Co-transformed roots express DsRED1 as visible marker.
(A and B) Nodulated wild-type root (left), control root transformed with pRedRoot lacking the silencing cassette (middle), and non-nodulated DgRNAi knockdown root (right) (A) under white light and (B) with transgenic roots showing DsRED1 fluorescence.
(C–H) AM phenotype of D. glomerata (Datisca) wild type, transgenic control, and DgSYMRK knockdown roots. (C and D) Wild-type and (E and F) transgenic control roots are well colonized and show arbuscules in inner cortical cells. (G and H) Typical DgSYMRK knockdown root with no AM formation but extraradical mycelium and aborted fungal infections (H and arrow in G). Such features were not seen in Datisca wild-type or transgenic control roots and are reminiscent of those observed on L. japonicus symrk mutant roots (Figure 3). Roots were inoculated simultaneously with Frankia bacteria and G. intraradices (8 wk). Transgenic and regenerated nontransgenic roots of 27 (control) and 23 (DgSYMRK RNAi construct) plants from three independent experiments were tested. Independent transformed roots examined were n = 42 (control) and n = 55 (DgSYMRK RNAi).
Scale bars: (A and B) 2 mm; (C, E, and G) 0.1 mm; (D, F, and H) 0.02 mm.

All putative SYMRK genes encode an N-terminal signal peptide, an extracellular region with two or three LRR motifs and one imperfect LRR, a transmembrane domain, and an intracellular serine/threonine protein kinase. SYMRK regions encoding putative kinase domains exhibit conserved intron positions and phases. Bars illustrate the exon-intron and predicted protein domain structure of representative SYMRK candidates. Positions of introns are indicated by black arrowheads. Predicted protein domains are SP, signal peptide; NEC, N-terminal extracellular region; CEC, conserved extracellular region preceeding LRRs; LR(R), leucine-rich repeats; TM, transmembrane domain; and PK, protein kinase domain. Names refer to species sampled and are shaded according to their root endosymbiotic capabilities: black, endosymbiosis with Frankia bacteria (Actinorhiza) and AM formation; gray, endosymbiosis with rhizobia and AM formation; white, AM formation only. Pictograms symbolize AR, Actinorhiza; RLS, Rhizobium-Legume Symbiosis; AM, Arbuscular Mycorrhiza. Dashed frames have no phylogenetic implications. The cladogram depicts relationships of angiosperm orders as deduced by molecular markers [53,54]. The four orders containing nodulating taxa are shaded light gray. Squares at the tips of branches indicate the presence of taxa with particular root endosymbiotic phenotypes (colour code is as for sampled plants). Filled and white wedges indicate branches where taxa on order and family level have been omitted, respectively. Popular species designations refer to Alder, Alnus glutinosa; Poplar, Po. trichocarpa; Tomato, Ly. esculentum; Poppy, P. rhoeas; Rice, O. sativa; Maize, Z. mays.

Transgenic roots were identified via fluorescence of eGFP encoded on the transfer-DNA.
(A–D) L. japonicus (Lotus) symrk-10 roots transformed with the respective control vector lacking a SYMRK expression cassette.
(E–AB) Lotus symrk-10 roots transformed with Lotus (E–J), Me. truncatula (Medicago) (K–P), D. glomerata (Datisca) (Q–V) and T. majus (Tropaeolum) (W–AB) SYMRK under control of the LjSYMRK promoter region.
(A, B, E, F, K, L, Q, R, W, and X) Roots co-cultivated with G. intraradices for 3 wk. (A and B) Transgenic control roots devoid of intraradical hyphae or arbuscules, with aborted fungal infection structures within epidermal cells (B and arrow in A). Roots carrying Lotus (E and F), Medicago (K and L), Datisca (Q and R), and Tropaeolum (W and X) SYMRK with dense fungal colonization of the inner root cortex (E, K, Q, and W) and arbuscule formation in inner cortical cells (F, L, R, and X).
(C, D, G–J, M–P, S–V, and Y–AB) Root systems inoculated with M. loti expressing DsRED for 4 wk. (C and D) Transgenic control roots showing no nodules. symrk-10 root systems transformed with Lotus (G–J), Medicago (M–P), Datisca (S–V), and Tropaeolum (W–AB) SYMRK develop nodules on transgenic roots. Nodules exhibit pink coloration in white light, indicating the presence of symbiosis-specific leghemoglobins (I, O, U, and AA) and DsRED fluorescence in inner nodule tissue indicating the presence of M. loti (J, P, V, and AB).
Scale bars: (A, E, K, Q, and W) 0.1 mm; (B, F, L, R, and X) 0.02 mm; (C, D, G, H, M, N, S, T, Y, and Z) 2 mm; (I, J, O, P, U, V, AA, and AB) 0.5 mm.