Involvement of WRKY, MYB and DOF DNA-binding proteins in interaction with a rice germin-like protein gene promoter

Abstract

Expression of germin-like proteins (GLPs) is reportedly modulated during exposure to pathogens and abiotic stresses. Nevertheless, little is known about the transcription factors and their modulatory role in the mechanism of the regulation of GLP genes. The promoter of Oryza sativa Root Expressed GLP2 (OsRGLP2) gene reportedly showed strong expression in transgenic tobacco during salinity, dehydration and wounding stresses. In the present study, an effort has been made to characterize the cis-regulatory elements of OsRGLP2 promoter and their binding proteins. The putative stress-responsive regulatory elements in the promoter and corresponding binding proteins (OsWRKY71, OsDOF18 and OsMYB1) were identified by in silico analysis. The DNA-binding domains of selected proteins were cloned, overexpressed and purified. Electrophoretic mobility shift assays (EMSAs) demonstrated that these recombinant domains were able to bind with DIG-labeled OsRGLP2 promoter fragments containing W-box, AAAG and WAACCA motifs. Binding was confirmed by competitor EMSA and EMSA with mutant oligonucleotides. These regulatory elements were also active in binding with nuclear factors from rice nuclear protein extract in vitro as confirmed by competitive EMSA. It can be concluded that OsWRKY71, OsMYB1 and OsDOF18 proteins are involved in transactivation of OsRGLP2 gene expression under different abiotic stress conditions.