Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field

Abstract

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.

Keywords: Moniliophthora roreri; RNA-Seq; Theobroma cacao; disease tolerance; frosty pod rot; hemibiotroph; metabolite.

Figure 1

Venn diagrams showing the numbers of genes differentially expressed between susceptible clones Pound‐7 and CATIE‐1000 and tolerant clones CATIE‐R7 and CATIE‐R4.

Figure 2

Principal coordinate analysis (PCoA) of RNA‐Seq reads (log₁₀) for three libraries for clones CATIE‐R4, CATIE‐R7, CATIE‐1000 and Pound‐7. (A) Comparisons using the top 200 highly expressed genes based on the mean number of reads averaged over all libraries. (B) Comparisons using genes differentially expressed between CATIE‐R4 and susceptible clones CATIE‐1000 and/or Pound‐7. (C) Comparisons using reads for genes differentially expressed between CATIE‐R7 and susceptible clones CATIE‐1000 and/or Pound‐7.

Figure 3

Fungal load (A), pod symptoms (B) and Moniliophthora roreri gene expression levels (C) for 43 M. roreri genes analysed over 114 naturally infected pods representing two susceptible clones (Pound‐7 and CATIE‐1000) and three tolerant clones (UF‐273, CATIE‐R7 and CATIE‐R4). Graphs show relative expression of M. roreri reference genes (Fr, % Theobroma cacao  Ref) in each pod, % chlorosis (Ch) and % necrosis (Nc) for each pod, and log₁₀ of M. roreri gene expression (%Pr) for 43 M. roreri genes for each pod.

Figure 4

Principal coordinate analysis (PCoA) of real‐time quantitative reverse transcription polymerase chain reaction expression levels for 43 M. roreri genes analysed over 114 naturally infected pods representing susceptible clones Pound‐7 and CATIE‐1000 and tolerant clones UF‐273, CATIE‐R7 and CATIE‐R4. (A) Comparisons between coordinates 1 and 2. (B) Comparisons between coordinates 1 and 3. (C) Comparisons between coordinates 2 and 3. Colours represent Pound‐7 (purple), CATIE‐1000 (red), UF‐273 (blue), CATIE‐R7 (yellow), and CATIE‐R4 (green). Shapes represent malformed green pods (squares), yellow pods with no necrosis (triangles) and necrotic pods (diamonds).

Figure 5

Moniliophthora roreri gene expression levels for MrMS and MrFR (A), MrAOX and MrLAC1 (B), MrCE8b and MrMGT (C) and MrAAO and MrTDAT (D) analysed over 114 naturally infected pods representing susceptible clones Pound‐7 and CATIE‐1000 and tolerant clones UF‐273, CATIE‐R7 and CATIE‐R4. Graphs also show % chlorosis and % necrosis (E) and relative expression for M. roreri reference genes (Fr, %Pr) (F).

Figure 6

Graphs showing pod weight (g), symptoms (chlorosis and necrosis), fungal load (Fr), and mannitol, fructose, fumaric acid, 2‐oxoglutaric acid and alanine concentrations (ng/mg dry weight) for pods of Pound‐7, CATIE‐1000, CATIE‐R7 and CATIE‐R4.