Additional file 1: of Phosphorylation of Arabidopsis SINA2 by CDKG1 affects its ubiquitin ligase activity

Figure S1. Amino acid sequence alignment between Arabidopsis SINAT1 (NP_181729.1), SINAT2 (NP_191363.1), SINAT3 (NP_567118.1), SINAT4 (NP_194517.1), SINAT5 (AAM11573) and SINA2. * indicates conserved amino acid moieties of the RING domain, ↓ indicates conserved amino acids of the putative B-box2 domain. Identical amino acid regions are shaded black and conserved sequences are shown in gray. Figure S2. (a) Reciprocal yeast two hybrid assay and (b) Yeast two hybrid deletion analysis of SINA2. Coding sequences were recombined into pDEST-AD and pDEST-BD, transformed into AH109 yeast cells and plated onto SD Trp−Leu− medium and replica plated on SD Trp−Leu/−His−Ade− medium. Figure S3. (a) Comparison of the PK domains from Arabidopsis thaliana (CDKG1, NP_201142 and CDKG2, NP_176925), A. lyrata (AlCDKG1, XP_002866550), Camelina sativa (CsCDKG1, XP_010484064), Brassica rapa (BrCDKG1, XP_009130249), B. oleracea var. oleracea (BoCDKG1, XP_013628692) and Taraenaya hassleriana (ThCDKG1, XP_010545957). Identical sequences are shown in black and areas conserved sequences are shaded gray. (b) Phylogenetic tree of PK domains of CDKG protein sequences was generated using the neighbor-joining method with MEGA. Bootstrap values (> 50%) from 500 replicates are shown. Figure S4. Sub-cellular localization of GFP-CDKG1 and GFP CFKG1. Agrobacterium strains carrying 35S:GFP, 35S:GFP-SINA2, and 35S:GFP-CDKG1 were infiltrated into Nicotiana benthamiana leaves, and the GFP signals from leaves that transiently express GFP-CDKG1 or GFP alone, as indicated, were analyzed by confocal microscopy. Figure S5. Germination rate of SALK_075762 cdkg1 seeds in response to treatment with 200 mM mannitol, 200 mM NaCl, or 100 μM ABA was scored 4 days after stratification. Data represent means of 60 seeds ± SD. Asterisks indicate significant differences from WT (P < 0.01). Figure S6. Failure of SINA2 to ubiquitinate CDKG1. Ubiquitination of CDKG1 by SINA2 was assayed in the presence of human E1, human E2 and ubiquitin and samples were resolved by SDS-PAGE and detected using anti-MBP. Table S1. Primers used in this study. (PDF 1326 kb