Transcriptome Profiling of Cucumber (Cucumis sativus L.) Early Response to Pseudomonas syringae pv. lachrymans

Abstract

Bacterial angular leaf spot disease (ALS) caused by Pseudomonas syringae pv. lachrymans (Psl) is one of the biological factors limiting cucumber open-field production. The goal of this study was to characterize cytological and transcriptomic response of cucumber to this pathogen. Plants of two inbred lines, B10 (susceptible) and Gy14 (resistant), were grown, and leaves were inoculated with highly virulent Psl strain 814/98 under growth chamber conditions. Microscopic and transcriptional evaluations were performed at three time points: before, 1 and 3 days post inoculation (dpi). Investigated lines showed distinct response to Psl. At 1 dpi bacterial colonies were surrounded by necrotized mesophyll cells. At 3 dpi, in the susceptible B10 line bacteria were in contact with degraded cells, whereas cells next to bacteria in the resistant Gy14 line were plasmolyzed, but apparently still alive and functional. Additionally, the level of H₂O₂ production was higher in resistant Gy14 plants than in B10 at both examined time points. In RNA sequencing more than 18,800 transcripts were detected in each sample. As many as 1648 and 2755 differentially expressed genes (DEGs) at 1 dpi as well as 2992 and 3141 DEGs at 3 dpi were identified in B10 and Gy14, respectively. DEGs were characterized in terms of functional categories. Resistant line Gy14 showed massive transcriptomic response to Psl at 1 dpi compared to susceptible line B10, while a similar number of DEGs was detected for both lines at 3 dpi. This suggests that dynamic transcriptomic response to the invading pathogen may be related with host resistance. This manuscript provides the first transcriptomic data on cucumber infected with the pathovar lachrymans and helps to elucidate resistance mechanism against ALS disease.

Keywords: DEGs; angular leaf spot disease; defense response; transcription factors.

Figure 1

Morphology and ultrastructure of leaves of susceptible B10 and resistant Gy14 cucumber lines upon infection with the Psl 814/98 strain (inoculum concentration of 1 × 10⁷ CFU mL⁻¹). (A) Morphology of inoculated cucumber leaves (arrows indicate selected lesions); (A1–A3), B10 plants at 0, 1 and 3 dpi, respectively; (A4–A6), Gy14 plants at 0, 1 and 3 dpi, respectively. (B) H₂O₂ accumulation in leaves as indicated by staining with DAB (arrows point to selected DAB precipitates); (B1–B3), B10 plants at 0, 1 and 3 dpi, respectively; (B4–B6), Gy14 plants at 0, 1 and 3 dpi, respectively. (C) Transmission electron microscopy micrographs of leaf mesophyll cells (arrowheads indicate selected bacteria; arrows indicate selected chloroplasts; asterisks indicate regions between plasma membrane and cell wall in plasmolyzed cells); (C1–C3), B10 plants at 0, 1 and 3 dpi, respectively; (C4–C6), Gy14 plants at 0, 1 and 3 dpi, respectively. Abbreviations: IS—intercellular space, N—nucleus, V—vacuole. Scale bars, 5 μm (C).

Figure 2

Hierarchical clustering and heatmap of Pearson’s correlation (R²) between B10 and Gy14 lines inoculated with Psl 814/98 strain at 0, 1 and 3 dpi. (A) Hierarchical clustering between samples; (B) correlation matrix of expression profiles, both X-axis and Y-axis represent each sample. Correlated profiles are colored from blue to white (high to low correlation). Abbreviations: B10_0–B10 line at 0 dpi (control), B10_1–B10 line at 1 dpi, B10_3–B10 line at 3 dpi, Gy14_0–Gy14 line at 0 dpi (control), Gy14_1–Gy14 line at 1 dpi, Gy14_3–Gy14 line at 3 dpi.

Figure 3

Summary of the differentially expressed genes (DEGs) identified for Gy14 and B10 lines at 1 and 3 dpi in comparison to 0 dpi. Numbers indicate DEGs detected in each pairwise comparison. Red color represents up-regulated genes, whereas green color corresponds to down-regulated genes. Abbreviations: B10_0–B10 line at 0 dpi (control), B10_1–B10 line at 1 dpi, B10_3–B10 line at 3 dpi, Gy14_0–Gy14 line at 0 dpi (control), Gy14_1–Gy14 line at 1 dpi, Gy14_3–Gy14 line at 3 dpi.

Figure 4

Venn diagrams representing the distribution of differentially expressed genes (DEGs) in the resistant Gy14 line and susceptible B10 line at 1 and 3 dpi with Psl 814/98. Abbreviations: B10_1–B10 line at 1 dpi, B10_3–B10 line at 3 dpi, Gy14_1–Gy14 line at 1 dpi, Gy14_3–Gy14 line at 3 dpi.

Figure 5

The expression of 14 selected differentially expressed genes (DEGs) for lines Gy14 and B10 at 0, 1 and 3 dpi. Diagrams show relative normalized gene expression level ±SEM revealed by RT-qPCR from 3 biological and 3 technical replicates. For data normalization, 2 reference genes CACS and TIP41 were used. Grey dashed line represents estimated numbers of fragments per kilobase million (FPKM). Full gene names are provided in Supplementary Table S3. Significance levels were calculated with Student’s t-test: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). Abbreviations: B10_0–B10 line at 0 dpi (control), B10_1–B10 line at 1 dpi, B10_3–B10 line at 3 dpi, Gy14_0–Gy14 line at 0 dpi (control), Gy14_1–Gy14 line at 1 dpi, Gy14_3–Gy14 line at 3 dpi.

Figure 6

Summary of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis in Gy14 and B10 lines at 1 and 3 dpi. Red and green indicate up- and down-regulated differentially expressed genes (DEGs), respectively.

Figure 7

The summary of differentially expressed transcription factors (TF-DEGs) in Gy14 and B10 lines at 1 and 3 dpi. Names of transcription factor families/subfamilies are based on classification implemented by the Plant Transcription Factor Database [31]. Red and green indicate up- and down-regulated TF-DEGs, respectively.