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Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity

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CSVd could not infect Nicotiana benthamiana when the plants were pretreated with crude leaf extract of Capsicum chinense ‘Sy-2’. C. chinense leaves were revealed to contain strong RNA-digesting activity.

Abstract

Several studies have identified active antiviral and antiviroid agents in plants. Capsicum plants are known to contain antiviral agents, but the mechanism of their activity has not been determined. We aimed to elucidate the mechanism of Capsicum extract’s antiviroid activity. Chrysanthemum stunt viroid (CSVd) was inoculated into Nicotiana benthamiana plants before or after treating the plants with a leaf extract of Capsicum chinense ‘Sy-2’. CSVd infection was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) 3 weeks after inoculation. When Capsicum extract was sprayed or painted onto N. benthamiana before inoculation, it was effective in preventing infection by CSVd. To evaluate CSVd digestion activity in leaf extracts, CSVd was mixed with leaf extracts of Mirabilis, Phytolacca, Pelargonium and Capsicum. CSVd-digesting activities were examined by quantifying undigested CSVd using qRT-PCR, and RNA gel blotting permitted visualization of the digested CSVd. Only Capsicum leaf extract digested CSVd, and in the Capsicum treatment, small digested CSVd products were detected by RNA gel blot analysis. When the digesting experiment was performed for various cultivars and species of Capsicum, only cultivars of C. chinense showed strong CSVd-digesting activity. Our observations indicated that Capsicum extract contains strong RNA-digesting activity, leading to the conclusion that this activity is the main mechanism for protection from infection by CSVd through spraying or painting before inoculation. To our knowledge, this is the first report of a strong RNA-digesting activity by a plant extract.

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Acknowledgments

We thank Dr. Susumu Yazawa for providing us ‘Sy-2’ seeds and Dr. Motoaki Doi and Dr. Soo-Jung Yang for providing assistance in conducting the experiments.

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Authors and Affiliations

  1. Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan

    Boubourakas Iraklis, Hiroko Kanda, Tomoyuki Nabeshima, Mayu Onda, Nao Ota & Munetaka Hosokawa

  2. Faculty of Agriculture, Kinki University, Nara, Nara, 631-8505, Japan

    Sota Koeda

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  1. Boubourakas Iraklis
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  2. Hiroko Kanda
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  3. Tomoyuki Nabeshima
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  7. Munetaka Hosokawa
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Correspondence to Munetaka Hosokawa.

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Communicated by K. K. Kamo.

Electronic supplementary material

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299_2016_1977_MOESM1_ESM.tif

Online Resource 1 Fractions of Capsicum extract with strong CSVd-digesting activities separated by LPLC with gel filtration column by AKTA. The y-axis represents signal strength (mAu). The x-axis represents fraction numbers (1–40). Using gel filtration chromatography, the fractions containing 75 kDa–158 kDa proteins (fractions 15–19) showed strong activities for CSVd digestion by qRT-PCR. The numbers, 158, 75, and 44 represented on the figure refer to the molecular weights calculated from the molecular markers (TIFF 649 kb)

299_2016_1977_MOESM2_ESM.tif

Online Resource 2 Result of second electrophoresis by substrate-based gel assay. (a) Different positions (a–e) were cut during the first time substrate-based gel assay. Molecular weight sizes were determined by molecular weight marker. (b) Second run substrate-based gel assay of each band (a–e; left panel). Silver staining result of each band (a–e; right panel) (TIFF 5503 kb)

299_2016_1977_MOESM3_ESM.tif

Online Resource 3 Boiling and phenol treatment of Capsicum extract. For boiling treatment, Capsicum extract was heated in boiling water for 10 min. For phenol treatment, the supernatant of Capsicum extract treated with phenol was used for CSVd digestion. For buffer treatment, phosphate buffer (50 mM, pH 6.5) was used instead of phenol. The y-axis represents CSVd concentration relative to the undigested control value on the basis of qRT-PCR. Each treatment was repeated three times, and the data are represented as mean ± standard deviation (bars) (TIFF 669 kb)

299_2016_1977_MOESM4_ESM.tif

Online Resource 4 Results of qRT-PCR determination of the undigested CSVd after mixing treatments with Capsicum extract. CSVd-containing total RNA was digested by protease K-treated Capsicum extract for 10 min at 37 °C. Protease K with 0 µl treatment refers to treatment with Capsicum extract with the same volume of water instead of protease K solution. Control denotes that CSVd-containing total RNA was mixed with phosphate buffer (50 mM, pH 6.5), which was used instead of Capsicum extract. The y-axis represents CSVd concentration relative to the undigested control value on the basis of qRT-PCR results. Each treatment was repeated three times, and the data are represented as mean ± standard deviation (bars) (TIFF 875 kb)

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Iraklis, B., Kanda, H., Nabeshima, T. et al. Digestion of chrysanthemum stunt viroid by leaf extracts of Capsicum chinense indicates strong RNA-digesting activity. Plant Cell Rep 35, 1617–1628 (2016). https://doi.org/10.1007/s00299-016-1977-z

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