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Abstract:
Salicylic acid (SA) belongs to the class of phenolic compounds and is composed of an aromatic ring with one carboxyl- and one hydroxyl group. In plants, it is a key signalling molecule for the regulation of local and systemic defence responses against biotrophic plant pathogens. One model organism to study biotrophic pathogens is Ustilago maydis, the causative agent of corn smut disease. U. maydis employs strategies to interfere with SA production and SA-associated signalling of its host Zea mays. These strategies are considered to supress defence responses and thereby contribute to a successful infection. Moreover, the U. maydis genome encodes three putative salicylate hydroxylases, UMAG_05230, UMAG_03408, and UMAG_05967, which are predicted to degrade SA. For one of these proteins, UMAG_05230 (Shy1), salicylate hydroxylase activity could already be experimentally confirmed. Based on the predicted enzymatic function of these proteins, it was hypothesized that by eliminating SA they could contribute to the suppression of host immunity. To provide insights into the biological role of SA degradation by salicylate hydroxylases in U. maydis, the functional characterization of these genes was continued in this study. It could be demonstrated that U. maydis is able to use SA as carbon source and that Shy1 is essential for SA utilization. Besides shy1 and the second salicylate hydroxylase-related gene UMAG_03408, which were previously shown to be induced during infection, also the third gene of this family, UMAG_05967, was strongly upregulated in these developmental stages. However, although induced during biotrophic growth, no involvement in virulence could be shown for these three genes. Transcriptional profiling revealed that shy1 and the two salicylate hydroxylase-related genes are induced in presence of SA, indicating that U. maydis is able to sense SA. To provide insights into the molecular mechanism of SA perception and signalling, a forward genetic screen was performed. This screen led to the identification of one key regulator for SA sensing, the binuclear zinc cluster transcription factor Rss1. Rss1 is important for SA sensing and modulates the expression of genes that are needed to metabolise SA and tryptophan. Rss1 most likely acts concomitantly as SA sensor and transcriptional activator. Although Rss1 is important for the regulation of SA-responsive genes in axenic culture, transcriptional profiling data provided evidence that additional cues and pathways could exist that regulate these genes during plant colonization. Moreover, virulence assays with rss1 deletion mutants showed that the deletion of rss1 had no impact on virulence in seedling infections.
