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 symptoms of the diseased stems of pitaya plants initially developed small yellow spots, which gradually spread, eventually leading to the rotting of entire stems. The occurrence of canker disease not only influences yields but also affects the quality of pitaya fruit. 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 clean RNA-Seq data and processed files were submitted to the Gene Expression Omnibus (GEO) database via the GenBank website (accession number GSE119976). 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
Sequenced raw data was filtered to obtained clean data and used for downstream bioinformatics analysis . A de novo transcriptome assembly analysis was used to identify LRR genes using the Trinity paired-end assembly method .The assembled clean reads were clustered and eliminated any redundancy in obtaining unigenes using the TIGR Gene Indices clustering tools (TGICL) . A Basic Local Alignment Search Tool (BLAST) analysis in the non-redundant protein sequences (nr/nt), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups of proteins (COG), InterPro, and SwissProt public protein databases was used to annotate and analyze these genes. 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 (Li, B, 2011). The formula of the FPKM method as following: FPKM (Gene A) = (106C) / (NL/103), which is given to be the expression of gene A; here, C is the number of fragments that are uniquely aligned to gene A, N is the total number of fragments that are uniquely aligned to all genes, and L is the number of bases on gene A . 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/, Fig 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 .
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 .
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 . After treated with DNase I (Thermo Fisher Scientific, USA), first strand cDNA synthesis was conducted according to the manufacturer’s protocols using the PrimeScript™ 1st strand cDNA synthesis kit (code No.6210A, Takara, Japan). The cDNA template of N2 and N3 samples were balanced to mixed as total N sample, same treatment with the cDNA of D1 and D3 to total D sample. qRT-PCR was performed using a 20-µL reaction system (ChamQTM Universal SYBR® qPCR Master Mix, Q711-02/03, Vazyme Biotech Co., Ltd, Beijing, China), containing 10 µL of ChamQ Universal SYBRqPCR Master Mix (2×), 2 µL cDNA templates, 0.4 µL PCR forward primer (10 µM), 0.4 µL PCR reverse primer (10 µM) and 7.2 µL of double distilled water. For 12 significantly up-regulated LRR genes with log2FoldChange (D/N) ≥1.0 (false discovery rate [FDR] ≤0.001), qRT-PCR assays (with three technical replicates) were carried out using a 7500 Applied Biosystems qRT-PCR System (Life Tech, 81 Wyman Street, Waltham, MA, 02454, USA). The primers for the 12 LRR genes were designed using Primer Premier 6 software (www.premierbiosoft.com). The pitaya ubiquitin gene (UBQ) was used as an internal reference for data normalization . The primer information for the 12 genes and UBQ is presented in Supplenmentary Information 3. The PCR conditions were performed as following: 50°C for 3 min, 95°C for 10 min, followed by 40 cycles at 95°C for 15 s, and 60°C for 1 min. Output data were generated using the Applied Biosystems 7500 software version 2.0.6 (ABI, America). The relative quantification method (Delta-Delta cycle threshold, 2-ΔΔCT) was conducted to evaluate quantitative variation 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) “Yuenanbairou” 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 3.
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 (pH5.7), 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.