A versatile and reliable two-component system for tissue-specific gene induction in Arabidopsis

Abstract

Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

Figure 1.

A schematic illustration of the Gateway-compatible constructs. A, Activator vector. B, Responder vectors. C, Activator/responder vector. The activator vector pMDC150 (A) contains a Gateway cloning cassette, flanked by unique AscI and PacI restriction recognition sites upstream of a CaMV 35S minimal promoter (min 35S) and chimeric transcription factor XVE. The inclusion of a minimal promoter allows the insertion of both enhancer sequences and promoters in the constructs (the inclusion of a minimal promoter does not interfere with the specificity of the promoters used in this study). The vector pMDC150 also contains a Nos promoter to drive the expression of a kanamycin-resistance gene for plant selection. All responder vectors (B) contain an XVE-responsive promoter (OlexA-TATA) upstream of a Gateway cloning cassette, also flanked by unique AscI and PacI restriction recognition sites for the easy diagnosis of DNA insertions. Vectors pMDC160, pMDC220, and pMDC221 contain a Nos promoter to drive the expression of the BAR- or hygromycin-resistance genes, respectively, for plant selection. The vectors pMDC160, pMDC220, and pMDC221 also contain the pBluescript vector sequence (CLONTECH), which can be used for plasmid rescue procedures because it encodes an ampicillin-resistance gene for bacterial selection and the ColE1 origin of replication. The pMDC220 vector also contains a second XVE-responsive promoter adjacent to the right border (RB) sequence, so that this vector can be used for conditional activation tagging experiments. The activator/responder vector pLB12 (C) contains both an activator unit and a responder unit, separated by a kanamycin-resistance gene driven by a Nos promoter for plant selection. The sequences and detailed maps of these vectors can be downloaded from http://www.unizh.ch/botinst/Devo_Website/curtisvector. LB, Left border; TE9, TE9 terminator; T3A, terminator; attR1 and attR2, att recombination sites; CMr, bacterial chloramphenicol resistance; ccdB, bacterial toxin gene for negative selection.

Figure 2.

Arabidopsis leaves showing sectors of induced GUS expression 24 h after induction with 2 μm 17-β-estradiol (0.01% Silwet 77) using an artist's paint brush.

Figure 3.

Cell-specific expression patterns of a GUS reporter gene after induction. Uninduced and induced GUS expression (uninduced shown on the left of each image, respectively) in Arabidopsis plant lines 24 h after induction (2 μm 17-β-estradiol in 0.01% [v/v] ethanol) or mock induction (0.01% [v/v] ethanol). A, Leaves of a plant line containing pMDC150-35S and pMDC160-GUS (induced ubiquitous expression can be seen across the leaf). B and C, GUS expression in plant lines containing pMDC150-GL2 and pMDC160-GUS. B, 3-d-old seedlings (induced expression is restricted to the atrichoblast cells). C, Mature leaves of 15-d-old plants (induced expression restricted to the trichomes). D, Whole plants at 7-to-10-d old (left and middle) and a flower (right) from plant lines containing pMDC150-RolC and pMDC160-GUS (induced expression is restricted to the vascular tissue). E, Mature leaves (left and middle) and a flower (right), including petals, from plant lines containing pMDC150-SUC2 and pMDC160-GUS (induced expression is observed in the vascular tissue [companion cells]). F and G, Roots in mature plant lines containing pMDC150-TobRB7 and pMDC160-GUS (induced expression is restricted to the tissue above the root apical meristem [RAM]). H, Plant line containing pLB12-EASE (induced expression is observed in the egg apparatus). Bars = 0.5 mm (G) and 20 μm (H).

Figure 4.

Stringent regulation of gene expression using the system is demonstrated by induction of DT-A in seedlings. Arabidopsis plant lines containing both the pMDC150-35S activator T-DNA and a pMDC221-DT-A responder T-DNA after 13 d of growth under uninduced conditions (A; mock inoculated Murashige and Skoog media) and under induced conditions (B; 2 μm 17-β-estradiol in Murashige and Skoog medium).

Figure 5.

Inducible gain-of-function phenotypes: Overexpression of KNAT1 leads to lobed leaf formation. A, 27 d of growth under uninduced conditions. B, 27 d of growth under induced conditions. A and B, Siblings from a plant line containing both the pMDC150-35S activator T-DNA and a pMDC221-KNAT1 responder T-DNA.

Figure 6.

Inducible expression of BBM leads to the formation of somatic embryos on cotyledons and leaves. A, 13 d of growth under uninduced conditions (mock inoculated Murashige and Skoog medium). B, 13 d of growth under induced conditions (5 μm 17-β-estradiol in Murashige and Skoog medium). A and B, Sibling plant lines containing both the pMDC150-35S activator T-DNA and a pMDC221-BBM responder T-DNA. C and D, Scanning electron micrographs of induced somatic embryos (C) in plant shown in B. D, Scanning electron micrographs of cotyledons and leaves with induced somatic embryos and a leaf-like outgrowth with a trichome on a cotyledon (arrow). E, 13-d-old plants that constitutively express BBM under the control of the CaMV 35S promoter (seeds kindly provided by Kim Boutilier).

Figure 7.

Induced ectopic LEC2 expression results in the formation of somatic embryos on cotelydons. A, 29 d of growth under uninduced conditions (mock inoculated Murashige and Skoog medium). B, 29 d of growth under induced conditions (5 μm 17-β-estradiol in Murashige and Skoog medium). A and B, Sibling plant lines containing both the pMDC150-35S activator T-DNA and a pMDC221-LEC2 responder T-DNA.

Figure 8.

The ectopic expression of FUSCA3 produces a seed dormancy phenotype. Plant lines containing both the pMDC150-35S activator T-DNA and a pMDC221-FUS3 responder T-DNA. A, Uninduced. B, Induced with 2 μm 17-β-estradiol. C, Induced with 5 μm 17-β-estradiol after 14 d.