Analysis of the structure and function of the LYK cluster of Medicago truncatula A17 and R108

Highlights

• The LYK gene cluster is highly divergent between Medicago truncatula genotypes.

• The essential role of LYK3 in nodulation in A17 is not conserved in R108.

• LYK2, LYK5 and LYK5bis may have accessory roles in nodulation.

• CRISPR-Cas9 allows targeting highly redundant gene families in Medicago truncatula.

Abstract

The establishment of the Legume-Rhizobia symbiosis is generally dependent on the production of rhizobial lipochitooligosaccharidic Nod factors (NFs) and their perception by plant Lysin Motif Receptor-Like Kinases (LysM-RLKs). In this study, we characterized a cluster of LysM-RLK genes implicated in strain-specific recognition in two highly divergent and widely-studied Medicago truncatula genotypes, A17 and R108. We then used reverse genetic approaches and biochemical analyses to study the function of selected genes in the clusters and the ability of their encoded proteins to bind NFs. Our study has revealed that the LYK cluster exhibits a high degree of variability among M. truncatula genotypes, which in A17 and R108 includes recent recombination events within the cluster and a transposon insertion in A17. The essential role of LYK3 in nodulation in A17 is not conserved in R108 despite similar sequences and good nodulation expression profiles. Although, LYK2, LYK5 and LYK5bis are not essential for nodulation of the two genotypes, some evidence points to accessory roles in nodulation, but not through high-affinity NF binding. This work shows that recent evolution in the LYK cluster provides a source of variation for nodulation, and potential robustness of signaling through genetic redundancy.

Introduction

The Fabaceae is one of the largest families among flowering plants and greatly contributes to animal and human nutrition through being second, behind the Poaceae, in terms of world crop production (Graham and Vance, 2003). This family is well-documented for its ability to form root-nodule, endophytic symbiotic interactions with soil nitrogen-fixing bacteria termed rhizobia, leading to their importance in sustainable agriculture and the natural environment.

The Legume-Rhizobia Symbiosis (LRS) is thought to have evolved from the more-widespread arbuscular mycorrhiza endosymbiosis (AM) (Parniske, 2008) and to have a common origin with other nitrogen–fixing nodule symbioses, including the rhizobia symbiosis with the non-legume Parasponia (van Velzen et al., 2018, Griesmann et al., 2018). Legumes and rhizobia exhibit partner specificity that is generally highly dependent on the structure of lipochitooligosaccharide (LCO) signals, called Nod factors (NFs) produced by bacteria under the control of a set of Nodulation (nod) genes (Andrews and Andrews, 2017). NFs are generally composed of three to five residues of N-acetyl glucosamine (chitin backbone), N-acylated on the terminal non-reducing sugar and they manifest strain-specific, diverse decorations at both ends of the molecule (D’Haeze and Holsters, 2002). Analysis of nodulation of different Sinorhizobium meliloti 2011 nod mutants affected in the structure of the long fatty acid chain (specified by nodFE) and the O-acetylation of the non-reducing sugar (specified by nodL) has revealed the significant effects of these substitutions for both infection and nodulation with various Medicago hosts (Ardourel et al., 1994). Purified or synthetic NFs elicit symbiotic responses on legume roots down to pM concentrations, suggesting that they are perceived by high affinity receptors (Ardourel et al., 1994).

From the plant side, the perception of NF involves Lysin Motif Receptor-Like Kinases (LysM-RLKs), a plant-specific RLK family (Bono et al., 2020). Members of this family have been shown to be located at the plasma membrane and are composed of an extracellular region (ECR) containing three LysMs, a single transmembrane-spanning helix (TM), which connects to an intracellular region (ICR) with a kinase-like domain (Buendia et al., 2018). Over the past two decades, the role of LysM-RLKs in LRS has been particularly studied in the model legumes Medicago truncatula (Mt) and Lotus japonicus (Lj), but also in legume crops such as pea (Pisum sativum – Ps) and soybean (Glycine max – Gm), and the non-legume Parasponia (Rutten et al., 2020). These studies suggest that legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobia LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation (Rutten et al., 2020). In each species, two pairs of orthologous LysM-RLKs, corresponding to MtNFP/LjNFR5 and MtLYK3/LjNFR1, have been shown to be essential for nodulation (Krönauer and Radutoiu, 2021). In M. truncatula NFP is required for all NF responses (Arrighi et al., 2006) and can be partially substituted by its orthologs from legumes and non-legumes (Bensmihen et al., 2011, Girardin et al., 2019) while LYK3 stringently regulates the perception of specific NFs for infection but is not essential for early NF responses (Catoira et al., 2001, Mitra et al., 2004, Smit et al., 2007, Bozsoki et al., 2020). NFP and LYK3 have been shown to physically interact in nodules (Moling et al., 2014), and co-expression in Nicotiana benthamiana leaves leads to a cell-death response ([Moling et al., 2014], Pietraszewska-Bogiel et al., 2013, Fliegmann et al., 2016), suggesting that they may function as a heteromer in plants. Recently, NFP and LYK3 have been shown to individually bind NFs with an affinity (Kd) in the µM range (Bozsoki et al., 2020, Gysel et al., 2021), which may suggest that a specific conformation of the two receptors or additional components is required for perception of NF.

In legumes, the MtLYK3/LjNFR1 gene is part of the orthogroup called LYK-I, which includes the CERK1 gene from non-legumes (Buendia et al., 2018). In rice (Oryza sativa), OsCERK1 plays a major role in the perception of chitin-oligomers (COs) produced by arbuscular mycorrhizal fungi (AMF) and fungal pathogens and is required for both AM and for defence responses (Miyata et al., 2014, Zhang et al., 2015, Carotenuto et al., 2017) The capacity to form AM has been lost in the Brassicaceae (Delaux et al., 2014) but studies on Arabidopsis thaliana has shown that CERK1 plays a major role in defence and in the perception of chitin-oligomers and other glucans (Delaux et al., 2014, Yang et al., 2022). In Parasponia andersonii duplication of the LYKI gene has led to PanLYK3 and PanLYK1, both of which are essential for AM and control nodulation with rhizobia, with PanLYK3 also retaining a role in CO perception (Rutten et al., 2020). In legumes the number of LYK-I genes has increased (9 genes in Mt and 5 in Lj) with many of them being located in a LYK gene cluster (Buendia et al., 2018, De Mita et al., 2014, Luu et al., 2022), which was initially identified through genomic synteny with the pea (Pisum sativum) SYM2 locus, in which variation allows this species to nodulate with specific rhizobia strains (Limpens et al., 2003). Evidence from molecular phylogeny strongly indicates that neofunctionalization of the MtLYK3/LjNFR1 gene in the LYK cluster, prior to legume speciation, allowed legumes to gain the ability to interact with specific rhizobia (Buendia et al., 2018, Bozsoki et al., 2017, Gibelin-Viala et al., 2019). M. truncatula has been chosen as one of the models for legume biology studies due to its relatively small genome (compared to other legumes) and the availability of a large number of genotypes, displaying genetic and geographic diversities (Young et al., 2011, Garmier et al., 2017). M. truncatula spp. truncatula cv Jemalong A17 (referred to here as A17) and M. truncatula spp. tricycla R108 (referred to here as R108) are the two most commonly-used genotypes for research as their genomes have been sequenced and tools have been developed for genetic and functional studies (Pecrix et al., 2018, Kaur et al., 2021). The two genotypes are very distinct as shown by phylogenomics of sequenced Medicago accessions (Zhou et al., 2017), and by analysis of various traits, including capacity to nodulate with different rhizobia strains (Young et al., 2011, Garmier et al., 2017). Recently, we have reported a variation in the LYK cluster of the two genotypes, involving the presence of a R108-specific gene, LYK2bis, which extends the nodulation specificity of R108 to S. meliloti 2011 nodL mutant producing non-O-acetylated NFs and to some natural strain isolates (Luu et al., 2022). In this article, we report on the divergence of the whole LYK cluster in the two genotypes and functional studies of specific LYK genes in the cluster.