RT Journal Article
SR Electronic
T1 A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 825190
DO 10.1101/825190
A1 André Alcântara
A1 Denise Seitner
A1 Fernando Navarrete
A1 Armin Djamei
YR 2019
UL http://biorxiv.org/content/early/2019/10/31/825190.abstract
AB 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.BiPluminol binding protein;bZIPbasic leucin zipper;DTTdithiothreitol;ERendoplasmic reticulum;ERSEER stress elements;HY5Elongated Hypocotyl 5;IRE1Inositol-requiring enzyme 1;LBLuria broth cell culture medium;mChmCherry;MES2-(N-morpholino)ethanesulfonic acid;OD600 nmoptical density at 600 nm;p35SCaMV 35S promoter;PCDprogrammed cell death;qRT-PCRquantitative real time polymerase chain reaction;S1Psite 1 protease;S2Psite 2 protease;SASalicylic acid;Skp1S-phase kinase-associated protein 1;Tmtunicamycin;UPRunfolded protein response;UPREUPR responsive elements;eYFPenhanced yellow fluorescent protein.