Transcriptome sequencing: a powerful tool for the identification of avirulence effector genes from the tomato leaf mould pathogen Cladosporium fulvum

P278 - Leaf mould disease of tomato (Solanum lycopersicum) is caused by the biotrophic fungal pathogen Cladosporium fulvum. Upon leaf entry, C. fulvum secretes a plethora of effector proteins to modulate host defence responses and promote successful infection. However, these effectors also trigger defence in tomato cultivars carrying cognate Cf disease resistance genes (avirulence effectors). Consequently, effector identification and functional characterization is required to further understand how C. fulvum facilitates infection, as well as the mechanisms involved in Cf-mediated effector perception and defence activation. Here, a combined bioinformatic and transcriptome sequencing approach was employed to identify avirulence effect or genes of C. fulvum. Firstly, we sought to clone the Avr5 avirulence effector gene, corresponding to the previously cloned Cf-5 resistance gene of tomato. RNA-Seq was performed on the sequenced race 0 strain (0WU; carries the Avr5 gene), as well as a race 5 strain (IPO 1979; lacks a functional Avr5 gene), during infection of susceptible tomato. Forty-four in planta-induced C. fulvum Candidate Effector (CfCE) genes of 0WU were identified that putatively encode a secreted, small cysteine-rich protein. An expressed transcript sequence comparison between strains revealed two polymorphic CfCE genes in IPO 1979. One of these conferred avirulence to IPO 1979 on Cf-5 tomato following complementation with the corresponding 0WU allele, confirming identification of Avr5. The remaining CfCE proteins were then tested for their ability to elicit a hypersensitive response upon transient expression in wild tomato species containing unknown resistance traits. Using this approach, several potentially novel avirulence determinants of C. fulvum were identified.