Figure 1.
Knockdown phenotype of CgCCaMK after Frankia inoculation.
(A-F) Longitudinal sections (6 µm) of nodule lobes stained with toluidine blue. (A, C and E) Transgenic control nodule. (B, D and F) CgCCaMK-RNAi 10 weeks after inoculation. Nodules in CgCCaMK-RNAi plants (B) were often smaller than in transgenic controls (A). The area between the meristem and the infection zone is wider in RNAi nodules (red arrow) (D). (E and F) Magnification of the fixation zone showing the presence of Frankia in cortical cells. FZ, fixation zone, IC, infected cells, IZ, infection zone, M, meristem. Bars = 100 µm. (G) Quantification of CgCCaMK mRNA levels in RNAi plants determined by real-time qPCR. Quantification was performed on 5 independent RNAi-CAM and 5 RNAi-UTR plants. CgUbi was used as a reference. The average of two independent non-transgenic control roots and three transgenic control roots is shown. Expression levels are relative to transgenic control roots. All error bars indicate standard errors of the mean of 3 technical replicates on different samples.
Table 1.
Actinorhizal nodulation in transgenic control (23 TC) and CgCCaMK knock down roots (31 RNAi-CAM and 24 RNAi-UTR).
Table 2.
Rhizophagus irregularis ( = Glomus intraradices) mycorrhization of transgenic control and CgCCamK knock-down roots.
Figure 2.
Complementation of M. truncatula dmi3 (TRV25) mutant with the CgCCaMK coding sequence under the control of the MtDMI3 promoter.
The mtdmi3 mutant was transformed with the control ProMtDMI3::MtDMI3 construct (A, C and E) or the ProMtDMI3::CgCCaMK construct (B, D and F). (A, B, C and D) semi-thin sections (6 µm) of nodules stained with toluidine blue. Nodules were obtained after inoculation with Sinorhizobium meliloti. Infected cells and infection threads are visible (arrows). (E and F) Cleared roots 6 weeks after inoculation with Rhizophagus irregularis stained with trypan blue. Fungal hyphae grew through the epidermis and exodermis and formed arbuscules and vesicles in the inner root cortex. IC: infected cells; IT: infection thread; IZ: infection zone; A: arbuscule; V: vesicle. Bars = 500 µm (A and B); 50 µm (C, D, E and F).
Figure 3.
Induction of spontaneous nodules in C. glauca roots expressing truncated CgCCaMK constructs.
(A, C and E) Frankia-infected nodule. (B, D and F) Spontaneous nodule. (A) Multi-lobed nodule 4 weeks after Frankia inoculation. (B) Small one-lobed nodule on hairy roots formed two months after transfer to hydroponics. (C, D, E and F) semi-thin sections (6 µm) of nodules stained with toluidine blue. (C) Section of a nodule lobe showing infected cortical cells and vascular system. (E) A close up of area in (C) showing hypertrophied cells containing Frankia. (D) Section of a spontaneous nodule harboring a poorly developed central vascular system and numerous cortical cell layers. (F) Close up of area in (D) showing that cortical cells are not hypertrophied and are free of bacteria. Phenolic compounds tend to accumulate in the form of droplets (arrow). CC, cortical cells; ICC, infected cortical cells; VS, vascular system. Bars = 500 µm (A and B); 100 µm (C, D, E and F).
Figure 4.
Induction of spontaneous nodules in D. trinervis roots expressing truncated CgCCaMK constructs.
(A) Multi-lobed nodules 4 weeks after inoculation with Frankia. (B) Multi-lobed spontaneous nodules on hairy roots formed two months after transfer to hydroponics. (C, D, E and F) semi-thin sections (6 µm) of nodules stained with toluidine blue. (C and E) Section of wild-type nodule harboring two lobes, a central vascular system (VS) and numerous infected cortical cells (ICC). (E) A close up of area in (C) showing the hypertrophied cells containing Frankia. (D and F) Section of a spontaneous nodule. Two lobes and a central vascular system are visible. (F) A close up of area in (D). Cortical cells are not hypertrophied and do not contain Frankia. CC, cortical cells; ICC, infected cortical cells; VS, vascular system. Bars = 500 µm (A and B); 100 µm (C, D, E and F).