Analyse der UPR vermittelten Stressantwort und ihrer Funktion während der biotrophen Entwicklung von<i> Ustilago maydis</i>

Abstract

The unfolded protein response (UPR) is a conserved eukaryotic signaling network that is activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER) to ensure protein homeostasis. In the baker's yeast <i>Saccharomyces cerevisiae</i>, the UPR signaling pathway is controlled by the ER stress sensor Ire1p and the bZIP transcription factor Hac1p or XBP1 in higher eukaryotes. In this study homologs of the central UPR regulators in the biotrophic fungus <i>Ustilago maydis</i> were characterized, identifying the UPR as an essential coordinator of pathogenic development. In <i>S. cerevisiae</i> the accumulation of un- or misfolded proteins leads to unconventional splicing of the <i>HAC1</i> mRNA by Ire1p allowing the translation of the central regulator Hac1p. The complementation of the <i>∆HAC1</i> mutant by <i>cib1</i> (homolog of <i>HAC1</i>) and comprehensive expression analysis showed that the regulatory mechanisms of the UPR in <i>U. maydis</i> are largely conserved. However, the range of regulated target genes includes secreted virulence factors that are essential for pathogenic development. <i>In silico</i> prediction of unfolded protein response elements (UPREs) in promoter regions identified the three previously characterized effector genes pit1/pit2 and tin1-1, as UPR target genes. Targeted deletion of the predicted UPRE abolishes cib1-dependent expression of pit2 and leads to significantly reduced virulence. Furthermore a functional UPR is necessary for elevated expression and correct processing of Pit2 in the ER. This study showed that the unspliced cib1 mRNA encodes Cib1u, a negative regulator of UPR signalling, which is in stark contrast to S. cerevisiae and the filamentous ascomycetes <i>Aspergillus niger</i> and <i>Trichoderma reseei</i>, but analagous to UPR regulation in higher eukaryotes. In addition we discovered a previously undescribed function of Cib1u in response to ER-stress that is likely conserved. The exact control of the UPR activity is required for the correct timing of the different steps within the pathogenic development of <i>U. maydis.</i> A premature activation of the UPR leads to an inhibition of the filamentous growth, which is necessary for plant infection. The specific activation of the UPR after successful penetration of the plant surface and their continuous activity during growth <i>in planta</i> is essential for the pathogenic development. Direct interaction between Cib1 and development regulator Clp1 during development leads to stabilization of Clp1 and modulation of Cib1-dependent gene expression, that triggers poliferation <i>in planta</i> and confers increased ER stress resistance during biotrophic growth. In summary, our data reveal that in U. maydis the UPR serves as a checkpoint to coordinate and align cellular physiology, developmental progress and secretion of effector proteins.