Figure 1. DIS and RAM2 are required for arbuscule branching and vesicle formation.
Arbuscule phenotype and complementation of dis (A) and ram2 (B) mutants. The fungus was stained with wheat-germ agglutinin (WGA)-AlexaFluor488. (C-D) Percent root length colonization of dis (C) and ram2 (D) mutants as compared to wild-type. Different letters indicate significant differences among treatments (ANOVA; posthoc Tukey). (C): n = 13; p≤0.1, F2,10 = 8.068 (total & int. hyphae); p≤0.001 F2,10 = 124.5 (arbuscules); p≤0.001, F2,10 = 299.1 (vesicles) (D): n = 15; p≤0.1, F2,12 = 10.18 (total & int. hyphae); p≤0.001 F2,12 = 57.86 (arbuscules); p≤0.001, F2,12 = 72.37 (vesicles). (A-D) Plants were inoculated with R. irregularis and harvested at 5 weeks post inoculation (wpi).
Figure 1—figure supplement 1. Identification of the dis mutation.
(A–B) Genetic map of the DIS locus on chromosome 4. Numbers next to marker positions refer to the proportion of recombinant individuals among the number of analyzed F2 mutant plants. Rough mapping had previously identified the position of the dis mutation on the south arm of chromosome 4 (Groth et al., 2013). (A) In the first fine-mapping round, the interval narrowed down by recombinants comprised 19 EMS-induced SNPs (red stars), that could be confirmed by re-sequencing the mutant genome using next generation sequencing. (B) Further fine mapping resulted in an interval with 3 of these confirmed SNPs. (C) Physical map of the DIS locus. LjT followed by a number refers to TAC clones. CM followed by a number refers to contigs. One of the three SNPs causes a G to A transition in exon 3 of chr.4.CM004.1640.r2.a resulting in an amino acid change from glycine to arginine at position 190 of the protein product, which shares 79% sequence identity with a β-keto-acyl ACP synthase I (KASI) from Arabidopsis thaliana. Black boxes indicate exons separated by introns. (D) The DIS gene is duplicated in tandem. (E) Gene structure of DIS, DIS-LIKE and KASI. Black boxes display exons separated by introns (black lines). Grey boxes indicate determined un-translated regions. (F) DIS, DIS-LIKE and KASI are predicted to contain a plastid transit peptide (green). The catalytic triad is shown in blue and the location of mutations identified by TILLING in the DIS gene are shown in red. We chose the dis-4 mutant for further analysis because the mutation resulted in a glycine replacement, which likely affects the functionality of the protein.
Figure 1—figure supplement 2. Protein sequence alignment of L. japonicus DIS with other KASI proteins.
(A) Sequence alignment of LjDIS, LjDIS-LIKE, LjKASI, AtKASI and E. coli KASI and KASII. (B) Identity matrix of LjDIS, LjDIS-LIKE, LjKASI and AtKASI.
Figure 1—figure supplement 3. Identification of mutation in the RAM2 gene.
(A) Genetic map of the red locus on chromosome 6. Numbers next to the marker position refer to the proportion of recombinant individuals among the number of analysed F3 (black) and F4 (grey) segregating and mutant plants respectively. Fine mapping narrowed down the interval between TM0553 and TM0302. Red arrows indicate the genomic interval that contains the causative mutation. (B) Gene structure of L. japonicus RAM2 with locations of the identified EMS-induced mutation at position 1663 (star, ram2-1) leading to an amino acid exchange from glycine to glutamic acid at position 555 of the RAM2 protein and LORE1 insertion (triangle, ram2-2). Black boxes indicate exons separated by intron (thin black line). Grey boxes indicate untranslated regions (UTRs) comprising 77 bp (5’UTR) and 151 bp (3’UTR). (C) Co-segregation analysis of arbuscule phenotype and mutation in the RAM2 gene in a number of F3 and F4 plants from segregating populations containing only the mutation on chromosome 6. The number of plants analysed per generation, arbuscule phenotype, genotype at markers TM0053 and TM0302 and the nucleotide observed at position 1663 in the RAM2 gene are indicated. The ram2 mutation at position 1663 clearly co-segregates with the stunted arbuscule phenotype.
Figure 1—figure supplement 4. Protein sequence alignment of L.
japonicus RAM2 with M. truncatula RAM2. Sequence alignment (A) and identity matrix (B) of LjRAM2, Lj1g3v2301880.1, MtRAM2 and Medtr7g067380.