The Recent Evolution of a Symbiotic Ion Channel in the Legume Family Altered Ion Conductance and Improved Functionality in Calcium Signaling

Abstract

Arbuscular mycorrhiza and the rhizobia-legume symbiosis are two major root endosymbioses that facilitate plant nutrition. In Lotus japonicus, two symbiotic cation channels, CASTOR and POLLUX, are indispensable for the induction of nuclear calcium spiking, one of the earliest plant responses to symbiotic partner recognition. During recent evolution, a single amino acid substitution in DOES NOT MAKE INFECTIONS1 (DMI1), the POLLUX putative ortholog in the closely related Medicago truncatula, rendered the channel solo sufficient for symbiosis; castor, pollux, and castor pollux double mutants of L. japonicus were rescued by DMI1 alone, while both Lj-CASTOR and Lj-POLLUX were required for rescuing a dmi1 mutant of M. truncatula. Experimental replacement of the critical serine by an alanine in the selectivity filter of Lj-POLLUX conferred a symbiotic performance indistinguishable from DMI1. Electrophysiological characterization of DMI1 and Lj-CASTOR (wildtype and mutants) by planar lipid bilayer experiments combined with calcium imaging in Human Embryonic Kidney-293 cells expressing DMI1 (the wild type and mutants) suggest that the serine-to-alanine substitution conferred reduced conductance with a long open state to DMI1 and improved its efficiency in mediating calcium oscillations. We propose that this single amino acid replacement in the selectivity filter made DMI1 solo sufficient for symbiosis, thus explaining the selective advantage of this allele at the mechanistic level.

Buffering Capacity Explains Signal Variation in Symbiotic Calcium Oscillations

“…A more detailed explanation of the nuclear localization is shown in Supplemental Figure S1. The K + channel represents the protein DMI1 (Venkateshwaran et al, 2012), which is modeled as a driver of changes in membrane voltage (Table I; Supplemental Fig. S5).…”

“…DMI1 is not the channel responsible for the release of Ca 2+ (Charpentier et al, 2008;Parniske, 2008;Venkateshwaran et al, 2012) but probably influences the activity of Ca 2+ channels. This is similar to how some K + channels act in other plants and yeast (Peiter et al, 2007).…”

“…DMI2 codes for a plasma membrane receptor-like kinase (Endre et al, 2002;Stracke et al, 2002) that is required to facilitate further signal transduction in the cell (Bersoult et al, 2005). DMI1 is a cation channel located preferentially on the inner nuclear envelope Edwards et al, 2007;Riely et al, 2007;Charpentier et al, 2008;Capoen et al, 2011;Venkateshwaran et al, 2012). DMI3 encodes a calcium calmodulin-dependent protein kinase that interacts with downstream components and is thought to be the decoder of the Ca 2+ oscillations Mitra et al, 2004;Hayashi et al, 2010).…”

A Laser Dissection-RNAseq Analysis Highlights the Activation of Cytokinin Pathways by Nod Factors in the Medicago truncatula Root Epidermis

“…SYMRK and DMI2 interact with proteins shown to be important for nodulation, which are, respectively, a mitogen-activated protein kinase kinase (MAPKK) called SYMRK-INTERACTING2 and a 3-HYDROXY-3-METHYLGLUTARYL COENZYME A REDUCTASE1 (MtHMGR1; Kevei et al, 2007). MtHMGR1 is thought to generate a secondary signal involved in triggering calcium oscillations within and around the nucleus, a key step of LCO signaling , which involves cation channels (Ané et al, 2004;Charpentier et al, 2008;Venkateshwaran et al, 2012), a calcium pump (Capoen et al, 2011), and nucleoporins (Kanamori et al, 2006;Saito et al, 2007;Groth et al, 2010). Calcium oscillations are decoded by a calcium-and calmodulin-dependent Ser/Thr protein kinase (CCamK, also known as DMI3 in M. truncatula), which interacts with and phosphorylates CYCLOPS (called INTERACTING PROTEIN OF DMI3 in M. truncatula), a DNA-binding transcriptional activator (Singh et al, 2014).…”

A role for the mevalonate pathway in early plant symbiotic signaling

“…Although these signaling components reside on the plasma membrane (10,11), the perception of symbiotic signals triggers sustained oscillations in Ca 2+ concentration both within the nucleus (nuclear Ca 2+ spiking) and around the nucleus (perinuclear Ca 2+ spiking) (12,13). As a result, the generation of second messengers transducing the signals from the plasma membrane to the nuclear envelope has long been hypothesized (12)(13)(14)(15)(16)(17)(18)(19).…”