Pdonor27 vector

The MtNIP5;1 promoter:: β-glucuronidase (GUS) construct was generated using the Gateway System (Life Technologies, Carlsbad, USA). The promoter fragment of the candidate gene (2.022 kb upstream of the MtNIP5;1 start codon, P - 1174 UT848 ) was ampli ed using the primers indicated in Additional le 1, cloned in the pDONOR27 vector (Invitrogen) and transferred to the pGWB3 plasmid (65).
Gus activity assay was performed in plants 4 weeks after inoculation following the protocol described by (66) with minor modi cations. Roots and nodules sections (100 µm) were incubated in a GUS buffer (0,69% PO 4 H 2 Na, 0,5 M EDTA pH 8, 0, 30% sarkosyl, 40 µl triton X-100 AND H 2 O) supplemented with X-Gluc (0,1 mg/ml) for 12-16 h at 28 °C in the dark, and then distained with bleach 50% and rinsed ve times in H 2 O. Afterwards, sections were observed with a Leica DM IRB microscope.

The MtNIP5;1 promoter::β-glucuronidase (GUS) construct was generated using the Gateway System (Life Technologies, Carlsbad, USA). The promoter fragment of the candidate gene (2,022 kb upstream of the MtNIP5;1 start codon, P -1174 UT848 ) was ampli ed using the primers indicated in Additional le 1, cloned in the pDONOR27 vector (Invitrogen), and transferred to the pGWB3 plasmid (68).
Gus activity assay was performed in plants 4 weeks after inoculation following the protocol described by Vernoud et al. (69) with minor modi cations. Roots and nodules sections (100µm) were incubated in a GUS buffer (0.69% PO 4 H 2 Na, 0.5M EDTA pH 8, 30% sarkosyl, 40µl triton X-100, and H 2 O) supplemented with X-Gluc (0.1 mg/ml) for 12-16h at 28°C in the dark, and then distained with bleach 50% and rinsed ve times in H 2 O. Afterwards, sections were observed with a Leica DM IRB microscope.