Selection of transformed lines.

<p>Tissues are analyzed by bright field microscopy (A, C, E, G, I, K) and fluorescent microscopy using DsRED1 filter settings (B, D, F, H, J, L). A+B: Discrimination between transformed strawberry calli (red arrow) and untransformed calli (white arrow) based on expression of DsRED1. C+D: Outgrowth of transgenic- (red arrow) and non-transgenic strawberry plants (white arrow). E+F: Non-transgenic strawberry plant (white arrow). G+H: Transgenic strawberry plant (red arrow). I+J: Outgrowth of marker free tobacco plantlet, (red arrow) on a DsRED1 expressing leave disk (white arrow). K+L: Mosaic picture showing wild type tobacco (left), transformed tobacco expressing DsRED1 before (middle) and after marker gene removal (right).</p

<i>MtNIN</i> induction upon Rhizobium application in legumes and non-legumes.

<p>Induction of <i>MtNIN</i> 24 h after exposure to flavonoid stimulated, compatible rhizobium bacteria (+) compared to water control (−) in Medicago (Med WT), transgenic poplar (line Pop 15.4 and Pop 18.1) and transgenic tobacco (line Tob 47 and Tob 65). Poplar <i>PtACT2</i> & <i>PtUBQ</i> and tobacco <i>NtGAPDH</i> were used as reference genes. Three biological replicates are shown for each line. Error bars show variation between three technical replicates.</p

Relative gene expression of legume Nod factor signaling genes in trans.

<p>Expression has been studied in transgenic poplar (line Pop 15.4 and Pop 18.1), tobacco (line Tob 47 and Tob 65) and tomato (Tom 15.4). Gene expression was compared to native gene expression in Medicago (Med WT) which was set to 1. <i>MtDMI2</i> has been used as interspecies reference gene. Error bars show variation between three technical replicates.</p

Construction of pHUGE-MtNFS and pHUGE-LjMtNFS.

<p>A: A Gene of interest, including 2–3 kb of up- and 2 kb downstream genomic region. B: The genomic region was cloned by PCR using primers introducing the endonuclease recognition sites I1 & I2. PCR products were cloned into pENTR vectors. Restriction sites I1 & I2, I2 & I3 or I3 & I4 introduced at the start and the end of different genomic regions have to be unique within the three vectors combined in step D. C: The resulting vector, containing the full length gene flanked by <i>att</i> recombination sites (A4 & A1) is combined with two alternative entry vectors and a pDEST vector. Both vectors are created in a similar fashion and contain an additional gene of interest flanked by the restriction sites I2 & I3 or I3 & I4. D: <i>att</i> recombination sites are removed in two rounds of digestion (I2, I3), heat inactivation of the restriction enzyme and subsequent re-ligation. E: The pDEST backbone is digested using the endonuclease recognition sites I1 & I4 and ligated into a predigested entry vector (either pENTR4-1, pENTR1-2 or pENTR2-3) without purification. Transformed library efficient DH10b cells are selected on kanamycin. Each resulting entry vector contains three DNA fragments. F: Finally, these vectors were combined into pHUGE-Red using multisite gateway. G: Final binary vector containing up to nine genes.</p

Plasmids used for transformation of strawberry, poplar, tobacco and tomato.

<p>Plasmids used for transformation of strawberry, poplar, tobacco and tomato.</p

Transformation efficiency of pHUGE-Red.

<p>Transformation efficiency of pHUGE-Red.</p

Nodulation of Medicago mutants complemented with the corresponding gene.

<p>Nodulation of Medicago mutants complemented with the corresponding gene.</p

GUS reporter studies in legumes and non-legumes.

<p>Histochemical blue staining of roots from Medicago (left row), Arabidopsis (center row) and tomato (right row) transformed with different Medicago Nod factor signaling gene promoter::GUS constructs.</p

Physical map of binary vector pHUGE-Red.

<p>Indicated are left border (LB), right border (RB), Rs-recombination sites (Rs), R-recombinase-LDB fusion gene (recLDB), the visual marker <i>DsRED1</i> (DsRED1) driven by <i>AtUBQ10</i> promoter, the fused selection markers <i>CodA-nptII</i> under control of <i>CaMV35S</i> promoter and the components required for multisite gateway; recombination sites (<i>attR3</i> and <i>attR4</i>) and two selection markers (CmR and ccdB). The backbone contains the P1 replicon as well as the pRiA4 replicon. Selection markers: kanamycin (KnR) and spectinomycin (SpR). In vector pHUGE-RedSeed the <i>AtUBQ10</i> promoter was exchanged for the seed coat specific napin promoter. Complete sequences are available at the GenBank database (pHuge-Red accession no. JN874480, pHuge-RedSeed accession no. JN874481).</p

Additional file 5: of The strigolactone biosynthesis gene DWARF27 is co-opted in rhizobium symbiosis

Nodule phenotype after knock-down of MtD27 through RNAi. (a) Relative transcript abundance as determined by qRT-PCR of MtD27 in M. truncatula transgenic roots expressing an empty vector control construct (EVi) or MtD27 RNAi construct (D27i). (b) Number of nodules formed on plants bearing transgenic roots harboring an empty vector control construct (EVi) or MtD27 RNAi construct (D27i). (c) Section through a nodule formed on a root expressing the empty vector control construct. (d) Section through a nodule formed on a root expressing the MtD27 RNAi construct. Scale bars are equal to 100 μm. Data shown in (a,b) represent means of 6 (a) or 5 (b) biological replicates ± SEM. Different letters above bars indicate statistical difference (p < 0.05, students’ t-test). (TIFF 4222 kb