Transcriptome and metabolome reprogramming in Vitis vinifera cv. Trincadeira berries upon infection with Botrytis cinerea

Abstract

Vitis vinifera berries are sensitive towards infection by the necrotrophic pathogen Botrytis cinerea, leading to important economic losses worldwide. The combined analysis of the transcriptome and metabolome associated with fungal infection has not been performed previously in grapes or in another fleshy fruit. In an attempt to identify the molecular and metabolic mechanisms associated with the infection, peppercorn-sized fruits were infected in the field. Green and veraison berries were collected following infection for microarray analysis complemented with metabolic profiling of primary and other soluble metabolites and of volatile emissions. The results provided evidence of a reprogramming of carbohydrate and lipid metabolisms towards increased synthesis of secondary metabolites involved in plant defence, such as trans-resveratrol and gallic acid. This response was already activated in infected green berries with the putative involvement of jasmonic acid, ethylene, polyamines, and auxins, whereas salicylic acid did not seem to be involved. Genes encoding WRKY transcription factors, pathogenesis-related proteins, glutathione S-transferase, stilbene synthase, and phenylalanine ammonia-lyase were upregulated in infected berries. However, salicylic acid signalling was activated in healthy ripening berries along with the expression of proteins of the NBS-LRR superfamily and protein kinases, suggesting that the pathogen is able to shut down defences existing in healthy ripening berries. Furthermore, this study provided metabolic biomarkers of infection such as azelaic acid, a substance known to prime plant defence responses, arabitol, ribitol, 4-amino butanoic acid, 1-O-methyl- glucopyranoside, and several fatty acids that alone or in combination can be used to monitor Botrytis infection early in the vineyard.

Keywords: Botrytis cinerea; grape ripening; grapevine; metabolome; plant defence; transcriptome..

Fig. 1.

Cluster of Trincadeira grapes at EL33 infected with B. cinerea. (A) Sporulation of the fungus was already observed by this stage in some berries of the cluster. (B) Magnification of (A). (This figure is available in colour at JXB online.)

Fig. 2.

Phenotypic and metabolic characterization of infected and mock-treated grape berries. (A, B) Fresh berry weight (A; g) and total anthocyanin content (B; absorbance at 520nm g^–1 of freeze-dried material) of infected and control berries at developmental stages of EL33 and EL35. Bars represent the standard error (SE). Statistically significant results are indicated: *, P<0.05; +, P<0.1. (C) Relative quantification of malic acid, tartaric acid, fructose, and glucose based on normalized responses. Specific chromatogram ion peaks were selected for relative quantification (based on peak height). Malate and tartrate content decreased in infected berries, whereas the content of fructose and glucose increased in these samples. Bars represent SE.

Fig. 3.

Analysis of infection- and veraison-responsive metabolites from developing berries of V. vinifera cv. Trincadeira. Soluble and volatile metabolites were profiled at developmental stages EL33 and EL35, i.e. veraison, after B. cinerea infection and were compared with non-infected berries. (A) Metabolites that were significantly increased or decreased in at least one of the comparisons (P<0.05, Wilcoxon test; *, significant). Response ratios were log₂ transformed and hierarchically clustered using Euclidian distance and complete linkage. Five clusters (CL_I–CL_V) represent the main response patterns of the monitored metabolites (Supplementary Table S3). (B) Venn diagram of infection- and veraison-responsive metabolites. (C) Independent component (IC) analysis of metabolite profiles at stages EL33 and EL35 with and without Botrytis infection. Five principal components representing 80% of the total variance were used to calculate independent components. The plot shows the sample scores of IC1 and IC3. (This figure is available in colour at JXB online.)

Fig. 4.

Analysis of potential positive metabolic markers of B. cinerea infection at stages EL33 and EL35 (veraison) of developing berries of V. vinifera cv. Trincadeira. (A) Metabolites that were significantly increased in both comparisons (P<0.05, Wilcoxon test) and metabolites that became detectable after infection were selected. Grey boxes indicate that the respective metabolites were not detectable. (B) Normalized response plot of selected metabolites that accumulated after infection. (This figure is available in colour at JXB online.)

Fig. 5.

Venn diagrams and functional analysis of differentially expressed genes. (A) Venn diagrams of significant transcripts (5% FDR in limma and ≥2-fold change; Supplementary Table S4) comparing infection-responsive transcripts between EL33 and EL35 berry developmental stages (left diagram) as well as veraison-responsive transcripts between infected and control berries (right diagram). (B) Summary of functional categories (Grimplet et al., 2012) significantly enriched (5% FDR in a Fisher’s exact test) within infection up- and downregulated transcripts in each developmental stage. Categories appear in order of significance. The whole list of significant categories can be found in Supplementary Table S5 at JXB online. (This figure is available in colour at JXB online.)

Fig. 6.

Overview of the major metabolic changes in response to B. cinerea infection in V. vinifera grapes at EL33 and EL35. Activation (+ shading in key) and inhibition (– shading in key) of the pathway as suggested by microarray data and increased content of metabolites at high and moderate levels are indicated. Ovals and hexagons represent metabolites and pathways, respectively. The expression profile of each individual gene and metabolite content is presented in Supplementary Fig. S8A and B for selected pathways. (This figure is available in colour at JXB online.)