Establishing proximity-dependent protein labeling to investigate co-chaperone mediated effector secretion in Ustilago maydis
by Nur Uddin Mahmud
Date of Examination:2025-08-22
Date of issue:2025-12-10
Advisor:Prof. Dr. Kai Heimel
Referee:Prof. Dr. Kai Heimel
Referee:Prof. Dr. Gerhard Braus
Persistent Address:
http://dx.doi.org/10.53846/goediss-11686
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Abstract
English
The complex process of protein folding within the endoplasmic reticulum (ER) of the corn smut fungi Ustilago maydis depends on the coordinated action of chaperone and their associated co-chaperone proteins. The unfolded protein response (UPR) is activated during ER stress and Dnj1 and Dnj2 co-chaperones are upregulated (Pinter et al., 2019). Dnj1 resides in the ER lumen and Dnj2 is predicted to be an ER transmembrane protein, both containing a functional J domain which is well known to interact with ER chaperones for proper protein folding. In U. maydis, the role of Dnj1 is studied to some extent but Dnj2 is fully uncharacterized. Here, we revealed that both Dnj1 and Dnj2 are required for full virulence of U. maydis and double deletion of Dnj1 and Dnj2 exacerbated the virulence compared to single deletions. Dnj1 was shown to physically interact with ER chaperone Bip1, to functionally interact with the lectin chaperone Calnexin (Cne1) and to mediate secretion of the Cmu1 effector (Lo Presti et al., 2016). The specific role in virulence of both Dnj1 and Dnj2 and how they contribute with overlapping or specific functions related to folding and processing of secreted effectors was so far unknown. To this end, we established proximity dependent labeling in U. maydis using two biotin ligases TurboID and UltraID to identify putative interaction partners and substrate proteins of Dnj1 and Dnj2. Under normal growth conditions, many proteins including central factors of the ER folding machinery were identified as proximally localized partners of Dnj1 and Dnj2. Only few secreted proteins were found in the closest proximity as the majority of secreted proteins and/or effectors are specifically expressed during the fungal-plant interaction. To induce expression of genes encoding secreted proteins in axenic culture, we overexpressed two master regulator transcription factors, Hdp2 and Biz1, leading to significantly increased expression of more than 40 % and of the whole secretome and more than 70% of the so-called second wave effectors, comprising all so far characterized effectors contributing to U. maydis virulence. In this strain background more than 80 secreted proteins were found to be enriched in Dnj1/Dnj2 BioID strains besides UPR core proteins, which were also found in normal growth conditions. Many of the secreted proteins were specifically enriched by either Dnj1 or Dnj2 and only few were enriched in both strain backgrounds. Hence, it is conceivable that Dnj1 and Dnj2 have specific sets of substrate proteins. However, both co-chaperones are centrally placed in a common set of core proteins of the ER folding machinery and were found in both Dnj1 and Dnj2 fusions as likely interaction partners. This strongly suggests a potential interdependent role for both proteins within the ER folding machinery and quality control.
Keywords: Ustilago maydis; protein folding; co-chaperone; unfolded protein response (UPR); endoplasmic reticulum (ER) stress; proximity dependent labeling
