Extended Data Fig. 6: Analyses of the efficiency of inactivation and down-regulation of MtAHLs in hairy roots of M. truncatula.

a and b, CRISPR/Cas9 mediated mutations introduced into MtAHL1 and MtAHL2 were investigated using Illumina sequencing, typically with about 5000–10000 reads for each sampled nodules and representative modified sequences in individual nodules are shown. The data were analyzed on the CRISPResso2 website⁴³ (https://crispresso.pinellolab.partners.org/). Representative sequences of mutated alleles of MtAHL1 and MtAHL2 are shown in a and b, respectively. The sgRNA sequences used are indicated in blue and the PAM site is in red. Nucleotide insertions are marked in bold. c, Photomicrographs of nodules formed on transgenic roots in which RNA interference was induced using RNAi constructs MtAHL1 RNAi or MtAHL2 RNAi; NCR169 RNAi used as a phenotype control. The upper images captured GFP fluorescence and the lower images were taken with a bright field. Scale Bar indicates 2 mm. d and e, Expression levels of both MtAHL1 and MtAHL2 were measured by quantitative reverse transcription PCR in nodules formed on roots transformed with MtAHL1 RNAi (d) or MtAHL2 RNAi (e). Six roots were chosen for each analysis and experiments were repeated three times. Expression levels were normalized against the Mt40S ribosomal gene. The columns demonstrate the mean ± SE of fold change in gene expression in different roots compared with control. Dots, individual data points. P values are indicated for comparing expression level with the control (unpaired t-test was used).