Nuclear-localized and deregulated calcium- and calmodulin-dependent protein kinase activates rhizobial and mycorrhizal responses in Lotus japonicus

Abstract

The common symbiosis pathway is at the core of symbiosis signaling between plants and soil microbes. In this pathway, calcium- and calmodulin-dependent protein kinase (CCaMK) plays a crucial role in integrating the signals both in arbuscular mycorrhizal symbiosis (AMS) and in root nodule symbiosis (RNS). However, the molecular mechanism by which CCaMK coordinates AMS and RNS is largely unknown. Here, we report that the gain-of-function (GOF) variants of CCaMK without the regulatory domains activate both AMS and RNS signaling pathways in the absence of symbiotic partners. This activation requires nuclear localization of CCaMK. Enforced nuclear localization of the GOF-CCaMK variants by fusion with a canonical nuclear localization signal enhances signaling activity of AMS and RNS. The GOF-CCaMK variant triggers formation of a structure similar to the prepenetration apparatus, which guides infection of arbuscular mycorrhizal fungi to host root cells. In addition, the GOF-CCaMK variants without the regulatory domains partly restore AMS but fail to support rhizobial infection in ccamk mutants. These data indicate that AMS, the more ancient type of symbiosis, can be mainly regulated by the kinase activity of CCaMK, whereas RNS, which evolved more recently, requires complex regulation performed by the regulatory domains of CCaMK.

Figure 1.

Symbiosis-Induced Gene Expression in Wild-Type and ccamk Mutant Roots Induced by Transformation with GOF-CCaMK Variants. Induction of NIN, SbtM4, SbtS, SbtM1, and PT4 in 2-week-old hairy roots carrying each of the GOF-CCaMK variants was analyzed. Fold induction levels in wild-type Gifu (A) and in ccamk (B) were calculated compared with that of transgenic hairy roots carrying a control vector (p35S:GFP) (expression level = 1). Total RNA was extracted from more than 10 roots per experiment. Error bars indicate sd (n = 3 to 6 independent experiments).

Figure 2.

Histochemical Detection of NIN and SbtM1 Induction by GOF-CCaMK. (A) to (F) CCaMK₃₁₄^TD-NLS was introduced into transgenic plants carrying NIN_pro:GUS ([A] to [C]) or SbtM1_pro:GUS fusions ([D] to [F]) by hairy root transformation. Activity of GUS was detected 1 week ([D] and [E]) or 2 weeks after transferring 10-d-old hairy roots to sterilized soil ([A] to [C] and [F]). (A) and (B) Cross section (A) and longitudinal section (B) (100-μm thick) of the hairy root carrying CCaMK₃₁₄^TD-NLS showed the NIN_pro:GUS activity in epidermal cells. (C) The activity was also detected in dividing cortical cells which would develop into a SPN (whole mount). (D) to (F) Cross section (D) and longitudinal sections ([E] and [F]) (100-μm thick) of the hairy root containing CCaMK₃₁₄^TD-NLS showed the SbtM1_pro:GUS activity in epidermal and cortical cells. Arrows indicate cortical cells with strong GUS activity (F). (G) to (I) SbtM1 promoter GUS plants without GOF-CCaMK were infected with G. intraradices for 2 weeks and examined for the GUS activity (whole mount). The AM infected roots were stained for the GUS activity ([G] and [H]), and the AM fungi were visualized with WGA Alexa Fluor 488 (I). Arrowheads indicate cortical cells with strong GUS activity ([G] and [H]). The GUS-positive cells in (H) correspond to the arbuscule-containing cells (indicated by white arrowheads in [I]). Bars = 100 μm.

Figure 3.

Induction of a PPA by AM Fungal Infection. Transgenic hairy roots carrying SbtM1_pro:SP:Venus ([A] to [C]) or SbtM1_pro:Venus ([D] to [F]) were infected with G. intraradices for 2 weeks. The images of Venus fluorescence ([A] and [D]) and fungal structure visualized by WGA Alexa Fluor 594 ([B] and [E]) are merged ([C] and [F]). Secreted Venus fluorescent protein was detected in the apoplastic space where the AM fungal hypha had penetrated into the host cell (C). A dense cytoplasmic bridge visualized by Venus fluorescence was detected in a cortical cell adjacent to an AM hypha (F). Bars = 25 μm.

Figure 4.

Induction of a PPA-Like Structure by GOF-CCaMK. (A) Hairy roots cotransformed with CCaMK₃₁₄^TD-NLS and SbtM1_pro:Venus were observed after 3 weeks of growth under aseptic conditions. Laser transmission and Venus fluorescence images were merged. (B) A stack of images obtained along the z axis of cells corresponding to box 1 region in (A) strongly expressed SbtM1_pro:Venus and showed dense cytoplasmic bridges. (C) to (E) Venus, DsRed, and merged images in the box 2 region in (A). (C) Cortical cells expressing SbtM1_pro:Venus showed the PPA-like structure. (D) Cytosol was visualized with a transformation marker DsRed under the control of the 35S promoter. (E) Merged image of Venus (C) and DsRed (D) showed cells (arrowheads) with dense cytoplasmic bridges compared with the neighboring cells. Bars = 100 μm in (A) and 25 μm in (B) to (E).