Axenic Culture of Sclerotinia sclerotiorum and Preparation of Sclerotinia Culture Filtrate Elicitor 1 (SCFE1)-containing Fractions, Triggering Immune Responses in Arabidopsis thaliana

• Published in: Bio-protocol Vol. 4; no. 17
• Main Authors: Malou Fraiture, Frédéric Brunner
• Format: Journal Article
• Language: English
• Published: Bio-protocol LLC 01.09.2014
• ISSN: 2331-8325
• DOI: 10.21769/BioProtoc.1232

The necrotrophic white mold fungus Sclerotinia sclerotiorum (S. sclerotiorum) is pathogenic to a broad range of plant species, including the Brassicaceae model plant Arabidopsis thaliana (Boland and Hall, 1994; Bolton et al., 2006). In Arabidopsis thaliana (A. thaliana), the semi-purified proteinaceous S. sclerotinorum elicitor SCFE1 (Sclerotinia culture filtrate elicitor 1) is sensed at the plasma membrane by the receptor-like protein RLP30 and triggers strong immune responses (Zhang et al., 2013), similar to the bacterial elicitor flagellin (Felix et al., 1999). Elicitation of plant defenses with SCFE1 is a tool to dissect the signaling pathway involving RLP30 and to study immunity to necrotrophic fungi. Here, we describe a simple protocol to axenically grow S. sclerotiorum. Further, we present a two-step liquid chromatography-based method for the partial purification of SCFE1 from culture filtrate (Figure 1A-B). Measurement by gas chromatography of the emission of the plant stress hormone ethylene is proposed as a bioassay to monitor elicitor activity in the fractions throughout the purification procedure (Figure 1C).Figure 1.  Two-step chromatographic fractionation of S. sclerotiorum culture filtrate to obtain semi-purified SCFE1. A. Purification scheme of SCFE1. Crude filtrate (CF) is loaded onto a Sepharose SP cation exchange chromatography column. The eluate (S1) is diluted 10-fold and loaded onto a Source 15S cation exchange chromatography column. Elution is performed with a linear gradient of 0 to 0.3 M KCl and elution fractions of 0.5 ml (F1 - F100) are collected. FT = flow-through. B. Chromatogram of the SCFE1-containing fractions eluted from a Source 15S cation exchange chromatography column. The black line represents the protein elution profile monitored with OD280 nm. The grey line shows the increasing conductivity of the elution buffer. ma.u. = milli-arbitrary units. mS = milli-Siemens. C. Ethylene response in Arabidopsis Col-0 to SCFE1-containing fractions obtained by two-step cation exchange chromatography. Arabidopsis Col-0 leaf pieces are treated with 15 µl CF, undiluted and 10-fold diluted S1 as well as the Source 15S elution fractions (Fx). Treatment with 0.5 µM flg22 is used as a positive control for ethylene production. No treatment and treatment with 15 µl buffers A and B are used as negative controls. In this representative purification, SCFE1 is contained in fractions F24 to F52. Bars represent average values (n=2) ± S.D.