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

<div><p>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 <i>Arabidopsis thaliana</i>, 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 <i>Medicago truncatula</i> 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 <i>Medicago truncatula</i>.</p></div

Comparison of GUS staining efficiency and tissue specificity of promoter:GUS and transactivatable (GAL4-VP16/ UAS) GUS constructs.

<p>Comparison of GUS staining efficiency and tissue specificity of promoter:GUS and transactivatable (GAL4-VP16/ UAS) GUS constructs.</p

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

<p>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.</p

New pCambia Golden Gate vector.

<p>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 <i>E</i>.<i>coli</i> 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 <i>A</i>. <i>rhizogenes</i> and transformed roots on selective medium. The T-DNA contains a <i>pAtUbi</i>:<i>DsRED</i> selection gene (red box and arrow) that allows detection of transformed roots using DsRED fluorescence. RB/LB: T-DNA right border and left border.</p

References and expected tissular expression of all tested promoters.

<p>References and expected tissular expression of all tested promoters.</p

Bacteria-induced cytoskeleton modifications of HeLa cells.

<p>HeLa cells untreated (A), inoculated with <i>S. meliloti</i> (B), <i>R. leguminosarum</i> (C), <i>A. caulinodans</i> (D), <i>C. taiwanensis</i> (E), <i>B. tuberum</i> (F), <i>C. crescentus</i> (G) and <i>E. coli</i> (H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hours after bacterial inoculation. Arrow: stress fiber.</p

Symbiotic phenotype of <i>S. meliloti</i> queuosine mutants.

<p>Dry weight of <i>M. truncatula</i> seedlings inoculated with <i>S. meliloti</i> 1021, different queuosine-deficient mutants and the <i>queF</i> complemented (GMI11186) strain at 40 dpi. Statistical significance (P<0.01) is shown with respect to strain 1021(*) and the <i>queF</i> mutant (^#), respectively. (B, C) Sections of <i>M. truncatula</i> 21–day old nodules induced by 1021 (B) and the <i>queF</i> isogenic mutant (C). (D, E) Electron micrographs of nodule cells infected with 1021 (D) or the <i>queF</i> mutant (E). <i>queF</i> mutant bacteroids are randomly organized within the infected cell whereas 1021 bacteroids show a radial organization. (Insert panel in E): arrows point to symbiosome membranes detached from <i>queF</i> bacteria (). Arrowhead, type 4/5 bacteroid. *, starch granules.</p

Determination of GTPases activation state in bacteria-treated HeLa cells.

<p>(A) Representative pull down assays of active Cdc42, Rac1 and RhoA GTPases at 48 hpi in non-inoculated- (control), <i>S. meliloti</i> 1021- and <i>queF</i>-inoculated HeLa cells. (B) Quantification of pull down assays using ImageJ software. Means ± S.D. were calculated from three independent experiments for Cdc42-GTP and mean from two independent experiments for Rac1-GTP and RhoA-GTP. Results were normalized to the corresponding total protein. Statistical significance (P<0.001) is shown (*) with respect to the control. (C) Immunoprecipitation of active and total CdC42 from non-inoculated (control) HeLa cells or cells inoculated with live and heat-killed wild-type bacteria 48 hpi. (D). Kinetics of Cdc42 activation. Actin, total Cdc42, Rac1 and RhoA or active GTP-bound forms of Cdc42, Rac1 or RhoA were detected by immuno-blotting of SDS-PAGE gels.</p

The <i>S. meliloti</i> queuosine biosynthetic pathway.

<p>preQ₀: 7-cyano-7-deazaguanine, preQ1: 7-(aminomethyl)-7-deazaguanine, AdoMet: S-adenosyl-L-methionine, EpoxyQ: epoxyqueuosine, Q: queuosine. Adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-IwataReuyl1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056043#pone.0056043-Reader1" target="_blank">[50]</a>.</p

Cytoskeleton modifications of HeLa cells infected with <i>S. meliloti</i> queuosine biosynthesis mutants.

<p>HeLa cells were incubated with <i>S. meliloti queC</i> (A, E), <i>queF</i> (B, F), <i>tgt</i> (C, G), <i>queA</i> (D, H), the wild type 1021 strain (I) and the <i>queF</i> complemented strain (GMI11186) (J) in 0.5% FCS culture medium alone (A–D, I, J) or supplemented with preQ1 (E–H). HeLa cells were stained with phalloidin-Texas red and observed by fluorescence microscopy 48 hpi. Scale bar: 10 µm.</p