Analysis of COI1-dependent signalling processes in Arabidopsis roots upon infection with the fungal pathogen Verticillium longisporum

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The ascomyceteVerticillium longisporum is a soil-borne vascular pathogen that causes major yield losses on rapeseed (Brassica napus). Our previous analysis of the V. longisporum/Arabidopsis thaliana interaction has unraveled two unexpected findings: (1) COI1, the receptor of the plant defense hormone jasmonic acid-isoleucine (JA-Ile), is required in roots for efficient proliferation of the fungus in the shoot. (2) The novel COI1 function operates even in the absence of plant-derived JA-Ile or fungus-derived JA-Ile mimics. The long-term goals of this proposal are to identify the disease-promoting root-to-shoot signal and to elucidate the COI1-dependent but JA-Ile-independent signal transduction pathway leading to its synthesis. During the proposed funding period, next generation sequencing (NGS) shall yield plant and/or fungal genes that are differentially expressed in infected aos (JA biosynthesis mutant) and coi1 (JA receptor mutant) roots. Translational fusions of selected plant and fungal genes to fluorescent proteins will be transformed into Arabidopsis and V. longisporum, respectively, and their expression will be monitored simultaneously by confocal laser microscopy. Information on the expression pattern (time point, cell-type-specificity) and coding region will be integrated to select candidate plant or fungal genes that might encode the susceptibility-enhancing signal. These genes will functionally characterized by analysis of corresponding mutants. To substantiate our hypothesis that COI1 can function independently from its ligand, we will complement the coi1 mutant with transgenes encoding mutant COI1 proteins that cannot bind JA-Ile. Moreover, the involvement of JAZ proteins will be analyzed using plants expressing a dominant negative JAZ protein under the control of a root-specific promoter. In order to identify novel COI1 interaction partners, several variants of the yeast two hybrid screen will be performed in a hi-throughput screening system. Analysis of transgenic plants or mutants will be done by monitoring the disease phenotype and the expression of the COI1-dependent but JA-Ile-independent genes identified by NGS.