A combination of conserved and diverged responses underlies Theobroma cacao's defense response to Phytophthora palmivora

Abstract

Background: Plants have complex and dynamic immune systems that have evolved to resist pathogens. Humans have worked to enhance these defenses in crops through breeding. However, many crops harbor only a fraction of the genetic diversity present in wild relatives. Increased utilization of diverse germplasm to search for desirable traits, such as disease resistance, is therefore a valuable step towards breeding crops that are adapted to both current and emerging threats. Here, we examine diversity of defense responses across four populations of the long-generation tree crop Theobroma cacao L., as well as four non-cacao Theobroma species, with the goal of identifying genetic elements essential for protection against the oomycete pathogen Phytophthora palmivora.

Results: We began by creating a new, highly contiguous genome assembly for the P. palmivora-resistant genotype SCA 6 (Additional file 1: Tables S1-S5), deposited in GenBank under accessions CP139290-CP139299. We then used this high-quality assembly to combine RNA and whole-genome sequencing data to discover several genes and pathways associated with resistance. Many of these are unique, i.e., differentially regulated in only one of the four populations (diverged 40 k-900 k generations). Among the pathways shared across all populations is phenylpropanoid biosynthesis, a metabolic pathway with well-documented roles in plant defense. One gene in this pathway, caffeoyl shikimate esterase (CSE), was upregulated across all four populations following pathogen treatment, indicating its broad importance for cacao's defense response. Further experimental evidence suggests this gene hydrolyzes caffeoyl shikimate to create caffeic acid, an antimicrobial compound and known inhibitor of Phytophthora spp.

Conclusions: Our results indicate most expression variation associated with resistance is unique to populations. Moreover, our findings demonstrate the value of using a broad sample of evolutionarily diverged populations for revealing the genetic bases of cacao resistance to P. palmivora. This approach has promise for further revealing and harnessing valuable genetic resources in this and other long-generation plants.

Keywords: Cacao; Evolution; Genomics; Phytophthora; Plant-defense; RNA-seq.

Fig. 1

T. cacao population centers include genotypes that are resistant and susceptible to P. palmivora. A Maximum likelihood phylogeny of T. cacao genotypes based on 23,439 SNPs. White and gray boxes beside the phylogeny indicate whether genotypes were considered resistant (gray) or susceptible (black) to P. palmivora according to Fister et al. [36]. Numbers on the nodes indicate bootstrap support and colors at the tips indicate population membership: Guiana (blue), Iquitos (red), Marañón (green), and Nanay (orange). B Map displaying approximate center of current distribution for each of the four populations sampled for this study (locations are from Cornejo et al. [15]). C Pairwise F_ST estimates for each population

Fig. 2

Different sets of genes are responsible for defense against P. palmivora across four cacao populations. A Overlap of differentially expressed genes for P. palmivora treatment versus control (top) and between resistant versus susceptible genotypes (bottom). The blue, red, green, and orange bars represent genes that are only differentially expressed in Guiana Iquitos, Marañón, or Nanay, respectively. The pink bar indicates genes that are differentially expressed across all four populations. Numbers above the bars indicate the number of differentially expressed genes in that specific intersection. B Pairwise Spearman correlations of log₂ fold changes for all genes investigated in this study, for P. palmivora treatment versus control (top) and between resistant versus susceptible genotypes (bottom). The bottom off-diagonal is the Spearman correlation coefficient. The top off-diagonal is the correlation coefficient depicted as an ellipse, the shape of which depends on the size of the coefficient. Asterisks indicate statistical significance (p < 0.001), tested using Spearman’s rho. C Overlap of enriched GO terms (Fisher’s exact test: FDR-adjusted p-value < 0.05) for P. palmivora treatment versus control (top) and resistant versus susceptible genotypes (bottom). The blue, red, green, and orange bars represent GO terms that are only enriched in Guiana Iquitos, Marañón, or Nanay, respectively. The pink bar indicates GO terms that are significantly enriched across all four populations. Numbers above the bars indicate the number of enriched GO terms in that specific intersection

Fig. 3

Common functional groups underlie different sets of pathogen-responsive genes. A Enriched GO terms (Fisher’s exact test: FDR-adjusted p-value < 0.05) and their median fold change for P. palmivora treatment versus control. Colored points indicate population membership: Guiana (blue), Iquitos (red), Marañón (green), or Nanay (orange). Point size is scaled to median fold change for the differentially expressed genes belonging to that term. B Enriched GO terms (Fisher’s exact test: FDR-adjusted p-value < 0.05) and their median fold change for resistant versus susceptible genotypes. Colored points indicate population membership: Guiana (blue), Iquitos (red), Marañón (green), or Nanay (orange). Point size is scaled to median fold change for the differentially expressed genes belonging to that term. C The percentage of genes from each population that are unique, calculated for each GO term that is enriched in all four populations. Terms that are significantly enriched in P. palmivora treatment versus control are on top, and terms that are significantly enriched in resistant versus susceptible genotypes are on bottom. Each point represents the proportion of differentially expressed genes belonging to a single GO term (indicated by color) that are unique to each population. For instance, Guiana has 22 differentially expressed genes in GO:0009834, 5 of which are not differentially expressed in any other population (5/22 = 22.7%). Means are shown as open diamonds

Fig. 4

TcCSE is involved in resistance of T. cacao to P. palmivora. A Expression of TcCSE (SCA6_Chr6v1_17513) across each population for control (blue) and treatment (yellow). In each population, expression is consistently higher after treatment. However, the difference in gene expression between control samples and treatment samples was only significant in the Nanay population (FDR-adjusted p-value < 0.05). Open diamonds indicate mean expression for susceptible genotypes and open circles indicate mean expression for resistant genotypes. The top and bottom of the box and whisker plots represent the 75th and 25th percentiles, respectively. Whiskers represent 1.5 times the interquartile range. B Relative abundance (intensity) of caffeic acid in N. benthamiana plants transformed with p35s:TcCSE or a control backbone (“empty”) vector control, at both 48 and 96 h post transformation. Means are shown as open triangles. Over-expression of TcCSE results in significantly higher caffeic acid accumulation relative to controls (t-test 48 hpi: p-value = 0.0164; t-test 96 hpi: p-value = 0.0174). C Mycelial area of P. palmivora cultures grown on plates of V8 media versus plates of V8 media amended with 2 mM caffeic acid. Means are shown as open triangles. Plates amended with 2 mM caffeic acid significantly inhibited mycelial growth (t-test: p-value < 0.001). D Relative abundance of theobromine from water (“mock”) or P. palmivora zoospore droplets placed on cacao leaves, or zoospores only (not in contact with leaf). Means are shown as open diamonds. Cacao leaves challenged with zoospores accumulated significantly more theobromine than either mock inoculated or zoospore-only controls (t-tests: p < 0.001). Mock inoculated leaves had significantly more theobromine than zoospore-only controls (t-test: p-value = 0.022). E Relative abundance of caffeic acid in samples challenged with plugs of V8 media (blue) versus plugs of P. palmivora mycelia (yellow). There were no significant differences between treatment, phenotype, or the treatment:phenotype interaction (one-way ANOVA, Intensity ~ Treatment + Phenotype + Treatment:Phenotype: p > 0.05)

Fig. 5

Population branch statistics identify differentially expressed genes under selection. A Population branch statistics can estimate lineage-specific selection leading to resistant genotypes. Branch lengths represent the magnitude of allele frequency change. For loci evolving neutrally in both resistant and susceptible genotypes, differences in allele frequency between resistant and susceptible individuals of the same population (S1, R1) will be smaller than allele frequency differences between susceptible individuals from two separate populations (S1, S2) (top). For loci under selection in resistant genotypes, differences in allele frequency between resistant and susceptible individuals of the same population (S1, R1) will be greater than allele frequency differences between susceptible individuals from two separate populations (S1, S2) (bottom). High PBS scores indicate genes that are under selection among resistant individuals from a given population. B Overlap of genic and non-genic regions designated as selection candidates (top 1% of their respective PBS distributions). PBS was estimated for 5 kb upstream of each gene (top), the gene body (middle), and 5 kb downstream of each gene (bottom). The blue, red, green, and orange bars represent genes that are only designated as selection outliers in Guiana, Iquitos, Marañón, or Nanay, respectively. Numbers above the bars indicate the number of selection outliers in that specific intersection. For all three regions, selection candidates are almost exclusively found in a single population. C Venn diagrams displaying the overlap between differentially expressed genes and genes under selection in resistant genotypes. Colors indicate population membership: blue (Guiana), red (Iquitos), green (Marañón), and orange (Nanay). Differentially expressed genes that are under selection in resistant individuals from a given population (intersection of the Venn diagrams) are high-quality candidates for further experimentation

Fig. 6

Transcriptome responses in wild Theobroma species reveal orthologous defenses. A Venn diagrams displaying overlap between genes that are differentially expressed in at least one population of T. cacao and supertranscripts that are differentially expressed in 4 (top), 2–3 (middle), or 1 (bottom) non-cacao Theobroma species. B Log₂ fold changes (± SE) for genes and superstranscripts from closely related Theobroma species in orthogroup 60, berberine bridge enzymes. Cladogram represents gene family relationships. C Log₂ fold changes (± SE) for genes and superstranscripts from closely related Theobroma species in orthogroup 361, WRKY transcription factors. Cladogram represents gene family relationships