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/ Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell (0092-8674) 2008 imex curation Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library orchard Only protein-protein interactions 8 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 Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu dinesh-kumar@yale.edu Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library Cell (0092-8674) 2008 orchard Only protein-protein interactions 8 imex curation Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell (0092-8674) 2008 imex curation Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library orchard Only protein-protein interactions 8 nicbe Nicotiana benthamiana bifc bimolecular fluorescence complementation bifc tag fluorescence tag visualisation by fluorescence tag fluorescence Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu dinesh-kumar@yale.edu Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library Cell (0092-8674) 2008 orchard Only protein-protein interactions 8 imex curation Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell (0092-8674) 2008 imex curation Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library orchard Only protein-protein interactions 8 nicbe Nicotiana benthamiana anti tag coip anti tag coimmunoprecipitation anti tag coip western blot western blot Immuno blot Immuno blot Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu dinesh-kumar@yale.edu Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library Cell (0092-8674) 2008 orchard Only protein-protein interactions 8 imex curation Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Chloroplastic protein NRIP1 mediates innate immune receptor recognition of a viral effector. Cell (0092-8674) 2008 imex curation Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library orchard Only protein-protein interactions 8 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 predetermined predetermined participant predetermined Caplan JL., Mamillapalli P., Burch-Smith TM., Czymmek K., Dinesh-Kumar SP. Tobacco leaves were transformed with expression constructs of epitope tagged proteins of interest, using Agroinfiltration using the GV2260 Agrobacterium strain. dinesh-kumar@yale.edu,savithramma.dinesh-kumar@yale.edu dinesh-kumar@yale.edu Accepted 2008-OCT-15 by ORCHARD. 16-Oct-2008: Contacted by JYOTI. Solanum lycopersicum (Tomato) cDNA library Cell (0092-8674) 2008 orchard Only protein-protein interactions 8 imex curation b2c7y6_nicbe NRIP1 protein protein nicbe Nicotiana benthamiana MRILSLPSTSFSLVDCHIPKNLTYGNVSSVTLTPMARSQFQPQKRRNFGNSNRTPGFSWMATVGEKVQVSTVPTSVPVRVALELLQAGHRYLDVRTAEEFSDGHAPGAINIPYMFRIGSGMIKNPNFAEQVLEHFGKDDEIIVGCQLGKRSFMAATDLLAAGFSGVTDIAGGYAAWTENGLPTDS D7510266D484DDC8 nrip1_sollc_protein N receptor interacting protein (NRIP1) (Tomato) protein protein sollc Solanum lycopersicum (Tomato) Lycopersicon esculentum Tomato An amino acid sequence which is not represented in UniProt The protein is a homologue of Tobacco NPIR1 protein UniProt Q45TE3 p03586-pro_0000041163 Replicase small subunit 183 kDa protein RNA-directed RNA polymerase protein protein tmv Tobacco mosaic virus (strain vulgare) (TMV) TMV Tobacco mosaic virus (strain U1) MAYTQTATTSALLDTVRGNNSLVNDLAKRRLYDTAVEEFNARDRRPKVNFSKVISEEQTLIATRAYPEFQITFYNTQNAVHSLAGGLRSLELEYLMMQIPYGSLTYDIGGNFASHLFKGRAYVHCCMPNLDVRDIMRHEGQKDSIELYLSRLERGGKTVPNFQKEAFDRYAEIPEDAVCHNTFQTMRHQPMQQSGRVYAIALHSIYDIPADEFGAALLRKNVHTCYAAFHFSENLLLEDSYVNLDEINACFSRDGDKLTFSFASESTLNYCHSYSNILKYVCKTYFPASNREVYMKEFLVTRVNTWFCKFSRIDTFLLYKGVAHKSVDSEQFYTAMEDAWHYKKTLAMCNSERILLEDSSSVNYWFPKMRDMVIVPLFDISLETSKRTRKEVLVSKDFVFTVLNHIRTYQAKALTYANVLSFVESIRSRVIINGVTARSEWDVDKSLLQSLSMTFYLHTKLAVLKDDLLISKFSLGSKTVCQHVWDEISLAFGNAFPSVKERLLNRKLIRVAGDALEIRVPDLYVTFHDRLVTEYKASVDMPALDIRKKMEETEVMYNALSELSVLRESDKFDVDVFSQMCQSLEVDPMTAAKVIVAVMSNESGLTLTFERPTEANVALALQDQEKASEGALVVTSREVEEPSMKGSMARGELQLAGLAGDHPESSYSKNEEIESLEQFHMATADSLIRKQMSSIVYTGPIKVQQMKNFIDSLVASLSAAVSNLVKILKDTAAIDLETRQKFGVLDVASRKWLIKPTAKSHAWGVVETHARKYHVALLEYDEQGVVTCDDWRRVAVSSESVVYSDMAKLRTLRRLLRNGEPHVSSAKVVLVDGVPGCGKTKEILSRVNFDEDLILVPGKQAAEMIRRRANSSGIIVATKDNVKTVDSFMMNFGKSTRCQFKRLFIDEGLMLHTGCVNFLVAMSLCEIAYVYGDTQQIPYINRVSGFPYPAHFAKLEVDEVETRRTTLRCPADVTHYLNRRYEGFVMSTSSVKKSVSQEMVGGAAVINPISKPLHGKILTFTQSDKEALLSRGYSDVHTVHEVQGETYSDVSLVRLTPTPVSIIAGDSPHVLVALSRHTCSLKYYTVVMDPLVSIIRDLEKLSSYLLDMYKVDAGTQ This protein is 50-kDa helicase domain of TMV's replicase protein (Interpro identifier IPR000606). 1 1116 864B352B25F4B6D3 tmvrn_nicgu TMV resistance protein N N protein protein nicgu Nicotiana glutinosa (Tobacco) MASSSSSSRWSYDVFLSFRGEDTRKTFTSHLYEVLNDKGIKTFQDDKRLEYGATIPGELCKAIEESQFAIVVFSENYATSRWCLNELVKIMECKTRFKQTVIPIFYDVDPSHVRNQKESFAKAFEEHETKYKDDVEGIQRWRIALNEAANLKGSCDNRDKTDADCIRQIVDQISSKLCKISLSYLQNIVGIDTHLEKIESLLEIGINGVRIMGIWGMGGVGKTTIARAIFDTLLGRMDSSYQFDGACFLKDIKENKRGMHSLQNALLSELLREKANYNNEEDGKHQMASRLRSKKVLIVLDDIDNKDHYLEYLAGDLDWFGNGSRIIITTRDKHLIEKNDIIYEVTALPDHESIQLFKQHAFGKEVPNENFEKLSLEVVNYAKGLPLALKVWGSLLHNLRLTEWKSAIEHMKNNSYSGIIDKLKISYDGLEPKQQEMFLDIACFLRGEEKDYILQILESCHIGAEYGLRILIDKSLVFISEYNQVQMHDLIQDMGKYIVNFQKDPGERSRLWLAKEVEEVMSNNTGTMAMEAIWVSSYSSTLRFSNQAVKNMKRLRVFNMGRSSTHYAIDYLPNNLRCFVCTNYPWESFPSTFELKMLVHLQLRHNSLRHLWTETKHLPSLRRIDLSWSKRLTRTPDFTGMPNLEYVNLYQCSNLEEVHHSLGCCSKVIGLYLNDCKSLKRFPCVNVESLEYLGLRSCDSLEKLPEIYGRMKPEIQIHMQGSGIRELPSSIFQYKTHVTKLLLWNMKNLVALPSSICRLKSLVSLSVSGCSKLESLPEEIGDLDNLRVFDASDTLILRPPSSIIRLNKLIILMFRGFKDGVHFEFPPVAEGLHSLEYLNLSYCNLIDGGLPEEIGSLSSLKKLDLSRNNFEHLPSSIAQLGALQSLDLKDCQRLTQLPELPPELNELHVDCHMALKFIHYLVTKRKKLHRVKLDDAHNDTMYNLFAYTMFQNISSMRHDISASDSLSLTVFTGQPYPEKIPSWFHHQGWDSSVSVNLPENWYIPDKFLGFAVCYSRSLIDTTAHLIPVCDDKMSRMTQKLALSECDTESSNYSEWDIHFFFVPFAGLWDTSKANGKTPNDYGIIRLSFSGEEKMYGLRLLYKEGPEVNALLQMRENSNEPTEHSTGIRRTQYNNRTSFYELING 1EC5B36D4C4BCBB4 p90211-pro_0000041189 Replicase small subunit RNA-directed RNA polymerase 183 kDa protein protein protein tmob Tobamovirus Ob MAHIQQSMQGALLDTVRGQNSLVNDLAKRRLYDTAVEEFNAKDRRPKINFSKSINEEQTLIVSQAYPEFQITFYNTQLAVHSLAAGLRSLELEYLMMQVPYGSLTYDIGGNFAAHLFKGRDYVHCCMPNLDLRDIMRHENQKDSVATYLSRLKARNKVLPAFQQEAFQRYSERSDEVVCNNTFQCCESNRYSSGGRVYAISLHSLYDIPADELGAALLRKNVHTLYAAFHFAEELLLEVSTVELPTIGGIFSRDGDKINFCFSNESTLNYSHSYSNLLKYVCKTYFPASNRFVYMKEFLITRVNTWFCKFTKLDTYTLYRGVYHRGCDQQEFYSAMEDAWHYKKTLAMLNSERIVLEDHSSVNYWFPKMKDMVIVPLFDVSLETQKRTKKEVIVSKDFVYTVLNHIRTYQAKALTYNNVLSFVESIRSRVIINGVTARSEWDVDKALLQSMAMTFFLITKLSMLKDELLVSKFTLSAKSVHEHVWDEIKRGCGNMFPSLKESLLRKKLISGSAEELEIEVPDMYVTFHDRFVAEYKASVEMPTIDISKDLSEAESYYSALSELSVLENSKDFDLEKFSRMCAINCVNPDIAAKIVVAVLSNESGVTLPFKEPTEGNMAEAMKSGEKDEVLTLGSQTDNTDLTSKSMVISGSLPLCGIASEISCDTFVRNEEINSLEEYHMLAAESVISNKMASIVYSGPLQVQQMQNYVDSLAASLSATVSNLKKLVKDSSVGFQDSLSKVGVFDVRKKMWLIKPTLKNHSWGVVQKFDGKCFLALLSYHNELPICDADWSKVAVSNESMVYSDMAKLRVLRKSIGEMPISVSSAKVTLVDGVPGCGKTKEILRRVNFSEDLVLVPGKEAAAMIRKRANQSGNIVANNDNVKTVDSFLMNLGKGPVCQFKRLFVDEGLMLHPGCVYFLVKLSLCNEAFVFGDTQQIPYINRVQNFPFPQHFSKLIVDETEKRRTTLRCPVDVTHFLNQCYDGAVTTTSKTQRSVGLEVVGGAAVMNPVTKPLKGKIVTFTQSDKLTMLSRGYQDVNTVHEIQGETYEEVSLVRLTPTPIHIISRESPHVLVGLTRHTRCFKYYTVVLDPLVKLVRDLECVSNFLLDVYMVDSVSA 1 1115 430157E4FDB573FF n-nrip1 1 6 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association To search for novel proteins that mediate the association between the N immune receptor with the p50 effector, we conducted a yeast two-hybrid screen with the TIR domain of N as bait. We identified N receptor-interacting protein 1 (NRIP1), which was represented by six identical, independent clones in the screen The TIR region coordinates are taken from the rerefence provided by the authors , Dinesh-kumar et al (2000). Structure-function analysis of the tobacco mosaic virus resistance gene N. PMID:11121079. Please note therse coordinates differ from the coordinates of the UniProt record. n-nrip1-1 4 5 unspecified role unspecified role prey prey 8 unspecified role unspecified role bait bait region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association Therefore, NRIP1 and N(TIR) interact directly in a yeast two-hybrid assay The TIR region coordinates are taken from the rerefence provided by the authors , Dinesh-kumar et al (2000). Structure-function analysis of the tobacco mosaic virus resistance gene N. PMID:11121079. Please note therse coordinates differ from the coordinates of the UniProt record. 1A n-nrip1-2 3 5 unspecified role unspecified role prey prey region enhanced cyan fluorescent protein (ECFP) tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 8 unspecified role unspecified role bait bait c-terminal tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged c-term range C-terminal range c-term range c-term range C-terminal range c-term range region sufficient to bind sufficient binding region sufficient to bind certain certain sequence position certain certain certain sequence position certain physical association physical association We also confirmed the association of N's TIR domain and NRIP1-Cerulean. NRIP1-Cerulean coimmunoprecipitated with N(TIR)-TAP in the absence and presence of p50, but not with TAP alone The TIR region coordinates are taken from the rerefence provided by the authors , Dinesh-kumar et al (2000). Structure-function analysis of the tobacco mosaic virus resistance gene N. PMID:11121079. Please note therse coordinates differ from the coordinates of the UniProt record. 1B p50-nrip1-3 4 5 unspecified role unspecified role prey prey 7 unspecified role unspecified role bait bait physical association physical association We found that yeast carrying p50 as bait and NRIP1 as prey activated expression of the LEU2 reporter gene (Figure 3A), suggesting that p50 and NRIP1 interact directly in a yeast two-hybrid assay. 3A p50-nrip1-4 3 5 unspecified role unspecified role prey prey region enhanced cyan fluorescent protein (ECFP) tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 7 unspecified role unspecified role bait bait region tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association NRIP1-Cerulean coimmunoprecipitated with p50-TAP but did not with TAP alone 3C p50-nrip1-5 3 5 unspecified role unspecified role prey prey region enhanced cyan fluorescent protein (ECFP) tag tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 9 unspecified role unspecified role bait bait region tap tagged calmodulin binding peptide plus protein a tag CBP-ProtA tagged CBP-ProtA tagged tap tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association NRIP1-Cerulean coimmunoprecipitated with p50-TAP but did not with TAP alone 3C p50-nrip1-6 2 5 unspecified role unspecified role neutral component neutral component region C-terminal YFP tag 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 7 unspecified role unspecified role neutral component neutral component region N-terminal YFP 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 physical association physical association Reconstituted fluorescence was detected in the cytoplasm, nuclei and chloroplasts 3D n-nrip1-3 2 5 unspecified role unspecified role neutral component neutral component region C-terminal YFP tag 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 7 unspecified role unspecified role ancillary ancillary 8 unspecified role unspecified role neutral component neutral component region N-terminal YFP 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 physical association physical association These results confirm that N and NRIP1 associate only in the presence of p50 in intact, living tissue. Figure 4