Molecular characterization and expression analysis of pitaya (Hylocereus polyrhizus) HpLRR genes in response to Neoscytalidium dimidiatum infection

Abstract

Background: Canker disease caused by Neoscytalidium dimidiatum is a devastating disease resulting in a major loss to the pitaya industry. However, resistance proteins in plants play crucial roles to against pathogen infection. Among resistance proteins, the leucine-rich repeat (LRR) protein is a major family that plays crucial roles in plant growth, development, and biotic and abiotic stress responses, especially in disease defense.

Results: In the present study, a transcriptomics analysis identified a total of 272 LRR genes, 233 of which had coding sequences (CDSs), in the plant pitaya (Hylocereus polyrhizus) in response to fungal Neoscytalidium dimidiatum infection. These genes were divided into various subgroups based on specific domains and phylogenetic analysis. Molecular characterization, functional annotation of proteins, and an expression analysis of the LRR genes were conducted. Additionally, four LRR genes (CL445.Contig4_All, Unigene28_All, CL28.Contig2_All, and Unigene2712_All, which were selected because they had the four longest CDSs were further assessed using quantitative reverse transcription PCR (qRT-PCR) at different fungal infection stages in different pitaya species (Hylocereus polyrhizus and Hylocereus undatus), in different pitaya tissues, and after treatment with salicylic acid (SA), methyl jasmonate (MeJA), and abscisic acid (ABA) hormones. The associated protein functions and roles in signaling pathways were identified.

Conclusions: This study provides a comprehensive overview of the HpLRR family genes at transcriptional level in pitaya in response to N. dimidiatum infection, it will be helpful to understand the molecular mechanism of pitaya canker disease, and lay a strong foundation for further research.

Keywords: Canker disease; Expression analysis; Leucine-rich-repeat genes; Neoscytalidium dimidiatum; Pitaya; Transcriptomics; qRT-PCR.

Fig. 1

Expression profiles of LRR-STK genes in four samples. N2: normal sample 2; N3: normal sample 3; D1: diseased sample 1; D3: diseased sample 3. LRR-STK: leucine-rich repeat receptor-like serine/threonine protein kinase

Fig. 2

Expression levels of FBXL, LRR-RLK, NBS-LRR, and other LRR subfamilies in four samples. N2: normal sample 2; N3: normal sample 3; D1: diseased sample 1; D3: diseased sample 3. FBXL: F-box/LRR-repeat protein; LRR: leucine-rich repeat; NBS: nucleotide-binding site; RLK: receptor-like kinase

Fig. 3

Phylogenetic analysis of 233 HpLRR genes with coding sequences (CDSs) divided to eight subgroups. The 9 red genes are LRR genes that were significantly up-regulated

Fig. 4

Gene structure and conserved motifs analysis of the 33 pitaya HpLRR transcriptional genes with coding sequences (CDSs) > 1.0 kb. a Gene structures: Yellow represents the gene CDS and blue represents the upstream or downstream gene sequences. b Conserved motifs of the CDS. The conserved motifs were using the MEME program. Different motifs were highlighted with different color boxes

Fig. 5

Verification of 12 differentially expressed LRR genes (DEGs) by qRT-PCR assays. Gene expression levels were measured by qRT-PCR and compared with RNA-Seq results. The histogram represents the fold changes of genes (diseased [D]/normal [N]) according to qRT-PCR, and the line chart represents gene expression according to RNA-Seq. All genes selected for qRT-PCR analysis were analyzed using three biological replicates. Error bars represent ±SD (2^-ΔΔCт) based on three experiments

Fig. 6

Conserved domains of the four LRR genes used in further research. Annotations: CL445.contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.contig2_All: LRR-STK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase

Fig. 7

Symptoms of different stages of N. dimidiatum infection of pitaya. Dpi: days post infection. a: Red-fleshed pitaya (Hylocereus polyrhizus) infected by N. dimidiatum; b: white-fleshed pitaya (Hylocereus undatus) infected by N. dimidiatum

Fig. 8

Expression profiles of four HpLRR transcriptional genes at different stages and different pitaya species of N. dimidiatum infection. (a): HpLRR genesexpression profiles in red-fleshed pitaya (Hylocereus polyrhizus) infected by N. dimidiatum; (b): HpLRR genes expression profiles in white-fleshed pitaya(Hylocereus undatus) infected by N. dimidiatum. The X-axis represents different stages of N. dimidiatum infection and the Y-axis represents the foldchange (2^-ΔΔCт) according to qRT-PCR. Annotations: CL445.Contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.Contig2_All: LRRSTK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase

Fig. 9

Expression profiles of four HpLRR genes in different pitaya tissues. The X-axis represents different pitaya tissues and the Y-axis represents the fold change (2^-ΔΔCт) according to qRT-PCR. Annotations: CL445.contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.contig2_All: LRR-STK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase

Fig. 10

Expression profiles of four HpLRR genes in response to SA, MeJA and ABA treatment. Annotations: CL445.contig4_All and Unigene2712_All: NBS-LRR; Unigene28_All and CL28.contig2_All: LRR-STK. LRR: leucine-rich repeat; NBS: nucleotide-binding site; STK: serine/threonine-protein kinase. Different letters (a, b, c) indicate significant difference of the expression of the target gene based on three technological replications [P < 0.05, Single factor ANOVA test (F test), n = 3]

Fig. 11

Conservation of functional motifs in non-angiosperm species of two genes (Unigene28_All and CL28.Contig2_All). Red arrows represent the typical conservation of LRR, while green arrows represent the typical conservation of the STKc_IRAK conserved sites. The representation of the domains is approximate and not to scale