Functional Analysis of RF2a, a Rice Transcription Factor

Abstract

RF2a is a bZIP transcription factor that regulates expression of the promoter of rice tungro bacilliform badnavirus. RF2a is predicted to include three domains that contribute to its function. The results of transient assays with mutants of RF2a from which one or more domains were removed demonstrated that the acidic domain was essential for the activation of gene expression, although the proline-rich and glutamine-rich domains each played a role in this function. Studies using fusion proteins of different functional domains of RF2a with the 2C7 synthetic zinc finger DNA-binding domain showed that the acidic region is a relatively strong activation domain, the function of which is dependent on the context in which the domain is placed. Data from transgenic plants further supported the conclusion that the acidic domain was important for maintaining the biological function of RF2a. The severe stunting symptoms of rice tungro disease are caused by infection of rice tungro bacilliform virus (RTBV), 1 a double-stranded DNA badnavirus. Understanding the transcriptional regulation of RTBV is an important factor to elucidate the basis of the disease. RTBV carries a single, vascular tissue-specific promoter with several defined DNA cis-elements (1-4). Box II, one of the DNA cis-elements in the promoter, is essential for phloem-specific expression of the promoter (3, 4).A bZIP type rice host transcription factor, RF2a, was identified by its interaction with Box II (5). Furthermore, overexpression of RF2a in transgenic plants is sufficient to activate expression of RTBV promoter in other than vascular tissues (6).Temporal and spatial regulation of gene expression relies largely on the function of gene-specific transcription factors and is achieved by the activity of multiple proteins that bind to regulatory elements and with other proteins to alter basal rates of transcription initiation and/or elongation (7-9). A typical gene-specific eukaryotic transcription factor includes a DNAbinding domain and one or more domains that influence the activation or repression of transcription through interactions with general transcription factors, co-factors, chromatin remodeling complexes, and components of RNA polymerase II holoenzyme, among others (7, 10 -13). Transacting domains are often characterized as having a high content of specific amino acids, including domains rich in the acidic amino acids, proline or glutamine (14 -16). Acidic domains have been reported to possess activation functions that include interactions with TATA-binding proteins (TBP) (13, 17), TBP-associated factors (TAFs) (18), TFIIA (19), TFIIB (20, 21), other general transcription complexes (13,22), and co-factors (12). Proline-rich and glutamine-rich domains typically act through interactions with TBP, TAFs, and other co-factors (14). Although proline-rich and glutamine-rich domains act as activation domains in most of the cases, they can also function as repression domains (23,24). RF2a contains three putative transacting domains, namely proline-rich and ac...

RF2b, a rice bZIP transcription activator, interacts with RF2a and is involved in symptom development of rice tungro disease

“…Proteins produced in E. coli strain BL21(DE3)pLysS were purified as described (20). EMSAs were carried out as described (20) by using 32 P-labeled Box IIm1 as probe (15).…”

“…The antibodies were further purified by using protein A Sepharose (Amersham Pharmacia Biosciences). Western blot analysis of different tissue protein samples prepared from 10-day rice seedlings was carried out as described (20) with anti-RF2b-⌬bZIP antibodies as primary antibodies and horseradish peroxidase-conjugated goat anti-rabbit antibodies as secondary antibody (Southern Biotechnology Associates).…”

“…BY2 protoplast transfection, Agrobacterium-medicated tobacco transformation, and particle bombardment-mediated transformation of rice were carried out as described (18,20,22).…”

“…We previously reported the isolation of rice bZIP transcription factor RF2a through its interaction with the Box II element (19). RF2a activates the promoter of RTBV in in vitro transcription reactions and in vivo assays (18)(19)(20). Expression of antisense gene constructs and dominant negative mutants of RF2a in transgenic rice plants caused abnormal developmental phenotypes, indicating that RF2a and potentially other factors are important for development of rice plants (18)(19)(20).…”

“…RF2a activates the promoter of RTBV in in vitro transcription reactions and in vivo assays (18)(19)(20). Expression of antisense gene constructs and dominant negative mutants of RF2a in transgenic rice plants caused abnormal developmental phenotypes, indicating that RF2a and potentially other factors are important for development of rice plants (18)(19)(20). Furthermore, data from electrophoretic mobility-shift assays (EMSAs) of purified RF2a and rice nuclear extracts suggested that proteins other than RF2a also bind to Box II (19).…”