An interaction between a MYC protein and an EREBP protein is involved in transcriptional regulation of the rice Wx gene.
We previously demonstrated that a 31-bp nucleotide sequence located upstream of the rice Wx gene played an important role in its expression. We further showed that this cis-acting regulator interacts with nuclear proteins extracted from developing rice endosperm. We used the 31-bp sequence as bait in a yeast one-hybrid system to isolate several cDNA clones from a rice cDNA expression library. One of these cDNAs encodes a MYC protein, designated OsBP-5, which is 335 amino acids long and contains a putative basic helix-loop-helix-ZIP DNA-binding domain. This domain exhibits 50% amino acid sequence identity with the R/B proteins that regulate the expression of genes involved in anthocyanin biosynthesis in plants. The results of electrophoretic mobility shift assays (EMSAs) and Southwestern gel blots indicate that this protein binds specifically to the CAACGTG motif within the 31-bp sequence. However, by itself, the OsBP-5 protein is unable to trans-activate a lacZ reporter gene controlled by the 31-bp sequence when tested in a yeast expression system. Interestingly, OsBP-5 can trans-activate this reporter gene when another protein, OsEBP-89, a member of the EREBP family of transcription factors, is present. Furthermore, in vitro pull-down experiments show that a protein isolated from developing rice endosperm interacts with the OsBP-5 protein, and Western blots confirm that the interacting protein is OsEBP-89. The formation of a supershift band in EMSAs also indicates that two proteins interact with each other. Interference of OsBP-5 gene expression by double-stranded RNA reduces the amylose content in mature seed of transgenic rice plants but has no visible effect on their phenotype. These results suggest that the OsBP-5 and OsEBP-89 proteins act synergistically, perhaps as a heterodimer, to regulate the transcription of the rice Wx gene.
Zhu
Ying
Y
National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, People's Republic of China.
Cai
Xiu-Ling
XL
Wang
Zong-Yang
ZY
Hong
Meng-Min
MM
D020816:Amino Acid Motifs; D000688:Amylose / Q000378:metabolism; D015139:Blotting, Southern; D026501:Blotting, Southwestern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D008957:Models, Genetic; D008969:Molecular Sequence Data; D012275:Oryza / Q000235:genetics*; D010641:Phenotype; D010940:Plant Proteins; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D012313:RNA / Q000378:metabolism; D034622:RNA Interference; D012639:Seeds / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D001616:beta-Galactosidase / Q000378:metabolism
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D010940:Plant Proteins; D016271:Proto-Oncogene Proteins c-myc; D015534:Trans-Activators; C093680:ethylene-responsive element binding protein; D012313:RNA; D000688:Amylose; D001616:beta-Galactosidase