Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

Abstract

SUMMARYThe nitrogen-fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix -mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix -mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix -mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE-INDUCED 1 (LjAPN1), encodes a nepenthesin-type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain-specific Fix -phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen-fixing) symbiosis in a rhizobial strain-dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

“…b), as described for nodules of many Fix − mutants (e.g. Hakoyama et al ., ; Yamaya‐Ito et al ., ; and references therein) and LbRNAi lines (Ott et al ., ). Vacuolization and vesicle formation are a result of membrane damage, which occurs in plastids and symbiosomes during nodule aging (Dupont et al ., ).…”

Rhizobial strain‐dependent restriction of nitrogen fixation in a legume‐Rhizobium symbiosis

“…How did the findings in the highlighted paper (Yamaya‐Ito et al ., ) come about? For over 10 years several Japanese groups had been isolating and characterizing Fix ‐ mutants of L. japonicus (e.g.…”

The Lotus japonicus nucleoporin GLE1 is involved in symbiotic association with rhizobia

“…However, the nitrogen‐fixation activities of the Ljgle1 mutant nodules were relatively higher than those of typical L. japonicus Fix − mutants such as fen1 (Imaizumi‐Anraku et al , Hakoyama et al ), sen1 (Suganuma et al , Hakoyama et al ), sst1 (Krusell et al ), Ljsym105 (Hossain et al ), ign1 (Kumagai et al ), syp71 (Hakoyama et al ) and apn1 (Yamaya‐Ito et al ). In addition, slightly larger numbers of nodules were formed in the Fix − mutants compared to the wild‐type plants, whereas nodulation was delayed in the Ljgle1 mutant compared to the wild‐type plant.…”

“…Moreover, the Fix − mutants have been reported to display a common symptom of the premature senescence of nodule cells characterized by irregularly shaped, enlarged symbiosomes, as a consequence of the increased symbiosome space between the symbiosome membrane and bacteroids, which contains the deteriorated bacteroids in mature nodules (Imaizumi-Anraku et al 1997, Suganuma et al 2003, Krusell et al 2005, Hossain et al 2006, Kumagai et al 2007, Hakoyama et al 2012a, Hakoyama et al 2012b, Yamaya-Ito et al 2018. In the Ljgle1 mutant, on the other hand, the symbiosome membrane attached to the bacteroid membrane and symbiosome space was hardly distinguishable.…”

“…We have studied the regulatory mechanisms of symbiotic nitrogen fixation by host plant genes using L. japonicus Fix − mutants that display lower or no nitrogen-fixation activity. In L. japonicus, it has been determined that the sulfate transporter SST1 (Krusell et al 2005), the ankyrin repeat domain-containing protein IGN1 (Kumagai et al 2007), the homocitrate synthase FEN1 (Hakoyama et al 2009), the putative transporter SEN1 (Hakoyama et al 2012a), the SNARE protein SYP71 (Hakoyama et al 2012b) and the aspartic peptidase APN1 (Yamaya-Ito et al 2018) are essential for symbiotic nitrogen fixation. In order to identify more plant genes required for symbiotic nitrogen fixation, we have been trying to isolate novel L. japonicus Fix − mutants.…”