Skip to main content

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.

Background

Plants suffer from various biotic and abiotic stresses during growth and development, and one of the most important stresses is disease caused by pathogens. Once attacked by a pathogen, plants perceive and recognize the pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) via cell surface receptors and trigger an immune response, which is known as PAMP or MAMP-triggered immunity (PTI/MTI) [1]. In pathogenic microorganism, flagellin (flg22), elongation factor Tu (EF-Tu), peptidoglycan (PGN) and lipopolysaccharide (LPS) from bacterial, with chitin, chitosan from fungal and β-glucans from oomycetes are typical PAMPs [2]. Once attacked by pathogens, plant first to activate defenses by plant pattern recognition receptors (PRRs) which located in cell surface. Surface-localized receptor kinases (RKs), especially the TM-LRR are the most known PRR proteins. One kind of the RKs is surface-localized receptor and the other are receptor-like proteins. These proteins can detect conserved PAMPs by various ligand-binding ectodomains to activate plant PTI/MTI [3]. Compared with PRR-RKs, the PRR-RLPs proteins have the same overall structures but lack an intracellular kinase domain.

In plants, there are two kinds of transmembrane receptor-like kinases (PRR-RLKs) are well-known. One kind of PRR-RKs are receptor-like serine/threonine kinases and the other are receptor histidine kinases. The RLKs are a large family of well-studied kinases with an extracellular domain, a transmembrane domain and an intracellular kinase domain in generally [4]. RLKs are sub-classed to leucine-rich repeat (LRR), lysine motifs receptor kinase (LYK) and Catharanthus roseus RLK1-like (CrRLK1L) according to its N-terminal extracellular domain and the leucine-rich repeat receptor like kinases (LRR-RLKs) were the best studied [5]. RLKs are cell surface localized and can recognize distinct ligands of microbial origin. For example, the flg22 and EF-Tu elf18 are detected by flagellin sensitive 2 (FLS2) and elongation factor Tu receptor (EFR) respectively which belong to LRR-RLKs. The chitin elicitor receptor kinases (CERK1) through homodimerization and phosphorylation mediate chitin-induced signaling [6]. The LysM-RK lysine motif receptor kinase5 (LYK5) binds fungal chitin to form a chitin inducible complex with CERK1 to induce plant immunity in Arabidopsis [7].

Not all kinases have extracellular or transmembrane domains, such as receptor-like cytoplasmic protein kinases (RLCKs). But these kinases also have catalytic domain in intracellular localization similar to RLKs [4]. Botrytis-induced kinase1 (BIK1), a plasma membrane-localized RLCK which defined as a serine/threonine kinase interact with FLS2 and brassinosteroid (BR) insensitive 1-associated kinase 1 (BAK1) to initiate plant immune responses to bacterial flagellin [8]. Another RLCK which named BR-signaling kinase 1 (BSK1) played as a substrate of the brassinosteroid receptor BR insensitive 1 (BRI1) to physically associates with the PAMP receptor FLS2 and positively regulates the PTI [9]. These results investigated that PRRs including receptor kinases (RKs), receptor-like proteins (RLPs) and their interacted kinases or proteins play important roles in plant defense of PTI process.

If PAMP-triggered immunity response is considerable, intracellular immunity is activated. Resistance (R) proteins in plants directly or indirectly recognize pathogen effectors and activate effector-triggered intracellular immunity (ETI). In plants, there is a large leucine-rich repeat (LRR) family of R proteins with the consensus amino acid (aa) sequence motif LxxLxLxxN/CxL (x denotes any aa) [10]. Most LRRs are immune receptors and mainly participate in growth, development, the ETI process of plant–pathogen interactions, and stress defense [11]. During ETI process, secreted virulence effectors are perceived by plants intracellular immune receptors. Among those immune receptors, nucleotide binding site leucine repeat receptors (NBS-LRRs) take up a large part [3]. The NBS-LRR type proteins are classified to toll/interleukin receptor (TIR)-NBS-LRR (TNL) and coiled-coil (CC)-NBS-LRR (CNL), and these are the majority resistance (R) proteins in response to pathogen infection in plants [12, 13].

The LRR family genes are mainly referenced to encode proteins with a leucine-rich repeat (LRR) conserved domain which including leucine-rich repeat receptor/receptor-like kinases (LRR-RKs/RLKs) subfamily, leucine-rich repeat receptor/receptor-like proteins (LRR-RPs/LRR-RLPs) subfamily, F-box/LRR-repeat protein (FBXL) subfamily and nucleotide binding site leucine rich repeat (NBS-LRR) subfamily that generally participated in plants immunity. In the aspect of structure, the main features of the LRR genes are their structural domain of 20–30 aa and their transmembrane domain containing 2–42 LRRs [14]. Previous studies showed that the LRR domain provides a versatile structural framework for protein–protein interactions and allows proteins to act as molecular switches in relation to pathogen recognition and defense activation [15, 16].

A typical LRR-RLK contains a N-terminal extracellular receptor domain, a single-pass transmembrane domain and a C-terminal intracellular kinase domain. The N-terminal domain perceives signals, the single-pass domain anchors the protein within the membrane and the C-terminal kinase domain transduces signals downstream via autophosphorylation [17]. In Arabidopsis thaliana, the LRR transmembrane receptor kinase FLS2 is a pattern-recognition receptor that determines the specificity of flagellin 22 (flg22) perception [18]. A Capsicum annuum (pepper) LRR protein (CaLRR1) was reported to regulate plant cell death and defense by interacting with two pathogenesis-related proteins (CaPR10 and CaPR4b) and hypersensitive induced reaction 1 protein (CaHIR1) [19]. Another C. annuum plasma membrane LRR protein (CaLRR51) acts as a positive regulator in response to bacterial Ralstonia solanacearum infection [20].

In the LRR-RLK family, the LRR receptor-like serine/threonine-protein kinase (LRR-STK) subfamily is the largest subfamily. The members play crucial roles in pathogen recognition signaling, the subsequent activation of plant defense mechanisms, and developmental control [21]. BRI1 and BAK1 are a pair of RLKs involved in BR signaling, which regulates plant growth, development, and stress responses [22, 23]. BAK1 is also an LRR-STK that acts as a signaling regulator by interacting with BRI1 in vitro and in vivo in Arabidopsis [22]. In contrast to LRR-RLKs, LRR-RPs lack a cytoplasmic kinase domain for initiation of downstream signal transduction in plants [24].

The FBXL subfamily is widely found in animals and plants, and there are approximately 40–50 aa in the N-terminus of the conserved F-box domain [25]. It was reported that F-box proteins are the substrate-recognition subunits of Skp1-Cullin-F-box protein (SCF) ubiquitin ligase complexes [26]. Genome-wide analyses showed that F-box genes respond to salt stress, heavy metals, and drought in soybean and the legume Medicago truncatula [27, 28]. Auxin-signaling F-box protein 4 (AFB4), which is similar to the auxin receptor known as Toll/interleukin-1 receptor (TIR1), plays a pivotal role in plant growth, development, and innate immunity, as shown by a combination of physiological, molecular, and genetic approaches [29]. An interesting study by Angot et.al. (2006) showed that seven type III secretion system (T3SS) effectors that contain both an LRR domain and an F-box domain promote disease in several host plants. The authors stated that this may be because they hijack their host SCF-type E3 ubiquitin ligases to interfere with the host ubiquitin/proteasome pathway to promote disease [30].

In mammals, FBXL2 was reported to interact with the pool of p85 beta subunits to control the phosphatidylinositol-3-kinase (PI3K) signaling cascade by harnessing the proteasomal degradation process [26]. Immunoprecipitation and tandem mass spectrometry (IP-MS) analysis showed that the fork head box M1 (FoxM1) transcription factor interacted with the FBXL2 protein in gastric cancer [31]. Meanwhile, Chen et al. showed that FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis [32]. Furthermore, research by Tosto et al. showed that FBXL7 overexpression was associated with Alzheimer’s disease in a study that compared Alzheimer’s disease-like transgenic mice to wild-type littermates [33]. These results show that FBXL proteins not only participate in growth, development, and disease in plants but also in mammals.

In plants, the NBS-LRR subfamily can directly associate with pathogen-derived effectors as receptors and indirectly sense effector-mediated modification of other host proteins [34]. Functioning as a molecular switch, NBS is a part of the NB-ARC (“nucleotide binding adaptor shared by APAF-1, resistance proteins, and CED-4”) domain [35]. Activated plant NBS-LRRs trigger a range of immune responses, such as the hypersensitive response (HR), which culminates in death of infected cells [36]. There are two major classes of NBS-LRRs in plants: the coiled-coil motif (CC)-NBS-LRR (CNL) subfamily and the TIR-NBS-LRR (TNL) subfamily [37]. The CC-NBS-LRR subfamily have an N-terminal CC domain, while the TIR-NBS-LRR subfamily have a TIR domain [38]. A wheat CC-NBS-LRR (TaRCR1) was reported to positively contribute to the defense response to the fungal pathogen Rhizoctonia cerealis by maintaining reactive oxygen species (ROS) homoeostasis [39]. Another CC-NBS-LRR protein Pm21 in wheat was proved to confer powdery mildew resistance [40]. In the plant Nicotiana benthamiana, overexpression of the novel fungal Plasmoparaviticola-induced TIR-NBS-LRR gene (VaRGA1) enhanced disease resistance and drought and salt tolerance [41]. Soybean resistance was improved to several mosaic virus strains by overexpression of soybean TIR-NBS-LRR type R gene GmKR3 [42]. Similar to other LRR genes, NBS-LRR genes play important roles in plant disease defense signaling.

Pitaya (Hylocereus polyrhizus and Hylocereus undatus) is an important tropical-subtropical fruit tree found in Central America, East Asia and Southeast Asia. Canker disease caused by the fungi Neoscytalidium dimidiatum is one of the most destructive and economically important diseases of in the pitaya industry [43, 44]. At present, due to the lack of reports on the pitaya genome, it is economic and efficient to study biotic and abiotic stress response genes using a high-throughput approach.

The fungal which named Neoscytalidium dimidiatum caused canker disease of pitaya has been isolated and identified in our previous work in Hainan Province, China [44]. Transcriptome-wide high-throughput RNA-Sequencing (RNA-Seq) about the expression profiles of resistant genes related to Neoscytalidium dimidiatum defense in normal and diseased stem tissues were made ensue [45]. Here, we analyzed and studied the pitaya (Hylocereus polyrhizus) LRR receptor-like (HpLRR) family resistant genes in response to N. dimidiatum infection based on previous RNA-Seq data detail. Transcriptomics, molecular characterization, and expression results regarding LRR subfamilies in pitaya after N. dimidiatum infection have not been previously reported. This study provides an overview of the subfamily classification of LRR genes in pitaya and provides a basis for future functional studies. The evolutionary history and functions of each LRR subfamily in pitaya remains to be understood in future research.

Results

Identification and phylogenetic analysis of pitaya HpLRR transcriptional genes

We have obtained the differentially expressed Hiseq data induced by pitaya canker disease through RNA-seq reported in detail in our previous study [45]. In this study, a total of 272 HpLRR transcriptional genes were identified based on previous de novo transcriptomic analysis. The heatmap analysis indicated that most LRR genes were up-regulated in diseased pitaya tissues. Among these genes, 12 (Unigene19327_All, Unigene21125_All, Unigene13635_All, CL1260.Contig2_All, CL2218.Contig1_All, Unigene19955_All, Unigene13405_All, Unigene18881_All, Unigene9087_All, Unigene13867_All, Unigene15298_All, and Unigene12636_All) were significantly up-regulated based on a log2FoldChange (D/N) ≥1.0 (FDR ≤0.001) (Supplemental Material 1). The 272 HpLRR transcriptional genes underwent a BLAST analysis (Supplemental Material 1) involving six protein databases (nr/nt, SwissProt, KEGG, COG, InterPro, and GO). Based on the BLAST analysis, these genes were annotated as belonging to the LRR-STK subfamily (135), FBXL subfamily (49), NBS-LRR subfamily (29), LRR-RLK subfamily (26), plant intracellular Ras group-related LRR (PIRL) subfamily (8), LRR transmembrane protein kinase subfamily (5), brassinosteroid LRR receptor kinase subfamily (3), and other LRR genes (17) (Table 1). Heatmaps of the 272 HpLRR gene expression levels in D1, D3, N2 and N3 samples are presented in Figs. 1 and 2. Although LRR-STK genes belong to the LRR-RLK subfamily, in order to distinguish them from common LRR-RLK genes, separate heatmaps were constructed.

Table 1 Classification of LRRs family genes based on six protein databases and phylogenetic analysis.
Fig. 1
figure1

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
figure2

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

Among the 272 HpLRR genes, 39 have no CDS, based on base sequence analysis using A plasmid Editor (ApE) software (http://jorgensen.biology.utah.edu/wayned/ape/). In contrast, some HpLRR genes had multiple CDSs. The 33 HpLRR genes with CDSs > 1.0 kb were selected for gene annotation and gene structure analysis, while the four HpLRR genes with the longest CDSs were selected for further expression analysis (at different fungal infection stages, in different pitaya tissues, and after plant hormone treatment). The aa sequence and molecular weight of the 233 HpLRR genes with CDSs varied greatly, from 30 aa/3.382 kDa to 1138 aa/128.777 kDa (Supplementary Material 2). The aa sequences of the 233 HpLRR genes were used for phylogenetic analysis using MEGA 6.0 [46], and the nine significantly up-regulated genes with CDSs are marked in red (Fig. 3). The phylogenetic analysis showed that the 233genes could be divided into eight subfamilies (Fig. 3 and Table 1). This indicated that these genes have some degree of aa-level similarity, suggesting evolutionary relationships. Nevertheless, the base and aa sequence evolutionary results were not consistent (Table 1), due to the degeneracy of codons.

Fig. 3
figure3

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

Gene structure of the 33 HpLRR transcriptional genes with CDSs > 1.0 kb

It was hard to conduct further research on all the LRR genes as there were many of them. Hence, we selected genes with CDSs > 1.0 kb for gene structure analysis and conserved motifs analysis (Fig. 4) for further study. The detailed information of these 33 genes with CDSs > 1.0 kb was showed in Table 2. All 33 HpLRR transcriptional genes have upstream and downstream sequences. CL1599.Contig2_All and CL1599.Contig1_All; CL971.Contig1_All and CL971.Contig2_All; CL1165.Contig6_All and CL1165.Contig3_All; Unigene11462_All, Unigene8930_All, and Unigene7537_All; and Unigene2712_All and CL332.Contig2_All have 99–100% similarity, indicating that each set of two or three genes have close evolutionary relationships.

Fig. 4
figure4

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

Table 2 Information of the 33 pitaya HpLRR transcriptional genes with CDSs > 1.0 kb

Verification of 12 differentially expressed LRR genes (DEGs) by qRT-PCR

Based on a PossionDis analysis, 12 genes were significantly up-regulated (|log2FoldChange (D/N)| ≥ 1.0, FDR ≤0.001) among the total set of 272 LRR genes. Among these 12 genes, seven (Unigene15298_All, Unigene21125_All, Unigene13635_All, CL1260.Contig2_All, Unigene13867_All, Unigene19327_All, and Unigene9087_All) were annotated as belonging to the LRR-STK subfamily, three (CL2218.Contig1_All, Unigene19955_All and Unigene13405_All) as belonging to the FBXL subfamily, one (Unigene18881_All) as a disease resistance gene, and one (Unigene12636_All) as belonging to the PIRL subfamily.

To verify the RNA-Seq results, qRT-PCR assays were performed. The qRT-PCR expression level trends of 11 of the 12 genes (not Unigene19955_All) were consistent with the RNA-Seq results (Fig. 5). In addition, three of the 12 (Unigene15298_All, Unigene21125_All, and CL2218.Contig1_All) have no CDS, and the number of aa of the remaining nine genes ranged from 35 to 290 (Supplementary Material 2).

Fig. 5
figure5

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

Expression profiles of four HpLRR genes under different stages of N. dimidiatum infection in different pitaya species

Four HpLRR genes (CL445.Contig4_All, Unigene28_All, CL28.Contig2_All, and Unigene2712_All) were selected for the subsequent expression analysis as they had the four longest CDSs (Supplementary Material 2). The conserved domain analysis indicated that CL445.Contig4_All and Unigene2712_All were NBS-LRR type resistance proteins, while Unigene28_All and CL28.Contig2_All were probable LRR-STKs (Fig. 6). The four genes have 3–9 LRR motifs (Fig. 6). In addition, CL445.Contig4_All and Unigene2712_All belong to the RX-CC_like family (“Coiled-coil domain of the potato virus X resistance protein and similar proteins”) and the NB-ARC family. Unigene28_All and CL28.Contig2_All have specific hits regarding the STKc_IRAK family (“Catalytic domain of the serine/threonine kinases, interleukin-1 receptor associated kinases, and related STKs”) and a common LRR-RLK (the PLN00113 superfamily). CL28.Contig2_All has a specific malectin-like domain; malectin is a novel endoplasmic reticulum carbohydrate-binding protein and a candidate player in the early steps of protein N-glycosylation [47].

Fig. 6
figure6

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

The pitaya stems presented obvious symptoms after 3 days of N. dimidiatum infection and rotted by day 15 (Fig. 7). However, compared with red-fleshed pitaya (Fig. 7a), the white-fleshed pitaya (Fig. 7b) possessed strong resistance. Due to the protective hard wax coat on the pitaya stems, the expression levels of the four LRR genes peaked after 3~4 days of infection in red-fleshed pitaya while 4~6 days in white-fleshed pitaya. The expression results (Fig. 8a) suggested that, in red fleshed pitaya (Hylocereus polyrhizus), compared with control level (0 h), CL445.Contig4_All expression first decreased and then suddenly increased on day 3, then decreased, and finally exhibited an increasing trend but remained lower than the control level. Unigene28_All and CL28.Contig2_All expression levels were increased after N. dimidiatum infection, but Unigene28_All reached a peak on day 3 while CL28.Contig2_All reached a peak on day 4. In contrast to the other three genes, Unigene2712_All expression always decreased compared with the control level. These results indicated that the four genes participated in the fungal infection response of pitaya, especially CL28.Contig2_All, which was significantly up-regulated on day 4. In contrast to the other three genes, Unigene2712_All may act as a negative regulator in plant–pathogen interactions. In the white-fleshed pitaya (Hylocereus undatus), the genes expression has the similarity trend. However, the infection time of the genes reached a peak in white-fleshed pitaya was later 1 day compared with red-fleshed pitaya (Fig. 8b). The four HpLRR genes expression files results were consistent with the infection symptoms of the two different pitaya species (Fig. 7).

Fig. 7
figure7

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
figure8

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

Tissue-specific expression profiles of the four HpLRR genes

Tissue-specific genes (also known as luxury genes) are genes whose products have specific functions in specific cell types. To investigate whether the four HpLRR genes we selected have specific functions in specific cells, tissue-specific expression profiles were obtained by qRT-PCR for 14 pitaya tissues (Fig. 9). CL445.Contig4_All was mainly expressed in the pericarp of a young green fruit; Unigene28_All was mainly expressed in the stamen, petal, and fruit pulp of both a young green fruit and a red fruit; CL28.Contig2_All was significantly expressed in the pericarp of both a young green fruit and a red fruit, and Unigene2712_All was mainly expressed in the flower bud. These results showed that Unigene28_All was significantly up-regulated and may play pivotal roles in pitaya flower and fruit growth and development.

Fig. 9
figure9

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

Expression profiles of the four HpLRR genes in response to SA, ABA, and MeJA treatments

In plants, hormones play important roles in response to a wide range of biotic and abiotic stress signaling networks. SA, ABA, jasmonates (JAs), and ethylene have crucial well-known roles in plant disease and pest resistance [48]. To better understand the four HpLRR genes’ responses to hormonal regulation of the plant–pathology interaction pathways, the expression patterns of these genes in response to SA, ABA, and MeJA treatments were assessed by qRT-PCR (Fig. 10). All four genes responded to the three hormones to some degree. Unigene28_All expression was significantly changed after 2 h of ABA treatment. CL28.Contig2_All was prominently expressed at 48 h of SA treatment. Unigene2712_All was significantly down-regulated by ABA, but significantly up-regulated by SA, reaching a peak at 24 h. The results showed that Unigene28_All and CL28.Contig2_All may play pivotal roles in the hormone-mediated disease resistance response.

Fig. 10
figure10

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]

Discussion

In the present study, we identified 272 LRR genes in pitaya in the de novo transcriptome assembly analysis, and 233 of them have CDSs. Most of these genes belonged to the LRR-RLK, NBS-LRR, or FBXL subfamilies and, within the LRR-RLK subfamily, the LRR-STK subfamily had the largest number of genes. The results of annotation and conserved domain analysis showed that LRR genes have the typical LxxLxLxxNxL domain and most of which were participated in plant disease. Despite having distinct evolutionary origins, most animals and plants need cell-surface or intracellular immune receptors with an LRR domain to detect pathogens and trigger defense responses [49]. In mammals, LRR kinases play key roles in disease, especially Parkinson’s and Crohn’s diseases [50, 51]. In plants, most of these LRRs are resistance proteins and can directly associate with pathogen-derived effectors or indirectly sense effector-mediated modification of other host proteins [52]. Diverse classes of cell-surface immune LRR receptors share important features for the initiation of resistance. The LRR genes identified in this study may play crucial roles in the pitaya immune response. The phylogenetic analysis showed that the base and aa sequence results were not consistent (Table 1) due to the degeneracy of codons. Additionally, genes with similar base sequences do not necessarily have similar functions.

Besides the three large LRR subfamilies, we also found eight PIRL genes, five LRR transmembrane protein kinase genes, three brassinosteroid related LRR receptor kinase genes, and 17 other LRR genes, such as genes for CLAVATA (CLV)-like LRR receptor kinases, the LRR protein SHOC-2-like, and the LRR receptor-like protein FASCIATED EAR2 (Fig. 2). PIRLs are a plant-specific class of LRR proteins that take part in developmental cell signaling and gene regulation, for example, in A. thaliana, PIRL1 and PIRL9 have redundant roles that are essential at a key transition point early in pollen development [53].

Most LRR-RLKs are transmembrane kinase and phosphorylate serine/threonine residues (also known as LRR-STK), and most LRR-RLKs is recognition of an extracellular ligand, which leads to activation of the intracellular kinase domain and subsequent transduction of downstream signaling pathways [54]. The five LRR transmembrane protein kinases genes that we identified may belong to the LRR-RLKs subfamily.

It has been reported that the LRR-RLK gene BAK1/SERK3 is involved in brassinosteroid signaling and is a major modulator of PAMP-triggered immunity in A. thaliana and N. benthamiana [55]. The three brassinosteroid LRR receptor kinase genes in this study may play critical roles in the brassinosteroid signaling pathway in response to N. dimidiatum infection of pitaya.

It has been reported that CLV1 is an RLK composed of an LRR-containing extracellular domain (with putative receptor function) and a cytoplasmic STK domain (linked via a transmembrane domain); it is thought to play an important role in meristem maintenance and flower development [54, 56]. Compared with CLV1, CLV2 is structurally similar but with a very short predicted cytoplasmic tail [57]. CLV3 is a stem cell-specific protein that activates the Arabidopsis CLAVATA stem cell signaling pathway [58]. These reports indicate that CLV proteins mainly participate in the growth and development of plants.

Four genes (CL445.Contig4_All, Unigene28_All, CL28.Contig2_All, and Unigene2712_All) were selected (because they had the four longest CDSs) for a further expression analysis. Among them, CL445.Contig4_All and Unigene2712_All were NBS-LRR resistance proteins, while Unigene28_All and CL28.Contig2_All were probable LRR receptor-like STK genes (Fig. 6). CL28.Contig2_All was significantly up-regulated on day 4 after N. dimidiatum infection while Unigene2712_All was significantly down-regulated in all infection stages. However, the time of the gene expression reached a peak in white-fleshed pitaya was later 1 day than that of red-fleshed pitaya (Fig. 8b). These two genes may play positive or negative roles in plant disease responses. Unigene28_All was significantly expressed on day 3 after N. dimidiatum infection and, based on the tissue-specific expression profiles, also significantly expressed in the stamen, petal, and fruit pulp of both a young green fruit and a red fruit. These results showed that Unigene28_All not only participates in pitaya flower and fruit growth and development but also in the disease resistance response. In addition, the expression levels of the four LRR genes peaked 3~4 days post infection in red-fleshed pitaya while 4~6 days in white-fleshed pitaya, suggested that it probably displayed different resistance among different pitaya species.

Plant hormones, which include SA, JAs, ABA, brassinosteroid, ethylene, and auxin, act as signals to trigger and mediate plant immune responses [59]. In plants, SA is a secondary metabolite with a central role as a signaling molecule in the activation of plant resistance to pathogens [60, 61]. Research has shown that SA accumulation was increased in infected leaves and uninfected leaves that mediated systemic acquired resistance (SAR) [62]. SA accumulation can parallel the increase in pathogenesis-related protein gene expression [61]. For example, BAK1 overexpression caused SA accumulation and deregulation of cell death control genes [63].

JAs, including jasmonic acid and the MeJA derivatives, have proved effective at improving plant stress tolerance [64]. It was reported that JAs are key signaling compounds in hypersensitive response cell death and accumulate in response to bacterial flg22 in the grape Vitis rupestris [65].

Similar to SA and JAs, ABA (a weak acid that contains 15 carbon atoms) is an important stress hormone and is involved in physiological processes [66]. ABA participates in embryogenesis, seed germination, floral transition, and so on [67, 68].

However, plant growth, development, and stress responses need multiple phytohormones involved in complex signaling network crosstalk [69]. In this study, pitaya stems were treated with SA, MeJA, and ABA. Although all four genes responded to the three hormones, CL28.Contig2_All expression reached a peak at 48 h after SA treatment and Unigene28_All was clearly regulated by ABA at 2 h. The results of hormones treatments showed that the expression of LRR family genes was regulated by plant hormones. The LRR family resistant genes and hormones related genes probably form a cross network to regulated plant disease together. Taking all the expression results together, we selected these two genes for further functional research. The subsequent analysis of conserved functional motifs in Unigene28_All and CL28.Contog2_All indicated that these two genes both have the typical LxxLxLxxNxL domain and the STRVGTIGYMAPE or SSVAxGTL/VGYLDPE conserved sites of STKc-IRAK (x denotes any aa) (Fig. 11). These domains or conserved sites may play important roles in protein function and signaling. However, in some cases, an LRR protein requires another helper or partner protein for functionality, so a complete functional study (including screening for interacting proteins) regarding Unigene28_All and CL28.Contog2_All is being carried out and the results will be published in the future.

Fig. 11
figure11

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

Canker disease caused by N. dimidiatum is one of the most serious diseases of pitaya in the main growing regions [44]. The N. dimidiatum is a fungal which has a strong specificity infection of its host pitaya. Currently, because there is a very hard technological bottleneck in genetic transformation system of pitaya, it is very difficult to investigate the gene function in pitaya by transgenic methods just like in model plants. The fungal also can’t infect the model plants like Arabidopsis thaliana, tobacco and tomato by our exploratory testing. So, it is a very hard work to identify the genes function with really deep experiments. At present, there are no reports on transcriptomics, molecular characterization, and expression analyses of LRR genes in pitaya induced by N. dimidiatum infection. From the expression profiles, we can identify the HpLRR genes in response to pitaya canker disease preliminary. These results provide important research information for a relatively poor studied pathosystem at this moment, provide a framework for further functional investigation of the pitaya LRR subfamilies, and contribute to a better understanding of the complexity of LRR genes in higher plants.

Conclusions

Canker disease caused by the fungus N. dimidiatum is one of the most important diseases because of its rapid spread and strong specificity infection in its host pitaya. The occurrence of canker disease not only poses a significant threat in yields but also affects the quality of pitaya. Currently, it is an important job to excavate the resistant genes and explain the functions of them. This study mainly analyzed the HpLRR family resistant genes structures by bioinformatics analysis and expression profiles in different pitaya tissues, different infected stages of red-fleshed pitaya (Hylocereus polyrhizus) and white-fleshed pitaya (Hylocereus undatus), and plant hormone treatments of HpLRR family resistant genes. Results of this study provided a comprehensive overview of the HpLRR family genes at transcriptional level in pitaya in response to N. dimidiatum infection and provided a basis for further in-depth functional studies.

Methods

Plant materials and growth conditions

The normal (N1, N2, and N3) and diseased (D1, D2, and D3) tissues of pitaya (Hylocereus polyrhizus) stems used for RNA-seq were collected from Ledong County, Hainan Province, China that had been grown for about 5 years under natural conditions on a plantation. The normal (N) group were healthy plants while the disease (D) group were diseased plants infected by N. dimidiatum fungal. The pitaya stems and fruits which infected by N. dimidiatum fungal appear small yellow spot initially, then spread quickly and almost all of the stems will go to rot finally. De novo transcriptome assembly and RNA-Seq were conducted using an Illumina HiSeq System by Shenzhen BGI Tech Company (Shenzhen, China). Principal component analysis (PCA) and correlations analysis were used to evaluate the repeatability of samples. After analysis, the N1 and D2 samples were abandoned due to poor repeatability. Therefore, the bioinformatics analysis and follow-up experiments were carried out based on four samples (N2, N3, D1, and D3). The accession number of RNA-sequencing data and profiles was GSE119976 in Gene Expression Omnibus database of NCBI. In addition, the pitaya stem tissues for N. dimidiatum fungal infection, hormone treatment and tissues specific expression were collected in a plantation of Hainan University not same with those used for RNA-seq. Five pitaya plants were used in each condition and displayed similar symptoms. All five pitaya plants were merged together to use for qRT-PCR experiments.

Sequences assembled and RNA-Seq bioinformatics analysis

Clean data was used for bioinformatics analysis which obtained by filtering from sequenced raw data [45]. Trinity paired-end assembly method was used to identify pitaya HpLRR family genes [70]. TIGR Gene Indices clustering tools was used to obtain unigenes by clustering the assembled clean reads and eliminating redundancy [71]. These unigenes were blasted to public proteins databases to annotate and analyze. Which including. The Expectation Maximization (RSEM) software (Version: V1.2.12; Parameter: default; http://deweylab.biostat.wisc.edu/ RSEM) was used to obtain expression level expressed in FPKM. The calculation method of unigenes’ FPKM was described as Li B [72]. The up- or down-regulated unigenes with an adjusted P-value ≤0.05 and fold change |(log2FC) ≥ 1| were defined as differentially expressed genes (DEGs) using NOIseq and PossionDis methods.

Identification and phylogenetic analysis of pitaya HpLRR transcriptional genes

The assembled unigenes were blasted to nr, GO, KEGG, COG, InterPro and SwissProt databases and the unigenes annotated to LRR family genes were selected for the next analysis. The expression levels of all HpLRR genes were analyzed by heatmap analysis using the free online data analysis platform OmicShare tools (http://www.omicshare.com/tools/, Figs. 1 and 2). Thereafter, all the sequences of 233 LRR genes with coding sequences (CDSs) were used to construct a phylogenetic tree based on 1000 bootstrap replicates and the neighbor-joining (NJ) method using MEGA 6.0 software [46].

Gene structure and phylogenetic analysis of 33 HpLRR transcriptional genes with CDSs> 1.0 kb

There were 33 HpLRR transcriptional genes with CDSs> 1.0 kb. Gene structure analysis of these genes was performed using the free online platform Gene Structure Display Server (GSDS v2.0, http://gsds.cbi.pku.edu.cn) by uploading the genes’ CDSs and assembly sequences. Conserved motifs analysis was carried on MEME program based on the amino acid sequences of the CDS. The phylogenetic analysis was carried out based on 1000 bootstrap replicates and the neighbor-joining (NJ) method using MEGA 6.0 software [46].

Verification of 12 significantly up-regulated differentially expressed LRR genes (DEGs) by quantitative reverse transcription (qRT)-PCR

In order to verify the reliability and consistency of the up or down regulated genes, all of the 12 differentially expressed LRR genes based on a PossionDis analysis were selected to perform qRT-PCR assay. The total RNA extraction of N2, N3, D1 and D3 pitaya samples were used improved cetyltrimethylammonium bromide (CTAB) method [73]. First strand cDNA synthesis of RNA was conducted according to manufacturer’s protocols (No.6210A, Takara, Japan) after treated with DNase I (Thermo Fisher Scientific, USA). Then, the N2 and N3 cDNA template were balanced mixed together to obtain total N sample, same with D1 and D3 to obtain total D sample. qRT-PCR assay was performed using a 20-μL reaction system according to the manufacturer’s protocols of ChamQ™ Universal SYBR® qPCR Master Mix kit (Q711–02/03, Vazyme Biotech Co., Ltd., Beijing, China). Twelve significantly up-regulated HpLRR family genes were selected for qRT-PCR assays using a 7500 Applied Biosystems qRT-PCR System (Life Tech, 81 Wyman Street, Waltham, MA, 02454, USA). It was proved that the pitaya ubiquitin gene (UBQ) was an great internal reference for data normalization [74]. The 12 HpLRR family genes and UBQ primers were designed using Primer Premier 6 software (www.premierbiosoft.com). The qRT-PCR conditions were performed as previous study [45]. The Delta-Delta cycle threshold (2-ΔΔCT) method was used to output data analysis based on three technical replicates.

Expression profiles of four HpLRR transcriptional genes under different stages of N. dimidiatum infection in different pitaya species

Among the total set of LRR genes, four HpLRR transcriptional genes (CL445.Contig4_All, Unigene 28_All, CL28.Contig2_All, and Unigene 2712_All) were selected for further expression research as they had the four longest CDSs. Red-fleshed pitaya (Hylocereus polyrhizus) “Jinduyihao” and white-fleshed pitaya (Hylocereus undatus) “Vietnam Bairou” tender stems were used for N. dimidiatum infection. The 5 healthy individual pitaya plants were used as control which collected from a plantation in Hainan University. Another 5 healthy individual pitaya plants were used for infected. The infected experiments were performed as following: N. dimidiatum hypha were collected after growing them in potato dextrose agar medium for 7–10 days. Before inoculation, the stems were sterilized twice using 75% ethyl alcohol. The hyphae were diluted, with 20–40 conidia in suspension, using 400X microscope magnification (Olympus Corporation, Tokyo, Japan). Inoculation was conducted by spraying the conidial suspension onto the pitaya stems. After spraying, stems were wrapped in absorbent wool with distilled water to retain moisture. The plants were then placed in an illuminated incubator at 28 °C under a 16/8 h light/dark cycle for 15 days. The pitaya stem tissues were collected for total RNA extraction after 0 h, 4 h, 12 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, and 15 days of N. dimidiatum infection. The total RNA was reversed transcribed into cDNA and the cDNA was used for qRT-PCR (as in the previous steps). The qRT-PCR primers for the four LRR genes were designed using Primer Premier 6.0 software (www.premierbiosoft.com). The primer details for the four genes are presented in Supplenmentary Information 1, 2, 3, 4.

Expression profiles of the four HpLRR genes in different tissues of pitaya

The following 14 healthy pitaya tissues were studied: (1) flower bud, (2) stamen, (3) pistil, (4) simple style, (5) petal, (6) calyx, (7–12) pericarp and fruit pulp of a young green, mature green, and red fruit, (13) tender stems, and (14) older stems were collected in pitaya plantation, Hainan province. The total RNA of the 14 tissues was extracted, reversed transcribed into cDNA, and used for qRT-PCR to obtain the expression profiles of the four selected genes.

Response of the four HpLRR genes to salicylic acid (SA), methyl jasmonate (MeJA), and abscisic acid (ABA) treatments

Healthy pitaya stems were collected from the plantation and sterilized twice using 75% ethyl alcohol before being treated with 5 mM SA solution (pH 5.7), 1 mM MeJA solution, and 100 uM ABA solution. The three solutions were all added with 0.01% Silwet-77 (Code: DE0025, Beijing BioDee Biotechnology Co. Ltd., China) to reduce the water’s surface tension. The stem tissues were collected after 0 h, 0.5 h, 1 h, 2 h, 4 h, 12 h, 24 h, and 48 h of hormone treatment. RNA extraction and qRT-PCR assays were performed as in the previous steps.

Availability of data and materials

The accession number of RNA-sequencing data and profiles was GSE119976 in Gene Expression Omnibus (GEO) database via NCBI website (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119976). The datasets generated and analyzed during the current study are available from the GEO website or corresponding author on reasonable request.

Abbreviations

LRRs:

Leucine-rich repeat genes

LRR-RLKs:

Leucine-rich repeats receptor-like kinases

NBS-LRRs:

Nucleotide binding site leucine-rich repeats

LRR-STKs:

Leucine-rich repeats receptor-like serine/threonine-protein kinases

BRI1:

Brassinosteroid-insensitive 1

BAK1:

BRI1-associated receptor kinase 1

NB-ARC:

Nucleotide binding adaptor shared by Apaf-1 resistance proteins, CED-4 domain

CC-NBS-LRR:

Coiled-coil motif nucleotide-binding site leucine-rich repeat

SA:

Salicylic acid

JA:

Jasmonates

MeJA:

Methyl jasmonate

ABA:

Abscisic acid

SAR:

Systemic acquired resistance

PR:

Pathogenesis related protein

References

  1. 1.

    Thomma BP, Nurnberger T, Joosten MH. Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell. 2011;23(1):4–15.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Newman MA, Sundelin T, Nielsen JT, Erbs G. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Front Plant Sci. 2013;4:139.

    PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Zipfel C. Plant pattern-recognition receptors. Trends Immunol. 2014;35(7):345–51.

    CAS  PubMed  Article  Google Scholar 

  4. 4.

    Jurca ME, Bottka S, Feher A. Characterization of a family of Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI). Plant Cell Rep. 2008;27(4):739–48.

    CAS  PubMed  Article  Google Scholar 

  5. 5.

    Greeff C, Roux M, Mundy J, Petersen M. Receptor-like kinase complexes in plant innate immunity. Front Plant Sci. 2012;3:209.

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Liu T, Liu Z, Song C, Hu Y, Han Z, She J, Fan F, Wang J, Jin C, Chang J, et al. Chitin-induced dimerization activates a plant immune receptor. Science. 2012;336(6085):1160–4.

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Cao Y, Liang Y, Tanaka K, Nguyen CT, Jedrzejczak RP, Joachimiak A, Stacey G. The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1. eLife. 2014;3.

  8. 8.

    Lin W, Li B, Lu D, Chen S, Zhu N, He P, Shan L. Tyrosine phosphorylation of protein kinase complex BAK1/BIK1 mediates Arabidopsis innate immunity. Proc Natl Acad Sci U S A. 2014;111(9):3632–7.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Shi H, Shen Q, Qi Y, Yan H, Nie H, Chen Y, Zhao T, Katagiri F, Tang D. BR-SIGNALING KINASE1 physically associates with FLAGELLIN SENSING2 and regulates plant innate immunity in Arabidopsis. Plant Cell. 2013;25(3):1143–57.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10.

    Kobe B, Deisenhofer J. Proteins with leucine-rich repeats. Curr Opin Struct Biol. 1995;5(3):409–16.

    CAS  PubMed  Article  Google Scholar 

  11. 11.

    Kim SH, Kwon SI, Saha D, Anyanwu NC, Gassmann W. Resistance to the Pseudomonas syringae effector HopA1 is governed by the TIR-NBS-LRR protein RPS6 and is enhanced by mutations in SRFR1. Plant Physiol. 2009;150(4):1723–32.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Van der Biezen EA, Jones JD. Plant disease-resistance proteins and the gene-for-gene concept. Trends Biochem Sci. 1998;23(12):454–6.

    PubMed  Article  Google Scholar 

  13. 13.

    Sekhwal MK, Li P, Lam I, Wang X, Cloutier S, You FM. Disease resistance gene analogs (RGAs) in plants. Int J Mol Sci. 2015;16(8):19248–90.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Song D, Li G, Song F, Zheng Z. Molecular characterization and expression analysis of OsBISERK1, a gene encoding a leucine-rich repeat receptor-like kinase, during disease resistance responses in rice. Mol Biol Rep. 2008;35(2):275–83.

    CAS  PubMed  Article  Google Scholar 

  15. 15.

    Bella J, Hindle KL, McEwan PA, Lovell SC. The leucine-rich repeat structure. Cell Mol Life Sci. 2008;65(15):2307–33.

    CAS  PubMed  Article  Google Scholar 

  16. 16.

    Williams SJ, Yin L, Foley G, Casey LW, Outram MA, Ericsson DJ, Lu J, Boden M, Dry IB, Kobe B. Structure and function of the TIR domain from the grape NLR protein RPV1. Front Plant Sci. 2016;7:1850.

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Gou X, He K, Yang H, Yuan T, Lin H, Clouse SD, Li J. Genome-wide cloning and sequence analysis of leucine-rich repeat receptor-like protein kinase genes in Arabidopsis thaliana. BMC Genomics. 2010;11:19.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  18. 18.

    Chinchilla D, Bauer Z, Regenass M, Boller T, Felix G. The Arabidopsis receptor kinase FLS2 binds flg22 and determines the specificity of flagellin perception. Plant Cell. 2006;18(2):465–76.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19.

    Hong JK, Hwang IS, Hwang BK. Functional roles of the pepper leucine-rich repeat protein and its interactions with pathogenesis-related and hypersensitive-induced proteins in plant cell death and immunity. Planta. 2017;246(3):351–64.

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Cheng W, Xiao Z, Cai H, Wang C, Hu Y, Xiao Y, Zheng Y, Shen L, Yang S, Liu Z, et al. A novel leucine-rich repeat protein, CaLRR51, acts as a positive regulator in the response of pepper to Ralstonia solanacearum infection. Mol Plant Pathol. 2017;18(8):1089–100.

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Afzal AJ, Wood AJ, Lightfoot DA. Plant receptor-like serine threonine kinases: roles in signaling and plant defense. Mol Plant Microbe Interact. 2008;21(5):507–17.

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC. BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell. 2002;110(2):213–22.

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Nam KH, Li J. BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell. 2002;110(2):203–12.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Albert I, Zhang L, Bemm H, Nurnberger T. Structure-function analysis of immune receptor AtRLP23 with its ligand nlp20 and Coreceptors AtSOBIR1 and AtBAK1. Mol Plant Microbe Interac. 2019;32(8):1038–46.

    CAS  Article  Google Scholar 

  25. 25.

    Cui HR, Zhang ZR, Lv W, Xu JN, Wang XY. Genome-wide characterization and analysis of F-box protein-encoding genes in the Malus domestica genome. Mol Genet Genomics. 2015;290(4):1435–46.

    CAS  PubMed  Article  Google Scholar 

  26. 26.

    Kuchay S, Duan SS, Schenkein E, Peschiaroli A, Saraf A, Florens L, Washburn MP, Pagano M. FBXL2-and PTPL1-mediated degradation of p110-free p85 beta regulatory subunit controls the PI (3) K signalling cascade. Nat Cell Biol. 2013;15(5):472.

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Jia Q, Xiao ZX, Wong FL, Sun S, Liang KJ, Lam HM: Genome-Wide Analyses of the Soybean F-Box Gene Family in Response to Salt Stress. Int J Mol Sci. 2017;18(4):818–34.

  28. 28.

    Song JB, Wang YX, Li HB, Li BW, Zhou ZS, Gao S, Yang ZM. The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula. Funct Integr Genomics. 2015;15(4):495–507.

    CAS  PubMed  Article  Google Scholar 

  29. 29.

    Hu Z, Keceli MA, Piisila M, Li J, Survila M, Heino P, Brader G, Palva ET, Li J. F-box protein AFB4 plays a crucial role in plant growth, development and innate immunity. Cell Res. 2012;22(4):777–81.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  30. 30.

    Angot A, Peeters N, Lechner E, Vailleau F, Baud C, Gentzbittel L, Sartorel E, Genschik P, Boucher C, Genin S. Ralstonia solanacearum requires F-box-like domain-containing type III effectors to promote disease on several host plants. Proc Natl Acad Sci U S A. 2006;103(39):14620–5.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Li LQ, Pan D, Chen H, Zhang L, Xie WJ. F-box protein FBXL2 inhibits gastric cancer proliferation by ubiquitin-mediated degradation of forkhead box M1. FEBS Lett. 2016;590(4):445–52.

    CAS  PubMed  Article  Google Scholar 

  32. 32.

    Chen BB, Glasser JR, Coon TA, Mallampalli RK. Skp-cullin-F box E3 ligase component FBXL2 ubiquitinates Aurora B to inhibit tumorigenesis. Cell Death Dis. 2013;4:e759.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Tosto G, Fu H, Vardarajan BN, Lee JH, Cheng R, Reyes-Dumeyer D, Lantigua R, Medrano M, Jimenez-Velazquez IZ, Elkind MS, et al. F-box/LRR-repeat protein 7 is genetically associated with Alzheimer's disease. Ann Clin Transl Neurol. 2015;2(8):810–20.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  34. 34.

    Tran DTN, Chung EH, Habring-Muller A, Demar M, Schwab R, Dangl JL, Weigel D, Chae E. Activation of a plant NLR complex through Heteromeric association with an autoimmune risk variant of another NLR. Curr Biol. 2017;27(8):1148–60.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Takken FL, Albrecht M, Tameling WI. Resistance proteins: molecular switches of plant defence. Curr Opin Plant Biol. 2006;9(4):383–90.

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Bentham A, Burdett H, Anderson PA, Williams SJ, Kobe B. Animal NLRs provide structural insights into plant NLR function. Ann Bot. 2017;119(5):827–702.

    PubMed  Google Scholar 

  37. 37.

    Chakraborty J, Jain A, Mukherjee D, Ghosh S, Das S. Functional diversification of structurally alike NLR proteins in plants. Plant Sci. 2018;269:85–93.

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Zhao Y, Weng Q, Song J, Ma H, Yuan J, Dong Z, Liu Y. Bioinformatics analysis of NBS-LRR encoding resistance genes in Setaria italica. Biochem Genet. 2016;54(3):232–48.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  39. 39.

    Zhu X, Lu C, Du L, Ye X, Liu X, Coules A, Zhang Z. The wheat NB-LRR gene TaRCR1 is required for host defence response to the necrotrophic fungal pathogen Rhizoctonia cerealis. Plant Biotechnol J. 2017;15(6):674–87.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Xing L, Hu P, Liu J, Witek K, Zhou S, Xu J, Zhou W, Gao L, Huang Z, Zhang R, et al. Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat. Mol Plant. 2018;11(6):874–8.

    CAS  PubMed  Article  Google Scholar 

  41. 41.

    Li X, Zhang Y, Yin L, Lu J. Overexpression of pathogen-induced grapevine TIR-NB-LRR gene VaRGA1 enhances disease resistance and drought and salt tolerance in Nicotiana benthamiana. Protoplasma. 2017;254(2):957–69.

    CAS  PubMed  Article  Google Scholar 

  42. 42.

    Xun H, Yang X, He H, Wang M, Guo P, Wang Y, Pang J, Dong Y, Feng X, Wang S, et al. Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean. Plant Mol Biol. 2019;99(1–2):95–111.

    CAS  PubMed  Article  Google Scholar 

  43. 43.

    Chuang MF, Ni HF, Yang HR, Shu SL, Lai SY, Jiang YL. First report of stem canker disease of pitaya (Hylocereus undatus and H. polyrhizus) caused by Neoscytalidium dimidiatum in Taiwan. Plant Dis. 2012;96(6):906.

    CAS  PubMed  Article  Google Scholar 

  44. 44.

    Xu M, Peng Y, Qi Z, Yan Z, Yang L, He M-D, Li Q-X, Liu C-L, Ruan Y-Z, Wei S-S, et al. Identification of Neoscytalidium dimidiatum causing canker disease of pitaya in Hainan, China. Australas Plant Pathol. 2018;47(5):547–53.

    CAS  Article  Google Scholar 

  45. 45.

    Xu M, Liu CL, Luo J, Qi Z, Yan Z, Fu Y, Wei SS, Tang H. Transcriptomic de novo analysis of pitaya (Hylocereus polyrhizus) canker disease caused by Neoscytalidium dimidiatum. BMC Genomics. 2019;20(1):10.

    PubMed  PubMed Central  Article  Google Scholar 

  46. 46.

    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  47. 47.

    Schallus T, Jaeckh C, Feher K, Palma AS, Liu Y, Simpson JC, Mackeen M, Stier G, Gibson TJ, Feizi T, et al. Malectin: a novel carbohydrate-binding protein of the endoplasmic reticulum and a candidate player in the early steps of protein N-glycosylation. Mol Biol Cell. 2008;19(8):3404–14.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Robert-Seilaniantz A, Navarro L, Bari R, Jones JD. Pathological hormone imbalances. Curr Opin Plant Biol. 2007;10(4):372–9.

    CAS  PubMed  Article  Google Scholar 

  49. 49.

    Padmanabhan M, Cournoyer P, Dinesh-Kumar SP. The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities. Cell Microbiol. 2009;11(2):191–8.

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Kang UB, Marto JA. Leucine-rich repeat kinase 2 and Parkinson's disease. Proteomics. 2017;17:1–2.

    Google Scholar 

  51. 51.

    Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, Almer S, Tysk C, O'Morain CA, Gassull M, et al. Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease. Nature. 2001;411(6837):599–603.

    CAS  PubMed  Article  Google Scholar 

  52. 52.

    Dangl JL, Jones JD. Plant pathogens and integrated defence responses to infection. Nature. 2001;411(6839):826–33.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  53. 53.

    Forsthoefel NR, Dao TP, Vernon DM. PIRL1 and PIRL9, encoding members of a novel plant-specific family of leucine-rich repeat proteins, are essential for differentiation of microspores into pollen. Planta. 2010;232(5):1101–14.

    CAS  PubMed  Article  Google Scholar 

  54. 54.

    Torii KU. Leucine-rich repeat receptor kinases in plants: structure, function, and signal transduction pathways. Int Rev Cytol. 2004;234:1–46.

    CAS  PubMed  Article  Google Scholar 

  55. 55.

    Chaparro-Garcia A, Wilkinson RC, Gimenez-Ibanez S, Findlay K, Coffey MD, Zipfel C, Rathjen JP, Kamoun S, Schornack S. The receptor-like kinase SERK3/BAK1 is required for basal resistance against the late blight pathogen phytophthora infestans in Nicotiana benthamiana. PLoS One. 2011;6(1):e16608.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  56. 56.

    Clark SE, Running MP, Meyerowitz EM. CLAVATA1, a regulator of meristem and flower development in Arabidopsis. Development. 1993;119(2):397–418.

    CAS  PubMed  Google Scholar 

  57. 57.

    Jeong S, Trotochaud AE, Clark SE. The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase. Plant Cell. 1999;11(10):1925–34.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  58. 58.

    Rojo E, Sharma VK, Kovaleva V, Raikhel NV, Fletcher JC. CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLAVATA stem cell signaling pathway. Plant Cell. 2002;14(5):969–77.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  59. 59.

    Liu W, Liu J, Triplett L, Leach JE, Wang GL. Novel insights into rice innate immunity against bacterial and fungal pathogens. Annu Rev Phytopathol. 2014;52:213–41.

    CAS  PubMed  Article  Google Scholar 

  60. 60.

    Yi SY, Kwon SY. How does SA signaling link the Flg22 responses? Plant Signal Behav. 2014;9(11):e972806.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  61. 61.

    An C, Mou Z. Salicylic acid and its function in plant immunity. J Integr Plant Biol. 2011;53(6):412–28.

    CAS  PubMed  Article  Google Scholar 

  62. 62.

    Aarts N, Metz M, Holub E, Staskawicz BJ, Daniels MJ, Parker JE. Different requirements for EDS1 and NDR1 by disease resistance genes define at least two R gene-mediated signaling pathways in Arabidopsis. Proc Natl Acad Sci U S A. 1998;95(17):10306–11.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  63. 63.

    Kim SY, Shang Y, Joo SH, Kim SK, Nam KH. Overexpression of BAK1 causes salicylic acid accumulation and deregulation of cell death control genes. Biochem Biophys Res Commun. 2017;484(4):781–6.

    CAS  PubMed  Article  Google Scholar 

  64. 64.

    Ahmad P, Rasool S, Gul A, Sheikh SA, Akram NA, Ashraf M, Kazi AM, Gucel S. Jasmonates: multifunctional roles in stress tolerance. Front Plant Sci. 2016;7:813.

    PubMed  PubMed Central  Google Scholar 

  65. 65.

    Chang X, Seo M, Takebayashi Y, Kamiya Y, Riemann M, Nick P. Jasmonates are induced by the PAMP flg22 but not the cell death-inducing elicitor Harpin in Vitis rupestris. Protoplasma. 2017;254(1):271–83.

    CAS  PubMed  Article  Google Scholar 

  66. 66.

    Dong T, Park Y, Hwang I. Abscisic acid: biosynthesis, inactivation, homoeostasis and signalling. Essays Biochem. 2015;58:29–48.

    PubMed  PubMed Central  Article  Google Scholar 

  67. 67.

    Li Y, Wang C, Liu X, Song J, Li H, Sui Z, Zhang M, Fang S, Chu J, Xin M, et al. Up-regulating the abscisic acid inactivation gene ZmABA8ox1b contributes to seed germination heterosis by promoting cell expansion. J Exp Bot. 2016;67(9):2889–900.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  68. 68.

    Yang W, Zhang W, Wang X. Post-translational control of ABA signalling: the roles of protein phosphorylation and ubiquitination. Plant Biotechnol J. 2017;15(1):4–14.

    CAS  PubMed  Article  Google Scholar 

  69. 69.

    Ning Y, Liu W, Wang GL. Balancing immunity and yield in crop plants. Trends Plant Sci. 2017;22(12):1069–79.

    CAS  PubMed  Article  Google Scholar 

  70. 70.

    Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011;29(7):644–52.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  71. 71.

    Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B, et al. TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics. 2003;19(5):651–2.

    CAS  PubMed  Article  Google Scholar 

  72. 72.

    Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011;12:323.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  73. 73.

    White EJ, Venter M, Hiten NF, Burger JT. Modified Cetyltrimethylammonium bromide method improves robustness and versatility: the benchmark for plant RNA extraction. Biotechnol J. 2008;3(11):1424–8.

    CAS  PubMed  Article  Google Scholar 

  74. 74.

    Fengxia YXW, Guoli G, Jin T. Cloning and sequence analysis of housekeeping genes actin and UBQ from pitaya. Guizhou Agric Sci. 2013;41(9):4.

    Google Scholar 

Download references

Acknowledgements

We thank professor Yongchuan Deng in Hainan University for providing experimental help for us.

Funding

This work was supported by the Construction of World First Class Discipline of Hainan University (RZZX201904), Species and Varieties Resources Protection Project of Ministry of Agriculture and Rural Affairs of China (18190033–1. Germplasm Protection of Pitaya), the Crop Science Postgraduate Innovation Project of Hainan University Tropical Agriculture and Forestry College (ZWCX2018007). The funders had no role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript.

Author information

Affiliations

Authors

Contributions

TH* conceived the study and participated in the design of all experiments. HJQ* took part in the experiments design and provided good idea during the research process, including the manuscript writing. XM performed the qRT-PCR analysis, statistical analysis and wrote the manuscript. LCL carried out RNA extraction, qRT-PCR validation and statistical analysis. FY, LZW and GPY conducted the bioinformatics studies. XR, CY and WSS participated in data analysis and discussion about the experiments. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Jia-Quan Huang or Hua Tang.

Ethics declarations

Ethics approval and consent to participate

Permissions were obtained from the pitaya orchard of Ledong Country, Hainan Province for the sampling and experiments used in this study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Additional file 1 Supplenmentary Information 1:

Information of all of the 272 genes.

Additional file 2 Supplenmentary Information 2:

Details information about the sequence characteristics of 233 LRR related-genes with the longest CDS.

Additional file 3 Supplenmentary Information 3:

Primers used for the genes in the manuscript.

Additional file 4 Supplenmentary Information 4:

Sequences were identified as LRR family genes on each database.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Xu, M., Liu, C., Fu, Y. et al. Molecular characterization and expression analysis of pitaya (Hylocereus polyrhizus) HpLRR genes in response to Neoscytalidium dimidiatum infection. BMC Plant Biol 20, 160 (2020). https://doi.org/10.1186/s12870-020-02368-6

Download citation

Keywords

  • Pitaya
  • Canker disease
  • Neoscytalidium dimidiatum
  • Transcriptomics
  • Leucine-rich-repeat genes
  • Expression analysis
  • qRT-PCR