Comparative Transcriptome and Metabolome Analysis of Resistant and Susceptible Piper Species Upon Infection by the Oomycete Phytophthora Capsici

Abstract

Phytophthora capsici is a destructive oomycete pathogen that causes devastating disease in black pepper, resulting in a significant decline in yield and economic losses. Piper nigrum (black pepper) is documented as susceptible to P. capsici, whereas its close relative Piper flaviflorum is known to be resistant. However, the molecular mechanism underlying the resistance of P. flaviflorum remains obscure. In this study, we conducted a comparative transcriptome and metabolome analysis between P. flaviflorum and P. nigrum upon P. capsici infection and found substantial differences in their gene expression profiles, with altered genes being significantly enriched in terms relating to plant-pathogen interaction, phytohormone signal transduction, and secondary metabolic pathways, including phenylpropanoid biosynthesis. Further metabolome analysis revealed the resistant P. flaviflorum to have a high background endogenous ABA reservoir and time-course-dependent accumulation of ABA and SA upon P. capsici inoculation, while the susceptible P. nigrum had a high background endogenous IAA reservoir and time-course-dependent accumulation of JA-Ile, the active form of JA. Investigation of the phenylpropanoid biosynthesis metabolome further indicated the resistant P. flaviflorum to have more accumulation of lignin precursors than the susceptible P. nigrum, resulting in a higher accumulation after inoculation. This study provides an overall characterization of biologically important pathways underlying the resistance of P. flaviflorum, which theoretically explains the advantage of using this species as rootstock for the management of oomycete pathogen in black pepper production.

Keywords: Phytophthora capsici; black pepper; pathogen defense; phenylpropanoid; phytohormones.

Figure 1

Significant differences in gene expression profiles between resistant (R; P. flaviflorum) and susceptible (S; P. nigrum) species of Piper. Expression profile clusters were identified by the maSigPro package from time-course RNA-Seq data of root samples at 0, 4, 12, 24, and 48 h post-Phytophthora capsici inoculation. x-axis: time; y-axis: gene expression. Red color indicates the resistant species, and green color indicates the susceptible species; lines represent the average of each time-group so as to visualize cluster-wide trends over time. Gene Ontology (GO) enrichment analysis was performed for the genes in each cluster; enriched biological process terms are visualized as word clouds below the cluster expression plots. Word clouds were generated by the WocEA software; font size and color denote the -log (P-value) and enrichment ratio (E-ratio), respectively.

Figure 2

Profiles of differentially expressed genes in the plant-pathogen interaction pathway. (A) KEGG pathway enrichment analysis of genes in cluster 6 from Figure 1 revealed genes higher-expressed in resistant Piper (P. flaviflorum) to be significantly enriched in plant-pathogen interaction pathway members. (B) The KEGG plant-pathogen interaction pathway map, annotated with differentially expressed genes. Red arrows indicate higher and green arrows indicate lower expression in resistant Piper; numbers indicate the number of putative genes with differential expression. (C) Heatmap of the differentially expressed genes from panel b. For the detailed gene list, refer to Supplementary Table S2.

Figure 3

Time-course-dependent accumulation of ABA and SA and patterns of differential expression for genes involved in phytohormone signaling and response in resistant Piper (R; P. flaviflorum) upon P. capsici inoculation. (A) Number and proportion of differentially expressed genes relative to all putative genes involved in phytohormone signaling. (B) Pathway distribution of the 155 differentially expressed genes involved in plant hormone signal transduction. Red color indicates higher and blue color indicates lower relative expression in resistant Piper compared with susceptible Piper. (C) Heatmap of genes from (B). (D) Principal component analysis plots of significant cluster genes involved in plant hormone signal transduction, based on RNA-seq data. For clarity, each dot represents the average expression from three replicates. (E) Time-course LC-MS/MS evaluating the plant hormone content of root samples at 0, 4, 12, 24, and 48 hpi, including abscisic acid (ABA), salicylic acid (SA), jasmonates (JA), auxins, and cytokinin (CK).

Figure 4

Enriched accumulation of lignin precursors in resistant Piper (P. flaviflorum). (A) Heatmap of all differentially expressed genes in the lignin biosynthesis pathway. (B) Diagram of the lignin biosynthesis pathway, annotated with differentially expressed genes. Red arrows indicate higher and green arrows lower expression in resistant Piper; numbers indicate the number of putative genes with differential expression. ND, not detected. (C) Time-course-dependent accumulation of lignin precursors in the LC-MS/MS data of root samples at 0, 4, 12, 24, and 48 hpi. R: resistant Piper (P. flaviflorum); S: susceptible Piper (P. nigrum).

Figure 5

Co-expression networks showing the relationships of lignin biosynthesis genes having differential expression in resistant (P. flaviflorum) and susceptible (P. nigrum) species of Piper with transcription factor, phytohormone response, and stress response genes. (A) Network for P. flaviflorum. (B) Network for P. nigrum.

Figure 6

Schematic summarizing the different transcriptome and metabolome profiles in resistant P. flaviflorum and susceptible P. nigrum upon P. capsici infection. Significant differences were observed in the expression of PTI and EIT pathway genes, endogenous phytohormone levels, phytohormone signaling and response, and also in the downstream phenylpropanoid biosynthesis pathway and lignin levels, leading to different postinfection phenotypes in resistant vs. susceptible Piper. Time-course ABA, SA, JA-Ile, and IAA levels were normalized and are presented as the relative content in resistant Piper compared with susceptible Piper; arrows after relative ratios indicate a significant increase (P < 0.05) in phytohormone levels at the indicated time point relative to 0 h for that species.