Genome-wide analysis and characterization of the LRR-RLK gene family provides insights into anthracnose resistance in common bean

Abstract

Anthracnose, caused by the hemibiotrophic fungus Colletotrichum lindemuthianum, is a damaging disease of common beans that can drastically reduce crop yield. The most effective strategy to manage anthracnose is the use of resistant cultivars. There are many resistance loci that have been identified, mapped and associated with markers in common bean chromosomes. The Leucine-rich repeat kinase receptor protein (LRR-RLK) family is a diverse group of transmembrane receptors, which potentially recognizes pathogen-associated molecular patterns and activates an immune response. In this study, we performed in silico analyses to identify, classify, and characterize common bean LRR-RLKs, also evaluating their expression profile in response to the infection by C. lindemuthianum. By analyzing the entire genome of Phaseolus vulgaris, we could identify and classify 230 LRR-RLKs into 15 different subfamilies. The analyses of gene structures, conserved domains and motifs suggest that LRR-RLKs from the same subfamily are consistent in their exon/intron organization and composition. LRR-RLK genes were found along the 11 chromosomes of the species, including regions of proximity with anthracnose resistance markers. By investigating the duplication events within the LRR-RLK family, we associated the importance of such a family with an expansion resulting from a strong stabilizing selection. Promoter analysis was also performed, highlighting cis-elements associated with the plant response to biotic stress. With regard to the expression pattern of LRR-RLKs in response to the infection by C. lindemuthianum, we could point out several differentially expressed genes in this subfamily, which were associated to specific molecular patterns of LRR-RLKs. Our work provides a broad analysis of the LRR-RLK family in P. vulgaris, allowing an in-depth structural and functional characterization of genes and proteins of this family. From specific expression patterns related to anthracnose response, we could infer a direct participation of RLK-LRR genes in the mechanisms of resistance to anthracnose, highlighting important subfamilies for further investigations.

Figure 1

Phylogenetic analysis of LRR-RLK proteins of Phaseolus vulgaris. The different colors represent protein classification into subfamilies obtained by HMMER. Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) identification was maintained for all proteins. The outer group consists of an LRR-RLK protein from the alga Chlamydomonas reinhardtii, represented in the figure by Cr.LRR- RLK. The figure was created using MEGA-X v10.2 software (https://www.megasoftware.net).

Figure 2

Conserved motifs, LRR domains, and consensus sequences of Phaseolus vulgaris LRR-RLK proteins. If the bit value of amino acid at this position is smaller than 1, it is represented with x; $2>\text{bits}\ge 1$, with lowercase; $3>\text{bits}\ge 2$, with capital letter; $\text{bits}\ge 3$, with bold capital. The figure was created using MEME Suite v5.5.3 software (https://meme-suite.org/meme/tools/meme).

Figure 3

Chromosomal location of the genes encoding LRR-RLKs of Phaseolus vulgaris in the 11 chromosomes, highlighting their link to the subfamily represented by the colors. The figure was created using Phenogram tool (http://visualization.ritchielab.org/phenograms/plot).

Figure 4

Analysis of duplicated LRR-RLK proteins in Phaseolus vulgaris. In the circle, in blue, the chromosomes of P. vulgaris are represented by numbers from one to ten. Duplicated proteins are identified by the colored lines inside the circle, indicating their location on the chromosomes. The figure was created using TBtools v1.130 software (https://github.com/CJ-Chen/TBtools).

Figure 5

Synteny analysis between the LRR-RLK genes of Phaseolus vulgaris and Glycine max. The chromosomes of P. vulgaris are identified by Chr followed by the chromosome number. The chromosomes of G. max are identified by Gm followed by the chromosome number. Thus, Chr 01 corresponds to chromosome 1 of P. vulgaris, whereas Gm 01, corresponds to chromosome 1 of G. max. The figure was created using TBtools v1.130 software (https://github.com/CJ-Chen/TBtools).

Figure 6

Analysis of LRR-RLK genes of Phaseolus vulgaris involved in the response of resistant (Puebla 152) and susceptible (Jaguar) lines to race 73 of Colletotrichum lindemuthianum at 72 and 96 h after inoculation (hpi) compared with the time control 0 hpi. IR72 represents the resistant line inoculated at 72 hpi. IR96 represents the resistant line inoculated at 96 hpi. CRO represents the control resistant line inoculated at 0 hpi. IS72 represents the susceptible line inoculated at 72 hpi. 1S96 represents the susceptible line inoculated at 96 hpi. CS0 represents the control susceptible line at 0 hpi. The volcano plots were created using the R statistical environment and the Venn diagrams were created using the website https://bioinformatics.psb.ugent.be/webtools/Venn/.

Figure 7

Heatmap analysis of differentially expressed LRR-RLK genes of Phaseolus vulgaris in resistant (Puebla 152) and susceptible (Jaguar) lines inoculated with race 73 of Colletotrichum lindemuthianum at 72 and 96 after inoculation (hpi). From a clustering analysis, groups (A), (B), (C), (D), and (E) were defined. Hierarchical cluster and heatmap analysis were performed using the Log2(Fold change) of each comparison (IR72hai, IS72hai, IR96hai, and IS96hai compared to their respective controls) in R software version 4.3.1. The analysis employed the circlize R package with the k-means algorithm considering 5 clusters.