Elucidating Salicylic Acid Sensing in Biotrophic Smut Fungi

Ustilago maydis is a biotrophic pathogenic fungus that causes tumours on all aerial parts of maize plants. With the help of numerous small secreted proteins, so called effectors, this pathogen is able to subvert plant defence responses and to manipulate the host metabolism for its own needs. While the perception of the pathogen by the plant is quite well investigated, little is known how the pathogen senses the defence status of its host in order to secrete the appropriate effector cocktail keeping the balance in the biotrophic interaction. We recently discovered that U. maydis is able to sense the important plant defence hormone salicylic acid (SA). This opens up the possibility that the fungal invader reads along the plant defence status via SA. With an SA-responsive promoter reporter construct in U. maydis we performed a mutagenesis screen to identify components of the fungal SA sensing machinery. Preliminary results with several mutant candidates provided a proof-of-concept. In this project we suggest strategies to dissect this novel SA signalling cascade and to functionally characterize its components. We will further include the related smut fungus Ustilago bromivora that infects the model plant Brachypodium distachyon, to test if fungal SA signalling components have orthologous functions in other smuts as well. This will allow additional experimental approaches and enables us in the future to explore fungal SA perception as a target for plant protection.

Salicylic acid (SA) is a plant hormone with an important role in plant immunity, especially upon biotrophic microbial attack. The maize infecting fungus Ustilago maydis is as a biotrophic pathogen also confronted with the salicylic acid mediated immune response of the host plant. By a genetic screen, the factor Required for SA Sensing 1 (Rss1) was identified to play a crucial role in fungal SA sensing. It is a binuclear transcription factor. In a yeast based transcriptional activation assay Rss1 shows only transcriptional activation in the presence of salicylic acid in the medium. Interestingly, the tryptophan degradation product anthranilic acid stimulates as well transcriptional activating activity of Rss1. By genome wide transcriptional analysis of the Rss1 mutants on SA containing medium, a number of Rss1- dependent regulated genes in axenic culture of U. maydis were identified. The identified differentially regulated genes encode for proteins which are either involved in the salicylic acid degradation or the tryptophan degradation pathway of the fungus. The deletion mutant of Rss1 does not show an obvious virulence phenotype on infected maize seedlings. RT-PCR studies of Rss1 target genes in the context of the infected plant show that factors like the salicylate hydroxylase Shy1 are not solely regulated in an Rss1 dependent fashion, leaving the possibility that additional signal transduction pathways beside the salicylic acid perception pathway are integrated during infection to regulate these target genes. Results concerning the identification of Rss1 led to a peer reviewed publication (Rabe et al., 2016) and a doctoral thesis.