Figure 5. Epistatic relationships between symbiotic RLK genes and common symbiosis genes.
Hairy roots of L. japonicus Gifu wild-type and different symbiosis defective mutants transformed with pUB:SYMRK-mOrange (SYMRK) or pSYMRK:SYMRK-RFP (pS SYMRK) (upper panel), or the empty vector (EV), pUB:NFR1-mOrange (NFR1) or pUB:NFR5-mOrange (NFR5) (lower panel) were generated. Plots represent the numbers of nodules (grey) and nodule primordia (white) per nodulated plant formed in the absence of rhizobia at 40 (SYMRK) and 60 (NFR5 + NFR1) dpt. White circles indicate individual organogenesis events. Black dots, data points outside 1.5 IQR of the upper/lower quartile; bold black line, median; box, IQR; whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. Table, fraction of nodulated per total number of plants. Plants transformed with pSYMRK:SYMRK-RFP or the empty pUB vector did not develop spontaneous nodules.
Figure 5—figure supplement 1. SYMRK-mediated spontaneous organogenesis events in nfr1-1, nfr5-2, and common symbiosis mutants.
Hairy roots of different symbiosis defective mutants transformed with pUB:SYMRK-mOrange (SYMRK) or pSYMRK:SYMRK-RFP (pS
SYMRK) were generated. Plot represents the numbers of organogenesis events (nodules and nodule primordia) per plant formed in the absence of rhizobia at 40 dpt. Bold black line, median; box, IQR; whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. A Kruskal–Wallis test followed by false discovery rate correction was performed. Different letters indicate significant differences. p < 0.05.
Figure 5—figure supplement 2. NFR5-mediated spontaneous organogenesis events in Gifu wild-type, nfr1-1, nfr5-2, and common symbiosis mutants.
Hairy roots of L. japonicus Gifu wild-type and different symbiosis defective mutants transformed with the empty vector (EV) or pUB:NFR5-mOrange (NFR5) were generated. Plot represents the number of organogenesis events (nodules and nodule primordia) per plant formed in the absence of rhizobia at 60 dpt. Black dots, data points outside 1.5 IQR of the upper quartile; bold black line, median; box, IQR; whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. A Kruskal–Wallis test followed by false discovery rate correction was performed. Different letters indicate significant differences. p < 0.05.
Figure 5—figure supplement 3. NFR1-mediated spontaneous organogenesis events in Gifu wild-type, nfr1-1, nfr5-2, symrk-10, and symrk-3.
Hairy roots of L. japonicus Gifu wild-type and different symbiosis defective mutants transformed with the empty vector (EV) or pUB:NFR1-mOrange (NFR1) were generated. Plot represents the number of organogenesis events (nodules and nodule primordia) per plant formed in the absence of rhizobia at 60 dpt. Black dots, data points outside 1.5 IQR of the upper quartile; bold black line, median. A Kruskal–Wallis test followed by false discovery rate correction was performed. Different letters indicate significant differences. p < 0.05.
Figure 5—figure supplement 4. SYMRK-mediated activation of the symbiosis-specific T90 reporter in symbiosis-defective mutants.
Hairy roots of three stable transgenic L. japonicus Gifu reporter lines homozygous for the T90 reporter fusion and the indicated mutant alleles transformed with pUB:CCaMK
T265D (CCaMK
T265D, a deregulated version of CCaMK), pUB:SYMRK-mOrange (SYMRK), or pSYMRK:SYMRK-RFP (pS
SYMRK) were generated and kept on agar plates for a total of 38 dpt (see ‘Materials and methods’). The vast majority of transgenic root systems did not develop spontaneous nodules at this time point under these growth conditions. GUS activity was analysed by histochemical staining with X-Gluc at 38 dpt. Representative root sections are shown. Number of plants with detectable GUS activity per total plants is indicated. Bars, 500 μm.
Figure 5—figure supplement 5. NFR-mediated activation of the symbiosis-specific T90 reporter in the nfr1-1 mutant background.
Hairy roots a stable transgenic L. japonicus Gifu reporter line homozygous for the T90 reporter fusion and the nfr1-1 mutant allele transformed with the empty vector (EV), pUB:NFR1-mOrange (NFR1), pUB:NFR5-mOrange (NFR5), or pUB:SYMRK-mOrange (SYMRK) were generated. GUS activity was analysed by histochemical staining with X-Gluc at 60 dpt. Representative root sections are shown. Number of plants with detectable GUS activity per total number of plants is indicated. Bars, 500 μm.