TY - JOUR

T1 - Powdery mildew effectors AVRA1 and BEC1016 target the ER J-domain protein HvERdj3B required for immunity in barley

AU - Li, Zizhang

AU - Velásquez-Zapata, Valeria

AU - Elmore, J. Mitch

AU - Li, Xuan

AU - Xie, Wenjun

AU - Deb, Sohini

AU - Tian, Xiao

AU - Banerjee, Sagnik

AU - Jørgensen, Hans J.L.

AU - Pedersen, Carsten

AU - Wise, Roger P.

AU - Thordal-Christensen, Hans

N1 - Funding Information: We thank our late and good friend, Dr Patrick Schweizer (Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany) for providing the RNAi vector and the Mlo RNAi construct, Dr Masumi Robertson (Commonwealth Scientific and Individual Research Organization Plant Industry) for the Gateway Y2H vectors (pDEST-ACT2 and pDEST-AS2-1), Greg Fuerst (USDA-ARS at Iowa State University) for conducting the Bh time-course infection experiment and expert isolation of RNA for the 3-frame Y2H library, Associate Professor Shoi Mano for the BiFC-vectors, and Drs Pietro Spanu (Imperial College, U.K.) and Ralph Panstruga (RWTH Aachen, Germany) for CSEP clones. Research supported in part by China Scholarship Council for PhD-student scholarship 201708340064 to Z.Z., Novo Nordisk Foundation Challenge grant NNF19OC0056457 to H.T.C., Villum Fonden Experiment Programme grant 00028131 to H.T.C., Fulbright \u2013 Minciencias 2015 & Schlumberger Faculty for the Future fellowships to V.V.Z., USDA-ARS Postdoctoral Research Associateship and USDA-NIFA-ELI Postdoctoral Fellowship 2017-67012-26086 to J.M.E., Oak Ridge Institute for Science and Education under U.S. Department of Energy contract number DE-SC0014664 to S.B., and National Science Foundation \u2013 Plant Genome Research Program grant 13-39348, USDA-National Institute of Food and Agriculture grant 2020-67013-31184, USDA-Agricultural Research Service projects 3625-21000-067-00D and 5030-21220-068-000-D to R.P.W. and Marie Sk\u0142odowska-Curie Actions Postdoctoral Fellowship project 101104193 to S.D. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the CSC, NNF, VF, USDA, NIFA, ARS, DOE, ORAU/ORISE, NSF or MSCA. USDA is an equal opportunity provider and employer. Publisher Copyright: © 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

KW - barley

KW - ER quality control

KW - ERdj3b

KW - immunity

KW - pathogen effectors

KW - powdery mildew

KW - signal peptide-independent ER-uptake

KW - yeast two-hybrid next-generation interaction screening

KW - barley

KW - ER quality control

KW - ERdj3b

KW - immunity

KW - pathogen effectors

KW - powdery mildew

KW - signal peptide-independent ER-uptake

KW - yeast two-hybrid next-generation interaction screening

U2 - 10.1111/mpp.13463

DO - 10.1111/mpp.13463

M3 - Journal article

C2 - 38695677

AN - SCOPUS:85192035338

VL - 25

JO - Molecular Plant Pathology

JF - Molecular Plant Pathology

SN - 1464-6722

IS - 5

M1 - e13463

ER -