Characterization of nfh1 Mutants.
(A)
Tnt1 insertion sites of the M. truncatula R108 mutants nfh1-1 (NF16587), nfh1-2 (NF12841), and nfh1-3 (NF11260). The box indicates the coding region of MtNFH1. The location of Tnt1 insertions in the promoter region are marked by indicated arrows.
(B) Analysis of MtNFH1 expression by RT-qPCR in wild-type and nfh1 mutant seedlings (nfh1-1, nfh1-2, and nfh1-3). Roots of seedlings were immersed in 1-mL syringes filled with Jensen medium containing 0.1 μM NodSm-IV(C16:2, Ac, S) for 18 h. Roots from 20 seedlings were used for each RNA extraction (3 RNA extractions per genotype; n = 3). Data indicate means ± se of normalized expression values (mean value of wild-type plants set to one). Statistically different transcript levels of mutants compared with wild-type plants are marked by asterisks (Student’s t test, P ≤ 0.05; Supplemental File 1).
(C) Formation of the lipodisaccharide NodSm-II(C16:2, Ac) released from NodSm-IV(C16:2, Ac, S) in the rhizosphere of wild-type and nfh1 mutant seedlings. Data indicate means ± se (1 plant per sample; n ≥ 9). Asterisks indicate significantly reduced hydrolytic activity in nfh1 mutants compared with wild-type plants (Kruskal-Wallis test, P ≤ 0.05; Supplemental File 1).