Engineered CaM2 modulates nuclear calcium oscillation and enhances legume root nodule symbiosis

The key physiological event essential to the establishment of nitrogen-fixing bacteria and phosphate-delivering arbuscular mycorrhizal symbioses is the induction of nuclear calcium oscillations that are required for endosymbioses. These regular fluctuations in nucleoplasmic calcium concentrations are generated by ion channels and a pump located at the nuclear envelope, including the CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15). However, how the CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown. Here, we demonstrate that the CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2), which, upon release of calcium, provides negative feedback to close the CNGC15s. Combining structural and evolutionary analyses of CaM residues with bioinformatic analysis, we engineered a holo-CaM2 with an increased affinity for CNGC15s. In planta, the expression of the engineered holo-CaM2 accelerates the calcium oscillation frequency, early endosymbioses signaling and is sufficient to sustain over time an enhanced root nodule symbiosis but not an increased arbuscular mycorrhization. Together, these results reveal that holo-CaM2 is a component of endosymbiosis signaling required to modulate CNGC15s activity and the downstream root nodule symbiosis pathway.

Nutrient acquisition is fundamental to life. Plants have evolved strategies to overcome soil phosphate limitation and gain access to atmospheric dinitrogen by developing beneficial associations with arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria, respectively. Unlike other crops, the vast majority of legumes have mastered associations with both endosymbionts, positioning them as key crops to develop sustainable agricultural practices in both developed and developing countries (1).The entry of nitrogen-fixing bacteria, known as rhizobia, and AM fungi into legume roots is initiated by the recognition of the endosymbiont. Host plants have plasma-membrane receptor-like kinases (2–6) that recognize rhizobial elicitors, lipochitooligosaccharides (LCOs), also known as Nod factors (7), and mycorrhizal factors composed of derivatives of LCOs and shorter chain chitooligosaccharides (8, 9). Although rhizobial and AM elicitors are recognized by different complexes of receptor-like kinases (10, 11), both symbionts require the activation of calcium oscillations in root epidermal nuclei (9, 12, 13) to set off the endosymbiosis program. In the model legume Medicago truncatula, two types of nuclear envelope localized ion channels are required to generate the calcium oscillation; the DOESN’T MAKE INFECTIONS1 (DMI1) channel and paralogs of CYCLIC NUCLEOTIDE GATED CHANNEL 15 (CNGC15) (14), and the calcium pump, MCA8 (15). Similar to the animal CNGCs, plant CNGCs are tetrameric ion channels that can include different CNGC units (16, 17). In M. truncatula, CNGC15a, CNGC15b, and CNGC15c are all involved in nuclear calcium oscillation in the root epidermis, nodulation, and arbuscular mycorrhization, suggesting that the three units could assemble into a heterocomplex at the nuclear envelope (14). However, how CNGC15s are regulated in planta to sustain a calcium oscillatory mechanism remains unknown.In this study, we demonstrate that CNGC15s are regulated by the calcium-bound form of the calmodulin 2 (holo-CaM2) in planta, which shapes the oscillatory pattern of nucleoplasmic calcium concentration by providing negative feedback on CNGC15s to cause its closure. By engineering CaM2 to generate CaM2^R91A, which specifically increased holo-CaM2 binding affinity to each CNGC15 unit, we accelerated closure of CNGC15s and increased the calcium oscillation frequency. We further show that accelerating the calcium oscillation frequency was sufficient to accelerate the early endosymbiosis signaling and that the expression of CaM2^R91A resulted in an enhanced root nodule symbiosis but not enhanced AM colonization. Our data reveal differential regulation of rhizobia and AM endosymbioses by CaM2^R91A and suggest that modulating calcium signaling can be used as a strategy to positively impact symbiosis with nitrogen-fixing bacteria.