Dual requirement of the LjSym4 gene for mycorrhizal development in epidermal and cortical cells of Lotus japonicus roots.

• The LjSym4 mutation leads to Lotus japonicus plants that are defective in arbuscular mycorrhiza (AM) development. • Two alleles of LjSym4 with different phenotypic strength are compared here. The development of AM was assessed by considering five parameters related to fungal structures present in root segments from wild-type and mutant plants. The distribution of intercellular hyphae was determined using semithin sections from resin-embedded roots. Cellular interactions were investigated ultrastructurally, whereas cell wall components from the host plant were identified using immunogold labeling. • In roots of Ljsym4-1 mutant, fungal hyphae were mostly restricted to the intercellular spaces of the cortex, indicating a block to infection by mutant cortical cells, which resulted in a very low number of arbuscules. • This observation suggests the presence of an additional, genetically defined 'checkpoint' for mycorrhizal development, located at the wall of cortical cells. The LjSym4 gene is therefore required for infection of both epidermal and cortical cells by AM fungi.