Endogenous suppressor(s) in Arabidopsis thaliana

Abstract

An ethyl acetate extract of Arabidopsis thaliana plants was tested for the presence of endogenous suppressor(s) (ES), and the active fraction, which partitioned into water phase contained a molecule(s) < 3000 Da based on a rough estimate using sized membrane filters. Foliar application of the ES enabled typically nonpathogenic fungi (non-adapted pathogens) to cause disease symptoms on A. thaliana. Consistently, the ES fraction severely suppressed the oxidative burst and the expression of defense-related genes such as FRK1, NHO1, WRKY22, WRKY29, PEN2, and PEN3 in plants challenged with non-adapted fungus Colletotrichum gloeosporioides or the fungal elicitor chitin.

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Acknowledgements

TLM thanks the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) for financial support during the doctoral program. We acknowledge Prof. Dr. Yoshitaka Takano (Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Kyoto, Japan) for generously providing Colletotrichum gloeosporioides strain S9275. This research was supported in part by the Grants-in-Aid for Scientific Research (18K05645) from the Japan Society for Promotion of Science (JSPS).

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Correspondence to Kazuhiro Toyoda.

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Supplementary material 1 Fig. S1 Purification procedure of endogenous suppressor (ES) from Arabidopsis thaliana Col-0 plants. Expanded leaves from 5- to 6-week-old healthy plants were harvested to extract the ES (Nasu et al. 1992). Briefly, 100 g of fresh leaves were ground in liquid nitrogen for 5 min, then added to 200 ml of distilled water and filtered through Miracloth (Calbiochem, Merck, Darmstadt, Germany) and centrifugated at 14,000 × g for 15 min at 4°C. The supernatant was added to ethyl acetate, and the aqueous phase was collected and lyophilized, then transferred to a size exclusion chromatography (SEC) column (Tosoh Toyopearl HW-50S; Tosoh Corp., Tokyo, Japan). The active fraction (ES-1) after SEC was confirmed in bioassays. Next, a cartridge-based ion-exchange (SPE) (Oasis MCX 6 cc/500 mg LP extraction cartridges; Waters, Milford, MA, USA) was used for solid-phase extraction to fractionate the ES-1 according to the instructions. Eluate 2 was harvested and tested for induction of susceptibility. The active fraction from eluate 2 (ES-2) was then added to an Amicon Ultra 3 K centrifugal filter unit (3000 Da cutoff; Millipore, Billerica, MA, USA), and centrifuged at 5000 × g for 45 min in a fixed angle rotor. The flow-through solution was subjected to size exclusion in a high-performance liquid chromatography (HPLC) column (TSKgel G3000SWXL; Tosoh) to obtain active fractions that were confirmed with bioassays. If necessary, reverse-phase HPLC chromatography (YMC-Pack ODS-AQ HPLC column; YMC, Kyoto, Japan) was used to further purify fractions from ES-2. The confirmed active fraction (F4) was lyophilized; this highly purified ES was called ES-3. Fig. S2 Characterization and biochemical properties of ES. a Rough estimate of molecular mass of ES using sized membrane filters. ES-2 was filtered Amicon Ultra-0.5 ml centrifugal filter units (100 K, 50 K, 30 K, 10 K, 3 K, 2 K), and each filtrate was bioassayed with Colletotrichum gloeosporioides. ES-2 (10 mg/ml) and distilled water (DW) were used as a positive and negative control, respectively. Conidial concentration was adjusted to 5 × 105 spores/ml. Image was taken at 4 dpi. Scale bar = 1 cm. b Dilution-end point assay. After separation with the Amicon units (less than 3 K), the filtrate was tested for induction of susceptibility. Susceptibility to non-adapted C. gloeosporioides was induced even at a low concentration (0.1 mg/ml). Image was taken at 3 dpi. c Heat stability of ES. Ten microliters of ES-2 (10 mg/ml) was heated at 65°C or 80°C for 20 min before the bioassay. Conidial suspension of C. gloeosporioides was used for bioassays. Image was taken at 3 dpi. d Proteinase K digestion assay. ES-2 (10 mg/ml) was mixed with Proteinase K (final concentration: 0–2 mg/ml; Takara Bio, Otsu, Japan). The mixture was incubated overnight at 37°C, then centrifuged in an Amicon Ultra unit (3 K) to eliminate proteinases. The filtrate was bioassayed with C. gloeosporioides. The ES was tolerant to Proteinase K digestion. e Highly purified ES (ES-3) obtained by reverse-phase HPLC chromatography. Samples (ES-2; 10 µl of 3 mg/ml in Milli-Q water) were separated repeatedly, and the active fraction (F4; RT = 6.32 min) was collected and lyophilized. This active F4 fraction, which was positive in the ninhydrin test, was regarded as a highly purified ES (ES-3) (DOCX 21 kb)

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Mai, T.L., Kawasaki, T., Fitrianti, A.N. et al. Endogenous suppressor(s) in Arabidopsis thaliana. J Gen Plant Pathol 86, 100–106 (2020). https://doi.org/10.1007/s10327-019-00897-z

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Keywords

  • Arabidopsis
  • Endogenous suppressor (ES)
  • Defense response
  • Induced susceptibility
  • Mycosphaerella supprescins