Background

Potato common scab is an economically important crop disease that is caused by Gram-positive, filamentous, soil bacteria from the genus Streptomyces. The first described and best characterized scab - causing Streptomyces spp. is Streptomyces scabies (syn. S. scabiei), which has a worldwide distribution (Bignell et al., 2010). Current control practices for common scab disease management include crop rotation, irrigation and soil fumigation; however, these strategies often fail, produce inconsistent results or are environmental unfriendly (Dees and Wanner, 2012). In order to develop better control strategies for the disease, we must first understand the molecular mechanisms used by S. scabies to infect the plant and to induce disease symptoms. Research has shown that the ability of S. scabies to cause disease is due to the production of virulence factors that play different roles during the infection process. Among the known or potential virulence factors that are produced by S. scabies is a family of plant toxins referred to as the N-coronafacoyl phytotoxins (also known as the COR-like metabolites), which resemble the plant hormone jasmonic acid and may function to suppress the plant immune response during pathogen infection (Bignell et al., 2010; Fyans et al., 2015). The primary coronafacoyl phytotoxin produced by S. scabies is N-coronafacoyl-L-isoleucine (CFA-L-Ile; Figure 1), which consists of the polyketide metabolite coronafacic acid linked via an amide bond to L-isoleucine. In addition, other N-coronafacoyl phytotoxins containing different isoleucine isomers or different amino acids (e.g., valine) can be produced in minor amounts (Fyans et al., 2015; Bown et al., 2016).


Figure 1. Structure of N-coronafacoyl-L-isoleucine (CFA-L-Ile) produced by Streptomyces scabies


The protocol described here was developed to isolate and purify N-coronafacoyl phytotoxins and their biosynthetic intermediates from large-scale cultures of S. scabies for purposes of structural and functional characterization. Previously, Fyans et al. (2015) described a protocol that was based in part on a published procedure for the isolation of the related N-coronafacoyl phytotoxin coronatine (COR) from cultures of the Gram-negative plant pathogenic bacterium Pseudomonas syringae (Palmer and Bender, 1993). As outlined by Fyans et al. (2015), strains of S. scabies are cultured in a soy flour mannitol broth (SFMB) medium, which promotes the production of the coronafacoyl phytotoxins, and then the culture supernatants are subjected to a two-step extraction with organic solvent under basic and acidic conditions in order to selectively isolate the phytotoxin - containing carboxylic acid fraction of the culture supernatants. The phytotoxins are then further purified using a combination of preparative thin layer chromatography (TLC) and semi-preparative reverse phase - high performance liquid chromatography (RP - HPLC). More recently, we described a modified version of this protocol in which we incorporated additional extraction steps using an aqueous solution of potassium bicarbonate (Bown et al., 2016). This modification was based on the procedure described by Mitchell and Frey (1986) for the isolation of P. syringae N-coronafacoyl phytotoxins, and we found that the incorporation of the additional extraction steps significantly improved the purity of the final S. scabies phytotoxin preparations. Moreover, we modified the organic solvent for the N-coronafacoyl phytotoxins by the addition of a small amount of acid, which significantly improved the solubility and yield of the purified phytotoxins for downstream structural and functional studies.

Here, we present the detailed step-by-step protocol for how we currently purify the S. scabies N-coronafacoyl phytotoxins in our laboratory.