Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

Martin Hampel,Kai Heimel,Ute Meyer,Mareike Jakobi,Florian Finkernagel,Gunther Doehlemann,Lara Schmitz,Olaf Kniemeyer

doi:10.1371/journal.pone.0153861

Abstract

The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker’s yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

Highlights

The infection process of plant pathogenic fungi and their host plants requires effective strategies to subvert plant defense responses and foster pathogenic growth
We have previously demonstrated that expression of U. maydis Cib1 suppressed endoplasmic reticulum (ER) stress sensitivity of Saccharomyces cerevisiae HAC1 deletion strains [31], suggesting that Cib1 is able to bind to the same or similar potential unfolded protein response elements (UPREs) that are bound by Hac1
UPR-dependent gene regulation is mediated by binding of the central UPR regulatory protein Hac1 to the UPREs in the promoter region of target genes

Summary

Introduction

The infection process of plant pathogenic fungi and their host plants requires effective strategies to subvert plant defense responses and foster pathogenic growth. The genome of the corn smut fungus Ustilago maydis contains 536 genes, which are predicted to encode secreted proteins that might function as effectors [1] Many of these putative effector-encoding genes are organized in clusters and are highly upregulated during plant colonization [9]. ER stress is manifested by accumulation of un- or misfolded proteins in the ER that are sensed by the ER luminal domain of Ire resulting in multimerization, trans-autophosphorylation, activation of the endoribonuclease domain and subsequent cleavage of the unconventional intron of the Hac encoding mRNA [21] This process, referred to as unconventional cytoplasmic splicing gives rise to the Hac transcription factor, which subsequently induces UPR target gene expression by binding to unfolded protein response elements (UPREs) in their promoters [22,23,24,25]. UPR target genes typically encode ER chaperones, proteins involved in phospholipid and fatty acid synthesis and the ER associated degradation (ERAD) pathway [27]

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Conclusion