Cysteine-Rich Receptor-Like Kinase Gene Family Identification in the Phaseolus Genome and Comparative Analysis of Their Expression Profiles Specific to Mycorrhizal and Rhizobial Symbiosis

Abstract

Receptor-like kinases (RLKs) are conserved upstream signaling molecules that regulate several biological processes, including plant development and stress adaptation. Cysteine (C)-rich receptor-like kinases (CRKs) are an important class of RLK that play vital roles in disease resistance and cell death in plants. Genome-wide analyses of CRK genes have been carried out in Arabidopsis and rice, while functional characterization of some CRKs has been carried out in wheat and tomato in addition to Arabidopsis. A comprehensive analysis of the CRK gene family in leguminous crops has not yet been conducted, and our understanding of their roles in symbiosis is rather limited. Here, we report the comprehensive analysis of the PhaseolusCRK gene family, including identification, sequence similarity, phylogeny, chromosomal localization, gene structures, transcript expression profiles, and in silico promoter analysis. Forty-six CRK homologs were identified and phylogenetically clustered into five groups. Expression analysis suggests that PvCRK genes are differentially expressed in both vegetative and reproductive tissues. Further, transcriptomic analysis revealed that shared and unique CRK genes were upregulated during arbuscular mycorrhizal and rhizobial symbiosis. Overall, the systematic analysis of the PvCRK gene family provides valuable information for further studies on the biological roles of CRKs in various Phaseolus tissues during diverse biological processes, including Phaseolus-mycorrhiza/rhizobia symbiosis.

Keywords: CRKs; Cysteine (C)-rich receptor-like kinases; Phaseolus; RLK; Rhizobium; common bean; genome-wide identification; legume; mycorrhizal fungi.

Figure 1

Phylogenetic analysis and chromosomal distribution of Phaseolus vulgaris CRK genes. (a) Protein sequences of 46 P. vulgaris Cysteine (C)-rich receptor-like kinase (CRK) homologs were identified in the Phytozome database. The phylogenetic tree was constructed using MEGA 7 software with the neighbor-joining (NJ) tree method with 1000 bootstrap values. (b) CRK genes localized to Phaseolus chromosomes. The chromosomes are represented by blue bars that are distributed numerically. The orange bands with black triangles indicate the CRK position on the chromosome.

Figure 2

Gene structure analysis of Phaseolus cysteine-rich receptor-like kinases (CRKs). The intron–exon structures of PvCRK genes were analyzed using the Gene Structure Display Server (GSDS) database. Exons/Coding sequence (CDS) are represented by orange bars, introns by grey lines, and upstream (5′)/downstream (3′) untranslated regions (UTRs) are blue bars.

Figure 3

Identification of motifs in CRK protein sequences. MEME was used to identify motifs in the 46 P. vulgaris CRKs. Significantly overrepresented motifs are graphically depicted by bars corresponding to their predicted position. The dark blue bars are analogous to salt stress response/antifungal domain (PF01657), and the corresponding sequence logo is shown in the lower section, in which conserved amino acids are represented by one-letter abbreviations. The red boxes represent kinase domains (PF00069) and light blue represents Pkinase_Tyr (PF07714).

Figure 4

In silico expression profiles of P. vulgaris CRKs. Heat map expression profiles of CRK family genes in various tissues of P. vulgaris. The transcriptome data across different tissues were extracted by Phytozome (P. vulgaris v2.1) and the P. vulgaris gene expression atlas (PvGEA). The heat map was generated by R using the Fragments per kilobase of exon model per million reads mapped (FPKM) values of each CRK gene.

Figure 5

Gene ontology (GO) annotation and RT-qPCR validation of RNA sequencing (RNA-Seq) data from symbiont-colonized P. vulgaris roots. (a) GO term annotation of PvCRKs were summarized in three main GO categories, biological process, molecular function and cellular component. GO enrichment analysis performed using AgriGO and REVIGO platforms. Bars indicates the frequency of genes with the same term. (b) RT-qPCR analysis showing relative expression of Phaseolus CRK3, CRK12, PT-4, MYB73, and ENODL12 genes. Candidate genes were selected and corresponding transcript accumulation under mycorrhized and nodulated conditions was quantified by RT-qPCR. RT-qPCR data are the averages of three biological replicates (n > 9). Statistical significance of differences between mycorrhized and nodulated roots was determined using an unpaired two-tailed Student’s t-test (** p < 0.01). Error bars represent means ± Standard error mean (SEM). (c) Heat map of the transcriptomic data obtained through RNA-Seq showing the expression profiles of CRK3, CRK12, PT-4, MYB73 and ENODL12. Color key in red and blue color represents upregulated and downregulated genes respectively whereas, yellow represents no transcript accumulation.

Figure 6

Consolidated representation of genome-wide expression profiling of differentially expressed genes (DEGs) and CRKs in response to root symbionts in P. vulgaris. Expression pattern of DEGs in response to mycorriza or rhizobia in P. vulgaris roots tissues were obtained based on p-values of ≤0.05 and fold changes of ≥2.0 (upregulated and downregulated). (a) Global transcriptome profile of mycorrhizal fungi and rhizobia activated and repressed genes, and the number of upregulated and downregulated CRKs under each symbiotic condition. (b) Venn diagram showing the number of overlapping expression (upregulated and downregulated) of CRK genes in mycorrhized and nodulated roots (clustered into four comparison groups represented by four rounded rectangles) (http://bioinformatics.psb.ugent.be/webtools/Venn/). (c) Heat maps showing the unique and overlapping CRK gene expression patterns specific to AM and rhizobial colonization. Colour bar shows the fold-change range, with red and blue representing upregulation and downregulation, respectively.