A high-throughput screening method to identify proteins involved in unfolded protein response of the endoplasmic reticulum in plants.

BACKGROUND:The 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. RESULTS:We 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. CONCLUSIONS:We 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.