Haruko Imaizumi-Anraku¹, Yoshikazu Shimoda¹, Teruyuki Hayashi², Mari Banba¹, Hiroshi Kouchi¹, Lu Han¹, Toshimasa Yamazaki³, Rintaro Suzuki³, Naoya Takeda⁴, Takaki Maekawa⁵, Makoto Hayashi¹

¹Plant Symbiosis Research Unit, NIAS, ²Nara Institute of Science and Technology, ³Biomolecular Research Unit, NIAS, ⁴National Institute for Basic Biology, 5Ludwig-Maximilians-Universität München

［Abstract］

The function of CCaMK was investigated in order to reveal molecular mechanisms in symbiosis between plants and soil microbes. Interaction between CCaMK and calmodulin was found to be essential for symbiosis between plants and rhizobia but not between plants and arbuscular mycorrhizal (AM) fungi. CCaMK also induces formation of a cytosolic structure, which is important for infection by AM fungi.

［Keywords］

Plant-microbe symbiosis, rhizobia, arbuscular mycorrhizal fungi, CCaMK, Lotus japonicus

［Background］

Nitrogen-fixing bacteria, collectively called rhizobia, infect legume plant roots to induce formation of nodules in which bacteria fix atmospheric nitrogen to ammonia that is supplied to host plants. This allows legume plants to survive in nitrogen-poor soil environments. AM fungi, on the other hand, can establish symbiosis with roughly 80% of land plant species and thereby supply host plants with phosphorus and other nutrients through hyphae developed between host roots and the rhizosphere. These symbioses are regulated by a series of host genes called common symbiosis genes. In order to better utilize the symbiosis between host plants and soil microbes for agriculture, we are investigating the molecular mechanisms involved in symbiosis. In this regard, we identified a gene, CCaMK, that plays central roles in symbiosis, among common symbiosis genes, and investigated its function.

［Results and Discussion］

1. A periodic change of cytosolic calcium concentration, calcium spiking, has been known to be one of the physiological responses of plants to symbionts. Common symbiosis genes can be classified by their roles as “upstream genes” that are essential for the calcium spiking, and as “downstream genes” that are not required for calcium spiking.
2. One of the “downstream genes”, CCaMK, causes host plants to form nodule structures even in the absence of rhizobia when the gene has been mutated to a gain-of-function state. We combined gain-of-function CCaMK with loss-of-function forms of other common symbiosis genes to clarify their roles in symbiosis. We found that “upstream genes” were only required for activation of CCaMK.
3. CCaMK consists of a kinase domain, a calmodulin-binding domain, and a calcium-binding domain. When we conferred a CCaMK mutation such that CCaMK failed to bind calmodulin, rhizobia could not infect plants, but AM fungi could. We concluded that binding between CCaMK and calmodulin is essential for symbiosis of rhizobia (Fig. 1).
4. Given the fact that roles of CCaMK are different between the two symbioses, we investigated function of CCaMK in AM symbiosis. We found that induction of host genes essential for infection of AM fungi required nuclear localization of CCaMK (Fig. 2). This fact strongly suggests that CCaMK is a decoder of calcium spiking. Furthermore, CCaMK was essential and sufficient to develop the pre-penetration apparatus, which is important for infection by AM fungi.

1. The hypothesis that CCaMK is important in symbiosis has been verified by our present studies.
2. We can now regulate host-gene expression regardless of the type of symbiont through the action of gain-of-function CCaMK. This will facilitate identification of genes that are functioning downstream of CCaMK.
3. It is expected that AM fungi and rhizobia can be more effectively utilized in agriculture by presenting the whole picture of symbiosis genes.

Fig. 1. Activation of CCaMK Step 1: Calcium ion binds to the calcium-binding domain. Step 2: The kinase domain is phosphorylated. At this step, mycorrhiza can infect host plants. Step 3: Calmodulin binds to the calmodulin-binding domain (CaMBD). At this step, rhizobia can infect host plants.	Fig. 2. Induction of mycorrhiza-symbiosis host gene SbtM1 expression by the common symbiosis gene CCaMK Localization of gain-of-function CCaMK in the cell nucleus resulted in more than 100 times expression of SbtM1 (black bar), whereas exclusion of gain-offunction CCaMK from the cell nucleus resulted in almost no induction (white bar).

[Reference]

1. Hayashi T, Banba M, Shimoda Y, Kouchi H, Hayashi M, Imaizumi-Anraku H (2010) A dominant function of CCaMK in intracellular accommodation of bacterial and fungal endosymbionts The Plant Journal 63(1):141-154
2. Shimoda Y, Han L, Yamazaki T, Suzuki R, Hayashi M, Imaizumi-Anraku H (2012) Rhizobial and fungal symbioses show different requirements for calmodulin binding to calcium calmodulin-dependent protein kinase in Lotus japonicus The Plant Cell 24(1):304-321
3. Takeda N, Maekawa T, Hayashi M (2012) Nuclear-localized and deregulated calcium- and calmodulin-dependent protein kinase activates rhizobial and mycorrhizal responses in Lotus japonicus The Plant Cell 24(2):810-822