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 . 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 Fig 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.
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 , 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.
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.
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 35–290 (Supplementary Material 2).
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 .
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 one 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).
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 well-known roles in plant disease and pest resistance . 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.