Transcriptome analysis reveals ethylene-mediated defense responses to Fusarium oxysporum f. sp. cucumerinum infection in Cucumis sativus L

Abstract

Background: Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (Foc), is a severe disease affecting cucumber (Cucumis sativus L.) production worldwide, but mechanisms underlying Fusarium wilt resistance in cucumber remain unknown. To better understand of the defense mechanisms elicited in response to Foc inoculation, RNA sequencing-based transcriptomic profiling of responses of the Fusarium wilt-resistant cucumber line 'Rijiecheng' at 0, 24, 48, 96, and 192 h after Foc inoculation was performed.

Results: We identified 4116 genes that were differentially expressed between 0 h and other time points after inoculation. All ethylene-related and pathogenesis-related genes from the differentially expressed genes were filtered out. Real-time PCR analysis showed that ethylene-related genes were induced in response to Foc infection. Importantly, after Foc infection and exogenous application of ethephon, a donor of ethylene, the ethylene-related genes were highly expressed. In response to exogenous ethephon treatment in conjunction with Foc inoculation, the infection resistance of cucumber seedlings was enhanced and endogenous ethylene biosynthesis increased dramatically.

Conclusion: Collectively, ethylene signaling pathways play a positive role in regulating the defense response of cucumber to Foc infection. The results provide insight into the cucumber Fusarium wilt defense mechanisms and provide valuable information for breeding new cucumber cultivars with enhanced Fusarium wilt tolerance.

Keywords: Cucumber; Exogenous ethylene; Fusarium wilt; RNA-seq.

Fig. 1

Hierarchical clustering of ethylene- related DEGs. Fold- difference was designated as fragments per kilobase of transcript per million fragments mapped. The maximum value was ‘1’ for each gene. The darker the shade of red, the higher the expression level. 0 h, 24 h, 48 h, 96 h, and 192 h indicate the sampling time points after inoculation

Fig. 2

Changes in relative expression level of six DEGs analyzed by qPCR and RNA-seq. The left vertical axis indicates the relative expression level determined by qPCR, the right vertical axis indicates the FPKM value determined by RNA-seq. 0 h, 24 h, 48 h, 96 h, and 192 h indicate time points after Foc inoculation. The entire cucumber roots were the samples. R, ‘Rijiecheng’, a Foc-resistant cucumber line, inoculated with Foc; CK, ‘Rijiecheng’ inoculated with sterile water. At least three biological replicates were performed in each experiment group. * P < 0.05, ** P < 0.01

Fig. 3

The phenotypic differences in vitro of Foc with ethephon. In vitro phenotypic differences between the Foc strain cultured on PDA medium supplemented with (a) sterile water and (b) 1000 ppm ethephon

Fig. 4

Phenotypic differences and disease index of cucumber seedlings. a, b Phenotypic differences of cucumber seedlings. The one third of seedlings roots treated with mock solutions, one third were inoculated with a Foc strain (spore concentration 10⁶ conidia/mL), and the others were inoculated with Foc and sprayed with exogenous ethephon on leaves (concentration 10 ppm, Foc + ET) at the same time. c, d Disease index for the phenotypes of the cucumber seedlings. The Y-axis indicates the disease index. The typical phenotypic differences and disease index were recorded and calculated 3 weeks after treatment. R, ‘Rijiecheng’ (Foc-resistant line), S, ‘Superina’, (Foc-sensitive line)

Fig. 5

Expression profiles of six candidate ethylene- related genes. These genes were calculated in cucumber at 0 h, 48 h, and 96 h (a) inoculation with Foc, b spray treatment with exogenous ET, and (c) after inoculation with Foc and spray treatment with exogenous ET. R, ‘Rijiecheng’ (Foc-resistant line), S, ‘Superina’ (Foc-sensitive line)

Fig. 6

Endogenous ethylene production by the root of cucumber seedlings. These seedlings were treated at 0, 48, and 96 h (a) inoculation with Foc, b after inoculation with Foc and spray treatment with exogenous ET. R, ‘Rijiecheng’ (Foc-resistant line), S, ‘Superina’ (Foc-sensitive line)