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. 2004 Jan 13;101(2):687-92.
doi: 10.1073/pnas.0307687100. Epub 2004 Jan 2.

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

Shunhong Dai  1 , Zhihong Zhang, Shouyi Chen, Roger N Beachy
Affiliations

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

Shunhong Dai et al. Proc Natl Acad Sci U S A. .

Abstract

The phloem-specific promoter of rice tungro bacilliform virus (RTBV) is regulated in part by sequence-specific DNA-binding proteins that bind to Box II, an essential cis element. Previous studies demonstrated that the bZIP protein RF2a is involved in transcriptional regulation of the RTBV promoter. Here we report the identification and functional characterization of a second bZIP protein, RF2b. RF2b, identified by its interaction with RF2a, binds to Box II in in vitro assays as a homodimer and as RF2a/RF2b heterodimers. Like RF2a, RF2b activates the RTBV promoter in transient assays and in transgenic tobacco plants. Both RF2a and RF2b are predominantly expressed in vascular tissues. However, RF2a and RF2b have different DNA-binding affinities to Box II, show distinctive expression patterns in different rice organs, and exhibit different patterns of subcellular localization. Furthermore, transgenic rice plants with reduced levels of RF2b exhibit a disease-like phenotype. We propose that the regulation of phloem-specific expression of the RTBV promoter and potentially the control of RTBV replication are mainly achieved via interactions of the Box II cis element with multiple host factors, including RF2a and RF2b. We also propose that quenching/titration of these and perhaps other transcription factors by RTBV is involved in the development of the symptoms of rice tungro disease.

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Figures

Fig. 1.
Fig. 1.
Predicted amino acid sequence and putative domain structure of RF2b. (A) Deduced amino acid sequence of RF2b. The bZIP domain region is indicated by underlining. These sequence data have been submitted to GenBank under accession no. AY466471. (B) Schematic domain structure of RF2b. Domain structure of RF2a is presented for comparative purposes. (C) Multiple amino acid sequence alignment of the bZIP domains of RF2b and RF2a (19), PKSF1 (26), PosF21 (27), RSG (28), VIP1 (29), VSF1 (30), and representatives from each major subgroup of bZIP proteins, ATB2 (42), ABI5 (43), GBF1 (44), and TGA1 (45), of Arabidopsis thaliana (31). Residues identical to RF2b are highlighted. Solid rectangles indicate positions of conserved leucine residues in the bZIP proteins. Solid circle shows the –10 position in bZIP domain related to the first conserved leucine residue. Amino acids that are important for DNA-binding specificity are indicated by a solid pentagon (36).
Fig. 4.
Fig. 4.
Gene copy number and expression of RF2b. (A) Southern blot analysis of RF2b. Ten micrograms of TP309 genomic DNA was digested with the enzymes indicated, and the blot was hybridized with RF2b probe. (B) Accumulation of mRNA of RF2a and RF2b in different tissues of rice. RNA gel blot analysis was performed by using total RNA from different tissues of 10-day-old seedlings of rice cultivar TP309. Ten micrograms of RNA was loaded per lane and stained with ethidium bromide (rRNA). The blots were hybridized with either RF2b- or RF2a-specific probes. (C) Accumulation of RF2b in different tissues of 10-day-old rice seedlings. Forty micrograms of protein samples from each tissue type was separated by SDS/PAGE. Equal loading was monitored by staining the blot with Ponceau S (Sigma) before the antibody reaction. Antibodies were prepared against RF2b mutant that lacked the bZIP domain. Arrow indicates the position of RF2b.
Fig. 2.
Fig. 2.
DNA-binding and dimer formation of RF2b. EMSAs of RF2b and the bZIP domain of RF2b and their cobinding with RF2a by using 32P-labeled Box IIm1 probe are shown. Proteins used in each reaction are labeled on top of each lane. The unlabeled probe was used as competitor in reactions as indicated. DNA–protein complexes are labeled at both sides of the image to show the formation of homodimers and heterodimers.
Fig. 3.
Fig. 3.
Activity of RF2b in regulation of RTBV promoter. (A Upper) Diagram of plasmids used in the BY-2 cell cotransfection assays. The E promoter in reporter construct of pE::GUS that contains five defined DNA cis elements (15), including Box II, with which RF2a and RF2b interact. In the effector constructs pCs::RF2a and pCs::RF2b, RF2a and RF2b coding sequences were driven by Cs (32, 33). (Lower) GUS activity of transfected BY-2 protoplasts samples. The E::GUS reporter gene was cotransfected into BY-2 protoplasts with Cs::RF2b, Cs::RF2a, Cs::RF2b/Cs::RF2b, and controls, as labeled. The results are the average with SD of three independent experiments, three samples per experiment, after normalization with 35S::GFP that was cointroduced and served as internal control. (B Upper) diagram of T-DNA regions of plasmids used in Agrobacterium-mediated tobacco transformation. (Lower) Histochemical localization of GUS in leaf tissues from transgenic tobacco plants. GUS activity is indicated in transgenic tissue by an indigo dye precipitate after staining with 5-bromo-4-chloro-3-inodyl-β-d-glucuronic acid. (1 and 3) Leaf with pGA-E::GUS construct; (2, 4, and 5) leaf with pGA-E::GUS/Cs::RF2b; (4) highlighting the expression of GUS in mesophyll cells; (5) highlighting the expression of GUS in epidermal cells.
Fig. 5.
Fig. 5.
Localization of RF2a and RF2b transcripts in rice seedlings. Longitudinal sections of the 5-day-old seedlings were hybridized with antisense and sense probes of RF2a and RF2b labeled with digoxigenin-UTP. Hybridization signal is visualized by red color; arrows indicate strong signals. (A) Antisense probe of RF2b; (B) antisense probe of RF2a; (C) sense RF2b probe; (D) sense probe of RF2a. AR, adventitious root; CO, coleoptile; L1, first leaf; L2, second leaf; M, mesocotyl; SC, scutellum; VB, vascular bundle.
Fig. 6.
Fig. 6.
Accumulation of RF2a and RF2b in tobacco BY-2 protoplasts. BY-2 protoplasts were transfected with plasmids encoding RF2b:GFP (a–c) and RF2a:GFP (d–f) fusion proteins. (a and d) Images of GFP fusion proteins visualized with blue light excitation. (c and f) Images visualized in white field. (b and e) Overlay of a and c and d and f, respectively.
Fig. 7.
Fig. 7.
Impact of RF2b on rice development. Photograph was taken 1.5 mo after transgenic rice plants were transplanted to soil. The two transgenic plants on the left carry control plasmid pLau-6-GUS, in which a GUS (uid A) gene is driven by CsVMV promoter. The four transgenic plants on the right carry the Cs:RF2b(–) antisense gene.
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