Development of a GAL4-VP16/UAS trans-activation system for tissue specific expression in Medicago truncatula

Abstract

Promoters with tissue-specific activity are very useful to address cell-autonomous and non cell autonomous functions of candidate genes. Although this strategy is widely used in Arabidopsis thaliana, its use to study tissue-specific regulation of root symbiotic interactions in legumes has only started recently. Moreover, using tissue specific promoter activity to drive a GAL4-VP16 chimeric transcription factor that can bind short upstream activation sequences (UAS) is an efficient way to target and enhance the expression of any gene of interest. Here, we developed a collection of promoters with different root cell layers specific activities in Medicago truncatula and tested their abilities to drive the expression of a chimeric GAL4-VP16 transcription factor in a trans-activation UAS: β-Glucuronidase (GUS) reporter gene system. By developing a binary vector devoted to modular Golden Gate cloning together with a collection of adapted tissue specific promoters and coding sequences we could test the activity of four of these promoters in trans-activation GAL4/UAS systems and compare them to "classical" promoter GUS fusions. Roots showing high levels of tissue specific expression of the GUS activity could be obtained with this trans-activation system. We therefore provide the legume community with new tools for efficient modular Golden Gate cloning, tissue specific expression and a trans-activation system. This study provides the ground work for future development of stable transgenic lines in Medicago truncatula.

Fig 1. New pCambia Golden Gate vector.

Schematic backbone of the pCAMBIA_CR1 vector showing the BsaI cloning sites (in red) that allow insertion of the oriented blocks using the Golden Gate strategy. Cloning sites (single cutter restriction enzymes or BsaI cloning sites) disrupt the LacZ gene (blue arrow) upon cloning, allowing blue/ white screening with the X-Gal substrate. For E.coli selection, a chloramphenicol (Cm) resistance gene can be used (yellow arrow outside the T-DNA fragment). A kanamycin resistance (kanR) gene, driven by a NOS promoter (yellow box and arrow), enables both selection for the presence of the plasmid in A. rhizogenes and transformed roots on selective medium. The T-DNA contains a pAtUbi:DsRED selection gene (red box and arrow) that allows detection of transformed roots using DsRED fluorescence. RB/LB: T-DNA right border and left border.

Fig 2. GoldenGate cloning strategy and consensus “sticky end” adapters used.

Schematic representation of the consensus sequences used as “sticky ends” for oriented cloning of each specific block. The pCAMBIA_CR1 binary vector backbone is shown in purple. Here, “AB” blocks are promoter regions, the “BC” block was separated as “BN” and “NC” fragments (where N is a chosen sequence, here TTCA) for the GAL4-VP16 chimeric transcription factor and for the “transcriptional terminator 5xUAS_minimal promoter” blocks, respectively. The β-glucuronidase (GUS) coding region was introduced as a CD block for UAS constructs or BD block for the direct promoter fusions. Note that the “B” adapter was designed to provide an optimized dicotyledon start codon context and the “C” adapter to provide a linker for in frame tag fusions, respectively. X is a chosen spacer nucleotide that will be removed after BsaI digeston. GOI: gene of interest.

Fig 3. Whole root and root section patterns of tissular GUS activity obtained in promoter:GUS fusions.

Whole M. truncatula transgenic roots and representative root sections showing GUS activity driven by the (A,B) pAtCO2, (C,D) pAtPEP, (E,F) pAtCASP1, (G,H) pLaSCR1 and (I,J) pAtSUC2 promoters using direct fusions with GUS coding sequence. GUS staining is shown in blue. Scale bar is 1 mm in (A,C,E,G,I) and 100 μm in (B,D,F,H,J). e: epidermis; c: cortex; en: endodermis; p: pericycle. Arrow head in (J) marks phloem companion cell.

Fig 4. Whole roots and root sections showing patterns of tissular GUS activity obtained in the GAL4-VP16/UAS trans-activation system.

Whole M. truncatula transgenic roots and representative root sections (10 μm) showing GUS activity driven by the (A,B) pSlEXT1, (C,D) pAtCO2 (E,F) pAtPEP and (G,H) pAtCASP1 promoters using GAL4-VP16/UAS transactivatable system. GUS staining is shown in blue. Scale bar is 1 mm in (A, C, E,G) and 100 μm in (B,D,F,H). e: epidermis; c: cortex; en: endodermis; p: pericycle.