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. 2001 Jun 19;98(13):7635-40.
doi: 10.1073/pnas.121186398. Epub 2001 Jun 5.

Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants

S Petruccelli  1 , S Dai, R Carcamo, Y Yin, S Chen, R N Beachy
Affiliations

Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants

S Petruccelli et al. Proc Natl Acad Sci U S A. .

Abstract

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides -164 to +45, result in phloem-specific expression of beta-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2-20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.

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Figures

Figure 1
Figure 1
(A) Schematic representation of the RTBV full-length promoter and of the E fragment of the promoter. The cis elements GATA, ASL Box, Box II, and Box I of E, as described by Yin et al. (10), are also indicated. Box II, the cis element recognized by RF2a, is indicated in black. Schematic representations are shown of the RF2a transcription factor and of the 3Δ mutant. The potential activation domains are indicated as follows: P, proline-rich region; A, acidic region; Q, glutamine-rich region. The dimerization and DNA-binding domain is indicated as bZIP. (B) Diagram of the constructs used for Agrobacterium-mediated transformation of tobacco (N. tabacum). The uidA gene (GUS-encoding gene) is driven either by the full-length RTBV promoter or E fragment (−164 to +45). The genes encoding the rice transcription factor RF2a and 3Δ mutant are driven by the 35S promoter of cauliflower mosaic virus.
Figure 2
Figure 2
Histochemical localization of GUS in tissues from transgenic tobacco plants. The results shown are representative of those observed in the 15 independent transgenic lines developed for each gene construct. Results with P-E∷GUS and P-FL∷GUS constructs were identical, and the figure is compiled from both sets of transgenic plants. (A, C, E, G, I, and K) Plants containing either P-E∷GUS or P-FL∷GUS gene. (B, D, F, H, J, and L) Plants containing either P-E∷GUS/P-35S∷RF2a or P-FL∷GUS/P-35S∷RF2a. GUS activity is indicated in transgenic tissue by an indigo dye precipitate after staining with X-Gluc. (A and B) Seedlings. (C and D) Juvenile leaves. (E and F) Roots. (G and H) Leaf sections showing vascular tissues of the midrib and leaf lamina. (I and J) Vascular tissue of the leaf midrib. (K and L) Cross section of lamina. c, cotyledon; cr, cortex; e, epidermis; ep, external phloem; g, guard cell; ip, internal phloem; l, leaf; m, mesophyll; p, phloem; pm, palisade mesophyll; py, parenchyma; rt, root tip; rv, root vein; sm, spongy mesophyll; t, trichome; vb, vascular bundle; x, xylem.
Figure 3
Figure 3
GUS activity in extracts of leaves from transgenic tobacco plants carrying the P-E∷GUS gene, P-E∷GUS/P-35S∷RF2a, P-FL∷GUS, or P-FL∷GUS/P35S∷RF2a. The amount of GUS (pmol MUG min−1 mg−1 protein) is indicated. The mean level of GUS activity for each construct is indicated by the thick, horizontal bar.
Figure 4
Figure 4
Correlation between GUS activity and the amount of RF2a determined by ELISA for transgenic T0 plant lines that carry P-E∷GUS/P-35S∷RF2a (●) or P-FL∷GUS/P-35S∷RF2a (⧫).
Figure 5
Figure 5
EMSA of RF2a and the 3Δ mutant of RF2a. Oligonucleotides containing the Box IIml sequences (10) were used as 32P-labeled probe or an unlabeled competitor in an EMSA with 500 ng of E. coli protein containing 3Δ or RF2a. Unbound probe is located near the bottom of the gel. The x band in lanes 1, 2, 4, and 5 is presumed to result from binding with an uncharacterized protein from E. coli. Lane 1, protein prepared from E. coli. Lane 2, reaction with extracts of E. coli that produce RF2a. Lane 3, reaction with equimolar amounts of RF2a and 3Δ plus 80× molar excess of unlabeled competitor probe relative to the labeled probe. Lane 4, reaction with equimolar amounts of RF2a and 3Δ. Lane 5, reaction with 3Δ.
Figure 6
Figure 6
Phenotypes of plants that contain the gene encoding the 3Δ mutant of RF2a. (A) The plants on the left contain the transgene and grew more slowly than the plant that lacked the transgene due to segregation in the T1 generation (Right). (B) intermediate phenotype, showing shoot elongation, curvature of leaves, and a decrease in apical dominance. (C) Size of roots in plants with the 3Δ gene (Right) compared with nontransgenic T1 progeny (Left). (D and E) Close-up of leaves showing downward curvature. (F) Abnormal plant showing the phenotype of severe stunting with thick leaf lamina.
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