Arabidopsis defense against Botrytis cinerea: chronology and regulation deciphered by high-resolution temporal transcriptomic analysis

Abstract

Transcriptional reprogramming forms a major part of a plant's response to pathogen infection. Many individual components and pathways operating during plant defense have been identified, but our knowledge of how these different components interact is still rudimentary. We generated a high-resolution time series of gene expression profiles from a single Arabidopsis thaliana leaf during infection by the necrotrophic fungal pathogen Botrytis cinerea. Approximately one-third of the Arabidopsis genome is differentially expressed during the first 48 h after infection, with the majority of changes in gene expression occurring before significant lesion development. We used computational tools to obtain a detailed chronology of the defense response against B. cinerea, highlighting the times at which signaling and metabolic processes change, and identify transcription factor families operating at different times after infection. Motif enrichment and network inference predicted regulatory interactions, and testing of one such prediction identified a role for TGA3 in defense against necrotrophic pathogens. These data provide an unprecedented level of detail about transcriptional changes during a defense response and are suited to systems biology analyses to generate predictive models of the gene regulatory networks mediating the Arabidopsis response to B. cinerea.

Figure 1.

Time Series of B. cinerea Infection on Arabidopsis Leaf 7. (A) Ten-microliter droplets of a suspension of B. cinerea spores (1 × 10⁵ spores mL⁻¹) were placed on detached leaf 7 from 4-week-old Arabidopsis plants. Images show the same leaf every 2 h after inoculation until 48 h. (B) A mock-inoculated leaf at 2 HAI (left) and 48 HAI (right). Bars = 10 mm.

Figure 2.

Growth of B. cinerea in Arabidopsis Leaves. (A) Growth measured by expression of the B. cinerea tubulin gene. Expression levels were determined using real-time PCR and are shown as the log₂ ratio of expression of B. cinerea Tubulin relative to Arabidopsis PUX1 (At3g27310). Error bars indicate se of three biological replicates. A one-way analysis of variance was performed to determine which pairs of adjacent time points differed significantly from each other. Significantly different groups (P ≤ 0.05) are labeled a to e. (B) Trypan blue staining of Arabidopsis leaves infected with B. cinerea 24 HAI. Red arrows: I, filamentous hyphae; II, large claw-like structures. Bar = 25 μm

Figure 3.

Infected Leaf Expression Profiles of the Gene Members of Each of the 44 Clusters Generated by SplineCluster. The individual gene profiles are shown as gray lines with the mean profile in dark blue. The dashed blue lines indicate the mean ± 1 sd of the cluster. The y axes indicate log₂ expression normalized on a per gene basis. hpi, hours post inoculation.

Figure 4.

Selected GO Terms Overrepresented in Clusters of Genes Differentially Expressed after B. cinerea Infection of Arabidopsis Leaves. GO terms are aligned with the time of gradient change and/or time of first differential expression of the cluster (in italics), with red boxes containing GO terms from upregulated genes and blue boxes containing GO terms from downregulated genes.

Figure 5.

GO Terms Relating to Abiotic Stress Responses Overrepresented in Clusters of Genes Differentially Expressed after B. cinerea Infection of Arabidopsis Leaves. GO terms are aligned with the time of gradient change and/or time of first differential expression of the cluster, with the cluster expression profile shown. All “response to abiotic stimulus” and nonredundant individual abiotic stress GO terms are shown.

Figure 6.

Number and Function of Genes Differentially Expressed during Both Natural Senescence and B. cinerea Infection. The number of genes up- and downregulated during senescence and B. cinerea infection and overlaps between these is shown in the Venn diagram. Selected overrepresented GO terms are shown for each subset of genes. [See online article for color version of this figure.]

Figure 7.

B. cinerea Infection Dampens Oscillations of Clock Gene Expression. The green line indicates the mean expression profile of four biological replicates of mock-inoculated leaves, while the red line indicates the mean expression profile of four biological replicates of infected leaves. Error bars indicate se (n = 4).

Figure 8.

Known cis-Regulatory Sequences Associate with Groups of Coexpressed Genes. Regulatory motifs (represented by sequence logos where character size indicates nucleotide frequency) are differentially enriched in the promoters of genes clustered on the basis of their expression during B. cinerea infection. The blue shaded boxes correspond to raw P value. Expression profiles from selected gene clusters that are enriched for TF binding motifs are shown on the right. Full results used to derive this figure are shown in Supplemental Table 9 online.

Figure 9.

TF Families Significantly Overrepresented for DEGs, Indicating Distinct Periods of Regulation. The plots show a number of TF families significantly upregulated ([A], red) and downregulated ([B], green) following B. cinerea infection. Color bars indicate P values (after FDR correction; Benjamini and Hochberg, 1995) with a range of significance thresholds (0.01, 0.05, 0.1, 0.25, and 0.5).

Figure 10.

The Number of Genes First Differentially Expressed at Each Time Point. TOFDE for all 9838 DEGs following infection with B. cinerea (HAI [hpi]) is shown. The inset shows early time points (5 to 15 HAI) in more detail. [See online article for color version of this figure.]

Figure 11.

Inferred Network Model Using the Discrete-Time Causal Structure Identification Algorithm. Numbered nodes represent a cluster from the SplineCluster clustering of genes differentially expressed during B. cinerea infection. The expression profile of B. cinerea tubulin was used as a proxy for pathogen growth. Selected TFs present in clusters are indicated under nodes. Colored boxes adjacent to nodes indicate motifs enriched in the promoter sequences of cluster genes, with TFs from the corresponding binding family highlighted in the same color. The color of nodes indicates the proportion of cluster genes that are differentially expressed in the tga3-2 mutant compared with the wild type (either in the 193 or 1233 set of potential target genes).

Figure 12.

Susceptibility of tga3 Mutants to B. cinerea. Altered susceptibility of tga3-2 and tga3-3 T-DNA insertion lines compared with Col-0 control. Lesion perimeters are a mean of 20 leaves drop inoculated with 10 μL of 10⁵ spores/mL of B. cinerea. Significantly different lesion perimeters in insertion lines compared with their respective Col-0 control were determined using a two-tailed Student’s t test assuming equal variance. **P value < 0.001 and ***P value < 0.0001. Error bars show se. hpi, hours post inoculation.