Abstract
Ca(2+)/calmodulin (CaM)-dependent protein kinase (CCaMK) is a key regulator of root nodule and arbuscular mycorrhizal symbioses and is believed to be a decoder for Ca(2+) signals induced by microbial symbionts. However, it is unclear how CCaMK is activated by these microbes. Here, we investigated in vivo activation of CCaMK in symbiotic signaling, focusing mainly on the significance of and epistatic relationships among functional domains of CCaMK. Loss-of-function mutations in EF-hand motifs revealed the critical importance of the third EF hand for CCaMK activation to promote infection of endosymbionts. However, a gain-of-function mutation (T265D) in the kinase domain compensated for these loss-of-function mutations in the EF hands. Mutation of the CaM binding domain abolished CaM binding and suppressed CCaMK(T265D) activity in rhizobial infection, but not in mycorrhization, indicating that the requirement for CaM binding to CCaMK differs between root nodule and arbuscular mycorrhizal symbioses. Homology modeling and mutagenesis studies showed that the hydrogen bond network including Thr265 has an important role in the regulation of CCaMK. Based on these genetic, biochemical, and structural studies, we propose an activation mechanism of CCaMK in which root nodule and arbuscular mycorrhizal symbioses are distinguished by differential regulation of CCaMK by CaM binding.
Highlights
Legumes have developed a unique mechanism of nitrogen fixation by symbiosis with soil bacteria that are collectively termed rhizobia
To investigate the roles of these functional domains in the activation of calmodulin (CaM)–dependent protein kinase (CCaMK), we created various deletion constructs of L. japonicus CCaMK that lacked one to three of the EF-hands (1-471, 1-429, and 1-340) and construct 1-314, which retains only the kinase domain (Figure 1). These constructs were placed under the control of the cauliflower mosaic virus 35S promoter and transformed into ccamk-3 mutant plants that were defective in both rhizobial and arbuscular mycorrhizal (AM) fungal infection (Tirichine et al, 2006) by Agrobacterium rhizogenes–mediated hairy root transformation
The construct with the kinase domain and CaM binding regulatory domain (CaMBD) (1-340) did not induce spontaneous nodule formation, the truncated 1-340 protein was expressed (Figure 1; see Supplemental Figure 1 online). These results indicate that the lack of CaMBD releases CCaMK from autoinhibition, thereby allowing nodule organogenesis on the roots of ccamk-3/1-314, and that the EF-hand motifs of CCaMK are indispensable for rhizobial infection
Summary
Legumes have developed a unique mechanism of nitrogen fixation by symbiosis with soil bacteria that are collectively termed rhizobia. The symbiotic interaction between legumes and rhizobia is initiated by the recognition of plant-derived flavonoids by rhizobia, which induces the biosynthesis and secretion of lipochito-oligosaccharide signal molecules, Nodfactors (NFs), from rhizobia. NFs trigger the rhizobial infection process through root hairs and trigger cortical cell division in compatible host legumes, leading to the formation of root nodules (RNs), in which the symbiotic rhizobia reside and fix atmospheric nitrogen (Kouchi et al, 2010). In addition to the nitrogen-fixing RN symbiosis, legumes are able to establish endosymbiotic associations with arbuscular mycorrhizal (AM) fungi, which are found in more than 80% of land plants. AM fungi secrete a mixture of sulfated and nonsulfated lipochito-oligosaccharide signals (LCOs), so-called Myc-LCOs, to stimulate formation of AM in host roots (Maillet et al, 2011).
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