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AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis.

Lin Z, Ho CW, Grierson D - J. Exp. Bot. (2009)

Bottom Line: This association was confirmed by in vivo co-immunoprecipitation.Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes.A model for AtTRP1 action is proposed.

View Article: PubMed Central - PubMed

Affiliation: Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.

ABSTRACT
Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter-GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed.

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Interaction assays of AtTRP1 with the ethylene receptors ERS1 and ETR1 in the yeast two-hybrid system and in planta. (A) Structure of ERS1 and ETR1 ethylene receptors. TMD, transmembrane domain; GAF, GAF domain; HK, histidine kinase domain; RD, receiver domain. Amino acid positions are numbered. (B) Domains used for interaction assays in the yeast two-hybrid system. DB, LexA DNA-binding-domain; AD, activation domain. Amino acids are numbered. (C) Activation analysis of the reporter gene LacZ to the bait/prey combination: Yeast transformed with DB-ERS1307-613/AD-AtTRP1, DB-NR117-635/AD-AtTRP1, DB-LeETR1132-754/AD-AtTRP1, and DB-LeETR1364-467/AD-AtTRP1 generated blue colour in 3 d when grown on minimal medium containing galactose and X-gal (Gala/x-gal), whereas yeast transformed with DB-ETR1350-738/AD-AtTRP1 remained white when grown on the same plate, indicating no interaction. (D) All the recombinants remained white when grown on medium containing glucose and X-gal (Glu/x-gal). P, positive control; N, negative control. (E) Top panel: western blot using anti-GFP to detect AtTRP1–GFP fusion protein in the total membrane protein fraction extracted from the transgenic (lines 3, 5, 6) and wild-type (Col) seedlings (15-d-old grown in the light in soil) or after immunoprecipitation with anti-GFP antibody using total membrane proteins from the transgenic seedlings (pull-down) prior to electrophoresis. One band was detected with size 60 kDa corresponding to AtTRP1–GFP protein. Lower panel: western blot using anti-NR antibody to detect ERS1 in the same samples as the top panel. In the total membrane protein fraction two bands of 130 and 68 kDa corresponding to the homodimer or monomer of the ERS1 receptor were detected. After immunoprecipitation prior to electrophoresis only the larger band (130 kDa) corresponding to the size of the ERS1 homodimer was detected in the pull-dwon pellet, suggesting that AtTRP1 forms a complex with ERS1 dimers and not monomers.
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fig9: Interaction assays of AtTRP1 with the ethylene receptors ERS1 and ETR1 in the yeast two-hybrid system and in planta. (A) Structure of ERS1 and ETR1 ethylene receptors. TMD, transmembrane domain; GAF, GAF domain; HK, histidine kinase domain; RD, receiver domain. Amino acid positions are numbered. (B) Domains used for interaction assays in the yeast two-hybrid system. DB, LexA DNA-binding-domain; AD, activation domain. Amino acids are numbered. (C) Activation analysis of the reporter gene LacZ to the bait/prey combination: Yeast transformed with DB-ERS1307-613/AD-AtTRP1, DB-NR117-635/AD-AtTRP1, DB-LeETR1132-754/AD-AtTRP1, and DB-LeETR1364-467/AD-AtTRP1 generated blue colour in 3 d when grown on minimal medium containing galactose and X-gal (Gala/x-gal), whereas yeast transformed with DB-ETR1350-738/AD-AtTRP1 remained white when grown on the same plate, indicating no interaction. (D) All the recombinants remained white when grown on medium containing glucose and X-gal (Glu/x-gal). P, positive control; N, negative control. (E) Top panel: western blot using anti-GFP to detect AtTRP1–GFP fusion protein in the total membrane protein fraction extracted from the transgenic (lines 3, 5, 6) and wild-type (Col) seedlings (15-d-old grown in the light in soil) or after immunoprecipitation with anti-GFP antibody using total membrane proteins from the transgenic seedlings (pull-down) prior to electrophoresis. One band was detected with size 60 kDa corresponding to AtTRP1–GFP protein. Lower panel: western blot using anti-NR antibody to detect ERS1 in the same samples as the top panel. In the total membrane protein fraction two bands of 130 and 68 kDa corresponding to the homodimer or monomer of the ERS1 receptor were detected. After immunoprecipitation prior to electrophoresis only the larger band (130 kDa) corresponding to the size of the ERS1 homodimer was detected in the pull-dwon pellet, suggesting that AtTRP1 forms a complex with ERS1 dimers and not monomers.

Mentions: It was previously reported that SlTPR1 interacts with the tomato ethylene receptors NR and to a lesser extent to LeETR1 in a yeast two-hybrid system (Lin et al., 2008a). To determine whether or not AtTRP1 functions in the same way as SlTPR1, the interactions of AtTRP1 with ERS1 and ETR1, the two Arabidopsis orthologues of the tomato ethylene receptors NR and LeETR1, were tested using the LexA-based yeast two hybrid system (Lin et al., 2008b). The cDNAs encoding ERS1 or ETR1, without the transmembrane domain and the GAF domain, were inserted into the bait vector pEG202 (ERS1307-613, ETR1350-738), and the coding sequence of AtTRP1 (nt: 1–830) was cloned in the prey vector pJG4-5 (Fig. 8A, B). All the constructs were confirmed by sequencing prior to transforming into yeast. Each bait construct was transformed into yeast strain EGY48 containing the LacZ reporter plasmid pSH18-34 and genetically integrated LEU2 reporter (Materials and methods). The suitability of the bait constructs was examined by testing their synthesis in yeast and the activation of the LacZ and LEU2 reporters prior to interaction assays (data not shown) (Lin et al., 2008c), and the prey construct AD-TRP1 and the prey vector pJG4-5 (as a negative control) were transformed with yeast containing each receptor construct. The interaction assays showed that AtTRP1 interacted with ERS1 and not with ETR1, whereas the prey vector pJG4-5 caused no interaction with either receptor (Fig. 9C). The interactions of AtTPR1 with the tomato ethylene receptors NR and LeETR1 were also examined. The NR partial cDNA encoding the NR protein without the transmembrane domain (NR117-635), together with the two partial cDNAs of LeETR1 encoding either the LeETR1 protein lacking the transmembrane domain (LeETR1132-754) or the histidine kinase domain alone (LeETR1364-647) were cloned into the bait vector pEG202 downstream of the LexA DNA-binding domain (DB) (Fig. 9B) (Lin et al., 2008c). All three tomato receptor constructs were shown to interact with AtTRP1 in yeast (Fig. 9C). The interactions of AtTRP1 with the ethylene receptors did not occur when yeast containing the combination of bait/prey constructs was grown on medium in the presence of glucose, which suppresses the expression of the prey protein AD-AtTRP1 (Fig. 9D).


AtTRP1 encodes a novel TPR protein that interacts with the ethylene receptor ERS1 and modulates development in Arabidopsis.

Lin Z, Ho CW, Grierson D - J. Exp. Bot. (2009)

Interaction assays of AtTRP1 with the ethylene receptors ERS1 and ETR1 in the yeast two-hybrid system and in planta. (A) Structure of ERS1 and ETR1 ethylene receptors. TMD, transmembrane domain; GAF, GAF domain; HK, histidine kinase domain; RD, receiver domain. Amino acid positions are numbered. (B) Domains used for interaction assays in the yeast two-hybrid system. DB, LexA DNA-binding-domain; AD, activation domain. Amino acids are numbered. (C) Activation analysis of the reporter gene LacZ to the bait/prey combination: Yeast transformed with DB-ERS1307-613/AD-AtTRP1, DB-NR117-635/AD-AtTRP1, DB-LeETR1132-754/AD-AtTRP1, and DB-LeETR1364-467/AD-AtTRP1 generated blue colour in 3 d when grown on minimal medium containing galactose and X-gal (Gala/x-gal), whereas yeast transformed with DB-ETR1350-738/AD-AtTRP1 remained white when grown on the same plate, indicating no interaction. (D) All the recombinants remained white when grown on medium containing glucose and X-gal (Glu/x-gal). P, positive control; N, negative control. (E) Top panel: western blot using anti-GFP to detect AtTRP1–GFP fusion protein in the total membrane protein fraction extracted from the transgenic (lines 3, 5, 6) and wild-type (Col) seedlings (15-d-old grown in the light in soil) or after immunoprecipitation with anti-GFP antibody using total membrane proteins from the transgenic seedlings (pull-down) prior to electrophoresis. One band was detected with size 60 kDa corresponding to AtTRP1–GFP protein. Lower panel: western blot using anti-NR antibody to detect ERS1 in the same samples as the top panel. In the total membrane protein fraction two bands of 130 and 68 kDa corresponding to the homodimer or monomer of the ERS1 receptor were detected. After immunoprecipitation prior to electrophoresis only the larger band (130 kDa) corresponding to the size of the ERS1 homodimer was detected in the pull-dwon pellet, suggesting that AtTRP1 forms a complex with ERS1 dimers and not monomers.
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fig9: Interaction assays of AtTRP1 with the ethylene receptors ERS1 and ETR1 in the yeast two-hybrid system and in planta. (A) Structure of ERS1 and ETR1 ethylene receptors. TMD, transmembrane domain; GAF, GAF domain; HK, histidine kinase domain; RD, receiver domain. Amino acid positions are numbered. (B) Domains used for interaction assays in the yeast two-hybrid system. DB, LexA DNA-binding-domain; AD, activation domain. Amino acids are numbered. (C) Activation analysis of the reporter gene LacZ to the bait/prey combination: Yeast transformed with DB-ERS1307-613/AD-AtTRP1, DB-NR117-635/AD-AtTRP1, DB-LeETR1132-754/AD-AtTRP1, and DB-LeETR1364-467/AD-AtTRP1 generated blue colour in 3 d when grown on minimal medium containing galactose and X-gal (Gala/x-gal), whereas yeast transformed with DB-ETR1350-738/AD-AtTRP1 remained white when grown on the same plate, indicating no interaction. (D) All the recombinants remained white when grown on medium containing glucose and X-gal (Glu/x-gal). P, positive control; N, negative control. (E) Top panel: western blot using anti-GFP to detect AtTRP1–GFP fusion protein in the total membrane protein fraction extracted from the transgenic (lines 3, 5, 6) and wild-type (Col) seedlings (15-d-old grown in the light in soil) or after immunoprecipitation with anti-GFP antibody using total membrane proteins from the transgenic seedlings (pull-down) prior to electrophoresis. One band was detected with size 60 kDa corresponding to AtTRP1–GFP protein. Lower panel: western blot using anti-NR antibody to detect ERS1 in the same samples as the top panel. In the total membrane protein fraction two bands of 130 and 68 kDa corresponding to the homodimer or monomer of the ERS1 receptor were detected. After immunoprecipitation prior to electrophoresis only the larger band (130 kDa) corresponding to the size of the ERS1 homodimer was detected in the pull-dwon pellet, suggesting that AtTRP1 forms a complex with ERS1 dimers and not monomers.
Mentions: It was previously reported that SlTPR1 interacts with the tomato ethylene receptors NR and to a lesser extent to LeETR1 in a yeast two-hybrid system (Lin et al., 2008a). To determine whether or not AtTRP1 functions in the same way as SlTPR1, the interactions of AtTRP1 with ERS1 and ETR1, the two Arabidopsis orthologues of the tomato ethylene receptors NR and LeETR1, were tested using the LexA-based yeast two hybrid system (Lin et al., 2008b). The cDNAs encoding ERS1 or ETR1, without the transmembrane domain and the GAF domain, were inserted into the bait vector pEG202 (ERS1307-613, ETR1350-738), and the coding sequence of AtTRP1 (nt: 1–830) was cloned in the prey vector pJG4-5 (Fig. 8A, B). All the constructs were confirmed by sequencing prior to transforming into yeast. Each bait construct was transformed into yeast strain EGY48 containing the LacZ reporter plasmid pSH18-34 and genetically integrated LEU2 reporter (Materials and methods). The suitability of the bait constructs was examined by testing their synthesis in yeast and the activation of the LacZ and LEU2 reporters prior to interaction assays (data not shown) (Lin et al., 2008c), and the prey construct AD-TRP1 and the prey vector pJG4-5 (as a negative control) were transformed with yeast containing each receptor construct. The interaction assays showed that AtTRP1 interacted with ERS1 and not with ETR1, whereas the prey vector pJG4-5 caused no interaction with either receptor (Fig. 9C). The interactions of AtTPR1 with the tomato ethylene receptors NR and LeETR1 were also examined. The NR partial cDNA encoding the NR protein without the transmembrane domain (NR117-635), together with the two partial cDNAs of LeETR1 encoding either the LeETR1 protein lacking the transmembrane domain (LeETR1132-754) or the histidine kinase domain alone (LeETR1364-647) were cloned into the bait vector pEG202 downstream of the LexA DNA-binding domain (DB) (Fig. 9B) (Lin et al., 2008c). All three tomato receptor constructs were shown to interact with AtTRP1 in yeast (Fig. 9C). The interactions of AtTRP1 with the ethylene receptors did not occur when yeast containing the combination of bait/prey constructs was grown on medium in the presence of glucose, which suppresses the expression of the prey protein AD-AtTRP1 (Fig. 9D).

Bottom Line: This association was confirmed by in vivo co-immunoprecipitation.Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes.A model for AtTRP1 action is proposed.

View Article: PubMed Central - PubMed

Affiliation: Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, UK.

ABSTRACT
Arabidopsis AtTRP1 is an orthologue of SlTPR1, a tomato tetratricopeptide repeat protein that interacts with the tomato ethylene receptors LeETR1 and NR in yeast 2-hybrid assays and in vitro, and modulates plant development. AtTRP1 is encoded by a single copy gene in the Arabidopsis genome, and is related to TCC1, a human protein that competes with Raf-1 for Ras binding, and distantly related to the immunophilin-like FK-binding proteins TWD1 and PAS1. The former is involved in auxin transport and the latter is translocated to the nucleus in response to auxin. AtTRP1 interacted preferentially with the Arabidopsis ethylene receptor ERS1 in yeast two-hybrid assays. This association was confirmed by in vivo co-immunoprecipitation. AtTRP1 promoter-GUS was highly expressed in vascular tissue, mature anthers, the abscission zone, and was induced by ACC. Overexpression of AtTRP1 in wild-type Arabidopsis resulted in dwarf plants with reduced fertility, altered leaf/silique morphology, and enhanced expression of the ethylene responsive gene AtChitB. Exogenous GA did not reverse the dwarf habit. Etiolated transgenic seedlings overexpressing AtTRP1 displayed enhanced sensitivity to low ACC and this was correlated with the transgene expression. Seedlings overexpressing AtTRP1 at high levels exhibited shortened and swollen hypocotyls, inhibited root growth, and an altered apical hook. Plants overexpressing AtTRP1 also showed a reduced response to exogenous IAA and altered expression of a subset of auxin early responsive genes. These results indicated that overexpression of AtTRP1 affects cross-talk between ethylene and auxin signalling and enhances some ethylene responses and alters some auxin responses. A model for AtTRP1 action is proposed.

Show MeSH
Related in: MedlinePlus