Characterisation of the Cf-Ecp2 gene encoding for recognition of the conserved fungal effector Ecp2 in Solanum pimpinellifolium and Nicotiana paniculata

Members of the Capnodiales class of fungi have evolved contrasting lifestyles on plants, ranging from biotrophy, through to endotrophy and necrotrophy. Many of these fungi contain homologs of the effector protein called Ecp2. The ‘currant’ tomato (Solanum pimpinellifolium) resistance (R) gene Cf-Ecp2 recognises Ecp2 produced by the pathogenic fungus, Cladosporium fulvum. In this study, Cf-Ecp2 was fine-mapped to the Orion locus, which contains eight Homologs of Cladosporium resistance gene Cf-9 (Hcr9s), four of which share 100% sequence identity within their open reading frame. The Cf-Ecp2 locus exemplifies the complex nature of many R gene loci brought about by successive rounds of tandem duplication. This extensive duplication renders R gene loci complex and difficult to resolve. As a consequence, the sequencing of a BAC minimal tiling path across the Cf-Ecp2 locus required use of both short read and long read sequencing technologies, with MinION providing vital scaffolding reads. A transposon mutagenesis experiment generated two deletion mutants. The mutants had lost the ability to recognise Ecp2 along with the OR2A (2A) gene from the Cf-Ecp2 locus. Wild-type tomato (Solanum lycopersicum) Cf0 stable transformants, overexpressing 2A via the cauliflower mosaic virus 35S promoter, partially recapitulated the S. pimpinellifolium CfEcp2 phenotype. Lack of penetrance of the phenotype in the transgenic plants was attributed to the functional interference of 2A overexpression. The non-host, Nicotiana paniculata TW99, also recognises Ecp2 from C. fulvum. N. paniculata CfEcp2 was characterised for its ability to recognise many homologs of Ecp2, including those from Mycosphaerella fijiensis (causal agent of Black Sigatoka on banana) and the wilt-causing fungi, Fusarium oxysporum and Verticillium dahliae. The ability of Cf-Ecp2 to code for the recognition of an effector from many plant pathogens provides an exciting opportunity to engineer resistance to such pathogens in important crops