Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley

Authors: LI, ZIZHANG - University Of Copenhagen; VELÁSQUEZ-ZAPATA, VALERIA - Iowa State University; Elmore, James - Mitch; LI, XUAN - University Of Copenhagen; XIE, WENJUN - University Of Copenhagen; DEB, SOHINI - University Of Copenhagen; TIAN, XIAO - University Of Copenhagen; BANERJEE, SAGNIK - Iowa State University; JØRGENSEN, HANS - University Of Copenhagen; PEDERSON, CARSTEN - University Of Copenhagen; Wise, Roger; THORDAL-CHRISTENSEN, HANS - University Of Copenhagen

Submitted to: Molecular Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/11/2024
Publication Date: 5/2/2024
Citation: Li, Z., Velásquez-Zapata, V., Elmore, J.M., Li, X., Xie, W., Deb, S., Tian, X., Banerjee, S., Jørgensen, H.J., Pederson, C., Wise, R.P., Thordal-Christensen, H. 2024. Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley. Molecular Plant Pathology. 25(5). Article e13463. https://doi.org/10.1111/mpp.13463.
DOI: https://doi.org/10.1111/mpp.13463

Interpretive Summary: Powdery mildew fungi infect more than 9,500 agronomic and horticultural plant species. In order to prevent economic loss due to diseases caused by powdery mildew, plant breeders incorporate disease resistance genes into varieties that are grown for food, feed, fuel and fiber. One of these resistance genes provides instructions for assembly of the MLA immune receptor, an ancestral protein that provides disease resistance to powdery mildew, stem- and stripe rust in small grain cereal crops, such as barley, wheat, and rye. In these systems, corresponding host and pathogen proteins interact during fungal infection to initiate plant defense, often relying on basic cellular processes, such as the endoplasmic reticulum (ER) and protein quality control systems elsewhere in the cell. USDA-ARS, Iowa State University, and University of Copenhagen scientists used a combination of computer- and laboratory-based methods to identify and characterize a barley J-containing protein, HvERdj3B, as a host target for two diverse powdery mildew virulence proteins. Their analyses showed, for the first time, that HvERdj3B is an ER-localized protein, and that that upon infection, the fungal proteins and their barley target translocate from the plant cytosol into the ER lumen. These observations support the involvement of these proteins in promoting disease resistance. Impact: These discoveries provide a foundation for further research into the complex molecular components that control disease resistance in crops.

Technical Abstract: The barley powdery mildew fungus, Blumeria hordei (Bh), secretes hundreds of candidate secreted effector proteins (CSEPs) to facilitate pathogen infection and colonization. One of these, CSEP0008, is directly recognized by the barley nucleotide-binding leucine-rich-repeat (NLR) receptor MLA1 and therefore is designated AVRA1. Here, we show that AVRA1 and the sequence-unrelated Bh effector BEC1016 (CSEP0491) suppress immunity in barley. We used yeast two-hybrid next-generation interaction screens (Y2H-NGIS), followed by binary Y2H and in planta protein–protein interactions studies, and identified a common barley target of AVRA1 and BEC1016, the endoplasmic reticulum (ER)-localized J-domain protein HvERdj3B. Silencing of this ER quality control (ERQC) protein increased Bh penetration. HvERdj3B is ER luminal, and we showed using split GFP that AVRA1 and BEC1016 translocate into the ER signal peptide-independently. Overexpression of the two effectors impeded trafficking of a vacuolar marker through the ER; silencing of HvERdj3B also exhibited this same cellular phenotype, coinciding with the effectors targeting this ERQC component. Together, these results suggest that the barley innate immunity, preventing Bh entry into epidermal cells, requires ERQC. Here, the J-domain protein HvERdj3B appears to be essential and can be regulated by AVRA1 and BEC1016. Plant disease resistance often occurs upon direct or indirect recognition of pathogen effectors by host NLR receptors. Previous work has shown that AVRA1 is directly recognized in the cytosol by the immune receptor MLA1. We speculate that the AVRA1 J-domain target being inside the ER, where it is inapproachable by NLRs, has forced the plant to evolve this challenging direct recognition.