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/ SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. J. Exp. Bot. (0022-0957) 2008 imex curation Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library 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 Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library J. Exp. Bot. (0022-0957) 2008 orchard Only protein-protein interactions imex curation SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. J. Exp. Bot. (0022-0957) 2008 imex curation Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library orchard Only protein-protein interactions in vitro In vitro pull down pull down anti tag western anti tag western blot anti tag western Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library J. Exp. Bot. (0022-0957) 2008 orchard Only protein-protein interactions imex curation SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. SlTPR1, a tomato tetratricopeptide repeat protein, interacts with the ethylene receptors NR and LeETR1, modulating ethylene and auxin responses and development. J. Exp. Bot. (0022-0957) 2008 imex curation Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library orchard Only protein-protein interactions allce Allium cepa (Onion) Onion confocal microscopy confocal microscopy tag fluorescence tag visualisation by fluorescence tag fluorescence Lin Z., Arciga-Reyes L., Zhong S., Alexander L., Hackett R., Wilson I., Grierson D. It was not possible to unambiguously identify other interacting proteins mentioned in Table 1 of the paper. Therefore, these protein interactions were not curated. donald.grierson@nottingham.ac.uk Accepted 2009-NOV-29 by ORCHARD. 23-Dec-2009: Contacted by JYOTI. Tomato ripening fruit cDNA library J. Exp. Bot. (0022-0957) 2008 orchard Only protein-protein interactions imex curation etr1_sollc Ethylene receptor 1 ETR1 protein protein sollc Solanum lycopersicum (Tomato) Lycopersicon esculentum Tomato MGSLLRMNRLLSSIVESCNCIIDPQLPADDLLMKYQYISDFFIALAYFSIPVELIYFVKKSAVFPYRWVLVQFGAFIVLCGATHLINLWTFNMHTRNVAIVMTTPKALTALVSCITALMLVHIIPDLLSVKTRELFLKKKAAQLDREMGIIRTQEETGRHVRMLTHEIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTLRHQNPVGLTVPIQLPVINQVFGTNHVVKISPNSPVARLRPAGKYMPGEVVAVRVPLLHLSNFQINDWPELSTKRYALMVLMLPSDSARQWHVHELELVEVVADQVAVALSHAAILEESMRARDLLMEQNVALDLARREAEMAVRARNDFLAVMNHEMRTPMHAIIALSSLLQETDLTPEQRLMVETILKSSNLLATLINDVLDLSRLEDGSLQLDIGTFNLHALFREVHSLIKPIASVKKLFVTLSLSSDLPEYVIGDEKRLMQILLNVVGNAVKFSKEGNVSISAFVAKSDSLRDPRAPEFFAVPSENHFYLRVQIKDTGIGITPQDIPNLFSKFTQSQALATTNSGGTGLGLAICKRFVNLMEGHIWIESEGLGKGSTAIFIIKLGIPGRANESKLPFVTKLPANHTQMSFQGLKVLVMDENGVSRMVTKGLLTHLGCDVTTVGSRDECLRVVTHEHKVVIMDVSMQGIDCYEVAVVIHERFGKRHGRPLIVALTGNTDRVTKENCMRVGMDGVILKPVSVYKMRSVLSELLEHGVVLES D7443B6517781D0A q41341_sollc Never-ripe ETR3 544279 protein protein sollc Solanum lycopersicum (Tomato) Lycopersicon esculentum Tomato MESCDCIEALLPTGDLLVKYQYLSDFFIAVAYFSIPLELIYFVHKSACFPYRWVLMQFGAFIVLCGATHFISLWTFFMHSKTVAVVMTISKMLTAAVSCITALMLVHIIPDLLSVKTRELFLKTRAEELDKEMGLIIRQEETGRHVRMLTHEIRSTLDRHTILKTTLVELGRTLDLAECALWMPCQGGLTLQLSHNLNNLIPLGSTVPINLPIINEIFSSPEAIQIPHTNPLARMRNTVGRYIPPEVVAVRVPLLHLSNFTNDWAELSTRSYAVMVLVLPMNGLRKWREHELELVQVVADQVAVALSHAAILEDSMRAHDQLMEQNIALDVARQEAEMAIRARNDFLAVMNHEMRTPMHAVIALCSLLLETDLTPEQRVMIETILKSSNLLATLINDVLDLSRLEDGILELENGTFNLHGILREAVNLIKPIASLKKLSITLALALDLPILAVGDAKRLIQTLLNVAGNAVKFTKEGHISIEASVAKPEYARDCHPPEMFPMPSDGQFYLRVQVRDTGCGISPQDIPLVFTKFAESRPTSNRSTGGEGLGLAICRRFIQLMKGNIWIESEGPGKGTTVTFVVKLGICHHPNALPLLPMPPRGRLNKGSDDLFRYRQFRGDDGGMSVNAQRYQRSL 40E40D2858EB170C b6gva2_sollc int106 protein protein sollc Solanum lycopersicum (Tomato) Lycopersicon esculentum Tomato MVSIEQVESPAAGKTSTAAEAPSGYVSDGYETASDSELNGAENGSNRESENTINTASSSSDGDFLKEKTQEQQPEVNQEQLNEKALAQANDAKVEGNALFKDGLYEEALSKYELALQVAADIPSSTEIRSICHANRAACFTKLGKHEETIKECTKALELNPTYIKALVRRAEAHEKLEHFEEAITDMTKILELEPSHDQARRTVILLKPLADEKREKMKEEMIGKLKEMGNSILGRFGMSVDNFKTVKDPNTGSYSVQFQK 7EC2776AB86B7BE3 nr-sitpr1 1 6 unspecified role unspecified role prey prey 5 unspecified role unspecified role bait bait 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 Figure 1A Figure S1 The NR partial cDNA sequence (NR117-635, lacking the N-terminal membrane spanning region), was inserted in the yeast two-hybrid bait vector pEG202 (containing the LexA DNA-binding domain, DB) (Fig. 1A). In the first round, 2x106 independent yeast colonies were screened and 47 positive interacting cDNA clones belonging to four families were recovered and sequenced. BLAST searches showed the encoded proteins (INT proteins; Table 1) have homologies to a TPR protein (initially called INT106; Arciga et al., 2003; named here as SlTPR1: Solanum lycopersicum tetratricopeptide repeat protein 1) etr1-sitpr1-1 1 6 unspecified role unspecified role prey prey 4 unspecified role unspecified role bait bait 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 Figure 1A SlTPR1 also interacted with the tomato LeETR1 ethylene receptor, but not with LeETR2, 4, 5, and 6 ethylene receptors in yeast two-hybrid assays nr-sitpr1-1 2 6 unspecified role unspecified role prey prey region LexA tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 1F Protein�protein interactions of SlTPR1 with NR and LeETR1 in vitro assays. etr1-sitpr1-2 2 4 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst 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 yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 6 unspecified role unspecified role prey prey region lexA tag tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast physical association physical association Figure 1F nr-sitpr1-2 3 6 unspecified role unspecified role neutral component neutral component region cfp tag cyan fluorescent protein tag CFP cyan fluorescent protein cyan fluorescent protein CFP cfp tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 unspecified role unspecified role neutral component neutral component 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 direct interaction direct interaction Figure 10 When two fluorescent constructs, SlTPR1 fused to CFP and NR fused to YFP (pDH51-GW-YFP) were transiently expressed in the same onion epidermal cell by co-bombardment, the two florescent fusion proteins were found to co-localize at the plasmalemma and nuclear membranes