Data from: Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity Wenlin Liao Mads E. Nielsen Carsten Pedersen Wenjun Xie Hans Thordal-Christensen https://orcid.org/0000-0001-8813-0157 Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark Correspondence: htc@plen.ku.dk First published 13th of October, 2022, Journal of Experimental Botany: https://doi.org/10.1093/jxb/erac403 Abstract Encasements formed around haustoria and biotrophic hyphae as well as hypersensitive reaction (HR) cell death are essential plant immune responses to filamentous pathogens. In this study we examine the components that may contribute to the absence of these responses in susceptible barley attacked by the powdery mildew fungus. We find that the effector CSEP0162 from this pathogen targets plant MONENSIN SENSITIVITY1 (MON1), which is important for the fusion of multivesicular bodies to their target membranes. Overexpression of CSEP0162 and silencing of barley MON1 both inhibit encasement formation. We find that the Arabidopsis ecotype No-0 has resistance to powdery mildew, and that this is partially dependent on MON1. Surprisingly, we find the MON1-dependent resistance in No-0 not only includes an encasement response, but also an effective HR. Similarly, silencing of MON1 in barley also blocks Mla3-mediated HR-based powdery mildew resistance. Our results indicate that MON1 is a vital plant immunity component, and we speculate that the barley powdery mildew fungus introduces the effector CSEP0162 to target MON1 and hence reduce encasement formation and HR. Methods Plant material The following barley (Hordeum vulgare) lines were used in this work. Ror2, a syntaxin mutant in cv. Ingrid w. low penetration resistance (Collins et al., 2003). The near-isogenic cv. Pallas lines, P-01 and P-02, with the powdery resistance genes, Mla1 and Mla3, respectively. Barley plants were grown under 16 h (20¡C) day (150 ?E m-2 s-1), 8 h night (15¡C). The following Arabidopsis thaliana lines were used in this work. Ecotype Columbia-0 (Col-0) and its mutants, mon1-1, eds1-2 and ndr1-1. Ecotype Nossen-0 (No-0) and its mutant mon1-2 (Ds transposon line 54-4894-1 received from RIKEN. Arabidopsis plants were grown under 8 h (21¡C) day (125 ?E m-2 s-1), 16 h night (15¡C). Arabidopsis germination rates were determined on ? MS phytoagar. Mutant allele genotypes were determined by PCR using primers listed in Supplementary Table S1. Fungal material The barley powdery mildew fungus (Bh) isolates, A6 and C15, were propagated on P-01 and P-02, respectively, by weekly transfer. The Arabidopsis powdery mildew fungus, Golovinomyces orontii (Go) isolate MPIPZ (Max-Planck-Institut fŸr PflanzenzŸchtungsforschung) was propagated on eds1-2 by bi-weekly transfer. Barley epidermal cell transient induced gene silencing (TIGS) and over-expression HvMON1 was transiently induced gene-silenced (TIGS). An HvMON1 RNAi fragment (316 bp) was designed by the siRNA-Finder (si-Fi) Software and introduced twice in opposing orientation in the destination vector pIPKTA30N to produce a hairpin transcript. A construct for over-expression of YFP-CSEP0162 was previously generated. Each of these pIPKTA30N-HvMON1i and pUbi-YFP-CSEP0162-nos constructs were co-transformed with pUbi-GUS-nos as marker into barley epidermal cells by particle bombardment. Subsequently, the leaves were placed in closed 1% phytoagar plates w. 10 ?g ml-1 benzimidazole at 16 h (20¡C) day (150 ?E m-2 s-1), 8 h night (15¡C). To study transformed cells, leaves were stained with X-Gluc for GUS activity. Immune response scorings To induce encasements around Bh haustoria, barley leaves were sprayed with 100 ?g ml-1 tetraconazole in 20% acetone, 0.04% Tween-20, 2 hours before inoculation with Bh. To study encasements, either alone or in combination with GUS staining, they were visualized 5 days after inoculation by their callose content after staining of the leaves with 0.01% aniline blue in 1 M glycine, pH 9.5, followed by UV epifluorescence microscopy. Penetration rate, encasement formation, HR cells and fungal development in Arabidopsis were scored by light and UV epifluorescence microscopy as described by Nielsen et al. (2017). In short, for scoring of penetration success, leaf material was trypan blue stained 2 days after Bh or Go inoculation. For each leaf, a minimum of 50 penetration attempts (presence of a fungal appressorium) were scored using light microscopy. Penetration was determined by the presence of a fungal haustorium. Callose staining was performed as above. Quantification of gene expression by qRT-PCR and powdery mildew biomass by qPCR Total RNA was extracted using Monarch¨ RNA Cleanup Kit (NEB). Reverse transcription and cDNA synthesis were performed using NEBNext¨ RNA First Strand Synthesis Module (NEB). Transcript quantification was carried out using Stratagene MX3000P real-time PCR detection system (Agilent Technologies) with FIREPol¨ EvaGreen¨ Mix (Solis BioDyne). Primers used to amplify PCR products of maximum 200 bp are described in Supplemental Table S1. The ubiquitin conjugating factor (UBC2) was used as barley reference gene. The level of gene expression was calculated using the relative quantification (??Ct) algorithm by Livak and Schmittgen (2001, Methods 25, 402-408) by merging data from three separate experiments each with two technical repeats. Total genomic DNA of Go-inoculated Arabidopsis and Bh-inoculated barley was extracted using DNeasy¨ Plant Mini Kit (QIAGEN). Go and Bh were quantified relative to plant DNA using primers described in Supplemental Table S1. Barley Stripe Virus Induced Gene Silencing (VIGS) The tripartite genome of Barley Stripe Mosaic Virus (BSMV) was used as basis for barley gene silencing. The binary Ti-constructs pCaBS-?, pCaBS-?, pCa-?bLIC and pCaBS-?b-TaPDS were described by Yuan et al. (2011). The Gateway cassette was inserted into the ligation-independent cloning site of pCa-?bLIC, and an RNAi fragment of HvMON1 (the 316 bp fragment also used for TIGS) and a full-length coding sequence of mYFP were inserted by Gateway LR clonase (Invitrogen) reactions. All constructs were transformed into A. tumefaciens strain EHA105 by selection on rifampicin (25/?g ml) and kanamycin (100 ?g/ml). Confirmed strains were co-infiltrated into N. benthamiana with A. tumefaciens containing pCaBS-? and pCaBS-? according to the method described above. When BSMV symptoms appeared on upper leaves approximately 10 dpi, then BSMV infected leaves were collected and ground in 20 mM Na-phosphate, pH 7.2 with 1% silica. The homogenates were smeared onto first leaves of 7-day-old barley seedlings by rubbing gently with fingers. The third leaves of treated barley plants were collected about two weeks later for qRT-PCR or Bh inoculation. Silencing of phytoene desaturase using pCaBS-?b-TaPDS was used as a positive indicator for the VIGS system, while pCa-?b-mYFP was used as negative control. Usage notes The data in sheets ÔFig 2BÕ and ÔFig 2CÕ are obtained by light microscopy to quantify the effect of MON1 silencing (2B) and CSEP0162 over-expression (2C) on encasement of Bh (C15) haustoria in transiently transformed epidermal cells of barley ror2. Leaves of eight-day-old barley plants were transformed by particle bombardment with a construct for GUS staining and either a construct for MON1 RNAi or a construct for CSEP0162 overexpression. Two days later, they were treated with tetraconazole and Bh inoculation. Encasement scorings were made after another 5 days in transformed cells visualized by GUS staining. Mock: data from untransformed epidermal cells of the bombarded leaves. The data in sheet ÔFig 3BÕ are qPCR-based quantification of Go fungal biomass relative to plant biomass 6 days after inoculation. Calculations were made using the relative quantification (??Ct) algorithm by Livak and Schmittgen (2001, Methods 25, 402-408). The data in sheet ÔFig 4Õ are light microscope scorings of Go fungal success rates in different Arabidopsis genotypes. Initially, ten categories of single spore/plant epidermal cell interactions we recorded: 1, unpenetrated; 2, unpenetrated+HR; 3, haustorium; 4, haustorium+HR; 5, haustorium+sec. hypha; 6, haustorium+sec. hypha+HR; 7, encased haustorium; 8, encased haustorium+HR; 9, encased haustorium+sec. hyphae; 10, encased haustorium+sec. hyphae+HR. Subsequently, fungal-epidermal cell interactions were calculated by making different combinations of these categories. The data in sheet ÔFig 5AÕ are qPCR-based quantification of the barley MON1 transcript after virus-induced gene silencing relative to the barley tubulin transcript, calculated using the relative quantification (??Ct) algorithm by Livak and Schmittgen (2001, Methods 25, 402-408). The data in sheet ÔFig 5DÕ are qPCR-based quantification of Bh fungal biomass relative to barley plant biomass 6 days after inoculation. Calculations were made using the relative quantification (??Ct) algorithm by Livak and Schmittgen (2001, Methods 25, 402-408). The data in sheet ÔFig 6AÕ are scoring of germination rates of seeds from different Arabidopsis genotypes. The data in sheet ÔFig S3Õ are measurements of lengths (um) of Go secondary hyphae developed from single germinated spores that penetrated and formed haustoria in the epidermal cells of different Arabidopsis genotypes. Funding This research support was supported by the China Scholarship Council (PhD-student scholarship 201706850092 to WL), the Novo Nordisk Foundation (Challenge grant NNF19OC0056457 to HTC), and by Villum Fonden (Experiment Programme grant 00028131 to HTC).