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Gherbi et al. 10.1073/pnas.0710618105.

Supporting Information

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Fig. 5. CgSymRK mRNA levels in C. glauca roots and nodules determined by quantitative RT-PCR. CgSymRK expression levels in (A) inoculated roots and (B) mature nodules (gray bar) are given relative to noninoculated roots. CgUbi was used as reference gene. ni, noninoculated roots. Error bars indicate standard errors of the mean of three technical replicates.

Fig. 6. Acetylene reduction activity (ARA) assays to assess the effects of CgSymRK RNAi on nitrogen fixation. Values obtained with two nontransgenic control root systems, two transgenic control root systems and five CgSymRK RNAi root systems are compared. NT: non-transgenic control; TC: transgenic control; 1-5: CgSymRK RNAi root systems with small unilobed nodules (1-3) and nodules showing 2-3 lobes each (4-5). Error bars indicate standard errors of the mean.

Fig. 7. Quantification of CgSymRKmRNA levels in five independent CgSymRKRNAi plants determined by real-time qPCR. CgUbi was used as reference. The average of two independent non-transgenic control roots and three transgenic control roots is shown. Expression levels are presented relative to transgenic control roots. All error bars indicate standard errors of the mean of 3 technical replicates on different samples. NT, non-transgenic control roots; TC, transgenic control roots. RNAi, CgSymRKRNAi plants.

Fig. 8. Intraradical colonization of C. glauca roots 4 or 8 weeks after inoculation with G. intraradices. Presence of intraradical hyphae, arbuscules/coiled hyphae and vesicles was assessed in roots from non-transgenic control (NT) and composite plants transformed with the control vector (TC) or with a CgSymRK RNAi vector (RNAi). Each bar represents quantitative analysis of one root system.

Fig. 9. Colonization of C. glauca roots by G. intraradices 8 weeks after inoculation. (A-B) Non-transgenic control roots and (C-D) roots transformed with the control vector show high colonization levels, arbuscules (Ar) vesicles (V) and intraradical hyphae (Ih) are present at high frequencies. (E-H) CgSymRK knockdown (RNAi) roots. Overall colonization levels are very low, but extensive extraradical hyphal growth can be observed on few roots (E). Hyphae tend to develop at the root surface following the epidermal cells forming appressoria (Ap) and aberrant hyphal swellings. (G-H) Penetration succeeds rarely, but can lead to the formation of vesicles (G) and arbuscules (H) morphologically similar to those formed in control plants. Scale bars: A, C, E, G: 50 mm; B, D, F, H: 5 mm.

Table 1. Reduced nodulation in CgSymRK RNAi composite plants

Nodulation was scored 12 weeks after inoculation with Frankia.

Table 2. Complementation of Nodulation and AM formation in Lotus symrk mutants carrying CgSYMRK

Nodulation was scored 4, 8, or 15 weeks after inoculation with M. loti, and AM after 3 weeks of cocultivation with G. intraradices. Results are compiled from two independent experiments.

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