DOI: 10.5061/dryad.73n5tb30w
title: Barley endosomal MONENSIN SENSITIVITY1 is a target of the powdery mildew effector CSEP0162 and plays a role in plant immunity

description: 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 reduces
 encasement formation and HR.
description: <strong>Plant
 material</strong>: The following barley (<em>Hordeum
 vulgare</em>) lines were used in this work.
 <em>Ror2</em>, a syntaxin mutant in cv. Ingrid w. low
 penetration resistance (Collins <em>et al</em>., 2003). The
 near-isogenic cv. Pallas lines, P-01, and P-02, with the powdery
 resistance genes, <em>Mla1</em> and <em>Mla3</em>,
 respectively. Barley plants were grown under 16 h (20°C) day (150
 μE
 m<sup>-2</sup> s<sup>-1</sup>), 8 h night (15°C).
 The following <em>Arabidopsis thaliana</em> lines were used in
 this work. Ecotype Columbia-0 (Col-0) and its mutants,
 <em>mon1-1</em>, <em>eds1-2,</em> and
 <em>ndr1-1</em>. Ecotype Nossen-0 (No-0) and its mutant
 <em>mon1-2</em> (Ds transposon line 54-4894-1 received from
 RIKEN. Arabidopsis plants were grown under 8 h (21°C) day (125
 μE
 m<sup>-2</sup> s<sup>-1</sup>), 16 h night (15°C).
 Arabidopsis germination rates were determined on ½ MS phytoagar. Mutant
 allele genotypes were determined by PCR using primers listed in
 Supplementary Table S1. <strong>Fungal material</strong>:
 The barley powdery mildew
 fungus (<em>Bh</em>) isolates, A6 and C15, were propagated on
 P-01 and P-02, respectively, by weekly transfer. The Arabidopsis powdery
 mildew fungus, <em>Golovinomyces orontii</em>
 (<em>Go</em>) isolate MPIPZ (Max-Planck-Institut für Pflanzenzüchtungsforschung) was propagated on <em>eds1-2</em> by bi-weekly transfer.  <strong>Barley epidermal cell transient induced gene silencing (TIGS) and over-expression</strong>: <em>Hv</em>MON1 was transiently induced gene-silenced (TIGS). An <em>Hv</em>MON1 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-<em>HvMON1i</em> 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 μg ml<sup>-1</sup> benzimidazole at 16 h (20°C) day (150 μE m<sup>-2</sup> s<sup>-1</sup>), 8 h night (15°C). To study transformed cells, leaves were stained with X-Gluc for GUS activity. <strong>Immune response scorings</strong>: To induce encasements around <em>Bh</em> haustoria, barley leaves were sprayed with 100 μg ml<sup>-1</sup> tetraconazole in 20% acetone, 0.04% Tween-20, 2 hours before inoculation with <em>Bh</em>. 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 <em>et al</em>. (2017). In short, for scoring of penetration success, leaf material was trypan blue stained 2 days after <em>Bh</em> or <em>Go</em> 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. <strong>Quantification of gene expression by qRT-PCR and powdery mildew biomass by qPCR</strong>: 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 (<sup>ΔΔ</sup>Ct) algorithm by Livak and Schmittgen (2001, Methods <strong>25</strong>, 402-408) by merging data from three separate experiments each with two technical repeats.  Total genomic DNA of <em>Go</em>-inoculated Arabidopsis and <em>Bh</em>-inoculated barley was extracted using DNeasy® Plant Mini Kit (QIAGEN). <em>Go</em> and <em>Bh</em> were quantified relative to plant DNA using primers described in Supplemental Table S1. <strong>Barley Stripe Virus Induced Gene Silencing (VIGS)</strong>: The tripartite genome of Barley Stripe Mosaic Virus (BSMV) was used as basis for barley gene silencing. The binary Ti-constructs pCaBS-α, pCaBS-β, pCa-γbLIC and pCaBS-γb-TaPDS were described by Yuan et al. (2011). The Gateway cassette was inserted into the ligation-independent cloning site of pCa-γbLIC, and an RNAi fragment of <em>HvMON1</em> (the 316 bp fragment also used for TIGS) and a full-length coding sequence of <em>mYFP</em> were inserted by Gateway LR clonase (Invitrogen) reactions. All constructs were transformed into <em>A. tumefaciens</em> strain EHA105 by selection on rifampicin (25/μg ml) and kanamycin (100 μg/ml). Confirmed strains were co-infiltrated into <em>N. benthamiana</em> with <em>A. tumefaciens</em> containing pCaBS-α and pCaBS-β 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-γb-<em>TaPDS</em> was used as a positive indicator for the VIGS system, while pCa-γb-mYFP was used as negative control.
description: The data in sheets ‘Fig 2B’ and ‘Fig 2C’ are
 obtained by light microscopy to quantify the effect of
 <em>MON1</em> silencing (2B) and CSEP0162 over-expression (2C)
 on encasement of <em>Bh</em> (C15) haustoria in transiently
 transformed epidermal cells of barley <em>ror2</em>. 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 <em>Bh</em> 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 ‘Fig 3B’ are qPCR-based quantification of Go fungal biomass
 relative to plant biomass 6 days after inoculation. Calculations were made
 using the relative quantification
 (<sup>ΔΔ</sup>Ct) algorithm by Livak and Schmittgen (2001,
 Methods <strong>25</strong>, 402-408).
 The data in sheet ‘Fig 4’ 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 ‘Fig 5A’ 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
 (<sup>ΔΔ</sup>Ct) algorithm by Livak and Schmittgen (2001,
 Methods <strong>25</strong>, 402-408).
 The data in sheet ‘Fig 5D’ are qPCR-based
 quantification of Bh fungal biomass relative to barley plant biomass 6
 days after inoculation. Calculations were made using the relative
 quantification (<sup>ΔΔ</sup>Ct) algorithm by Livak and Schmittgen (2001,
 Methods <strong>25</strong>, 402-408).
 The data in sheet ‘Fig 6A’ are scoring of
 germination rates of seeds from different Arabidopsis
 genotypes. The data
 in sheet ‘Fig S3’ are measurements of lengths (um) of
 <em>Go</em> secondary hyphae developed from single germinated
 spores that penetrated and formed haustoria in the epidermal cells of
 different Arabidopsis genotypes.