One-step Agrobacterium mediated transformation of eight genes essential for rhizobium symbiotic signaling using the novel binary vector system pHUGE

Abstract

Advancement in plant research is becoming impaired by the fact that the transfer of multiple genes is difficult to achieve. Here we present a new binary vector for Agrobacterium tumefaciens mediated transformation, pHUGE-Red, in concert with a cloning strategy suited for the transfer of up to nine genes at once. This vector enables modular cloning of large DNA fragments by employing Gateway technology and contains DsRED1 as visual selection marker. Furthermore, an R/Rs inducible recombination system was included allowing subsequent removal of the selection markers in the newly generated transgenic plants. We show the successful use of pHUGE-Red by transferring eight genes essential for Medicago truncatula to establish a symbiosis with rhizobia bacteria as one 74 kb T-DNA into four non-leguminous species; strawberry, poplar, tomato and tobacco. We provide evidence that all transgenes are expressed in the root tissue of the non-legumes. Visual control during the transformation process and subsequent marker gene removal makes the pHUGE-Red vector an excellent tool for the efficient transfer of multiple genes.

Figure 1. Physical map of binary vector pHUGE-Red.

Indicated are left border (LB), right border (RB), Rs-recombination sites (Rs), R-recombinase-LDB fusion gene (recLDB), the visual marker DsRED1 (DsRED1) driven by AtUBQ10 promoter, the fused selection markers CodA-nptII under control of CaMV35S promoter and the components required for multisite gateway; recombination sites (attR3 and attR4) 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 AtUBQ10 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).

Figure 2. GUS reporter studies in legumes and non-legumes.

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.

Figure 3. Construction of pHUGE-MtNFS and pHUGE-LjMtNFS.

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 att 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: att 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.

Figure 4. Selection of transformed lines.

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).

Figure 5. Relative gene expression of legume Nod factor signaling genes in trans.

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. MtDMI2 has been used as interspecies reference gene. Error bars show variation between three technical replicates.

Figure 6. MtNIN induction upon Rhizobium application in legumes and non-legumes.

Induction of MtNIN 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 PtACT2 & PtUBQ and tobacco NtGAPDH were used as reference genes. Three biological replicates are shown for each line. Error bars show variation between three technical replicates.