Signals and metabolic consequences during the interaction of Brassicaceae and <i>Verticillium longisporum</i>

Abstract

The soil-borne, hemibiotrophic pathogen Verticillium longisporum is a severe threat for oilseed rape (Brassica napus) cultivation. In order to develop effective, defensive treatments for infected plants a thorough understanding of the plant-pathogen interaction is necessary. Therefore this work was conducted to find and identify metabolic changes and involved metabolite pathways in the pathogenic fungus or in the invaded host plant. For that a metabolite fingerprinting approach was optimized by adjusting the analytical parameters and adapting extraction methods to the analysed types of samples. In order to identify extracellular metabolites that are produced by V. longisporum in the xylem vessel, an in vitro assay was established resulting in the identification of seven metabolites from the tryptophan metabolism that accumulates in a provided xylem like environment. Nevertheless none of the identified substances produced by the fungus in vitro was detectable in planta after V. longisporum infection. Hence all accumulating metabolites in planta were analysed. A comprehensive analysis of apoplastic fluids and different plant organs in a time course from 5 to 35 days past infection in B. napus revealed a strong change in the metabolite pattern caused by V. longisporum infection. The identification of ubiquitously accumulating infection markers resulted in the detection of the phytoalexin cyclobrassinin and 22 related newly described substances. Structure elucidation was performed by MS/MS and pseudo-MS/MS/MS fragmentation analysis. The distribution of the cyclobrassinin related compounds within the plant correlated with the amount of fungal DNA detected in the plant organs pointing to a high contamination and a strong metabolic response in the hypocotyl tissue. By combining the obtained structural information and a proposed pathway for the phytoalexin camalexin in Arabidopsis thaliana an analogue model for the biosynthetic pathway of cyclobrassinin is proposed. A subgroup of the identified infection markers hints to a degradation of cyclobrassinin resulting in 2-mercapto-indole-3-carboxylic acid (MICA) derivatives. When samples from Camelina sativa – whose infection process with V. longisporum was accessed for the first time in this work - and A. thaliana were included in the comprehensive analysis of Brassicaceae it was found, that other groups of infection markers are more general than the species specific cyclobrassinin related markers. Raphanusamic acid (RA), a substance putatively related to phytoalexin or glucosinolate metabolism, pipecolic acid and three glycosylated salicylic acid (SA) derivatives proved to accumulate in the three Brassicaceae. Two trihydroxy fatty acids were identified infection markers from apoplastic fluids of B. napus and A. thaliana. Furthermore, five groups of infection markers were identified exclusively in the apoplastic wash fluid (AWF). The saturated dicarboxylic acids and their monoamide derivatives are infection markers in B. napus as well as in A. thaliana. Furthermore, diamides, aldehydes and alcohol derivatives were found to accumulate in B. napus plants. The mono- and diamide derivatives were unequivocally identified by chemically synthesized authentic standards. Three polyamine infection markers were additionally measured in a targeted analysis whereas the glucosinolates often involved in plant-pathogen interactions were not found to be reliable infection markers in B. napus. Preliminary results from a priming experiment of B. napus with the C9 dicarboxylic acid, azelaic acid, indicated only a shift in cyclobrassinin biosynthesis but no enhanced resistance to V. longisporum infection. An infection resembling metabolite pattern could not be achieved by flotation on several identified infection markers like SA or RA. The elicitation of B. napus leaves with CuCl2 demonstrated that the cyclobrassinin related substances were plant derived substances. They as well as markers like RA, SAG and pipecolic acid accumulate upon biotic and abiotic stress in B. napus. In summary more than 70 metabolites were unequivocally identified either as fungal substances that accumulate in in vitro assays or as plant-derived metabolites in Brassicaceae upon V. longisporum infection.