Transcriptome and Coexpression Network Analyses Provide Insights into the Resistance of Chinese Cabbage During Different Stages of Plasmodiophora brassicae Infection

Abstract

Clubroot is a destructive soilborne disease caused by Plasmodiophora brassicae that threatens the production of Chinese cabbage. The molecular mechanisms underlying the resistance of Chinese cabbage to clubroot remains unclear, making the identification and analysis of resistance genes crucial for developing resistant varieties. Comparative transcriptome analysis of roots from the resistant line "JJ S5-1" and the susceptible line "SYY10-1" revealed significant differences in gene expression profiles at various stages after inoculation. Weighted gene coexpression network analysis revealed midnight blue and green modules as substantially associated with disease response, with each showing positive regulatory patterns. Several defense-related genes and transcription factors important for resistance to Plasmodiophora brassicae were identified, including disease resistance proteins, PR1, PBS1, and TGA, and WRKY transcription factors, most of which were upregulated following inoculation. Key genes associated with trait-related expression patterns were analyzed and a working model was proposed to explain the mechanism of clubroot disease resistance to Plasmodiophora brassicae infection in Chinese cabbage. These findings offer a valuable resource for further investigation of the immune response in the resistance of "JJ S5-1" to clubroot disease.

Keywords: Chinese cabbage; clubroot; regulatory network; weighted gene coexpression network analysis.

Figure 1

Phenotypic observations and transcriptome overview of Chinese cabbage upon P. brassicae inoculation. (a) Clubroot symptoms in the roots of S- and R-lines at 0, 2, 3, 4 and 5 weeks (marked as CK, 2W, 3W, 4W and 5W) after P. brassicae inoculation; (b) The relative content of P. brassicae was detected at 2, 3, 4 and 5 weeks of roots of R- and S-lines. The values represent the average of three biological replicates (n = 3). Error bars represent the mean ± standard deviation (SD) of three independent experiments. Distinct letters indicate significant differences at p < 0.05; (c) Expression levels of all samples; (d) Pearson correlation analysis; (e) Principal component analysis.

Figure 2

DEGs analysis of R- and S-line in response to P. brassica inoculation. (a) DEGs at 3W and 5W after P. brassicae inoculation in R-line; (b) DEGs at 3W and 5W after P. brassicae inoculation in S-line; (c) Comparison of DEGs at 3W and 5W after P. brassicae inoculation in R- and S-line; (d) Venn diagram of DEGs among R-3W vs. R-CK, R-5W vs. R-CK and R-5W vs. R-3W; (e) Venn diagram of DEGs among S-3W vs. S-CK, S-5W vs. S-CK and S-5W vs. S-3W; (f) Venn diagram of DEGs among R-CK vs. S-CK, R-3W vs. S-3W and R-5W vs. S-5W; (g) Classification of differentially expressed transcription factors; (h) Number of upregulated and downregulated genes of MYB, NAC, AP2, bHLH and WRKY transcription factors.

Figure 3

GO analysis of DEGs. The Y-axis represents GO terms grouped by functional classification: biological process (pink), molecular function (blue), cellular component (orange). The X-axis represents the percentage of DEGs enriched in each pathway relative to total annotated genes.

Figure 4

Top 15 terms of KEGG analysis of DEGs for each group. Bubble plot showing pathway enrichment analysis, with the X-axis indicating comparison groups, the Y-axis displaying enriched pathways; Dot size representing the number of enriched genes, and dot color reflecting the statistical significance of enrichment (FDR < 0.05).

Figure 5

WGCNA of DEGs in R- and S-line at 0, 3, 5 WPI. (a) Hierarchical clustering tree generated by WGCNA; (b) Module-sample group association analysis. Each row represents a module (labeled with the same color as in (a)), and each column corresponds to a sample group. The color intensity in each cell reflects the correlation coefficient between the module and the sample group; (c) Correlation analysis between modules and P. brassicae infected samples of resistant (R) and susceptible (S) lines. The value in each cell indicates the correlation coefficient between the module genes and infected samples, with the corresponding p-value displayed beside; (d) Expression trends of DEGs in midnight blue module; (e) Expression trends of DEGs in green module.

Figure 6

qRT-PCR analysis of DEGs. The values represent the average of three biological replicates. Error bars represent the mean ± standard deviation (SD) of three independent experiments. Distinct letters indicate significant differences at p < 0.05.

Figure 7

qRT-PCR expression validation for 10 selected genes. The gene names corresponding to each histogram are displayed above their respective bars. Left Y-axis represents qRT-PCR values, and right Y-axis represents FPKM values. The values represent the means ± SDs (n = 3) of three biological replicates.

Figure 8

Speculation on the clubroot disease resistance mechanism of P. brassicae infection in Chinese cabbage.