Promoter elements required for phloem-specific gene expression from the RTBV promoter in rice

Previous studies indicated that a DNA fragment comprising nucleotides (nt) ?164 to +45 of the RTBV promoter is sufficient to drive phloem-specific expression of a reporter gene in transgenic rice plants. In addition, two potential cis elements, Box I (nt ?3 to +5) and Box II (nt ?53 to ?39) were identified by DNA-protein interaction assays. In this study, the results of further in vivo studies involving mutagenesis of selected DNA sequences and analysis of expression of a reporter gene in transgenic rice plants revealed that, in addition to Box I and Box II, other elements are required for phloem-specific gene expression, among which are a direct repeat (ASL Box, nt ?98 to ?79) and a GATA motif (nt ?143 to ?135). All the these elements bind rice nuclear factors specifically, and mutations of the elements were identified that resulted in loss-of-competition in electrophoretic mobility shift assays. A DNA fragment comprising nt ?164 to ?32, which contains Box II, the ASL Box and the GATA motif, conferred phloem-specific reporter gene expression independent of Box I when fused to a heterologous CaMV 35S minimal promoter and introduced to transgenic rice plants. Studies that introduced point mutations suggested that in the context of the ?103 to +45 promoter fragment, Box II and the ASL Box act synergistically to confer tissue-specific gene expression. Similar studies in the ?164 to +45 promoter fragment indicated that the ?164 to ?103 region, which includes the GATA motif, contains sequences that are functionally redundant with those in the ?103 to ?32 region, including the ASL Box and Box II. It is concluded that these regions act additively to direct phloem-specific gene expression.