The protein elicitor Hrip1 enhances resistance to insects and early bolting and flowering in Arabidopsis thaliana

Abstract

The elicitor Hrip1 isolated from necrotrophic fungus Alternaria tenuissima, could induce systemic acquired resistance in tobacco to enhance resistance to tobacco mosaic virus. In the present study, we found that the transgenic lines of Hrip1-overexpression in wild type (WT) Arabidopsis thaliana were more resistant to Spodoptera exigua and were early bolting and flowering than the WT. A profiling of transcription assay using digital gene expression profiling was used for transgenic and WT Arabidopsis thaliana. Differentially expressed genes including 40 upregulated and three downregulated genes were identified. In transgenic lines of Hrip1-overexpression, three genes related to jasmonate (JA) biosynthesis were significantly upregulated, and the JA level was found to be higher than WT. Two GDSL family members (GLIP1 and GLIP4) and pathogen-related gene, which participated in pathogen defense action, were upregulated in the transgenic line of Hrip1-overexpression. Thus, Hrip1 is involved in affecting the flower bolting time and regulating endogenous JA biosynthesis and regulatory network to enhance resistance to insect.

Fig 1. Analysis of Hrip1 expression in transgenic plants.

The Hrip1 RNA and protein expression levels in wild-type (WT) and transgenic Arabidopsis were quantified through (A) real-time PCR and (B) Western blot analysis. 35S:Hrip1-1, 35S:Hrip1-2, 35S:Hrip1-3, 35S:Hrip1-4, 35S:Hrip1-5 and 35S:Hrip1-6: transgenic Arabidopsis lines overexpressing Hirp1; Col-0: WT Arabidopsis lines. Actin2 and Actin7 were used as internal controls in real-time PCR analysis. Actin2 was used as the internal control in Western blot analysis. Each experiment was repeated more than thrice, and similar results were obtained per run. Data are presented as the means ± SD of three independent experiments. Asterisks indicate significant differences between transgenic and WT Arabidopsis (Student’s t-test: *P < 0.05; **P < 0.01).

Fig 2. Effects of Hrip1 overexpression on the bolting time of transgenic Arabidopsis.

(A) Bolting phenotype, (B) bolting time; bars represent standard deviation (n ≥ 30) and (C) FT expression levels in wild-type (WT) and Hrip1-overexpressing transgenic lines quantified through qRT-PCR analysis. Actin2 and Actin7 were used as internal controls in real-time PCR analysis. All experiments were repeated more than thrice. Data are presented as the means ± SD of three independent experiments. Asterisks indicate significant differences between transgenic and WT Arabidopsis lines (Student’s t-test: *P < 0.05; **P < 0.01).

Fig 3. Suppressive effects of transgenic plants on the growth of Spodoptera exigua.

(A) S. exigua individuals fed with 2-week-old transgenic plants (growing on nutrient soil) for 6 days. (B) Weight of S. exigua individuals fed with transgenic plants for 6 days. The red vertical line is shown for scale and represents 1 cm. Bars represent standard deviation (n≥30). Each experiment was repeated more than thrice. Data are presented as the means ± SD of three independent experiments. Asterisks indicate significant differences between transgenic and WT Arabidopsis lines (Student’s t-test: *P < 0.05; **P < 0.01).

Fig 4. Comparison of expression levels in WT (control, AtRNA_C) and transgenic (treat, AtRAN_H) Arabidopsis.

(A) Comparison of RPKM distribution between WT and transgenic Arabidopsis. The y-axis indicates the log₁₀(RPKM+1) values of genes. The x-axis indicates samples: WT (control) and transgenic (treat) Arabidopsis lines. (B) Summary of the RPKM density distribution of WT and transgenic Arabidopsis lines. The y-axis indicates density values. The x-axis indicates the log₁₀(RPKM+1) values of genes. Red and blue represent WT and transgenic Arabidopsis lines, respectively. (C) Volcano plot of differentially expressed genes. The y-axis indicates −log₁₀(padj) values with significant differences. The x-axis indicates the log₂(fold-change) values. Blue points represent genes that are not considerably differentially expressed, red points represent considerably upregulated genes, and green points represent considerably downregulated genes.

Fig 5. Verification of differentially expressed DGE results using qRT–PCR.

(A–E) The relative expression level of each gene significantly fold-changed in transgenic Hrip1-overexpressing Arabidopsis lines. Actin2 and Actin7 were used as internal controls in real-time PCR analysis. Each experiment was repeated more than thrice. Data are presented as the means ± SD of three independent experiments. Asterisks indicate significant differences between transgenic and WT Arabidopsis (Student’s t-test: **P < 0.01).

Fig 6. Jasmonic acid contents of rosette leaves from wild-type (WT) and Hrip1-overexpressing transgenic lines.

Jasmonic acid contents of 2-week-old rosette leaves from WT, 35S:Hrip1-1, 35S:Hrip1-2 and 35S:Hrip1-3 transgenic plants (growing on nutrient soil). Data are presented as the means ± SE of three biological replicates. Asterisks represent a significant difference between transgenic and WT Arabidopsis lines (Student’s t-test *P < 0.05, **P < 0.01).