Genetic Resources for Legume Research

The L. japonicus ecotypes Gifu B-129 and Myakojima (MG-20) and mutant lines nin-2, ern1-2 [57 (link)], and cerberus-12 [22 (link)] were used in this study. For M. truncatula, the mutant line sunn-1 [58 (link)] was used. The mutant lines rpg-1 (SL5706-3), rpg-2 (SL454-2), and rpg-6 (SL0181) were isolated from forward genetic screening of an EMS mutagenesis population of L. japonicus Gifu B-129. Other rpg allelles were obtained from a LORE1 retrotransposon insertion mutagenesis pool [29 (link)]. The transposon insertion in each gene was verified by PCR product sequencing; primers are shown in S1 Table. Meshorhizobium loti R7A, constitutively expressing GFP or lacZ (referred to as R7A GFP or R7A LacZ), or M. loti MAFF303099 carrying RFP, or DsRED were used for L. japonicus nodulation experiments, and Sinorhizobium meliloti 1021-mCherry was used for M. truncatula nodulation experiments. Spores of the mycorrhizal fungus Rhizophagus irregularis were used for analysis of AM symbiotic phenotypes. For hairy root transformation of L. japonicus or M. truncatula roots, Agrobacterium rhizogenes strain AR1193 was used. A. tumefaciens strain EHA105 or GV3101 (pSoup) were used for N. benthamiana transient expression and stable transformation of L. japonicus as previously described [59 ]. Plasmids were cloned in Escherichia coli DH10B or DH5α. Saccharomyces cerevisiae strain AH109 was used for the yeast two-hybrid assay.