Angot et al. 10.1073/pnas.0509393103. |
Supporting Materials and Methods
GALA and SKP1-Like cDNA Cloning.
For ASKs accession nos., see ref. 1. ASK1, ASK20, and ASK21 were PCR amplified from an Arabidopsis Col-0 cDNA preparation. ASK2, 3, and 4 were PCR amplified from EST clones U23024, U13779, and U14059 obtained from the Nottingham Arabidopsis Stock Center (Nottingham, U.K.). ASK5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, and 19, having no intron, were all PCR amplified from Arabidopsis Col-0 genomic DNA (oligonucleotide table). The cloned ASK20 and ASK21 sequences correspond to the two longer splice variants described (1) The Medicago truncatula SKP1-like cDNAs MSKa and MSKb were PCR amplified from root cDNA preparations and cloned like ASK cDNAs.For the complementation test, we PCR amplified the GALA7 gene with oligonucleotides oNP530 and oNP532 to amplify from the whole-promoter region (410 bp) to the last codon and clone the PCR fragment in KpnI/BamHI in pSC154 (2). The downstream oligonucleotide oNP532 contains one degenerated site (M), the final construct encoding either a glycine (GGA) or a stop codon (TGA) as the last codon of GALA7 ORF before the Bordetella pertusis adenylate cyclase CyaA' ORF (3) in pSC154. Oligonucleotides oNP533 and oNP534 enabled us to construct the deleted GALA7 by recombinant PCR. pNP219, 220, 221, and 222 are pSC154-derived plasmids cloned into the KpnI site of pLAFR6 (4). pNP220 contains the GALA7 ORF in fusion with the CyaA', and pNP219 contains the GALA7DF-box ORF in fusion with CyaA'. pNP221 and pNP222 are, respectively, the equivalent of pNP220 and pNP219 with a stop codon as the last codon of GALA7 or GALA7DF-box.
GST Pull-Down.
The GALA6 and GALA6DF-box coding sequences were Gateway-recombined into the pDEST15 (Invitrogen, Carlsbad, CA). Recombinant GST fusion proteins were extracted from BL21 (DE3) pLysE Escherichia coli (Stratagene, La Jolla, CA) under nondenaturing conditions by sonication in extraction buffer (150 mM NaCl, 0.1 M Tris·Hcl, pH 7.5, 10 mM b-glycerolphosphate, 1 mM DTT, 0.5%NP40, 1 mM NAF, 1 mM PMSF, and Complete Antiprotease). The GST fusion proteins were then purified on bulk Glutathione-Sepharose 4B (Amersham Pharmacia, Piscataway, NJ) by a 4-h incubation at 4°C under agitation, followed by three washes with the same buffer. Two-week-old Arabidopsis Col-0 seedlings expressing 6myc-tagged ASK2 (5) were ground in extraction buffer (100 mM NaCl, 50 mM Tris·HCl, pH 7.5, 10% glycerol, 0.1% Tween 20, 10 mM b-glycerolphosphate, 1 mM NAF, 1 mM PMSF, and Complete Antiprotease added with PolyClar AT and quartz). After a 15-min 20,000 × g centrifugation, the protein content of the supernatant was determined by Bradford assay. The GST pull-down assays were realized by incubating for 4 h at 4°C the Glutathione-Sepharose-bound GST-GALA6 and GST-GALA6DF-box with 5 mg of total plant protein extract under agitation. After three washes with extraction buffer, bound proteins were eluted by the addition of SDS-loading buffer and incubation for 10 min at 90°C. To detect the ASK proteins, we used a 1:5,000 dilution of a purified rabbit polyclonal antibody raised against a peptide of the Arabidopsis ASK1 protein (6). Immunolabeled proteins were then detected by using peroxidase-conjugated goat anti-rabbit IgGs and ECL (Amersham Biosciences, Piscataway, NJ).
Multiple GALA-Mutant Strain.
For the disruption of GALA2, 4, 5, 6, and 7 by the cre-lox approach, upstream and downstream fragments were PCR-amplified and cloned in the pCM184 disruption vector digested with appropriate restriction sites. Disruption vectors for GALA2 (plasmid pSG402), GALA4-GALA5 (pGG19), and GALA6-GALA7 (pSG400) genes were constructed. The gene-deletion procedure followed the method described, except that the cre gene from pCM157 (7) was cloned into pLAFR6 (4). The resulting plasmid (pGG15) was then introduced in the recipient strains by electroporation. At every disruption step, the genomic structure of the transformants was verified by both PCR analysis and Southern hybridization.1. Risseeuw EP, Daskalchuk, TE, Banks TW, Liu E, Cotelesage J, Hellamnn H, Estelle M, Somers DE, Crosby, WL (2003) Plant J 34:753-767.
2. Cunnac S, Occhialini A, Barberis P, Boucher C, Genin S (2004) Mol Microbiol 53:115-128.
3. Sory MP, Cornelis GR (1994) Mol Microbiol 14:583-594.
4. Huynh TV, Dahlbeck D, Staskawicz BJ (1989) Science 245:1374-1377.
5. Xu L, Liu F, Lechner E, Genschik P, Crosby WL, Ma H, Peng W, Huang D, Xie D (2002) Plant Cell 14:1919-1935.
6. Potuschak T, Lechner E, Parmentier Y, Yanagisawa S, Grava S, Koncz C, Genschik P (2003) Cell 115:679-689.
7. Marx CJ, Lidstrom ME (2002) BioTechniques 33:1062-1067.