PMC

Phenotypes of Lotus accessions inoculated with B. elkanii USDA61, the T3SS-deficient BErhcJ mutant of B. elkanii, or M. japonicum MAFF303099. (A, B, and C) Nodule numbers and (D, E, and F) fresh weights of Lotus japonicus Gifu (A, D), L. burttii (B, E), and L. japonicus MG-20 (C, F) inoculated with the wild-type (USDA61) or T3SS-deficient mutant (BErhcJ) of B. elkanii USDA61, or Lotus symbiont M. japonicum MAFF303099 measured on day 30 post-inoculation. Measurements were performed three times with 6 to 16 plants each time. In panels B and C, brownish nodules were included in the count of white nodules. Error bars indicate standard deviations. The Student’s t-test was performed for fresh weight comparisons; ** P<0.01 vs. control (no inoculation). (G) Root nodules of the three Lotus accessions inoculated with the above bacteria. Scale bars=1 mm. (H) Brownish nodules of L. burttii and MG-20 inoculated with wild-type USDA61. Scale bars=1 mm.

Root length colonization after growth in sand-filled pots vs. tip-wick hydroponics. Comparison of (A) percent colonization of Lotus japonicus roots with Rhizophagus irregularis and (B) L. japonicus root fresh weight in sand-filled pot vs. tip-wick hydroponics at 4 weeks post-inoculation (wpi). Mann-Whitney tests were used for statistical comparison (n = 8–15 separate plants from one tip-box or two independent pots).

(A) Primary root length (PRL), post-embryonic root (PER) number and PER density of wild-type plants 2 wpg after treatment with solvent (M) or three different concentrations of KAR₁, KAR₂ or rac-GR24 (GR24) (n = 32–57). (B) PER density of wild-type plants at 2 wpg and treated with solvent (Mock) 1 μM KAR₁, 1 μM KAR₂, or 1 μM rac-GR24 (n = 43–51). Plants were transferred onto fresh hormone-containing medium after 5 days. (C-D) RT-qPCR-based expression of DLK2 normalized to Ubiquitin expression in roots at 2 wpg after 2 hours treatment with solvent (Mock), (C) 1 μM KAR₁ and 1 μM KAR₂, (D) 1 μM rac-GR24 (n = 4). (A and C) Letters indicate different statistical groups (ANOVA, post-hoc Tukey test). (B) Asterisks indicate significant differences (ANOVA, Dunnett test, N.S.>0.05, *≤0.05). (D) Asterisk indicate significant differences versus mock treatment (Welch t.test, *≤0.05, **≤0.01, ***≤0.001).

Complementation analysis of the Lotus japonicus lb123 mutant by LjGlb2-1. Phenotypes of (A) whole plants and (B) nodules of hairy root transformed plants at 6 weeks post-inoculation. Roots of the lb123 mutant were transformed with Agrobacterium rhizogenes strain LBA1334 harboring the LjLb2 promoter coupled to either LjLb2, GUS, or LjGlb2-1. The numbers in (B) indicate the proportion of plants bearing pink nodules. Scale bars are (A) 5 cm and (B) 1 mm. (C) Shoot fresh weight per plant. (D) Acetylene reduction activity (ARA) expressed per nodule fresh weight. Data are means (±SE) of 16–17 plants. Different letters indicate significant differences between means as determined by Duncan’s multiple range test (P<0.05).