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Specific expression of DR5 promoter in rice roots using a tCUP derived promoter-reporter system

PLoS ONE (2014) 9(1)
DOI: 10.1371/journal.pone.0087008
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Abstract

Variation of transgene expression caused by either position effect at the insertion site or the promoter/enhancer elements employed for the expression of selectable marker genes has complicated phenotype characterization and caused misinterpretation. We have developed a reporter system in rice to analyze the influence of vector configuration, spacer and selectable marker gene promoter on the expression of the promoterless GUS reporter and DR5 promoter. Our results indicate that a spacer inserted between the reversed 35S promoter and the GUS reporter could reduce leaky expression of the reporter but was unable to block the nonspecific expression of DR5::GUS. Stacking the selectable marker unit in head to tail with the GUS reporter aided the gene specific expression of the GUS reporter under the DR5 promoter even when the 35S promoter is used for expression of the selectable marker. Compared to 35S under this configuration, a quick and distinctive expression of DR5::GUS was observed in the root cap, quiescent center and xylem cells in the root apical meristem by using the tCUP derived promoter (tCUP1) for selection, that is similar to the pattern obtained by a sensitive DR5 variant (DR5rev) in Arabidopsis. These data suggest a conserved property of the tCUP promoter in preventing enhancer-promoter interactions in rice as it does in Arabidopsis, and also demonstrate that an analogous distal auxin maximum exists in roots of rice. Therefore, the tCUP promoter based selection system provides a new strategy for specific expression of transgenes in rice. © 2014 Zhou et al.

Figures

  • Figure 1. T-DNA constructs created to combine promoterless GUS reporter vectors with different configurations and promoters to express selectable markers. (A) Group of vectors constructed in head to tail orientation with selectable marker upstream of the GUS reporter. The selectable marker cassette is comprised of hygromycin phosphotransferase gene (hptII, 1026 bp, in gray box) driven by CaMV 35S (848 bp, a1), Nos (307 bp, a2) or tCUP1 (519 bp, a3) promoters and terminated by the same CaMV 35S terminator (225 bp). The b-glucuronidase reporter gene (GUS, 2053 bp, in blue box) with nopaline synthase (nos) terminator (268 bp) at the 39 end near the T-DNA right border (RB). (B) Group of vectors
  • Figure 2. GUS leaky expression in calli transformed by promoterless GUS reporter vectors containing different selectable marker promoters in different stacking configurations. GUS leaky expression in representative callus transformed by promoterless GUS reporter vectors in (A–D) configurations combined with 35S (a1,b1,c1,d1), Nos (a2,b2,c2,d2) or tCUP1 (a3,b3,c3,d3) selectable marker promoters as indicated in Figure 1. Fifty Transformed calli per vector were stained after 3 weeks selection and calli with the average numbers of staining spots were pictured individually at the same magnification. Pictures show representative calli for at least three replicates. Bars: 1 mm. doi:10.1371/journal.pone.0087008.g002
  • Figure 4. Construction of DR5::GUS reporter vectors on the basis of A and C configurations. (A) The synthetic auxin responsive promoter DR5 (267 bp, yellow arrow) was inserted in group A vectors before the GUS reporter to make a1-DR5, a2-DR5 and a3-DR5. (B) A similar strategy was used to construct the DR5 promoter in group C vectors and named c1-DR5, c2-DR5 and c3-DR5 respectively. doi:10.1371/journal.pone.0087008.g004
  • Figure 5. Specific and non-specific expression of DR5::GUS in adventitious roots of T0 transgenic plants. (A, B) Non-specific expression of DR5::GUS in root tip and stele in both weak (A) and strong (B) staining lines. (C) Staining pattern with DR5::GUS expressed in root cap and zone of elongation. (D, E) Specific localization of DR5::GUS in columella cells only (D) or in columella cells and quiescent center (E). (F, G) Specific expression of DR5::GUS in root cap, quiescent center and protoxylem cells in the root meristem. Roots positioned in a protophloem plane, xylem cells expressing GUS are arranged in a uni-cellular plate (F) or roots positioned in a protoxylem plane, two arrays of protoxylem cells expressing GUS (G) are shown. Bars: A–C, 200 mm, D–G, 100 mm. Adventitious roots of 1-month-old T0 plants were collected and stained in 1 mM X-gluc for 1 h (A–E) or 0.5 mM Xgluc for 4 h (F, G). Positions of columella cell (green arrow), quiescent center (red arrow) and xylem cell (yellow arrow) are indicated. Detailed organization of rice root tissue and cell types can be found in references [56,57]. doi:10.1371/journal.pone.0087008.g005
  • Figure 6. Summary of DR5::GUS staining patterns in primary roots of T1 transgenic plants. (A) Varied expression patterns of DR5::GUS (a-i) in primary root tips of 8-day-old T1 transgenic seedlings grown in IRRI rice solution. The most intense GUS expression was mainly observed in columella (a), in columella and zone of elongation (b), in root cap (c), in root cap and basal meristematic zone (d), in root cap and protoxylem (e), in root cap, protoxylem and meristematic zone (f), in basal meristematic and elongation zones, absent in root cap and promeristem (g), evenly stained in root cap and meristematic zone (h), in root cap, stele and meristematic zone (i). Samples were stained for 30 min for (c)-(i) or 16 h for (a) and (b). Bar: 100 mm. (B) Images (a-i) shown on the right are magnifications of the corresponding images shown on the left (A) to provide detailed staining patterns in the apical root. Positions of columella cell (green arrow), quiescent center (red arrow) and xylem cell (yellow arrow) are indicated. Bar: 100 mm. (C) Three major staining patterns in lateral roots (a-c) from all the analyzed T1 transgenic seedlings transformed by six DR5::GUS vectors. The most intense GUS expression was clearly observed in lateral root cap (a), in lateral root cap and xylem of both primary and lateral roots (b), in the whole vascular bundle and less specifically in the lateral root cap (c). Bars: 100 mm. (D) Patterns within T1 transgenic seedlings of each vector were classified and presented as a heat map of percentage. Number of GUS positive T1 seedlings observed for each vector is indicated in brackets. doi:10.1371/journal.pone.0087008.g006
  • Figure 7. Auxin responsiveness in DR5::GUS transgenic lines selected by vectors with hptII controlled by tCUP1 promoter. Both a3DR5 (A–D) and c3-DR5 (E–H) transgenic lines using the tCUP1 promoter for hptII gene expression were selected for the auxin responsive test. Representative images are shown for two independent lines each showing the DR5 promoter strongly induced in primary root tips (B, F) and lateral roots (D, H) after 1 mM NAA treatment for 24 h. In the mock treatment, specific but different DR5::GUS expression was observed between a3-DR5 and c3-DR5 lines in primary root tips (A, E) as well as in lateral roots (C, G). Bars: 100 mm. doi:10.1371/journal.pone.0087008.g007

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Zhou, J., Yu, F., Wang, X., Yang, Y., Yu, C., Liu, H., … Chen, J. (2014). Specific expression of DR5 promoter in rice roots using a tCUP derived promoter-reporter system. PLoS ONE, 9(1). https://doi.org/10.1371/journal.pone.0087008

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