Genome-Wide Characterization, Evolution, and Expression Analysis of the Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Gene Family in Medicago truncatula

Abstract

Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases (RLKs) in plants. They play roles in plant growth and developmental and physiological processes, but less is known about the functions of LRR-RLKs in Medicago truncatula. Our genome-wide analysis revealed 329 LRR-RLK genes in the M.truncatula genome. Phylogenetic and classification analysis suggested that these genes could be classified into 15 groups and 24 subgroups. A total of 321 genes were mapped onto all chromosomes, and 23 tandem duplications (TDs) involving 56 genes were distributed on each chromosome except 4. Twenty-seven M.truncatula LRR-RLK segmental duplication gene pairs were colinearly related. The exon/intron organization, motif composition and arrangements were relatively conserved among members of the same groups or subgroups. Using publicly available RNAseq data and quantitative real-time polymerase chain reaction (qRT-PCR), expression profiling suggested that LRR-RLKs were differentially expressed among different tissues, while some were expressed specifically in the roots and nodules. The expression of LRR-RLKs in A17 and 4 nodule mutants under rhizobial infection showed that 36 LRR-RKLs were highly upregulated in the sickle (skl) mutant [an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant] after 12 h of rhizobium inoculation. Among these LRR-RLKs, six genes were also expressed specifically in the roots and nodules, which might be specific to the Nod factor and involved in autoregulation of the nodulation signal. Our results provide information on the LRR-RLK gene family in M. truncatula and serve as a guide for functional research of the LRR-RLKs.

Keywords: M. truncatula; evolutionary analysis; expression profiling; leucine-rich repeat receptor-like kinase (LRR-RLKs); phylogenetic analysis.

Figure 1

Phylogenetic analysis of LRR-RLKs retrieved in M. truncatula. The coding sequences for 329 MtLRR-RLKs were aligned by clusterW and the phylogenetic tree was constructed using MEGA 7.0 by the neighbor-joining method with 1000 bootstrap replicates. All MtLRR-RLKs were classified into 15 distinct groups and 24 subgroups based on the nomenclature of Arabidopsis LRR-RLKs (from I to XV) and were distinguished by different colors.

Figure 2

Genomic distribution of LRR-RLK genes across M. truncatula chromosomes. Chromosomal locations of MtLRR-RLKs were indicated based on the physical position of each gene. The positions of genes on each chromosome were drawn with Mapgene software and the number of chromosome was on the top of each chromosome. Genes with the same color represent a pair of tandem duplicated genes.

Figure 3

The Circos figure for chromosome locations with MtLRR-RLK segmental duplication links. The red lines indicate segmented duplicated gene pairs.

Figure 4

The distribution of Ka/Ks values in all tandem and segmental duplicated MtLRR-RLKs. The Ka/Ks value of each duplicated gene pair was calculated by KaKs_Calculator2.0.

Figure 5

The distribution of Divergence Time (MYA) in all tandem and segmental duplicated MtLRR-RLKs. The divergence time of each duplicated gene pair was calculated by T = Ks/2γ (γ = 1.5 × 10⁻⁸). γ is the rate of divergence for nuclear genes of dicotyledonous plants.

Figure 6

The synteny of LRR-RLK genes in different genomes of M. truncatula, Arabidopsis and soybean. (a) The synteny of AtLRR-RLK and MtLRR-RLK gene pairs. (b) The synteny of GmLRR-RLK and MtLRR-RLK gene pairs.

Figure 7

Expression profiles of MtLRR-RLK gene Medtr3g078250. (a) Expression levels of MtLRR-RLK gene Medtr3g078250 in various tissues. Expression profiles of Medtr3g078250 gene was analyzed in nine different tissues: leaf (L), petiole (Pe), stem (St), vegetablive bud (V-b), flower (Fl), pod (Po), root (Rt), root tip (R-t), nodule (Nd). The data are from three independent biological replicates, and error bars indicate standard deviations. (b) Expression of MtLRR-RLK gene Medtr3g078250 after S. meliloti infection in five M. truncatula genotypes: wild type A17, nfp, lyk3, skl, and sunn mutants. Values in the line graphs show average Trimmed Mean of M component (TMM) counts normalized to cv Jemalong A17 at 0 hpi. Error bars represent SE calculated from three independent biological replicates.

Figure 8

Expression profile of the 36 representative genes are highly expressed after rhizobium inoculation 12 h in mutant skl (http://pages.discovery.wisc.edu/~sroy/Medicago_symbiosis_transcriptome/query.php). Red arrows indicate genes that highly expressed in root, nodule and skl mutant under rhizobium infection.

Figure 9

Organ expression patterns of the MtLRR-RLKs which are highly expressed in root and nodule (https://mtgea.noble.org/v3/). Red arrows indicate genes that highly expressed in root, nodule and skl mutant under rhizobium infection.