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Transgenic expression of an insect diapause-specific peptide (DSP) in Arabidopsis resists phytopathogenic fungal attacks

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Abstract

Diapause-specific peptide (DSP) is an insect antimicrobial peptide, composed of 41 amino acid residues including six cysteines and isolated from diapausing adults of the leaf beetle Gastrophysa atrocyaneais. Recent research results have been demonstrated that in vitro, DSP has selective antifungal activity against the higher animal pathogenic fungus Trichophyton rubrum and the plant phytopathogenic fungus Fusarium solani. To elucidate the effect of DSP on other phytopathogenic fungi, we respectively introduced a Cht1SP-DSP-FLAG fusion gene and Cht1SP-FLAG fusion gene into Arabidopsis ecotype col-0. Transgenic plants expressing Cht1SP-DSP-FLAG fusion gene showed significant resistance to Golovinomyces cichoracearum (biotrophic fungus), Botrytis cinerea (necrotrophic fungus) and Hyaloperonospora arabidopsidis (H. a.) Noco2 (oomycete), whilst transgenic plants expressing Cht1SP-FLAG fusion gene showed no resistance to these pathogens. These results indicated that for many plant pathogenic fungi, DSP has high antimicrobial activity, which suggests that DSP has a huge potential in protecting crops from damage from phytopathogenic fungi.

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Authors and Affiliations

  1. Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, China

    Tingquan Wu, Yu’e Lin & Zhaojun Liang

  2. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China

    Tingquan Wu, Yongfang Chen, Weida Chen, Shenghao Zou, Yongqing Zhang & Dingzhong Tang

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  1. Tingquan Wu
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  2. Yongfang Chen
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  3. Weida Chen
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  4. Shenghao Zou
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  5. Yongqing Zhang
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  6. Yu’e Lin
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  7. Zhaojun Liang
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Corresponding author

Correspondence to Tingquan Wu.

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Supplemental Fig. 1

Cht1SP -FLAG fusion gene in Arabidosis transformants. A. The schematic representation of plant transformation construction of Cht1SP sequence. The synthetic Cht1SP was constructed into PGWB11 vector. RB, right border;LB,left border;HPT,hygromycin-resistance gene;35S,CaMV 35S promoter;Nos-T, terminator of the nopaline synthase gene;FLAG,FLAG tag. B. Detection of the introduced Cht1SP in transformants by genomic PCR. The size of Cht1SP sequence is 60 bp which a red arrow indicates. L plus numbers indicates the names of the transformant lines. M, DNA marker; Col-0, wild type control. C. Detection by real-time PCR of transcript of the introduced Cht1SP in transgenic plant lines (SL1, SL2, SL3, SL4, SL5, SL6, SL8). Relative gene expressions of Cht1SP in these transgenic lines and Col-0 wild type (as a control) were examined using real-time PCR with actin2 as an internal control. (JPEG 909 kb)

Supplemental Fig. 2

Transgenic Arabidopsis plants with Cht1SP-FLAG fusion gene showed no resistance to G.cichoracearum . Four-week-old plants were inoculated with G. cichoracearum. A. Leaves were stained with trypan blue at 7 dpi. Red arrow indicates an initiation colony. B. Quantification of fungal growth by calculating the number of conidiophores per colony at 7 dpi. Bars represent the mean (n > 30). Different letters indicate significant differences between transgenic lines and the wild type at P < 0.05. Experiments were repeated three times and the results are similar. (JPEG 1849 kb)

Supplemental Fig. 3

Transgenic Arabidopsis plants with Cht1SP-FLAG fusion gene exhibited no resistance to B. cinerea . A. Leaves from 4-week-old Col-0 and transgenic plants were detached and inoculated with B.cinerea. Leaves were photographed 3 dpi. B. Lesion size induced by B.cinerea. Lesion size was measured at 3 dpi. Bar represents the mean (n > 30) and SD from ten samples. Different letters indicate significant differences between transgenic lines and the wild type at (P < 0.05, t-test). Experiments were repeated two times and the results are similar. (JPEG 1561 kb)

Supplemental Fig. 4

Transgenic Arabidopsis plants with Cht1SP -FLAG fusion gene displayed no resistance to H. arabidopsidis. Two-week-old plants were infected with H. arabidopsidis. Noco2. The number of spores was monitored at 7 dpi. Bars represent the mean (n > 30) and standard deviation. Different letters indicates statistically significant differences between the samples (P < 0.05, t-test). Experiments were repeated three times and the results are similar. (JPEG 320 kb)

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Wu, T., Chen, Y., Chen, W. et al. Transgenic expression of an insect diapause-specific peptide (DSP) in Arabidopsis resists phytopathogenic fungal attacks. Eur J Plant Pathol 137, 93–101 (2013). https://doi.org/10.1007/s10658-013-0219-2

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