A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts

Abstract

In legumes, Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is a component of the common symbiosis genes that are required for both root nodule (RN) and arbuscular mycorrhiza (AM) symbioses and is thought to be a decoder of Ca(2+) spiking, one of the earliest cellular responses to microbial signals. A gain-of-function mutation of CCaMK has been shown to induce spontaneous nodulation without rhizobia, but the significance of CCaMK activation in bacterial and/or fungal infection processes is not fully understood. Here we show that a gain-of-function CCaMK(T265D) suppresses loss-of-function mutations of common symbiosis genes required for the generation of Ca(2+) spiking, not only for nodule organogenesis but also for successful infection of rhizobia and AM fungi, demonstrating that the common symbiosis genes upstream of Ca(2+) spiking are required solely to activate CCaMK. In RN symbiosis, however, CCaMK(T265D) induced nodule organogenesis, but not rhizobial infection, on Nod factor receptor (NFRs) mutants. We propose a model of symbiotic signaling in host legume plants, in which CCaMK plays a key role in the coordinated induction of infection thread formation and nodule organogenesis.

Figure 1

Complementation of rhizobial infection phenotypes of non-nodulating mutants by CCaMK^T265D transformation. (a–h) Transformed plants were inoculated with DsRed-labeled Mesorhizobium loti. (a, c) Mature nodules formed on the roots of ccamk-3/CCaMK^T265D (ccamk/TD) and symrk-3/CCaMK^T265D (symrk-3/TD) after 4 weeks of inoculation. Scale bars are 1 mm. (b, d, g) Root hairs of ccamk/TD, symrk-3/TD and castor-4/CCaMK^T265D (castor/TD) 2 weeks after inoculation, shown as merged images of bright-field and fluorescence images (DsRed). Infection threads can be seen inside the curled root hairs. Scale bars are 100 μm. (e) A mature nodule section of symrk-3/TD stained with toluidine blue. The nodule was filled with differentiated bacteroids. Scale bar is 20 μm. (f, h) Nodule primordia with rhizobial infection on the roots of symrk-7/CCaMK^T265D (symrk-7/TD) and pollux-2/CCaMK^T265D (pollux/TD), shown as merged images of bright-field and fluorescence images (DsRed). Scale bars are 200 μm.

Figure 2

Complementation of mycorrhization phenotypes of ccamk-3 and upstream mutants by CCaMK^T265D or wt-CCaMK transformation. (a–j) Symbiotic phenotypes of transformed plants were observed 4 weeks after inoculation with Glomus intraradices. (a–f) Roots of ccamk-3/CCaMK^T265D (ccamk/TD), symrk-7/CCaMK^T265D (symrk/TD), castor-4/CCaMK^T265D (castor/TD), pollux-2/CCaMK^T265D (pollux/TD) and nup85-3/CCaMK^T265D (nup85/TD) as well as ccamk-3/wt-CCaMK (ccamk/TT) were filled with well developed arbuscules. (g–j) In the case of symrk-7/wt-CCaMK (symrk/TT), castor-4/wt-CCaMK (castor/TT), pollux-2/wt-CCaMK (pollux/TT) and nup85-3/wt-CCaMK (nup85/TT), mycorrhizal invasion was aborted in the epidermis and only running hyphae (g) and swollen appressoria (h, i) were observed. All scale bars are 100 μm.

Figure 3

Complementation of rhizobial infection phenotypes and mycorrhization phenotypes of cyclops mutant by CCaMK^T265D or wt-CCaMK transformation. Symbiotic phenotypes of transformed plants were observed 4 weeks after inoculation with lacZ-labelled Mesorhizobium loti (a–d) or Glomus intraradices (e–h). (a, c) Mature nodules on the roots of wild-type/CCaMK^T265D (Gifu/TD) and bump-like structures on the roots of cyclops-4/CCaMK^T265D (cyclops/TD) were formed. Scale bars are 1 mm. (b, d) Rhizobial infection was confirmed by lacZ staining. Effective nodules with rhizobial infection were formed on the roots of Gifu/TD (b), but rhizobial infection was aborted at the epidermis on bump-like structures on the roots of cyclops/TD (d). The inset shows magnified view of the aborted infection thread (arrow) and the micro-colony (arrowhead). Scale bars are 500 μm. (e–h) Roots of cyclops/TD were filled with arbuscules only occasionally (h), fungal invasion was aborted in the epidermis and only running hyphae (e) and swollen appressoria (g) were observed in the roots of cyclops-4/TD, as well as cyclops-4/wt-CCaMK (f). Scale bars are 200 μm (e,f) and 100 μm (g, h).

Figure 4

Complementation of rhizobial infection phenotypes of nfr1 and nfr5 mutants by CCaMK^T265D transformation. (a–f) Transformed plants were inoculated with DsRed-labelled M. loti. (a, b) The empty nodules formed on the roots of nfr1-4/CCaMK^T265D (nfr1/TD) and nfr5-2/CCaMK^T265D (nfr5/TD) after 4 weeks of inoculation. Scale bars are 1 mm. (c–f) Bright-field and fluorescence (DsRed) images were merged into single images. (c, d) Nodule primordia without rhizobial infection on the roots of nfr1/TD and nfr5/TD. Scale bars are 200 μm. (e, f) Root hairs of nfr1/TD and nin-2/CCaMK^T265D (nin/TD) 2 weeks after inoculation. Scale bars are 100 μm. (e) Neither bacterial colonization nor infection thread formation was observed on the roots of nfr1/TD. (f) Aberrant curled root hairs with micro-colonies were observed on the roots of nin/TD.

Figure 5

A model for regulation pathways responsible for RN and AM symbioses. (a) In response to Nod factors, the signal generated by NFR1/NFR5 splits into two pathways, one of these flows into the common symbiosis pathway (pink line). The input of another pathway (deep pink line) is prerequisite for successful infection of rhizobia. Epistasis between CYCLOPS and NSP2 on the pathway remains unclear. ITs were rarely, but initiated in cyclops (Yano et al., 2006, 2008), while no micro-colonies were observed in nsp2 (Murakami et al., 2006). Therefore, CYCLOPS appears to be downstream of NSP2 on the pathway leading to IT formation. One possible explanation is that NSP2 may be positioned on another pathway that originates from NFR1/NFR5. For nodule organogenesis, only one signal is sufficient for activation of the downstream pathway, in which LHK1, NSP1/2 and NIN are involved. CYCLOPS is not involved in nodule organogenesis. (b) In the AM symbiosis, a plausible AM pathway that bifurcates after putative receptors (grey arrow) might be converged with common symbiosis pathway (blue arrows).