LjPUB13 promoter activity in L. japonicus

For the promoter-GUS-terminator construction, a 1475 bp promoter with a 5′ untranslated region (UTR) and a 310 bp terminator region was amplified from L. japonicus genomic DNA (the primers are listed in Additional file 1: Table S3) and cloned into GoldenGate vectors [47]. The PCR fragments were firstly cloned into GoldenGate Level 0 vectors before being assembled as a construct (promoter:GUS:terminator) in a modified pIV10 vector [48]. The construct was transferred into A. rhizogenes AGL1.

Hairy root induction using A. rhizogenes was performed as described previously [49]. Chimeric plants were transferred into magenta growth boxes containing a sterilized 4:1 mix of clay granules and vermiculite as well as ¼ strength B&D medium supplemented with 1 mM KNO₃. For inoculation, liquid cultures of M. loti cv. R7A expressing DsRed were grown to an optical density of 0.02 and applied directly to the root systems (0.7 ml per plant). Plants were grown at 21 °C (16 h light, 8 h dark) and harvested at indicated times post inoculation. GUS staining was performed as described previously [50]. Whole roots were visualized on a Leica M165 FC stereomicroscope.