Abstract
BackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signaling in planta. A set of 35 genes from a library of secreted proteins from the maize pathogen Ustilago maydis were transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress in Nicotiana benthamiana plants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.
Highlights
The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development
The spliced mRNA translates into a functional transcription factor that shuttles to the nucleus and promotes the upregulation of genes that contain UPR responsive elements (UPREs) and endoplasmic reticulum (ER) stress elements (ERSEs) in their regulatory regions [12, 33, 46]
Fluorescence intensity is measured in a plate reader. pBIP1 regulatory region of the BiP1 protein from A. thaliana, eYFP enhanced yellow fluorescent protein, mCh mCherry, p35S CaMV 35S promoter, P2A porcine teschovirus-1 2A “self-cleaving” peptide
Summary
The unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. There, two proteases cleave the full length protein — the site 1 protease (S1P) in the C-terminal region inside the Golgi and the site 2 protease (S2P) in its cytosolic end — releasing the transcription factor which migrates to the nucleus and upregulates ER stress genes [10]. Both signaling pathways lead to the upregulation of genes to either correctly fold or degrade misfolded proteins, and to regulate transcription and translation to restore ER homeostasis [17, 44]. Transient ER stress can be relieved by the UPR, while persistent ER stress may lead to programmed cell death (PCD, [32])
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