For the overexpression constructs, the Xdh2 and Pxy genes were amplified from L. japonicus Gifu and MG20 genomic DNA by PCR using primers (Xdh2; 5`-TCCTA GCTATGAGTTCTCTCA-3`and 5´-CTAGCTAGCATTCCAAGGGA-3`, Pxy; 5`-ACCCCAAAACCATGAA CCT-3; and 5`-CATATAAACAGATTAATCAGC-3`) containing attB sites. The Smc6 CDS was amplified from L. japonicus Gifu and MG20 cDNA from root and nodule tissues, which were produced using the 5`-SMART RACE cDNA amplification kit (CLONTECH), by PCR using primers (Smc6; 5`-CCGGCGTTTGCAGAATGAAGCGGAGA-3` and 5`-GAGATTGAACATGATGAAACGCAG-3`) containing attB sites. The PCR amplification products were recombined into the pDONR207 (Invitrogen) using the Gateway BP reaction (Invitrogen) to create entry clone vectors. The entry clone vectors were recombined using Gateway LR reactions (Invitrogen) with the destination vector pIV10::Ubiquitin_promoter::GW, to create the constructs: pIV10::Ubiquitin_promoter::Gifu_Xth2, pIV10::Ubiquitin_promoter::MG20_Xth2, pIV10::Ubiquitin_promoter::Gifu_Pxy, pIV10::Ubiquitin_promoter::MG20_Pxy, pIV10::Ubiquitin_promoter::Gifu_Smc6 and pIV10::Ubiquitin_promoter::MG20_Smc6.
For complement analysis, Agrobacterium strain AR1193 was used for hairy root transformation experiments as described previously 62, 89
For complement analysis, Agrobacterium strain AR1193 was used for hairy root transformation experiments as described previously 62, 89