CYCLOPS, a mediator of symbiotic intracellular accommodation

Abstract

The initiation of intracellular infection of legume roots by symbiotic rhizobia bacteria and arbuscular mycorrhiza (AM) fungi is preceded by the induction of calcium signatures in and around the nucleus of root epidermal cells. Although a calcium and calmodulin-dependent kinase (CCaMK) is a key mediator of symbiotic root responses, the decoding of the calcium signal and the molecular events downstream are only poorly understood. Here, we characterize Lotus japonicus cyclops mutants on which microbial infection was severely inhibited. In contrast, nodule organogenesis was initiated in response to rhizobia, but arrested prematurely. This arrest was overcome when a deregulated CCaMK mutant version was introduced into cyclops mutants, conferring the development of full-sized, spontaneous nodules. Because cyclops mutants block symbiotic infection but are competent for nodule development, they reveal a bifurcation of signal transduction downstream of CCaMK. We identified CYCLOPS by positional cloning. CYCLOPS carries a functional nuclear localization signal and a predicted coiled-coil domain. We observed colocalization and physical interaction between CCaMK and CYCLOPS in plant and yeast cell nuclei in the absence of symbiotic stimulation. Importantly, CYCLOPS is a phosphorylation substrate of CCaMK in vitro. Cyclops mutants of rice were impaired in AM, and rice CYCLOPS could restore symbiosis in Lotus cyclops mutants, indicating a functional conservation across angiosperms. Our results suggest that CYCLOPS forms an ancient, preassembled signal transduction complex with CCaMK that is specifically required for infection, whereas organogenesis likely requires additional yet-to-be identified CCaMK interactors or substrates.BiFC ͉ map-based cloning ͉ plant-microbe symbiosis ͉ protein phosphorylation ͉ protein-protein interaction L egume plants can establish endosymbiotic interactions with nitrogen-fixing rhizobia and phosphate-delivering arbuscular mycorrhiza (AM) fungi. Plant root hairs form a tight curl in which rhizobia are entrapped. From this closed infection pocket, the bacteria are guided by plant membrane-delimited infection threads (ITs) into the root nodule, a specialized organ developed by the plant to provide an optimized environment for nitrogen fixation (1). AM fungal hyphae are guided through epidermal and cortical cells toward the inner cortex (2), where arbuscules, highly branched intracellular symbiotic structures, are formed (3). Intracellular infection by rhizobia and AM fungi is preceded by an exchange of specific signaling molecules. Rhizobia produce lipochito-oligosaccharides (Nod factors) that activate host plant responses including root hair deformation, and preinfection thread formation, which are structures that determine the path of IT growth through the root (4), and initiation of cortical cell division (1). One of the earliest plant responses to stimulation by Nod factors is Ca 2ϩ -spiking, which consists of perinuclear oscillations of calcium concentration in root cells (5). In the legume

Nuclear-Localized and Deregulated Calcium- and Calmodulin-Dependent Protein Kinase Activates Rhizobial and Mycorrhizal Responses in Lotus japonicus

“…We also tested the T265D substituted form of CCaMK 314 , designated CCaMK 314 TD . Analysis in L. japonicus revealed that green fluorescent protein (GFP)-fused CCaMK is localized in the nucleus (Yano et al, 2008), although there is no canonical NLS in CCaMK. However, GFP-fused CCaMK 314 TD was found both in the cytosol and in the nucleus of leaf epidermal cells of Nicotiana benthamiana (see Supplemental Figures 2A to 2C online), indicating that these proteins had lost the ability to specifically localize to the nucleus.…”

“…Indeed, previous studies have identified symbiosis mutants in legume plants that abolish both AMS and RNS. Several such symbiosis (Sym) genes have been identified through studies of these mutants (Endre et al, 2002;Stracke et al, 2002;Ané et al, 2004;Lé vy et al, 2004;ImaizumiAnraku et al, 2005;Kanamori et al, 2006;Tirichine et al, 2006;Saito et al, 2007;Yano et al, 2008;Groth et al, 2010;Murray et al, 2011). A signaling pathway composed of these symbiosis genes is termed the "common Sym pathway" (Parniske, 2008).…”

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…Therefore, LYK3 is thought to mediate bacterial entry into root hairs (Catoira et al, 2001;Limpens et al, 2003;Smit et al, 2007). Downstream components of the NF signal transduction pathway in M. truncatula include DMI2, a leucine-rich repeat receptor kinase (Esseling et al, 2004), DMI1, a potassium ion channel (Ané et al, 2004;Riely et al, 2007), NENA, a nuclear pore component (Groth et al, 2010), CCaMK/DMI3, a Ca 2+ and calmodulin-dependent protein kinase (Lévy et al, 2004;Mitra et al, 2004), the CCaMK-regulated transcriptional regulator CY-CLOPS/IPD3 (Messinese et al, 2007;Yano et al, 2008;Singh et al, 2014), and various transcriptional regulators such as NSP1, NSP2, ERN1, and NIN (Kaló et al, 2005;Smit et al, 2005;Marsh et al, 2007, Middleton et al, 2007. As a consequence of NF signaling, the expression of many symbiotic genes such as the early nodulin gene ENOD11 is induced (Andriankaja et al, 2007;Hirsch et al, 2009).…”

Deep Sequencing of the Medicago truncatula Root Transcriptome Reveals a Massive and Early Interaction between Nodulation Factor and Ethylene Signals

“…These include DOESN'T MAKE INFECTIONS1 (DMI1), DMI2, and DMI3, two nucleoporins, and the DMI3-interacting protein IPD3/CYCLOPS (Messinese et al, 2007;Yano et al, 2008). DMI2 (SYMRK in L. japonicus) encodes a Leu-rich repeat (LRR)-RLK required for calcium spiking and subsequent transmission of the Nod factor signal (Endre et al, 2002;Stracke et al, 2002).…”