Corrigenda for Adams and Turner (2010) J. Exp Bot 61: 4373–4386

The following section of the Materials and methods to be changed as follows Preparation of constructs and construction of transgenic lines Arabidopsis transgenic lines (background Col gl1) lines were prepared to identify whether distinct active sites on the COI1 protein were responsible for differential functional activation. To obtain the transgenic lines LRR and Fbox, different portions of the COI1 coding sequence were PCR-amplified as NcoI/SmaI fragments from the construct containing the entire COI1 coding sequence in pPily (Ferrando et al., 2000; intron-tagged COI1::HiA, Devoto et al., 2002), and cloned de novo into the vector pPily to obtain C-terminal translational fusions. The construct pCoiLRR was obtained by PCR amplification (from bp 328 to 1779) using the primers ADs47 (5′-CTTCCACCATGGAGATTTCTAACAACCTTA-3′) and ADa4 (5′-TAGCTACCCGGGTATTGGCTCCTTCAGGAC-3′). The construct pCoiFbox was derived by PCR amplification (from bp 1 to 168) using the primers ADs3 (5′-GATCTACCATGGAGGATCCTGATATC-3′) and ADa38b (5′-TAGCTACCCAGGAGTCACATGCTCTCTCGTCTC-3′). To obtain the transgenic line W44, the mutated COI1 coding sequence was PCR-amplified as NcoI/SmaI fragment from the construct pCOI1W44,A (Devoto et al., 2002) to obtain the construct pCoiW44 using the primers ADs3 and ADa4. Forward (s) and reverse (a) primers contained the restriction sites Nco1 and Sma1, respectively. The KpnI cassettes containing the fusions, a double CaMV35S promoter and a NOS terminator, were transferred from the vectors pCoiLRR, pCoiFbox, and pCoiW44 into the binary kanamycin-resistant plasmid pBin19PLUS (van Engelen et al., 1995). Binary vectors were transferred into Agrobacterium tumefaciens GV3101 by electroporation. All constructs were sequenced and the expression levels of the mutated proteins were verified by Western blot analysis using the antibody peroxidase-coupled monoclonal anti-HA antibody 3F10 (Roche) as described by Devoto et al. (2002) with the exception that the Protease Inhibitor Cocktail for plant cell and tissue extracts (SIGMA) had to be added to the protein extraction buffer to guarantee protein stability. In addition, heat treatment prior to electrophoresis had to be eliminated. Each construct line was used as the male parent in crosses to the male-sterile coi1-16 and coi1-1. The homozygous coi1 lines with each transgene were selected for MeJA and kanamycin resistance in F2 and F3 populations. Original version Construction of transgenic lines The transgenic lines LRR and W44 contained the constructs pΔF-box and pCOI1W44A, respectively, as previously described (Devoto et al., 2002). These were introduced into the vector pPily and introduced into plants by Agrobacterium-mediated transformation. The F-box of the transgenic lines contained the construct pCOI1F-box which was obtained by replacing an NcoI–SmaI fragment of pLexA-COI1 (Devoto et al., 2002) with a purified and digested PCR fragment amplified with the primers s3 (5′-GATCTACCATGGAGGATCCTGATATC) and a38b (5′-TAGCTACCCAGGAGTCACATGCTCTCTCGTCTC). This was introduced into the vector pPily and introduced into plants by Agrobacterium-mediated transformation. The expression level of each transgene was verified by immunoblot analysis. All the constructs carry a kanamycin-resistant marker. Each construct line was used as the male parent in crosses to the male-sterile coi1-16 and coi1-1. The homozygous coi1 lines with each transgene were selected for MeJA and kanamycin resistance in F2 and F3 populations. The legend of Figure 5 to be changed as follows Fig. 5. Complementation of coi1-16 with various COI1 constructs. (A) Diagrams of COI1 and the different constructs used for complementation studies. All the fusions are under the control of a double CaMV35S promoter and have a NOS terminator. An asterisk indicates a substitution of Trp44 to alanine. Original version Fig. 5. Complementation of coi1-16 with various COI1 constructs. (A) Diagrams of COI1 and the different constructs used for complementation studies. An asterisk indicates a substitution of Trp44 to alanine. The Acknowledgements section to be changed as follows Acknowledgements Special thanks go to Dr Janice Cooke for kindly providing facilities and equipment and to Dr Allen Good for providing a plant facility. We thank Mrs Elaine Patrick for the invaluable technical help, Mr Tom Cox for initial tests of coi1-16 on ACC, Dr Frances Robson for designing the primers for coi1-16, Dr Paul Thomas, Dr Liam Dolan, and Dr Paul Linstead for help with the microscopy, and Dr Paul Larsen, Dr Chi-Kuang Wen, Dr John Mullet, Dr Roberto Solano, and Dr Tatsuya Sakai for providing seeds mentioned in the Materials and methods section. This work was supported by the University of East Anglia (Norwich, UK), the Biotechnology and Biological Sciences Research Council (BBSRC grant 83/P18585 to JGT), and a Natural Sciences and Engineering Research Council of Canada Discovery Grant Award, Dr Michael Adams and Mrs Akemi Adams. Original version Acknowledgements Special thanks go to Dr Janice Cooke for kindly providing facilities and equipment, and to Dr Allen Good for providing a plant facility. We thank Dr Alessandra Devoto and Mr Tom Cox for initial tests of coi1-16 on ACC, Dr Frances Robson for designing the primers for coi1-16, Dr Paul Thomas, Dr Liam Dolan, and Dr Paul Linstead for help with the microscopy, Dr Alessandra Devoto for providing transgenic lines, and Dr Paul Larsen, Dr Chi-Kuang Wen, Dr John Mullet, Dr Roberto Solano, and Dr Tatsuya Sakai for providing seeds mentioned in the Materials and methods section. This work was supported by the University of East Anglia (Norwich, UK), a Natural Sciences and Engineering Research Council of Canada Discovery Grant Award, Dr Michael Adams, and Mrs Akemi Adams. The following additional references to be cited Ferrando A, Farras R, Jasik J, Schell J, Koncz C. 2000. Intron-tagged epitope: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells. The Plant Journal22, 553–560. Conner AJ, Nap J-P, Pereira A, Stiekema WJ. 1995. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Research4, 288–290.

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