. Destination vectors contain a gene (ccdB) that is lethal to most strains of E. coli. 'Empty' destination vectors are therefore selected against upon transformation of E. coli cells with the recombination reaction. This negative selection, combined with positive selection for an antibiotic resistance marker, ensures that resulting colonies contain plasmids that have undergone recombination. The ease and speed with which a captured target sequence can be shuttled simultaneously into a variety of destination vectors are great advantages for high-throughput functional genomics/proteomics investigations. Several laboratories have developed Gateway-compatible