Abstract
Coronatine is a polyketide phytotoxin produced by several pathovars of the plant pathogenic bacterium Pseudomonas syringae. It is one of the most important virulence factors determining the success of bacterial pathogenesis in the plant at both epiphytic and endophytic stages of the disease cycle. This protocol describes an optimized procedure to culture bacterial cells for coronatine production and to quantify the amount of coronatine secreted in the culture medium using an HPLC-based method.
Keywords: Coronatine, HPLC, Pseudomonas syringae, Plant pathogen, Virulence factor
Background
Coronatine (COR), a potent bacterial phytotoxin, is a molecular mimic of the plant hormone jasmonoyl-L isoleucine (JA-Ile). As such, COR activates jasmonic acid (JA) signaling, induces JA-responsive genes, and antagonizes the action of the immune signal salicylic acid. COR consists of two components, coronafacic acid (CFA) and coronamic acid (CMA). The genes that encode for CMA and CFA biosynthesis are not constitutively expressed in the bacterium. Instead, these genes are induced on the plant leaf surface, in planta or in vitro when the bacterium is grown in inducing medium (Palmer and Bender, 1993; Panchal et al. 2016). This article describes a method adapted from Panchal et al. (2016) to determine the ability of bacteria to produce coronatine, which can be used as an indication of virulence.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
We thank Dr. Carol Bender for useful advice on the coronatine extraction method. This study was supported by a grant from the US National Institute of Allergy and Infectious Disease (5R01AI068718) to Dr. Maeli Melotto.
References
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