IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Plant J. (0960-7412) 2009 imex curation Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu 27-Oct-2007: Contacted by JYOTI. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu Accepted 2009-NOV-5 by ORCHARD. 27-Oct-2007: Contacted by JYOTI. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Plant J. (0960-7412) 2009 imex curation Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu 27-Oct-2007: Contacted by JYOTI. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu Accepted 2009-NOV-5 by ORCHARD. 27-Oct-2007: Contacted by JYOTI. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Arabidopsis 14-3-3 lambda is a positive regulator of RPW8-mediated disease resistance. Plant J. (0960-7412) 2009 imex curation Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu 27-Oct-2007: Contacted by JYOTI. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. orchard Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Yang X., Wang W., Coleman M., Orgil U., Feng J., Ma X., Ferl R., Turner JG., Xiao S. xiao@umbi.umd.edu Accepted 2009-NOV-5 by ORCHARD. An Arabidopsis cDNA library constructed using mRNA extracted from leaf tissues challenged with several pathogens , details of pathogens not supplied by authors. Plant J. (0960-7412) 2009 orchard Only protein-protein interactions imex curation rpw82_arath Protein RESISTANCE TO POWDERY MILDEW 8.2 RPW8.2 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MIAEVAAGGALGLALSVLHEAVKRAKDRSVTTRFILHRLEATIDSITPLVVQIDKFSEEMEDSTSRKVNKRLKLLLENAVSLVEENAELRRRNVRKKFRYMRDIKEFEAKLRWVVDVDVQVNQLADIKELKAKMSEISTKLDKIMPQPKFEIHIGWCSGKTNRAIRFTFCSDDS FD01C3B3C73C4518 14336_arath 14-3-3-like protein GF14 lambda General regulatory factor 6 14-3-3-like protein RCI2 14-3-3-like protein AFT1 GRF6 AFT1 RCI2 F12B17_200 At5g10450 protein protein arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress MAATLGRDQYVYMAKLAEQAERYEEMVQFMEQLVTGATPAEELTVEERNLLSVAYKNVIGSLRAAWRIVSSIEQKEESRKNDEHVSLVKDYRSKVESELSSVCSGILKLLDSHLIPSAGASESKVFYLKMKGDYHRYMAEFKSGDERKTAAEDTMLAYKAAQDIAAADMAPTHPIRLGLALNFSVFYYEILNSSDKACNMAKQAFEEAIAELDTLGEESYKDSTLIMQLLRDNLTLWTSDMQEQMDEA 4017D23098E74891 rpw8_2-14_3_3 3 4 unspecified role unspecified role bait bait 5 unspecified role unspecified role prey prey c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association To identify proteins directly interacting with RPW8.2 and modulating its function, we screened an Arabidopsis Y2H LexA cDNA prey library (van der Biezen et al., 2000) using RPW8.2 as bait. From approximately 4 x 106 cDNA clones, we identified GF14λ (At5g10450) as one of several putative interactors. Of eight GF14λ cDNA clones identified, six encode proteins that differ in the length of the N-terminal part of the protein (Figure 1a), indicating that the first 51 amino acids at least of the N-terminus of GF14λ are not required for its interaction with RPW8.2. Figure 1a rpw8_2-14_3_3-1 2 4 unspecified role unspecified role bait bait c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role prey prey physical association physical association suggesting that the C-terminal 38 amino acids of RPW8.2 are required for its interaction with GF14λ. Second, we tested the interaction between GF14λ and the RPW8.2 Figure 1c rpw8_2-14_3_3-2 1 4 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal required to bind necessary binding region required to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role prey prey region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association The antibody detected YFP, GF14κ�YFP and GF14λ�YFP from the total protein extracts as expected (Figure 1d). In the pulldown assays, we detected the band corresponding to GF14λ�YFP only from the input prepared from GF14λ�YFP-expressing plants, and not from the YFP- and GF14κ�YFP-expressing plants (Figure 1d), indicating that ΔNtRPW8.2 was able to specifically bind the plant-expressed GF14λ, not GF14κ and YFP. Figure 1d rpw8_2-14_3_3-3 1 4 unspecified role unspecified role bait bait region his tag his tag Hexa-His-tag 6-His-tag Histidine-tag Hexa-His-tag Histidine-tag 6-His-tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined c-terminal sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain 5 unspecified role unspecified role prey prey region yfp tag yellow fluorescent protein tag YFP yellow fluorescent protein yellow fluorescent protein YFP yfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined arath Arabidopsis thaliana (Mouse-ear cress) Mouse-ear cress physical association physical association The antibody detected YFP, GF14κ�YFP and GF14λ�YFP from the total protein extracts as expected (Figure 1d). In the pulldown assays, we detected the band corresponding to GF14λ�YFP only from the input prepared from GF14λ�YFP-expressing plants, and not from the YFP- and GF14κ�YFP-expressing plants (Figure 1d), indicating that ΔNtRPW8.2 was able to specifically bind the plant-expressed GF14λ, not GF14κ and YFP. Figure 1e