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

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) proteins are a major family that play crucial roles in plant growth, development, and biotic and abiotic stress responses, especially in disease defence. 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 Hp LRR family genes at transcriptional level in pitaya in response to N. dimidiatum infection and provides a basis for further in-depth functional studies. NB-ARC: nucleotide binding adaptor shared 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: acid methyl ester; ABA: abscisic acid; SAR: systemic acquired resistance; PR: pathogenesis related protein.

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    protein Pm21 in wheat was proved to confer powdery mildew resistance [40]. In the plant Nicotiana benthamiana, overexpression of the novel fungal Plasmopara viticola-induced TIR-NBS-LRR gene (VaRGA1) enhanced disease resistance and drought and salt tolerance [41]. In soybean, overexpression of the TIR-NBS-LRR type R gene GmKR3 enhanced the plant's resistance to several strains of soybean mosaic virus (SMV), the closely related potyviruses bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), and the secovirus bean pod mottle virus (BPMV) [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]. [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 2). This indicated that these genes have some degree of aalevel similarity, suggesting evolutionary relationships. Nevertheless, the base and aa sequence evolutionary results were not consistent (Table 2), due to the degeneracy of codons.
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 informations of these 33 genes with CDSs >1.0 kb was showed in Table 3
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 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.
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 wellknown 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.

Discussion
In the present study, we identified 272 LRR genes in pitaya in the de novo transcriptome  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 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. 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, there is a very hard technological bottleneck to break through in building tissues culture system in pitaya. So, it is very difficult to investigate the genes functions according to transgene methods in pitaya limit to the technology of genetic transformation system. The fungal also can't infect the model plants