Two ineffective-nodulating mutants of Lotus japonicus - Different phenotypes caused by the blockage of endocytotic bacterial release and nodule maturation

Mutants defective in nodule development and nitrogen fixation of Lotus japonicus B-129 'Gifu' were obtained by induced mutagenesis with EMS (ethylmethane sulfonate) treatment. Using a symbiont of L. japonicus, Rhizobium loti JRL501, 17,000 M2 seeds were screened for plants affected in their symbiotic phenotype, resulting in the successful isolation of eleven stable mutants. In this paper, we report two ineffective nodulating mutants among them. Reciprocal crossing between wild type 'Gifu' and these mutants indicated that their phenotypes are under monogenic and recessive control. Furthermore, tests for allelism with these mutants showed that the mutated genes are non-allelic. Ultrastructural analysis revealed that these mutants were inhibited at different stages of nodule development and maturation. Based on histological characteristics of the nodules, two ineffective nodulating mutants were named alb1 (aberrant localization of bacteria inside nodule) and fen1 (fail in enlargement of infected cells), respectively. In the nodules of alb1, most of the bacteria failed to invade the cytoplasm of cortical cells and were tightly confined inside infection threads or localized in intercellular spaces of nodules. Following prolonged inoculation, alb1 mutant also formed pale-pink colored nodules with a low frequency, in which bacteria differentiated into bacteroid and fixed nitrogen normally. Although the infected cells in the nodules of fen1 mutant contained numerous differentiated bacteroids, they failed to enlarge by cell expansion and showed a low activity of nitrogen fixation.