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Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J - Plant Mol. Biol. (2011)

Bottom Line: Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo.Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box.Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Sylvius Laboratory, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

ABSTRACT
Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

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ORA59 and ERF1 bind to two GCC boxes in the PDF1.2 promoter in vitro. a After SDS–PAGE and Western blotting recombinant proteins were detected with anti-His HRP-conjugated antibodies. Positions of protein size markers are indicated in k Dalton. b EMSAs were performed with recombinant ORA59 and ERF1 proteins and radio-labeled SF, m1, m2 or dm fragments. ORA47 protein and wild-type or mutated AOC2 promoter fragments were used as control. The arrowheads mark the positions of protein-DNA complexes (C) and free probes (F)
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Fig3: ORA59 and ERF1 bind to two GCC boxes in the PDF1.2 promoter in vitro. a After SDS–PAGE and Western blotting recombinant proteins were detected with anti-His HRP-conjugated antibodies. Positions of protein size markers are indicated in k Dalton. b EMSAs were performed with recombinant ORA59 and ERF1 proteins and radio-labeled SF, m1, m2 or dm fragments. ORA47 protein and wild-type or mutated AOC2 promoter fragments were used as control. The arrowheads mark the positions of protein-DNA complexes (C) and free probes (F)

Mentions: To establish whether ORA59 and ERF1 indeed bind the GCC boxes in the PDF1.2 promoter as suggested by the trans-activation experiments, we produced recombinant proteins expressed in Escherichia coli with N- and C-terminal Strep and His tags, respectively. Levels of intact ORA59 protein in the soluble fraction of E. coli extracts were almost undetectable. However high amounts of ORA59 and ERF1 proteins were present in the insoluble fraction, presumably in inclusion bodies. Therefore proteins in the insoluble fraction were dissolved in buffer with 6 M urea, proteins were renatured by dilution and dialysis, and full-length ORA59 and ERF1 were isolated by sequential Ni–NTA and Strep-Tactin affinity chromatography. Analysis of the proteins by staining of an SDS-PAA gel with coomassie brilliant blue (not shown) or immunoblot analysis with anti-His antibodies conjugated to horseradish peroxidase (Fig. 3a) showed a single main reactive band in each protein preparation. Although the tagged ORA59 and ERF1 proteins have similar predicted sizes of 31 and 29 kDa, respectively, ORA59 migrated in the denaturing gel system at a position corresponding to 42 kDa, which might be due to a specific structure of the protein. ORA59 expressed in Arabidopsis protoplasts also migrated at the same position (results not shown), which makes it unlikely that the aberrant migration is an artifact due to the E. coli expression system.Fig. 3


Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis.

Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J - Plant Mol. Biol. (2011)

ORA59 and ERF1 bind to two GCC boxes in the PDF1.2 promoter in vitro. a After SDS–PAGE and Western blotting recombinant proteins were detected with anti-His HRP-conjugated antibodies. Positions of protein size markers are indicated in k Dalton. b EMSAs were performed with recombinant ORA59 and ERF1 proteins and radio-labeled SF, m1, m2 or dm fragments. ORA47 protein and wild-type or mutated AOC2 promoter fragments were used as control. The arrowheads mark the positions of protein-DNA complexes (C) and free probes (F)
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Related In: Results  -  Collection

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Fig3: ORA59 and ERF1 bind to two GCC boxes in the PDF1.2 promoter in vitro. a After SDS–PAGE and Western blotting recombinant proteins were detected with anti-His HRP-conjugated antibodies. Positions of protein size markers are indicated in k Dalton. b EMSAs were performed with recombinant ORA59 and ERF1 proteins and radio-labeled SF, m1, m2 or dm fragments. ORA47 protein and wild-type or mutated AOC2 promoter fragments were used as control. The arrowheads mark the positions of protein-DNA complexes (C) and free probes (F)
Mentions: To establish whether ORA59 and ERF1 indeed bind the GCC boxes in the PDF1.2 promoter as suggested by the trans-activation experiments, we produced recombinant proteins expressed in Escherichia coli with N- and C-terminal Strep and His tags, respectively. Levels of intact ORA59 protein in the soluble fraction of E. coli extracts were almost undetectable. However high amounts of ORA59 and ERF1 proteins were present in the insoluble fraction, presumably in inclusion bodies. Therefore proteins in the insoluble fraction were dissolved in buffer with 6 M urea, proteins were renatured by dilution and dialysis, and full-length ORA59 and ERF1 were isolated by sequential Ni–NTA and Strep-Tactin affinity chromatography. Analysis of the proteins by staining of an SDS-PAA gel with coomassie brilliant blue (not shown) or immunoblot analysis with anti-His antibodies conjugated to horseradish peroxidase (Fig. 3a) showed a single main reactive band in each protein preparation. Although the tagged ORA59 and ERF1 proteins have similar predicted sizes of 31 and 29 kDa, respectively, ORA59 migrated in the denaturing gel system at a position corresponding to 42 kDa, which might be due to a specific structure of the protein. ORA59 expressed in Arabidopsis protoplasts also migrated at the same position (results not shown), which makes it unlikely that the aberrant migration is an artifact due to the E. coli expression system.Fig. 3

Bottom Line: Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo.Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box.Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biology, Sylvius Laboratory, Leiden University, P.O. Box 9505, 2300 RA, Leiden, The Netherlands.

ABSTRACT
Plant defense against microbial pathogens depends on the action of several endogenously produced hormones, including jasmonic acid (JA) and ethylene (ET). In defense against necrotrophic pathogens, the JA and ET signaling pathways synergize to activate a specific set of defense genes including PLANT DEFENSIN1.2 (PDF1.2). The APETALA2/Ethylene Response Factor (AP2/ERF)-domain transcription factor ORA59 acts as the integrator of the JA and ET signaling pathways and is the key regulator of JA- and ET-responsive PDF1.2 expression. The present study was aimed at the identification of elements in the PDF1.2 promoter conferring the synergistic response to JA/ET and interacting with ORA59. We show that the PDF1.2 promoter was activated synergistically by JA and the ET-releasing agent ethephon due to the activity of two GCC boxes. ORA59 bound in vitro to these GCC boxes and trans-activated the PDF1.2 promoter in transient assays via these two boxes. Using the chromatin immunoprecipitation technique we were able to show that ORA59 bound the PDF1.2 promoter in vivo. Finally, we show that a tetramer of a single GCC box conferred JA/ethephon-responsive expression, demonstrating that the JA and ET signaling pathways converge to a single type of GCC box. Therefore ORA59 and two functionally equivalent GCC box binding sites form the module that enables the PDF1.2 gene to respond synergistically to simultaneous activation of the JA and ET signaling pathways.

Show MeSH