Grams of leaf sample was ground in liquid nitrogen and resuspended in 2 mL of immunoprecipitation (IP) buffer (50 mM HEPES, 50 mM NaCl, 10 mM EDTA, and 0.two Triton X-100, pH 7.5). The supernatant was incubated with 30 mL of anti-FLAG M2 affinity agarose (Sigma) for three h. Immunocomplexes have been washed three times with IP buffer and eluted in three three 50 mL of 3xFLAG peptide at a concentration of 500 mg mL21 (Sigma) in IPNuclear IsolationTomato `Moneymaker’ plants were vacuum infiltrated with 1 3 108 cfu mL21 Pto IV carrying empty pBRR1-MCS5 vector, pBBR1 expressing HopQ13xFLAG, or pBBR1 expressing HopQ1(S51A)-3xFLAG. Twenty grams of tomato leaves was collected for each and every sample 12 h post inoculation. Nuclei have been straight away isolated as described previously (Craig and Beavis, 2004). Plant Physiol. Vol. 161,The HopQ1 Effector Interacts with Tomato 14-3-3 ProteinsWestern BlottingSDS-PAGE and subsequent immunoblotting were performed as described previously (Liu et al., 2011). FLAG immunoblots were performed with monoclonal anti-FLAG conjugated to horseradish peroxidase (HRP; Sigma) at a concentration of 1:1,000. HA immunoblots have been performed with anti-HA-HRP (Roche) at a concentration of 1:1,000. GFP immunoblots were performed with anti-GFP-HRP (Miltenyi Biotec) at a concentration of 1:2,000. Anti-histone H3 immunoblots have been performed with anti-histone H3 antibody conjugated to HRP (Abcam) at a concentration of 1:1,000. Immunoblots detecting the chloroplast PSII subunit PsbO were performed with rabbit polyclonal anti-PsbO (Abcam) at a concentration of 1:1,000. Goat anti-rabbit IgG-HRP conjugate (Bio-Rad) was utilized at a concentration of 1:3,000 for the detection of PsbO by way of enhanced chemiluminescence. Luciferase western blotting was performed employing anti-luciferase (Sigma) at a concentration of 1:5,000. Sequence data from this article can be found inside the GenBank data libraries under accession numbers. 1182506 (HopQ1), 543565 (TFT1), X95903.1 (TFT5), 100301918 (GRAS2), 822098 (RIN4), 826728 (SGT1b), and AF192262.1 (RAR1).Supplemental DataThe following components are out there within the on line version of this article. Supplemental Figure S1. ClustalW alignment of HopQ1 and homologs from phytopathogenic bacteria. Supplemental Figure S2. Spectra of phosphorylated HopQ1 peptides. Supplemental Figure S3. Western blot of GFP- and HA-tagged proteins used in confocal microscopy. Supplemental Figure S4. HopQ1(S551D)-GFP and HopQ1-GFP both exhibit nuclear-cytoplasmic localization.4-Azidobutylamine MedChemExpress Supplemental Figure S5.2-Hydroxybutyric acid Description HopQ1 can reproducibly complement the Pto DC3000 DIV on tomato `Rio Grande 76R’. Supplemental Figure S6. No virulence impact for HopQ1 or HopQ1(S51A) is detected immediately after delivery from Pto DC3000 DIV on the susceptible cultivars Moneymaker or Rio Grande 76S.PMID:23613863 Supplemental Table S1. Total list of proteins identified by mass spectrometry with variety of unique spectra. Supplemental Table S2. Full list of proteins identified by mass spectrometry with number of total spectra. Supplemental Table S3. Primers utilised for cloning.ACKNOWLEDGMENTSAll mass spectrometry was performed at the University of California, Davis, Genome Center Proteomics Core Facility. We thank Brett Phinney for use with the mass spectrometry gear. We thank DongHyuk Lee for cloning HopQ1 into pBBR1-MCS5. We thank Kentaro Inoue for the sort gift of PsbO antiserum. Received November 29, 2012; accepted February 15, 2013; published February 15, 2013.LITERATURE CITEDAoyama T, Chua NH (1997) A glucocorticoid.
