Cellular Localization of Rice SUMO/SUMO Conjugates and in vitro Sumoylation Using Rice Components

doi:10.1016/j.rsci.2019.02.003

Abstract

Joo Joungsu, Hee Choi Dong, Hyon Kim Sang, Ik Song Sang. Cellular Localization of Rice SUMO/SUMO Conjugates and in vitro Sumoylation Using Rice Components[J]. Rice Science, 2020, 27(1): 1-4.

Fig. 1. Four SUMO members (OsSUMO1, OsSUMO2, OsSUMO3 and OsSUMO4) in rice. ===A, Amino acid sequence alignment among SUMO proteins of Oryza sativa, Arabidopsis thaliana, Saccharomyces cerevisiae and Homo sapiens by ClustalW. The consensus SUMO attachment sites are indicated by underlines. Arrow indicates the cleavage site. Symbols with backgroud indicate highly conserved amino acid. B, Schematic representation of rice SUMO genes. C, Phylogenic tree representation of amino acid sequence distance among SUMO genes. D, RNA gel blot analyses of OsSUMO genes. E, Expression levels of OsSUMO genes under ABA and stress conditions. ===Ca, Callus; L, Leaf; R, Root; BH, Panicles before heading; AH, Panicles after heading; S, Mature seed; NT, Non-treated; ABA, Abscisic acid.

Fig. 2. Subcellular localization analysis of GFP:OsSUMO fusion proteins. ===A, Over-expression plasmid consisted of the constitutive OsCc1 promoter. B, RNA gel blot analysis of GFP:OsSUMOs or GFP over-expression transgenic rice. C, Subcellular localization of GFP:OsSUMO fusion proteins in rice roots. D, Subcellular localization of GFP:OsSUMO3 fusion protein in leaves. ===GFP, Green fluorescent protein; WT, Wild type; DAPI, 4',6-diamidino-2-phenylindole.

Fig. 3. in vitro sumoylation assays. ===A, Schematic representation of plasmids for recombinant proteins. B, in vitro sumoylation assays:recombinant proteins were over- expressed in E. coli and purified using GST or nickel affinity column.===E1, OsSAE1- OsSAE2; E2, OsSCE2a; E3, OsSIZ1.===‘+’ and ‘-’ indicate with SAE1 and without SAE2, respectively.

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