Genome-wide transcriptional profiling of Botrytis cinerea genes targeting plant cell walls during infections of different hosts

Abstract

Cell walls are barriers that impair colonization of host tissues, but also are important reservoirs of energy-rich sugars. Growing hyphae of necrotrophic fungal pathogens, such as Botrytis cinerea (Botrytis, henceforth), secrete enzymes that disassemble cell wall polysaccharides. In this work we describe the annotation of 275 putative secreted Carbohydrate-Active enZymes (CAZymes) identified in the Botrytis B05.10 genome. Using RNAseq we determined which Botrytis CAZymes were expressed during infections of lettuce leaves, ripe tomato fruit, and grape berries. On the three hosts, Botrytis expressed a common group of 229 potentially secreted CAZymes, including 28 pectin backbone-modifying enzymes, 21 hemicellulose-modifying proteins, 18 enzymes that might target pectin and hemicellulose side-branches, and 16 enzymes predicted to degrade cellulose. The diversity of the Botrytis CAZymes may be partly responsible for its wide host range. Thirty-six candidate CAZymes with secretion signals were found exclusively when Botrytis interacted with ripe tomato fruit and grape berries. Pectin polysaccharides are notably abundant in grape and tomato cell walls, but lettuce leaf walls have less pectin and are richer in hemicelluloses and cellulose. The results of this study not only suggest that Botrytis targets similar wall polysaccharide networks on fruit and leaves, but also that it may selectively attack host wall polysaccharide substrates depending on the host tissue.

Keywords: Botrytis; CAZymes; RNAseq; grape; lettuce; noble rot; plant pathogenic fungi; tomato.

Figure 1

Botrytis transcript abundance in infected plant tissues. Relative composition of RNAseq transcript reads between Botrytis and the three plant hosts (i.e., lettuce leaves, tomato fruit and grape berries).

Figure 2

Comparisons of CAZyme gene expression during infections of lettuce leaves, tomato fruit and grape berries. (A) Venn diagrams showing the overlapping and unique sets of CAZyme Botrytis genes (left) and CAZyme Botrytis genes with potential signal peptides (right) detected in the transcriptomes of the three infected host tissues. (B) Scatterplots showing all possible pair-wise correlations between DESeq-normalized counts of CAZyme Botrytis genes in lettuce, tomato, and grape.

Figure 3

Botrytis genes encoding predicted secreted CAZymes and their relative expression levels during infections of three plant hosts. The outermost ring represents all of the secreted CAZymes predicted in the Botrytis cinerea genome (strain B05.10). The inner four rings represent the relative expression of each CAZyme gene during Botrytis infections of lettuce leaves (green, second ring from outside), ripe tomato fruit (red, third ring from outside) and ripe grape berries (purple, the innermost ring). The relative expression of each gene is the log2-transformed percentage of normalized reads among the total normalized reads from all CAZyme Botrytis genes that possess a secretion signal peptide.

Figure 4

Phylogenetic tree and expression levels of characterized and candidate enzymes that act on pectin backbones. Bootstrap consensus trees inferred from 1000 replicates using the Neighbor-Joining method for the Botrytis CAZymes with predicted signal peptides in the GH28 (A), PL1 and PL3 (B) and CE8 (C) subfamilies. The percentages (>50%) of replicated trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Colored-boxes in the phylogenetic trees indicate protein tribes (>2 members) determined by BLASTp alignments (e-value < 1e−6) and Tribe-MCL (Enright et al., 2002). Boxes in (A), gray = tribe 1, light blue = tribe 16, purple = tribe 26 and green = tribe 10. Boxes in (B) brown = tribe 8, yellow = tribe 24. In (C), pink box = tribe 11 (Supplemental Table S2). The colors in the heat maps represent the numbers of DESeq-normalized transcript counts (log10) of the Botrytis genes in infected lettuce leaves, ripe tomato fruit and grape berries.

Figure 5

qRT-PCR validation of the RNAseq expression results for BcPG1 and BcPG2 genes in ripe tomato fruit and grape berries. Relative expression levels of BcPG1 (A) and BcPG2 (B) measured in the biological replications of Botrytis-infected tomato fruit and botrytized-grape berries. (C) The scatterplot depicts the correlation between the qRT-PCR relative expression of BcPG1 and BcPG2 genes and their corresponding raw reads normalized against the same reference gene (BcRPL5) used in the qRT-PCR analyses. The data points indicate that the BcPG2 expression in grape berries were close to zero (average of 4.49e−5) and in tomato fruit the expression was 3.57e−2 on average; thus they overlapped in the graph. The Pearson correlation coefficient (r) is presented (P-value < 0.001). A linear trend is shown.

Figure 6

Phylogenetic tree and expression levels of predicted xyloglucan (XyG) backbone-degrading enzymes. Bootstrap consensus trees inferred from 1000 replicates using the Neighbor-Joining method for the Botrytis CAZymes with predicted signal peptides in the GH16 (A) and GH3 (B) subfamilies. The percentages (>50%) of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Colored-boxes in the phylogenetic trees indicate protein tribes (> 2 members) determined by BLASTp alignments (e-value < 1e−6) and Tribe-MCL (Enright et al., 2002). Gray box in (A) correspond to tribe 5, while the light blue box in (B) refers to tribe 4 (Supplemental Table S2). The colors in the heat maps represent the number of DESeq-normalized transcript counts (log10) of the Botrytis genes in infected lettuce leaves, ripe tomato fruit and grape berries.

Figure 7

Phylogenetic tree and expression levels of putative copper-dependent lytic polysaccharide monooxygenases (LPMOs) and cutinases. Bootstrap consensus trees inferred from 1000 replicates using the Neighbor-Joining method for the Botrytis CAZymes with predicted signal peptides in the AA9 (A) and CE5 (B) subfamilies. The percentages (>50%) of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. Colored-boxes in the phylogenetic trees indicate protein tribes (>2 members) determined by BLASTp alignments (e-value < 1e−6) and Tribe-MCL (Enright et al., 2002). In (A), gray box = tribe 13. Boxes in (B) light blue = tribe 15 and purple = tribe 31 (Supplemental Table S2). The colors in the heat maps represent the number of DESeq-normalized transcript counts (log10) of the Botrytis genes in infected lettuce leaves, ripe tomato fruit and grape berries.