Symbiotic Rhizobia Bacteria Trigger a Change in Localization and Dynamics of the Medicago truncatula Receptor Kinase LYK3

Abstract

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria. INTRODUCTIONPlants in the family Fabaceae (legumes) form symbiotic relationships with nitrogen-fixing rhizobial bacteria. The bacteria live in association with plant roots inside morphologically unique structures called nodules. In this mutualistic interaction, the bacteria reduce (or fix) molecular dinitrogen to ammonia, a form of nitrogen that plants can use; in exchange, the plants supply the bacteria with carbon sources. Through this intimate metabolic partnership, both plant and bacteria benefit.An exchange of signals initiates symbiosis between legumes and bacteria. Plant roots secrete flavonoids, which cause induction of bacterial genes required for synthesis of a lipochitooligosaccharide called Nod Factor (NF) (Fisher and Long, 1992). Within minutes of application of bacteria or purified NF, root hair membrane depolarization occurs (Ehrhardt et al., 1992). Within the first hour after NF treatment, cellular calcium levels begin to oscillate in root hairs and root epidermal cells (called calcium spiking) (Ehrhardt et al., 1996). A number of phenotypic changes occur in root hairs following calcium spiking. First, actively elongating root hairs stop growing and swell. The root hairs then reinitiate tip growth and grow to form a curl around a bacterial colony. Bacteria enter root hairs through plant-derived intracellular structures called infection threads. These events coincide with transcriptional changes that are largely NF dependent (Mitra e...

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

“…Therefore, it is plausible that the active GTP-bound ROP10 is partitioned into lipid rafts of the PM. Current evidence also supports the association of NF receptors with lipid rafts, such as the colocalization of LYK3 and the lipid raft-localized flotillin-like proteins FLOT2 and FLOT4 (Haney et al, 2011) and the interaction of a symbiotic remorin protein SYMREM1 with NFP and LYK3 in the raft during infection (Lefebvre et al, 2010). Thus, we hypothesize that GTPbound ROP10 is also recruited to lipid rafts during infection, where it forms a protein complex with NF receptors and associated proteins.…”

A Membrane Microdomain-Associated Protein, Arabidopsis Flot1, Is Involved in a Clathrin-Independent Endocytic Pathway and Is Required for Seedling Development

“…Apart from the well-studied clathrin-dependent endocytosis (Chen et al, 2011), other entry pathways, including membrane raftmediated endocytosis, also operate in plant cells (Murphy et al, 2005;Moscatelli et al, 2007;Bandmann and Homann, 2011). Recent studies suggested that plant flotillins play a critical role in symbiotic bacterial infection of M. truncatula (Haney and Long, 2010;Haney et al, 2011), and the recycling of PIN proteins may depend on structural sterols and membrane rafts in Arabidopsis Willemsen et al, 2003;Men et al, 2008). Although these studies have begun to provide insights into the role of endocytosis in plants, the molecular mechanisms underlying these events remain to be elucidated.…”

“…Recent studies reported that misexpression of the membrane raft protein remorin altered virus propagation (Borner et al, 2005;Raffaele et al, 2009;Titapiwatanakun et al, 2009), and plant flotillins appear to play a critical role in symbiotic bacterial infection in M. truncatula (Haney and Long, 2010;Haney et al, 2011). Arabidopsis flotillin1 (Flot1), a protein first identified in DRMs using subcellular fractionation, two-dimensional electrophoresis, and mass spectrometry, is considered to be one such membrane microdomain protein (Borner et al, 2005).…”

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

“…Bacteria enter the root hair following localized cell wall hydrolysis (Xie et al, 2012) within an inward-growing channel known as the infection thread. At the biochemical level, Nod factors elicit a complex series of changes, including ion fluxes at the root hair tip (Felle et al, 1998), periodic increases in intracellular calcium within and around the nucleus (Ehrhardt et al, 1996;de Ruijter et al, 1999;Wais et al, 2000), rearrangement of the root hair cytoskeleton (Cárdenas et al, 1998;Timmers et al, 1999), changes in phosphorylation profiles , modification of membrane protein dynamics (Haney et al, 2011), and induction of cortical cell divisions (Ehrhardt et al, 1992;Timmers et al, 1999, and refs. therein).…”

“…Nod factor receptors, which include Lysine motif domain-containing receptorlike kinase3 (LYK3) and Nodulation factor perception (NFP) in Medicago truncatula and Nodulation factor receptor1 (NFR1) and NFR5 in Lotus japonicus, are plasma membrane-localized receptor-like kinase and kinase-like (RLK) proteins (Amor et al, 2003;Smit et al, 2007;Haney et al, 2011;Moling et al, 2014) consisting of an intracellular kinase domain and an extracellular region with two or three chitin-binding Lys motifs (LysM). LysM domains bind Nod factors through their chitin backbone (Petutschnig et al, 2010;Broghammer et al, 2012) and are implicated in Nod factor recognition specificity .…”