Analysis of Clp1-dependent UPR modulation in Ustilago maydis
by Niko Pinter
Date of Examination:2019-06-06
Date of issue:2019-06-13
Advisor:Prof. Dr. Kai Heimel
Referee:Prof. Dr. Kai Heimel
Referee:Prof. Dr. Gerhard Braus
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Abstract
English
The unfolded protein response (UPR) is a conserved signaling pathway, that is present in all eukaryotic cells and ensures endoplasmic reticulum (ER) homeostasis under stress conditions. In the phytopathogenic fungus U. maydis, the UPR is activated after plant penetration as a result of increased demands on the secretory pathway during the fungal/plant interaction. However, prolonged activation of the UPR is deleterious for U. maydis and UPR activity needs to be modulated during plant colonization. This modulation is achieved by the physical interaction between Cib1 (Clp1 interacting bZip 1), the central regulator of the UPR and Clp1 (Clampless 1), an important developmental regulator of U. maydis and the decisive factor for the induction of fungal proliferation after successful host penetration. The interaction between both proteins leads to increased stability of Clp1 and alters UPR gene expression. In this study, the functional consequences of this interaction on the physical properties of Cib1, the impact on Cib1 DNA binding and the transcriptional output of the UPR were characterized. Expression of clp1 leads to elevated ER stress resistance, increased protein stability and altered phosphorylation patterns of Cib1. Transcriptome analysis (RNAseq) during ER stress identified a set of 65 upregulated UPR core genes, whose expression is differentially modulated upon clp1 induction. Chromatin immunoprecipitation of Cib1 with subsequent whole-genome sequencing (ChIPseq) identified UPR elements (UPRE) in promoters of the large majority of UPR core genes and revealed that Cib1 DNA-binding specificity is not altered by Clp1. In a comprehensive gene deletion analysis, a previously uncharacterized UPR target gene was identified that is specifically required for biotrophic growth of U. maydis. UMAG_02729 encodes an intramembrane cleaving signal peptide peptidase (spp1) that contains a conserved active site typical for aspartyl proteases. Plants inoculated with Δspp1 mutants or strains expressing enzymatically inactive Spp1 triggered massive plant defense responses as evidenced by reactive oxygen species (ROS) accumulation and strongly increased expression of pathogenesis-related plant genes. Complementation of the spp1 deletion strain with orthologous genes from Sporisorium reilianum and Ustilago hordei recovered virulence and expression of the well-characterized human ortholog HM13 suppressed the virulence defect of the spp1 deletion mutant in a dose-dependent manner. However, the virulence-specific function of Spp1 is not related to known functions of signal peptide peptidases, such as ER-associated degradation (ERAD), hypoxia adaptation or effector secretion. Deletion of predicted UPREs in the promoter of spp1 significantly reduced spp1 expression upon ER stress. Co-immunoprecipitation analysis of Spp1 with subsequent LC-MS analysis revealed members of the signal peptidase complex (SPC) and the O-mannosyltransferase Pmt4, as potential interaction partners or substrates of Spp1. In summary, the data of this study revealed a potential mechanism on how UPR in U. maydis is modulated by Clp1 and a novel factor important for plant defense suppression that is not connected to previously known pathways related to signal peptide peptidase function or plant defense suppression by phytopathogenic fungi.
Keywords: Ustilago maydis; UPR; unfolded protein response; SPP; signal peptide peptidase; Cib1; Clp1; Spp1; ERAD; ER-associated degradation; RNAseq; ChIPseq