Research

Mla- and Rom1-mediated control of microRNA398 and chloroplast copper/zinc superoxide dismutase regulates cell death in response to the barley powdery mildew fungus Weihui Xu1, Yan Meng1 and Roger P. Wise1,2 1

Department of Plant Pathology and Microbiology, Center for Plant Responses to Environmental Stresses, Iowa State University, Ames, IA 50011-1020, USA; 2Corn Insects and Crop Genetics

Research Unit, US Department of Agriculture-Agricultural Research Service, Iowa State University, Ames, IA 50011-1020, USA

Summary Author for correspondence: Roger Wise Tel: +1 515 294 9756 Email: [email protected] Received: 26 August 2013 Accepted: 8 October 2013

New Phytologist (2014) 201: 1396–1412 doi: 10.1111/nph.12598

Key words: barley, Blumeria graminis, hypersensitive reaction, microRNA, reactive oxygen species (ROS), superoxide dismutase (SOD).

 Barley (Hordeum vulgare L.) Mildew resistance locus a (Mla) confers allele-specific interac-

tions with natural variants of the ascomycete fungus Blumeria graminis f. sp. hordei (Bgh), the causal agent of powdery mildew disease. Significant reprogramming of Mla-mediated gene expression occurs upon infection by this obligate biotrophic pathogen.  We utilized a proteomics-based approach, combined with barley mla, required for Mla12 resistance1 (rar1), and restoration of Mla resistance1 (rom1) mutants, to identify components of Mla-directed signaling.  Loss-of-function mutations in Mla and Rar1 both resulted in the reduced accumulation of chloroplast copper/zinc superoxide dismutase 1 (HvSOD1), whereas loss of function in Rom1 re-established HvSOD1 levels. In addition, both Mla and Rom1 negatively regulated hvumicroRNA398 (hvu-miR398), and up-regulation of miR398 was coupled to reduced HvSOD1 expression. Barley stripe mosaic virus (BSMV)-mediated over-expression of both barley and Arabidopsis miR398 repressed accumulation of HvSOD1, and BSMV-induced gene silencing of HvSod1 impeded Mla-triggered H2O2 and hypersensitive reaction (HR) at barley–Bgh interaction sites.  These data indicate that Mla- and Rom1-regulated hvu-miR398 represses HvSOD1 accumulation, influencing effector-induced HR in response to the powdery mildew fungus.

Introduction Plants have developed two interdependent systems to defend against pathogen attack: basal defense, also referred to as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), and resistance (R)-gene-mediated defense, designated as effector-triggered immunity (ETI). Whereas basal defense is initiated by recognizing conserved PAMPs through plasma membrane receptors, R-gene-mediated resistance is triggered through specific recognition of effectors produced by pathogens (Ellis et al., 2000; Chisholm et al., 2006; Jones & Dangl, 2006; Bent & Mackey, 2007). The most prevalent class of R genes encode intracellular receptors containing an N-terminal coiled-coil (CC) or Toll/interleukin-1 receptor-like (TIR) domain, a nucleotidebinding domain (NB), and C-terminal leucine-rich repeats (LRRs; Jones & Dangl, 2006). Although significant achievements have been made in decrypting components of plant defense, much remains to be discovered regarding the mechanisms by which R genes regulate their output. Barley powdery mildew, caused by the obligate biotrophic fungus Blumeria graminis f. sp. hordei (Bgh), is a destructive foliar disease. The barley (Hordeum vulgare L.)–Bgh interaction has 1396 New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

become a model plant–fungal system; many powdery mildew R genes, as well as their downstream targets (Friedt & Ordon, 2007; Keller et al., 2007), typify plant immune responses (G€ohre & Robatzek, 2008). Among these is Mildew resistance locus a (Mla), which encodes c. 30 allele-specific variants of CC-NBLRR proteins (Halterman & Wise, 2004; Seeholzer et al., 2010). Plants harboring Mla exhibit a hypersensitive reaction (HR) and produce reactive oxygen species (ROS) in incompatible barley– Bgh interactions (H€ uckelhoven & Kogel, 1998; H€ uckelhoven et al., 2000; Vanacker et al., 2000). Subsequent to infection by Bgh, differentially expressed HvWRKY10, HvWRKY19, and HvWRKY28 act as positive regulators to control Mla-mediated immunity, as well as basal defense (Meng & Wise, 2012). In parallel, MLA proteins translocate into the nucleus upon recognition of appropriate Bgh Avirulence a (AVRa) effectors, where they associate with HvWRKY1/HvWRKY2 transcription factors and de-repress basal defense (Shen et al., 2007). HvWRKY1 interacts with MYB6 and sequesters its DNA-binding activity; this sequestration is released by interaction between activated MLA and MYB6, initiating MYB6-regulated resistance signaling (Chang et al., 2013). The CC domain of MLA10 is necessary and sufficient for inducing cell death, and that cell death is enhanced by No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

New Phytologist sequestration of MLA10 in the cytoplasm (Bai et al., 2012). By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against Bgh, indicating that cell death-promoting activity can be separated from resistance. Although MLA proteins exhibit high amino acid identity, different Mla alleles may or may not need a second independent gene, Required for Mla12 resistance1 (Rar1), to confer immunity (Shen et al., 2003; Bieri et al., 2004; Halterman & Wise, 2004). Rar1 encodes an intracellular Zn2+-binding protein (Shirasu et al., 1999) and rar1 mutations compromise Mla-specified resistance and HR (Shirasu et al., 1999; H€ uckelhoven et al., 2000). These Rar1 functions are antagonized by Restoration of Mla resistance1 (Rom1; (Freialdenhoven et al., 2005; Zellerhoff et al., 2008), where a rar1 rom1 double mutant restores HR upon inoculation with incompatible Bgh isolates (Freialdenhoven et al., 2005). Although direct interaction between RAR1 and MLA has not been observed, RAR1 does share the interactor heat shock protein 90 (HSP90) with MLA1 and MLA6, as well as suppressor of G2 allele of SKP1 (SGT1) with MLA1 (Azevedo et al., 2002; Bieri et al., 2004; Zhang et al., 2010). Hence, RAR1 appears to function as a co-chaperone in the RAR1–SGT1–HSP90 complex and positively modulates steadystate levels of MLA (Bieri et al., 2004; Shirasu, 2009). MicroRNAs are single-stranded RNA molecules 21–23 nucleotides in length that bind to the partially or fully complementary mRNAs to regulate gene expression post-transcriptionally (Bartel, 2004; Chen, 2005; Mallory & Bouche, 2008; Mendell & Olson, 2012). In plants, microRNAs play crucial roles in plant development, stress responses, and hormone signaling (JonesRhoades et al., 2006; Mallory & Vaucheret, 2006; Zhang et al., 2006, 2007; Voinnet, 2009; Jiao et al., 2010), as well as in host– pathogen interactions (Fahlgren et al., 2007; Katiyar-Agarwal & Jin, 2010; Zhang et al., 2011). For example, the PAMP flagellin induces Arabidopsis microRNA393 (miR393), which negatively regulates the expression of auxin receptors TIR1, auxin signaling F-box 2 (AFB2), and AFB3 (Navarro et al., 2006). By contrast, bacterial effectors AvrPtoB and AvrPto suppress miR393 (Navarro et al., 2008), and infiltration of avirulent strains of Pseudomonas syringae pv. tomato DC3000 (avrRpm1 or avrRpt2) down-regulates miR398 expression in Arabidopsis (Jagadeeswaran et al., 2009). In tomato (Solanum lycopersicum), miR482 and miR2118 target mRNAs encoding disease resistance proteins with NB and LRR motifs; infection by viruses or bacteria can suppress miR482-mediated silencing (Shivaprasad et al., 2012). Suppression of host microRNAs has also been observed in galled stems of pine (Pinus taeda L.) infected with fusiform rust disease caused by the fungus Cronartiumquercuum f. sp. fusiforme (Lu et al., 2007). Recent discoveries suggest that microRNAs directly target plant R genes in tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), and legumes (Medicago truncatula) (Zhai et al., 2011; Eckardt, 2012; Li et al., 2012; Shivaprasad et al., 2012). However, the role of R genes in the regulation of specific microRNA expression has not been reported to date. Highly parallel RNA profiling has revealed that transcripts are significantly differentially expressed upon infection of barley with Bgh (Caldo et al., 2004, 2006). Conserved Mla targets have been No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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postulated based on analysis of the transcript profiles of plants harboring Mla1, Mla6, and Mla12 alleles compared with the profiles of the respective loss-of-function mutants mla1-m508, mla6m9472, and mla12-m66 (Moscou et al., 2011). To extend these RNA-based investigations, identical samples from Barley1 GeneChip (Affymetrix, Santa Clara, CA, USA) transcript-profiling experiments were resolved using a proteomics-based approach. Using an integrated mutational approach, we show that barley miR398 is regulated by the disease resistance proteins MLA and ROM1. This miRNA regulatory element controls the chloroplast copper/zinc superoxide dismutase 1 (HvSOD1) protein, which then influences barley cell death in response to the powdery mildew fungus, establishing a previously unrecognized role for R-gene-mediated control of miRNAs to regulate plant defense.

Materials and Methods Fungal isolates Blumeria graminis f. sp. hordei 5874 (AVRa1, AVRa6, AVRa12) was propagated on Hordeum vulgare L. cv Manchuria (CI 2330) in a controlled growth chamber at 18°C with 16 h : 8 h light : dark. Plant material and loss-of function mutants The cereal introduction (CI) lines CI 16151 and CI 16137 were obtained by introgression of Mla6 and Mla1, respectively, into the susceptible cultivar Manchuria (Moseman, 1972). Mla6 mutants were generated by fast-neutron bombardment of CI 16151 and selected from 30 000 M2 families (Meng et al., 2009). Three mla6 mutant alleles, mla6-9472, mla6-9480, and mla611538, were confirmed by genetic complementation, Southern blot (Halterman et al., 2001), and Barley1 GeneChip analyses (Caldo et al., 2004, 2006). Mutant mla6-m18982 was isolated from mla6-11506, which also contained a Blufensin 1 (Bln1) deletion (Meng et al., 2009). To isolate this fourth mla6 mutation, mla6-11506 was backcrossed to CI 16151 (Mla6) and selfed. A total of 144 F2 individuals were genotyped for Mla6 and Bln1 to identify homozygous mla6-m18982 (mla6/mla6, Bln1/Bln1). The Mla1 mutant mla1-m508 was provided by Shauna Somerville (University of California, Berkeley, CA, USA; Zhou et al., 2001), and Sultan 5 and rar1-m100 were gifts from J. Helms Jørgensen (Risø National Laboratory, Roskilde, Denmark; Torp & Jørgensen, 1986). The rom1-1 mutant was provided by Andreas Freialdenhoven and Paul Schulze-Lefert (Max Planck Institute, Cologne, Germany; Freialdenhoven et al., 2005). For RNA and protein profiling, planting and inoculation with Bgh were performed as described by Caldo et al. (2004, 2006). Each experiment was replicated three times according to a standard split-split-plot design. Protein extraction and two-dimension electrophoresis Proteins were extracted according to the previously described procedure (Hurkman & Tanaka, 1986). One gram of 7-d-old New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

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seedling first leaves (PO:0007094) was used for protein extraction. One milligram of total protein, as measured by the Bradford assay (Sigma, St Louis, MO, USA), was loaded onto an immobilized pH gradient (IPG) strip (18 cm; pH 4–7; Amersham Biosciences, Piscataway, NJ, USA). Isoelectric focusing (IEF) was performed using the Ettan IPGphor II Isoelectric Focus System (Amersham Biosciences). IPG strips were equilibrated for 30 min in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 50 mM DTT, and 0.03% bromophenol blue). SDS-polyacrylamide gel electrophoresis (PAGE; 12% T, 2.6% C) was performed on an Ettan DALT II (Amersham Biosciences). Identification of differentially expressed proteins SDS-PAGE gels were stained with SYPRO Ruby (Molecular Probes, Eugene, OR, USA). Images were acquired using Typhoon 9400 (GE Healthcare, Pittsburgh, PA, USA) equipped with 532-nm laser and 610-nm band-pass emission filters and analyzed using PDQUEST 2-D analysis software (Bio-Rad, Hercules, CA, USA). Protein levels were normalized using the total quantity in valid spots (those not saturated, but still detected by PDQUEST). Protein spots that displayed at least three-fold change between wild-type and mutant samples were excised from gels. In-gel digestion and Matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry Candidate spots were excised from gels and washed with 500 ll of 100 mM NH4HCO3 for 1 h and then three times with 20 mM NH4HCO3 and 50% (v/v) acetonitrile. After dehydration in 100% acetonitrile, gel pieces were incubated with 10 mM DTT in 100 mM NH4HCO3 for 30 min at 60°C, and then with 55 mM iodoacetamide in 100 mM NH4HCO3 for 30 min at room temperature in the dark. Gel pieces were washed with 20 mM NH4HCO3 and dehydrated with 100% acetonitrile, and washing/dehydration procedures were repeated. Subsequent to drying at 37°C, gel pieces were rehydrated for 30 min at 4°C in 20 mM NH4HCO3 and 10 ng ll 1 of L-1-tosylamido-2phenylethyl chloromethyl ketone (TPCK)-treated sequencegrade trypsin digestion buffer (Promega, Madison, WI, USA). The supernatant was removed and replaced with 20 ll of 20 mM NH4HCO3 for overnight digestion at 37°C. The digestion solution was collected by extracting gel pieces twice with 1% acetic acid (v/v) in 50% acetonitrile (v/v). After drying, the peptides were then dissolved in matrix solution containing 10 mg ml 1 a-Cyano-4-hydroxycinnamic acid (CHCA) in 50% CH3CN (v/v) and 0.1% trifluoroacetic acid (TFA) (v/v) and applied to a MALDI target plate. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses were performed using a QSTAR XL quadrupole TOF mass spectrometer (AB/MDS Sciex, Toronto, ON, Canada) equipped with an MALDI ion source. Mass spectra for MS analysis were acquired over m/z 600– 2500. After every regular MS acquisition, MS/MS acquisition was performed against the most intensive ions in the MS scan. New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

Database search All spectra were processed in a MASCOT (Matrix Science, London, UK) database search. Peak lists were generated by ANALYST QS (AB/MDS Sciex) and were used for MS/MS ion searches with the following parameters: one maximum missing cleavage, fixed modification carboxyamidomethyl cysteine, and variable modification oxidation of methionine. Peptide mass tolerances were  100 ppm. Fragment mass tolerances were  1 Da. No restrictions on protein molecular weight were applied. Protein identification was based on the probability-based Mowse Score. The significance threshold was set to P < 0.05. BSMV-mediated microRNA over-expression and gene silencing The biolistic BSMV–virus-induced gene silencing (BSMV-VIGS) system (Meng et al., 2009) was adapted for over-expression of ath-miR398. The stem-loop fragment from Arabidopsis athMIR398a was introduced in an antisense orientation into PacI and NotI recognition sites downstream of the cb stop codon in the BSMV c sub-genome (designated BSMV:athMIR398OE; Supporting Information Table S1). For over-expression of hvumiR398, ath-miR398 sequences were replaced by hvu-miR398 in the stem-loop of Arabidopsis ath-MIR398a. The modified stem-loop was introduced into the BSMV c sub-genome (designated BSMV:hvuMIR398OE; Table S1). Barley HvSod1 cDNA was synthesized using Superscript III reverse transcriptase (Invitrogen, Grand Island, NY, USA). Fragments corresponding to HvSod1 cDNA 49–328 and 381–585 were amplified and introduced in an antisense orientation into PacI and NotI recognition sites as described previously (Meng et al., 2009). The resulting constructs were designated BSMV:319749-328 and BSMV:3197381-585. A bombardment-based strategy (Meng et al., 2009; Meng & Wise, 2012) was used to introduce BSMV into first leaves of 7-d-old barley cv Black Hull-less (CIho 2277) seedlings, which are susceptible to BSMV infection. Seven days after bombardment, second leaf (PO:0007094) sap, harboring amplified recombinant virions, was utilized to mechanically infect first leaves of CI 16151 seedlings. Carborundum (0.05 g; Sigma) in phosphate buffer (pH 7.5) without BSMV was used as a ‘mock’ control. After 12 d, third leaves (PO:0007106) with significant BSMV symptoms were used for Bgh inoculation, and also harvested for RNA/protein extraction. RNA gel blot analyses For microRNA detection, total RNA was extracted from 7-d-old seedlings as described previously (Caldo et al., 2004). To detect microRNA in miR398 over-expression experiments, total RNA was extracted from third leaves (PO:0007106) with significant BSMV symptoms 12 d after BSMV infection. Forty micrograms of total RNA was used for microRNA gel blot analysis as described by Sunkar et al. (2006). The membranes were exposed to storage phosphor screen (GE Healthcare) and scanned using a No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

New Phytologist Typhoon Scanner 9400 (GE Healthcare), or exposed to X-ray film (Thermo Scientific, Waltham, MA, USA). Image quantification was performed using IMAGEQUANT 5.0 (Molecular Dynamics, Sunnyvale, CA, USA). For profiling HvSod1 expression, five micrograms of total RNA from the parallel RNA samples extracted for miR398 detection was used. RNA gel blot analyses were performed based on Xi et al. (2009). HvActin was used as a loading control. HvSod1 accumulation from each plant was normalized by the ratio of HvSod1 to HvActin. Relative expression of HvSod1 for each plant was calculated against the HvSod1 level from control plants (specified in the figure legends), which was scaled to a uniform standard of 1.0. Western blot analysis Barley protein was extracted with buffer containing 50 mM TrisHCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1% SDS, and a protease inhibitor cocktail (1 : 100 dilution; Sigma). One microgram of total protein was fractionated by 12.5% SDS-PAGE gels and transferred to Immun-Blot PVDF membrane (Bio-Rad). HvSOD1 protein levels were evaluated by western blot using anti-AtCSD2 (Arabidopsis copper/zinc superoxide dismutase 2) antibodies (1 : 5000; Agrisera, Vännäs, Sweden), which has low cross-reactivity with Arabidopsis CSD1. Membranes were developed with ECL Plus western blotting detection reagents (GE Healthcare) and scanned using a Typhoon Scanner 9400 (GE Healthcare), or exposed to X-ray film (Thermo Scientific). The HvACTIN loading control was detected by western blot using anti-ACTIN (1 : 5000; Sigma). Image quantification was performed using IMAGEQUANT 5.0 (Molecular Dynamics). HvSOD1 accumulation from each plant was normalized by the ratio of HvSOD1 to ACTIN. Relative expression of HvSOD1 for each plant was calculated against the HvSOD1 level from control plants (specified in figure legends), which was scaled to a uniform standard of 1.0. Microscopic observation of Bgh-induced autofluorescence, histochemical detection of H2O2, and elongating secondary hyphae The observation of HR was performed as described by Xi et al. (2009). For comparison of HR between CI 16151 and mla6m9472, 7-d-old seedlings were inoculated with Bgh isolate 5874. First leaves were harvested at 24 h after inoculation (HAI). For observation of HR from HvSod1 silenced plants, third leaves (PO:0007106) with significant BSMV symptoms were placed on 1% phyto agar (Duchefa, Haarlem, the Netherlands) medium, inoculated with Bgh isolate 5874, grown under light, and harvested at 48 HAI. The apical 5 cm of each leaf was removed and the next 6 cm was fixed and decolorized for 48 h in ethanol: glacial acetic acid fixative (3 : 1, v/v) with one change with 70% ethanol (v/v). Following fixation, leaves were cleared in lacto: glycerol: water solution (1 : 1 : 1, v/v). Hypersensitive reactions were examined using a fluorescence microscope (LeitzFluovert, Wetzlar, Germany) equipped with a fluorescein filter No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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set H3 (excitation filter BP420-490; suppression filter LP 520). Images were acquired using the Olympus DP11 microscope digital camera system (Olympus America, Center Valley, PA, USA). Tissues identical to those used for measuring autofluorescence were used to quantify H2O2 at barley–Bgh interaction sites (Thordal-Christensen et al. 1997; H€ uckelhoven et al., 1999). First leaves were placed in 3,3′-diaminobenzidine (DAB; 1 mg ml 1 w/v, pH 3.8) at 14 HAI and harvested at 24 HAI. For analysis of HvSod1 silenced plants, leaf fragments were placed in DAB at 22 HAI. After 10 h, stained tissues were fixed and decolorized. DAB-stained tissues were examined with a Carl Zeiss Imager M1 microscope equipped with the AxioCam HRc imaging system (Carl Zeiss Inc., G€ottingen, Germany). Only type A and type B epidermal short cells (Koga et al., 1990) were evaluated for interaction sites. For examination of elongating secondary hyphae (ESH), Bgh-inoculated tissues identical to those used for measuring autofluorescence were harvested at 120 HAI. After fixation and decolorization, tissues were stained with Coomassie Brilliant Blue R-250 (0.05% Coomassie Brilliant Blue (w/v), 50% methanol (v/v), and 10% acetic acid (v/v)). ESH were observed with the Zeiss Axio Imager M.1 microscope. Each data set was analyzed independently using the R (version 2.15.2) package MULTCOMP (Hothorn et al., 2008). A linear model was fitted with number of cells (with whole-cell autofluorescence, DAB-stained brown cells, or elongating secondary hyphae) as the response variable and treatment (BSMV:00, BSMV:319749-328, and BSMV:3197381-585) as the predictor variable. An analysis of variance was performed on the fitted model and comparisons were tested using the Tukey–Kramer method, correcting for multiple testing. Data access GeneChip data are available at the PLEXdb gene expression resource (www.plexdb.org). Accession BB2 corresponds to the 180 GeneChips involving Sultan 5 and derived mutants and BB10 designates the 144 GeneChips from Manchuria near-isogenic lines and loss-of-function mutants. Data are also available at ArrayExpress (http://www.ebi.ac.uk/arrayex press) with accessions E-TABM-82 (Sultan 5) and E-TABM142 (Manchuria NILs; Caldo et al., 2006; Moscou et al., 2011). The HvSod1 genomic sequence from CI 16151, including the 5′ regulatory region, is annotated within GenBank accession number JQ277734. Analogous HvSod1 promoter sequences in CI 16151, mla6-m9472, mla6-m9480, mla6-m11538, mla6m18982, CI 16137, mla1-m508, Sultan 5, mla12-m66, rar1-m100, and rar1 rom1 are reported under GenBank accessions JQ364431, JQ364432, JQ364433, JQ364434, JQ364435, JQ364436, JQ364437, JQ364438, JQ364439, JQ364440, JQ364441, and JQ364442, respectively (Fig. S1). HvSod1 CDS are reported under GenBank accessions JQ364443, JQ364444, JQ364445, JQ364446, JQ364447, JQ364448, JQ364449, JQ364450, JQ364451, JQ364452, JQ364453, and JQ364454. New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

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Results Experimental concept and design Host gene expression in the Mla-specified response to Bgh has been well characterized at the transcriptional level (Caldo et al., 2004, 2006; Moscou et al., 2011). Although barley proteomics has been used to investigate organelle function and response to abiotic stress (Petersen et al., 2013), Mla-mediated mechanisms that influence translation are less clear (Moeller et al., 2012). We utilized a proteomics-based approach, combined with loss-offunction mla deletion mutants (Zhou et al., 2001; Meng et al., 2009), to identify downstream targets of Mla. Homozygous mla mutants manifest in a partially or fully susceptible phenotype to Bgh isolate 5874 (AVRa1, AVRa6, AVRa12), however, display an otherwise normal growth habit (Fig. 1a,b). The NaN3-derived, Bgh-susceptible rar1-m100 mutant (Torp & Jørgensen, 1986) and the NaN3-derived, Bgh-resistant double mutant rar1 rom1 (Freialdenhoven et al., 2005; Fig. 1b) were also used to interrogate Mla-mediated barley–Bgh interactions and HR cell death. Proteins were isolated from the same samples as for transcriptprofiling experiments, in which 7-d-old seedling first leaves were harvested at 0, 16, and 32 HAI with the Bgh isolate 5874 (AVRa6; Moscou et al., 2011). Noninoculated plants at these three timepoints were used as controls. Sixteen and 32 HAI were selected based on the observation that 16 HAI is the initial time-point for establishment of the perihaustorial interface between fungal

New Phytologist haustoria and host epidermal cells, and also where significant divergent expression between compatible and incompatible interactions begins (Caldo et al., 2004). The analysis presented here is based on 36 2D-PAGE gels (two genotypes 9 three time-points 9 two inoculation treatments 9 three biological replications). Mla1 and Mla6 alleles are required for accumulation of barley plastid Cu/Zn superoxide dismutase To identify differential accumulation of proteins regulated by Mla, we first focused on the comparison of CI 16151 (Mla6) and the loss-of-function mutant mla6-m9472. Individual proteins separated by 2D-PAGE that exhibited differential expression (Fig. S2) were assessed by MALDI-TOP-MS/MS (Table S2). Two low-molecular-weight proteins exhibited significantly reduced accumulation and one displayed increased accumulation in mla6-m9472, compared with CI 16151, in both inoculated (INOC) and noninoculated (NON-INOC) treatments at all three time-points (Figs 2a,b, S2). MS/MS ion analysis coupled with a MASCOT search against the National Center for Biotechnology Information (NCBI) nr database identified these as two glutathione S-transferase (GST; EC 2.5.1.18) proteins and a chloroplast copper/zinc superoxide dismutase (Cu/Zn-SOD; EC 1.15.1.1) protein. We focused on the chloroplast Cu/Zn SOD, because of its unique organelle location and essential function in defense against abiotic and biotic stresses (Gupta et al.,

(a)

(b)

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Fig. 1 Infection phenotypes of barley seedlings. Seven-day-old seedlings from 10 genotypes were inoculated with Blumeria graminis f. sp. hordei (Bgh) isolate 5874 (AVRa1 (Avirulence a1), AVRa6, and AVRa12) and photographed 7 d after inoculation. An infection type of 0 is immune (no sporulation), 1–2 is considered resistant, but with minor Bgh colonization, and an infection type of 3–4 is susceptible (abundant sporulation). 1n, few small necrotic flecks (0.5 mm); 1–2n, significant small necrotic flecks (1 mm); c, limited chlorosis. Plants are homozygous for all genotypes. (a) CI 16151 (Mla6 (Mildew resistance locus a allele 6), Rar1 (Required for Mla12 resistance1), Rom1 (Restoration of Mla resistance1)) and its fast neutron (FN)-derived loss-of-function mutants mla6-m9472 (mla6-m9472, Rar1, Rom1), mla6-m9480 (mla6-m9480, Rar1, Rom1), mla6-m11538 (mla6-m11538, Rar1, Rom1), and mla6m18982 (mla6-m18982, Rar1, Rom1). (b) CI 16137 (Mla1, Rar1, Rom1) and its gamma irradiation (GR)-derived Mla1 loss-offunction mutant mla1-m508 (mla1-m508, Rar1, Rom1); Sultan 5 (Mla12, Rar1, Rom1) and its sodium azide (NaN3)-derived mutants rar1-m100 (Mla12, rar1-m100, Rom1) and rar1 rom1 (Mla12, rar1-m100, rom1-1). No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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(a)

(c)

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Fig. 2 Profiling of barley superoxide dismutase 1 (HvSOD1) expression. Samples were extracted from 7-d-old seedling first leaves (PO:0007094) that were used for time course Barley1 GeneChip profiling (Caldo et al., 2006; Meng et al., 2009; Moscou et al., 2011), based on a split-split-plot design. Seedlings were inoculated with Blumeria graminis f. sp. hordei (Bgh) isolate 5874 (AVRa1, AVRa6, and AVRa12) and tissue was harvested at 0, 8, 16, 20, 24, and 32 h after inoculation (HAI). Noninoculated plants were included as controls for each treatment. In this proteomics analysis, parallel tissue from three timepoints (0, 16, and 32 HAI) was selected. Protein profiling was based on 36 gels (two genotypes 9 three time-points 9 two treatments 9 three replications). The molecular mass (kDa) and isoelectric points (pI) for each gel are noted. (a) Representative 2D gel for CI 16151 (Mla6, Rar1, Rom1) at 16 HAI. The magnified section highlights a differentially expressed HvSOD1 protein (arrow) with higher accumulation in CI 16151. (b) Representative 2D gel for mla6m9472 (mla6-m9472, Rar1, Rom1) at 16 HAI. The magnified section highlights a differentially expressed HvSOD1 protein (arrow) with lower accumulation in mla6-m9472. (c) Western blot analysis for HvSOD1 for CI 16151 (Mla6), mla6-m9472, mla6-m9480, mla6-m11538, mla6-m18982, CI 16137 (Mla1), and mla1-m508. Uniform loading was verified by probing with HvACTIN. (d) Relative levels of HvSOD1 based on (c) in CI 16151 (Mla6), mla6-m9472, mla6-m9480, mla6-m11538, mla6-m18982, CI 16137 (Mla1), and mla1-m508. Normalized HvSOD1 accumulation from each plant was calculated by the ratio of HvSOD1 to ACTIN. The relative HvSOD1 expression levels from each plant were calculated against the HvSOD1 level in CI 16151 (Mla6) for mla6 mutants, and against the HvSOD1 level in CI 16137 (Mla1) for mla1-m508. The relative HvSOD1 levels in CI 16151 (Mla6) and CI 16137 (Mla1) were scaled to a uniform standard of 1.0 (see the Materials and Methods section).

1993; Kliebenstein et al., 1999; Rizhsky et al., 2003; Sunkar et al., 2006). A total MS/MS ion search against our barley protein database found a single hit corresponding to chloroplast Cu/Zn SOD (Mowse score 260; P < 0.01; Table S2). We designated this protein HvSOD1, equivalent to maize (Zea mays; Poaceae) chloroplast Cu/Zn superoxide dismutase SOD1 (Kernodle & Scandalios, 2001). The gene encoding HvSOD1 was confirmed to be present as a single copy by a database search against the barley genome (http://webblast.ipk-gatersleben.de/barley/viroblast.php; International Barley Sequencing Consortium; Mayer et al., 2012) and whole-genome shotgun assemblies from cv Morex, Barke, and Bowman. This result was confirmed by Southern No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

blot analysis utilizing BamHI, KpnI, and DraI digested genomic DNA probed with the HvSod1 sequence (data not shown). Two cytoplasmic Cu/Zn SOD proteins were also identified via database searches; however, the unique chloroplast HvSod1 probe did not cross-hybridize to these additional genes, thus distinguishing the chloroplast from cytoplasmic Cu/Zn superoxide dismutases. The reduced accumulation of HvSOD1 in the mla6-m9472 deletion mutant (Fig. 2a,b) suggested that Mla influences the expression of HvSod1. As shown in Fig. 2(c,d), western blot analysis with anti-SOD antibody confirmed that HvSOD1 accumulation was reduced in the four independent mla6 loss-of-function deletion mutants mla6-m9472, mla6-m9480, New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

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mla6-m11538, and mla6-m18982, in addition to mla1-m508, derived from CI 16137 (Mla1; Fig. 1b), indicating that Mla is required for full HvSOD1 expression. Next, we compared time-course Barley1 GeneChip results with the protein profiles for CI 16151 (Mla6), CI 16137 (Mla1), and their respective loss-of-function mutants, mla-m9472 and mla1-m508, respectively. HvSod1 is represented by Barley1 probeset Contig3197_at, which is annotated to encode chloroplast Cu/Zn-SOD (Fig. 3). Indeed, HvSod1 transcripts were reduced in mla6-m9472 compared with wild-type CI 16151 plants (Fig. 3a), but still with a relatively high log expression value of 10. No significant difference was observed between CI 16137 (Mla1) and mla1-m508 (Fig. 3c). However, HvSOD1 protein accumulation was suppressed in both mla6-9472 and mla1-m508 plants across all three time-points (Fig. 3b,d, respectively). Influence of Rar1 on accumulation of HvSod1 transcripts and restoration of HvSOD1 accumulation in the rar1 rom1 double mutant Mla alleles differentially require Rar1 to effectively mediate resistance to Bgh (Fig. 1b). Mla6 and Mla12 require Rar1 to effect resistance, while Mla1 does not (Shen et al., 2003; Bieri et al., 2004; Halterman & Wise, 2004). As accumulation of HvSOD1 appeared to be Mla dependent, we next investigated whether Rar1 is also involved in regulating HvSod1 transcripts and/or HvSOD1 protein levels (Fig. 4). We assessed both Sultan 5 (Mla12, Rar1, Rom1) and its rar1-m100 mutant (Mla12, rar1m100, Rom1; Shirasu et al., 1999) and found that accumulation was significantly lower in rar1-m100 than in Sultan 5, wild-type plants (Fig. 4a,c). Rom1 antagonizes Rar1 function during barley–Bgh and barley–Magnaporthe oryzae interactions (Freialdenhoven et al., 2005; Zellerhoff et al., 2008). The rom1-1 mutation in a rar1-m100 background restores resistance and Mla-specified HR to Bgh (Fig. 1b; Freialdenhoven et al., 2005). To investigate whether the rom1-1 mutation also restores the compromised HvSod1 expression and the accumulation of HvSOD1, we assessed both transcript and protein levels at the 0, 16 and 32 HAI time-points, for both INOC and NON-INOC treatments. Comparison of the wild-type progenitor Sultan 5 (Mla12, Rar1, Rom1), rar1-m100 (Mla12, rar1-m100, Rom1), and the rar1 rom1 double mutant (Mla12, rar1-m100, rom1-1) demonstrated that the accumulation of HvSOD1 in the rar1 rom1 double mutant is restored from the compromised level in rar1-m100 to a level comparable to that in Sultan 5 (Fig. 4b). This is consistent with time-course Barley1 GeneChip profiling data in that rar1 rom1 completely restored HvSod1 transcript accumulation from the diminished level in rar1-m100 to a level equivalent to that of its Sultan 5 progenitor (Table 1, Fig. 4c). To verify that the differential expression of HvSod1 between wild-type and mutant plants was not caused by a mutation(s) in the HvSod1 gene, we sequenced both the promoter and proteincoding region for all 10 genotypes in Fig. 1. Comparison among wild types and their paired mutants demonstrated that there New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

New Phytologist were no differences in either the HvSod1 promoter or the protein-coding region (Fig. S1). De-regulation of hvu-miR398 by mutations in Mla and Rom1 Many microRNA species are conserved in the plant kingdom, as well as their corresponding targets. Among these is miR398, which has been identified in both monocot and dicot plants (Sunkar et al., 2006; Yao et al., 2007; Dugas & Bartel, 2008; Sunkar & Jagadeeswaran, 2008; Li et al., 2010). miR398 targets the chloroplast Cu/Zn SOD gene in Arabidopsis and rice (Oryza sativa) (Sunkar et al., 2006; Dugas & Bartel, 2008). In general, genes encoding chloroplast Cu/Zn SOD are widely conserved at both the RNA and amino acid levels among plant species. Comparison of Arabidopsis CSD2 and HvSod1 shows 89% identity at the nucleotide level. In addition, the miR398 target site is also conserved (Fig. 5a). These data prompted us to investigate whether the influence of Mla and Rar1 on the accumulation of HvSod1 and HvSOD1 manifests through miR398. Barley miR398, designated hvu-miR398, was ascertained by participation in the ‘Comparative sequencing plant small RNAs’ project (Mahalingam & Meyers, 2010). We then analyzed the level of hvu-miR398 in CI 16151 barley plants harboring Mla6, as well as in deletion mutants mla6-m9472, mla6-m9480, mla6m11538, and mla6-m18982. As shown in Fig. 5(b), hvu-miR398 accumulated to high levels in the four mla6 mutants, but was not observed in the wild-type progenitor, CI 16151, indicating that Mla6 has a negative regulatory effect on hvu-miR398. We also tested whether expression of hvu-miR398 is affected upon Bgh challenge by assaying its levels over a 0, 16, and 32 HAI time course. As shown in Fig. 3(b), hvu-miR398 accumulated in mla6-m9472, but not in wild-type CI 16151 (Mla6), further confirming the negative regulatory effect of Mla6 on hvu-miR398. To cross-reference these findings with additional Mla alleles, we measured the level of hvu-miR398 in wild-type CI 16137 (Mla1) and its mutant mla1-m508 (Fig. 3d). Data were consistent with the above results for the Mla6 allele, indicating that deletion mutations of Mla release control of hvu-miR398, and that this release is genetically coupled to reduced HvSOD1 accumulation. Lastly, to analyze the effect of Rar1 and Rom1 on hvu-miR398 expression, we examined wild-type Sultan 5 (Mla12, Rar1, Rom1), as compared with rar1-m100 (Mla12, rar1-m100, Rom1) and the rar1 rom1 double mutant (Mla12, rar1-m100, rom1-1). As shown in Fig. 4(a), hvu-miR398 expression was almost undetectable in Sultan 5 and its mutant rar1-m100. However, significant accumulation of hvu-miR398 was detected in the rar1 rom1 double mutant compared with Sultan 5 (Fig. 4b), suggesting a negative regulatory effect of Rom1 on hvu-miR398. Reduction of HvSOD1 by over-expression of miR398 The above loss-of-function mutant data suggest the possibility that the accumulation of HvSod1 transcripts and/or HvSOD1 protein is repressed by hvu-miR398. To further verify hvu-miR398 function, we adapted a BSMV-VIGS system (Meng No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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Fig. 3 Comparison between profiles of superoxide dismutase 1 (HvSod1) transcription, HvSOD1 accumulation, and barley microRNA398 hvu-miR398 expression in CI 16151 (Mla6, Rar1, Rom1), CI 16137 (Mla1, Rar1, Rom1) and paired mutants mla6-m9472 (mla6-m9472, Rar1, Rom1) and mla1-m508 (mla1-m508, Rar1, Rom1), respectively. Uniform loading was verified by probing with HvACTIN for western blot analysis and hvu-miR167 was used for microRNA. Barley1 GeneChip transcript profile data are from PLEXdb accession BB10 (Meng et al., 2009; Moscou et al., 2011). Barley HvSod1 is represented by probeset Barley1_03197. Expression levels of HvSOD1 and hvu-miR398 were normalized to corresponding ACTIN and hvu-miR167 levels, respectively. The normalized expression levels were used to calculate relative expression levels. (a) Transcript levels of HvSod1 in CI 16151 and mla6-m9472. Standard errors were calculated based on mean log expression values from three independent replications. (b) Western blot analysis for HvSOD1 (top panel), RNA gel blot analysis for hvu-miR398 (second panel), and RNA loading control hvu-miR167 (third panel) in CI 16151 and mla6-m9472. The relative expression levels of HvSOD1 and hvu-miR398 (fourth panel) from each treatment were calculated against noninoculated (NON-INOC) CI 16151 (Mla6) at 0 h after inoculation (HAI). (c) Expression profiles of HvSod1 and hvumiR398 in CI 16137 and mla1-m508. Standard errors were calculated based on mean log expression values from three independent replications. (d) Western blot analysis for HvSOD1 (top panel), RNA gel blot analysis for hvu-miR398 (second panel), and RNA loading control hvu-miR167 (third panel) in CI 16137 and mla1-m508. The relative expression levels of HvSOD1 and hvu-miR398 (fourth panel) from each treatment were calculated against noninoculated (NON-INOC) CI 16137 (Mla1) at 0 HAI.

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et al., 2009; Meng & Wise, 2012) to over-express miR398. BSMV is a tripartite, positive-sense RNA virus (Petty et al., 1990; Holzberg et al., 2002). We first inserted the stem-loop fragment from Arabidopsis ath-MIR398a downstream of the BSMV cb stop codon in the BSMV c sub-genome (driven by the Cauliflower mosaic virus 35S promoter). This construct was designated BSMV:athMIR398OE. We predicted that this construct would generate small RNAs identical to ath-miR398 via doublestranded RNA (dsRNA) replication intermediates (Zamore, 2001), or from the precursor stem-loop structure of athMIR398, positioned in the minus-sense strand during BSMV replication. BSMV constructs were introduced into barley (Meng et al., 2009; Feng et al., 2013). Mock (carborundum in phosphate buffer, pH7.2), BSMV:00 (empty vector), and BSMV:athNew Phytologist (2014) 201: 1396–1412 www.newphytologist.com

Fig. 4 Comparison between superoxide dismutase 1 (HvSod1) transcription, HvSOD1 accumulation, and hvu-miR398 in wild-type Sultan 5 (Mla12, Rar1, Rom1) and mutants rar1-m100 (Mla12, rar1-m100, Rom1) and rar1 rom1 (Mla12, rar1-m100, rom1-1). Expression levels of HvSOD1 and hvumiR398 were normalized to corresponding ACTIN and hvu-miR167 levels, respectively. The normalized expression levels were used to calculate relative expression levels. Uniform loading was verified by probing with HvACTIN for western blot analysis and hvumiR167 was used for microRNA. Barley1 GeneChip transcript profile data are from PLEXdb accession BB2 (Caldo et al., 2006; Meng et al., 2009; Moscou et al., 2011). (a) Western blot analysis for HvSOD1 (top panel), RNA gel blot analysis for hvu-miR398 (middle panel), and RNA loading control hvu-miR167 (bottom panel) for Sultan 5 and rar1-rom100. The relative expression levels of HvSOD1 and miR398 (fourth panel) from each treatment were calculated against noninoculated (NON-INOC) Sultan 5 (Mla12, Rar1, Rom1) at 0 h after inoculation (HAI). (b) Western blot analysis for HvSOD1 (top panel), RNA gel blot analysis for hvumiR398 (second panel), and RNA loading control hvu-miR167 (third panel) for Sultan 5 and rar1 rom1. The relative expression levels of HvSOD1 (fourth panel) for each treatment were calculated against NON-INOC Sultan 5 (Mla12, Rar1, Rom1) at 0 HAI, whereas the relative expression level of hvu-miR398 (fourth panel) is the expression ratio of miR398/miR167. (c) Barley1 GeneChip transcript accumulation of HvSod1 for Sultan 5, rar1-m100, and rar1 rom1. HvSod1 is represented by probeset Barley1_03197. Standard errors were calculated based on mean log expression values from three independent replications.

MIR398OE treatments were applied to eight plants each for three independent biological replicates. After 12 d, third leaves (PO:0007106) with significant BSMV symptoms were harvested to quantify miRNA, mRNA and protein. Eleven of 22 BSMV: athMIR398OE-infected third leaves (PO:0007106) displayed increased levels of ath-miR398a, compared with the mock or BSMV:00 controls, which remained unchanged (Fig. 6a). As illustrated in Fig. 6(b), HvSOD1 accumulation was concomitantly reduced in ath-miR398 over-expression plants, compared with mock or BSMV:00 controls. Nevertheless, RNA gel blot analysis demonstrated that there was no significant difference in HvSod1 transcript levels among mock, BSMV:00, and BSMV: athMIR398OE treated plants (Fig. 6c). These results suggest that ath-miR398 has a role in repressing the accumulation of HvSOD1 protein. No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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Table 1 Summary of effects of Mla, Rar1, and Rom1 variants on superoxide dismutase 1 gene (HvSod1) transcription and translation, barley microRNA hvu-miR398 accumulation, and infection type after challenge with Blumeria graminis f. sp. hordei (Bgh) isolate 5874

Plant genotype CI 16151 (Mla6) mla6-m9472 mla6-m9480 mla6-m11538 mla6-m18982 CI 16137 (Mla1) mla1-m508 Sultan 5 (Mla12, Rar1, Rom1) Mla12, rar1-m100e, Rom1 Mla12, rar1-m100e, rom1-1

Mla transcript accumulation

HvSod1 transcript accumulation

HvSOD1 protein accumulation

hvu-miR398 accumulation

Infection typea

+++b

+++ +++ +++ +++ +++ +++ +++ +++ + ++++

+++ + + + + +++ ++ +++ + +++

ND +++ +++ +++ +++ ND +++ ND ND ++

0 3–4 4 4 4d 0 4 0–1n 3nc 1nc

c

+++ +++ +++ +++

a Phenotypes of different barley–powdery mildew interactions at 7 d after inoculation with Bgh 5874. An infection type of 0 is immune (no sporulation), 1–2 is considered resistant, but with minor Bgh colonization, and an infection type of 3–4 is susceptible (abundant sporulation). 1n, few small necrotic flecks (0.5 mm); 1–2n, significant small necrotic flecks (1 mm); c, limited chlorosis. b ‘+’ designates the relative accumulation level: ‘+’, considered the lowest accumulation level; ‘+++’, considered the highest accumulation level. ‘ND’, not detectable. C ‘ ’designates background signal; no apparent transcript accumulation. d mla6-m18982 is slightly less susceptible than mla6-m9480 and mla6-m11538, but more susceptible than mla6-m9472. e In genotypes containing the rar1-m100 mutation, Rar1 transcript accumulation is equivalent to ‘+’.

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Fig. 5 Detection of hvu-miR398 in CI 16151 (Mla6, Rar1, Rom1) and four mla6 loss-of-function mutants. (a) Alignment of microRNA miR398 target sites in plastid Cu/Zn superoxide dismutase (Sod) genes in Arabidopsis copper/zinc superoxide dismutase 2 (At-CSD2) and barley (HvSod1) and comparison of miR398 sequences between Arabidopsis (ath-miR398) and barley (hvu-miR398). Watson–Crick pairing (vertical dashes) and G : U wobble pairing (colons) are indicated. The minimum free energy (mfe) was determined with the RNAhybrid server at http://bibiserv.techfak.unibielefeld.de/rnahybrid/submission. html (Rehmsmeier et al., 2004). (b) Expression of hvu-miR398 in CI 16151 (Mla6, Rar1, Rom1) and mla6 loss-of-function mutants mla6-m9472, mla6-m9480, mla6-m11538, and mla6-m18982. Uniform loading was verified by probing with hvu-miR167.

We next replaced ath-miR398 in the ath-MIRNA stemloop with hvu-miR398, designated BSMV:hvuMIR398OE, and BSMV-based miRNA over-expression experiments were performed as described above for Arabidopsis miR398 over-expression. RNA gel blot analysis of five independent replicates demonstrated that BSMV:hvuMIR398OE plants accumulated high levels of hvu-miR398, compared with mock or BSMV:00 controls (Fig. 6d). Moreover, these same plants also revealed significantly lower accumulation of HvSOD1 (P < 0.05; Fig. 6e), even though HvSod1 transcript levels remained unchanged (Fig. 6f). This result was corroborated by the analysis of 5′-rapid amplification of cDNA ends (5′ RACE) for the Mla6 mutant mla6-m9472, where no predominant HvSod1 cleavage site was identified in the miRNA398 target region (Fig. S3). No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

Bgh-induced HR is impeded by silencing of HvSod1 In plants, gene-for-gene disease resistance is often typified by HR. To assess HR in both CI 16151 (Mla6) and mla6-m9472, 7-d-old seedlings were challenged with Bgh isolate 5874 (AVRa6). First leaves (PO:0007094) were used to document the presence of autofluorescence as an indicator of HR (H€ uckelhoven et al., 2000; Vanacker et al., 2000). In wild-type CI 16151 (Mla6), HR-induced, whole-cell autofluorescence was observed in the epidermis challenged with Bgh isolate 5874, as well as the mesophyll underlying HR-containing epidermal cells at 24 HAI (Fig. 7a,b). The most intense autofluorescence was seen in epidermal cells neighboring stomata (Fig. 7b). By contrast, no whole-cell autofluorescence was detected in the compatible mla6m9472–Bgh interaction (Fig. 7c). New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

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Fig. 6 Over-expression of miR398 and repression of superoxide dismutase 1 (HvSOD1) accumulation. The Barley stripe mosaic virus–virus-induced gene silencing (BSMV-VIGS) system was adapted for microRNA over-expression (see the Materials and Methods section). The stem-loop fragment from Arabidopsis microRNA precursor ath-MIR398a was introduced in an antisense orientation into PacI and NotI recognition sites downstream of the cb stop codon in the Cauliflower mosaic virus 35S promoter-driven BSMV c sub-genome (designated BSMV:athMIR398OE). Twelve days after mechanical infection of first leaves (PO:0007094), third leaves with significant BSMV symptoms were harvested for RNA and protein extraction. Expression levels for HvSod1 and HvSOD1 from each plant were normalized to the corresponding HvActin and HvACTIN levels, respectively. The normalized expression levels were used to calculate relative expression levels. (a) RNA gel blot analysis for miR398 expression (upper panel) in mock, BSMV:00, and two BSMV: athMIR398OE plants in which the ath-miR398 is in the ath-MIR398a stem-loop fragment. Uniform loading was verified by probing with hvu-miR167 (lower panel). (b) Western blot analysis for HvSOD1 (lower band of upper panel). Uniform loading was verified by probing with HvACTIN (upper band of upper panel). The relative HvSOD1 expression levels (lower panel) for each plant were calculated against the HvSOD1 level for the mock control. (c) RNA gel blot analysis for HvSod1 in mock, BSMV:00, and two BSMV:athMIR398OE plants (top panel). Uniform loading was verified by probing with HvActin (middle panel). The relative HvSod1 expression levels (bottom panel) for each plant were calculated against the HvSod1 level for the mock control. (d) RNA gel blot analysis for miR398 expression (upper panel) in mock, BSMV:00, and BSMV:hvuMIR398OE plants in which the ath-miR398 is replaced with hvu-miR398 in the ath-MIR398a stem-loop fragment. Uniform loading was verified by probing with hvu-miR167 (lower panel). (e) Western blot analysis for HvSOD1 (lower band of upper panel). HvACTIN was used as a protein loading control (upper band of upper panel). The relative HvSOD1 expression levels (lower panel) for each plant were calculated against the HvSOD1 level for the mock-1 control. (f) RNA gel blot analysis for HvSod1 in mock, BSMV:00, and BSMV:hvuMIR398OE plants (top panel). Uniform loading was verified by probing with HvActin (middle panel). The relative HvSod1 expression levels (bottom panel) for each plant were calculated against the HvSod1 level for the mock-1 control.

To investigate whether HvSod1 is involved in HR, silencing was performed using our bombardment-based BSMV-VIGS system (Meng et al., 2009). Two constructs (BSMV:319749-328 and BSMV:3197381-585) were prepared using independent segments of the HvSod1 cDNA inserted into the BSMV-mediated silencing vector. First leaves from 7-d-old CI 16151 plants were mechanically infected by BSMV. After 12 d, third leaves New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

showing BSMV symptoms, but without BSMV-induced necrosis, were placed on 1% phyto agar plates and inoculated with Bgh isolate 5874. At 48 HAI, the leaves were sampled for determination of HR and HvSOD1 accumulation. Local autofluorescence was observed on all plants after Bgh inoculation (Fig. S4), which triggers the accumulation of autofluorogenic compounds in the barley epidermis. Upon silencing HvSod1, No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

New Phytologist Bgh-attacked epidermal cells exhibited significantly less autofluorescence compared with BSMV:00 controls (P = 0.0062 and P < 0.001 for BMSV:00 versus BSMV:319749-328 and BMSV:00 versus BSMV:3197381-585, respectively; Figs 7j, S4; Table S3). Accumulation of hydrogen peroxide (H2O2) is associated with HR in barley–Bgh interactions (H€ uckelhoven et al., 1999). We used DAB staining to further investigate whether the reduced autofluorescence in HvSod1-silenced plants is linked to H2O2 accumulation. Intense brown coloration, indicative of H2O2 formation, was observed in Bgh-challenged epidermal cells, as well as the mesophyll cells neighboring those epidermal cells (Fig. 7d,e). By contrast, only local, less intense staining was detected in the compatible mla6-m9472–Bgh interaction (Fig. 7f). Upon HvSod1 silencing, the number of epidermal cells with intense brown coloration was reduced significantly, compared with BSMV:00 controls (P = 0.00313 and P < 0.001 for BMSV:00 versus BSMV:319749-328 and BMSV:00 versus BSMV:3197381-585, respectively; Figs 7g,h,i,k, S4; Table S4). Western blot analysis showed that HvSOD1 was also reduced in silenced plants compared with mock and BSMV:00 controls (Fig. 7l), indicating a correlation between HvSOD1 levels and Bgh-triggered HR and accumulation of H2O2. These data indicate that HvSOD1 plays an important role in H2O2 accumulation and HR during early barley–Bgh interactions. Finally, we were interested to test if the HvSOD1-mediated generation of H2O2 and autofluorescence was coupled to resistance. To do this, ESH, an indicator of functional haustoria (Ellingboe, 1972), were quantified side-by-side in an equivalent set of BSMV:00 and HvSod1-silenced leaves as was tested for autofluorescence and accumulation of H2O2. No significant difference in ESH was detected among BSMV:00-, BSMV:319749-328- and BSMV:3197381-585-infected plants at 120 HAI (Table S5).

Discussion Modulation of hypersensitive reaction by HvSod1 Chloroplasts are one of the most powerful sources of ROS in plant cells. In addition, the thylakoid membrane-bound primary electron acceptor of photosystem I (PSI) is a major site for production of superoxide radicals (O2∙ ; Zurbriggen et al., 2009; Gill & Tuteja, 2010). The plastid Cu/Zn SOD is the main isoform of SOD in plants (Asada, 1999), which preferentially localizes on the stromal face of thylakoid membranes in chloroplasts and converts the PSI O2∙ into hydrogen peroxide (H2O2; Ogawa et al., 1995). Despite the association of plastid Cu/Zn SOD with plant defense responses (Gupta et al., 1993; Kliebenstein et al., 1999; Rizhsky et al., 2003; Sunkar et al., 2006), its expression and exact role in plant R-gene-mediated disease resistance remain unknown. Here, we used an integrated mutational approach to show that barley HvSod1 is regulated by Mla and Rom1, in conjunction with hvu-miR398 (Figs 2–6), and that HvSod1 influences Mla-mediated HR. Plants harboring wildtype Mla and Rar1 alleles, as well as rar1 rom1 double mutants, No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

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produce an HR upon inoculation with Bgh (H€ uckelhoven et al., 1999, 2000; Vanacker et al., 2000; Freialdenhoven et al., 2005; Zurbriggen et al., 2009; Fig. 7a,b). Expression of HvSod1 was elevated in these Bgh-resistant plants, and concomitantly reduced in susceptible mla and rar1 plants (Figs 3, 4). Moreover, the Mla-specified HR was attenuated in HvSod1-silenced plants (Figs 7j, S4). H2O2 and O2∙ are important ROS signaling molecules (Apel & Hirt, 2004). SOD reacts with O2∙ at nearly diffusion-limited rates to produce H2O2 and the increased concentration of H2O2 is known to inhibit SOD activity (Hodgson & Fridovich, 1975). Therefore, the homeostasis of O2∙ and H2O2 in chloroplasts should be elaborately maintained by chloroplast Cu/Zn SOD. Because H2O2 is relatively stable and diffusible across the membrane (Bienert et al., 2006), it has been suggested to be a signaling molecule in regulating multiple biological processes (Veal et al., 2007), including the HR in plant–pathogen interactions. Hence, we may presume that H2O2 produced by Cu/Zn SOD in chloroplasts functions in the HR, and the reduced SOD accumulation would be predicted to dampen H2O2 production and to abate the cellular HR. The cellular fluctuation resulting from pathogen attack may be surveyed by chloroplasts and eventually activate the HvSOD1-mediated H2O2 accumulation. Hence, chloroplast-produced H2O2 may function as intracellular and intercellular signal molecules to amplify the cellular ROS signals and trigger the HR (Liu et al., 2007; Zurbriggen et al., 2009; Straus et al., 2010). This hypothesis is supported by HR and H2O2 accumulation from mesophyll cells, which are rich in chloroplasts but do not physically interact with Bgh (Fig. 7a,d), as well as from epidermal cells neighboring chloroplast-containing stomatal guard cells (Fig. 7b,e). ROS accumulation has also been observed in barley Sultan 5 (Mla12) chloroplasts at Bgh interaction sites and is accompanied by the HR in mesophyll cells (H€ uckelhoven et al., 2000). VIGS-induced silencing of HvSod1 significantly reduced the HR and accumulation of H2O2 (Figs 7g–k, S4; Table S4). These data suggest that HvSOD1-generated H2O2 contributes to the ROS accumulation and enhances the response of barley to Bgh infection. However, silencing of HvSod1 did not break Mla6-mediated resistance (Table S5). These data suggest the uncoupling of resistance from the HvSod1-mediated HR, but we also cannot rule out the possibility that the immunity conferred by Mla6 is epistatic to HvSod1mediated accumulation of H2O2. MicroRNA-mediated regulatory loops and repression of HvSOD1 accumulation MicroRNA miR398 expression is affected by abiotic and biotic stresses (Dugas & Bartel, 2008; Hsieh et al., 2009; Jagadeeswaran et al., 2009; Jia et al., 2009), and responses to diurnal cycles (Sire et al., 2009). miR398 levels are also regulated by the Arabidopsis SPL7 (squamosa promoter binding protein-like 7) protein involved in copper homeostasis (Yamasaki et al., 2009). Here, we propose that hvu-miR398 is regulated by Mla and Rom1 and functions as a repressor of HvSOD1 protein accumulation (Table 1; Fig. 8). Both genetic and physical lines of evidence New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

New Phytologist

1408 Research (a)

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Fig. 7 Microscopic detection of hypersensitive reaction in response to Blumeria graminis f. sp. hordei (Bgh). (a) Whole-cell autofluorescence was observed from wild-type CI 16151 (Mla6), in both epidermal cells (e) and mesophyll cells (m) at 24 h after inoculation (HAI) with Bgh isolate 5874 (AVRa1, AVRa6, AVRa12). Bar, 50 lm. (b) Epidermal cells neighboring the stomatal complex (s) exhibit strong whole-cell autofluorescence. Bar, 50 lm. (c) Whole-cell autofluorescence from susceptible mutant mla6-m9472 plants was absent. Bar, 50 lm. (d) Intense brown coloration was observed in mesophyll cells (m) adjacent to epidermal cells (e) with attached conidium (c). Bar, 50 lm. (e) H2O2 accumulation was evidenced by intense brown coloration produced by 3,3′-diaminobenzidine (DAB) staining in epidermal cells (e) at 24 HAI with Bgh isolate 5874 (AVRa1, AVRa6, AVRa12). Conidia attempting penetration and the neighboring stomatal complex are designated c and s, respectively. Bar, 100 lm. (f) Local brown coloration around papilla (p) in the mla6-m9472 susceptible mutant. Bar, 25 lm. (g–i) Representative DAB-stained images for Barley stripe mosaic virus (BSMV)-induced superoxide dismutase 1 (HvSod1) gene silencing. Third leaves (PO:0007106) from plants infected with BSMV:00 (g), BSMV:319749-328 (h), and BSMV:3197381-585 (i) were inoculated with Bgh isolate 5874 (AVRa1, AVRa6, AVRa12). Decreased accumulation of H2O2, as indicated by fewer DAB-stained (brown) cells, was observed in BSMV:319749-328 (P < 0.0313) and BSMV:3197381-585 (P < 0.001) infected leaves compared with BSMV:00 controls. Bar, 200 lm. (j) Quantitative analysis of autofluorescing cells among plants infected with BSMV:00, BSMV:319749-328, and BSMV:3197381-585 and inoculated with Bgh isolate 5874 (AVRa1, AVRa6, AVRa12). Error bars,  SE. Significance: **, P = 0.0062; ***, P < 0.001. (k) Quantitative analysis of H2O2 accumulation among plants infected with BSMV:00, BSMV:319749-328, and BSMV:3197381-585 and inoculated with Bgh isolate 5874 (AVRa1, AVRa6, AVRa12). Error bars,  SE. Significance: *, P = 0.0313; ***, P < 0.001. (l) Reduced HvSOD1 accumulation in plants infected with BSMV:319749-328 and BSMV:3197381-585. Third leaves (PO:0007106) from mock- or BSMV-infected plants were harvested for protein extraction. Uniform loading was verified by probing with HvACTIN.

New Phytologist (2014) 201: 1396–1412 www.newphytologist.com

No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

New Phytologist support this hypothesis: in susceptible mla1 and mla6 deletion mutants, miR398 is up-regulated compared with resistant CI 16137 (Mla1) and CI 16151 (Mla6) progenitors, and HvSOD1 is concomitantly reduced (Table 1; Fig. 3b,d). This implies that miR398 expression is released from Mla-mediated, steady-state regulation, which then results in miR398-specified repression of HvSOD1 protein accumulation. The above genetic data are corroborated by BSMV-mediated over-expression of ath-miR398 and hvu-miR398, which resulted in less HvSOD1 protein, even though HvSod1 transcript levels remained constant (Fig. 6). Thus, the present evidence suggests that hvu-miR398 may function as a repressor of HvSOD1 translation. Nevertheless, c. 9% HvSod1 mRNA cleavage was identified in the hvu-miR398 target region (Fig. S3), thus supporting post-transcriptional regulation of HvSod1 by interconnected mechanisms. Alternatively, hvumiR398 may target genes encoding proteins required for HvSOD1 stability. In Arabidopsis, miR398 has been reported to direct both mRNA cleavage and translation for the gene encoding copper chaperone for SOD (CCS1) (Beauclair et al., 2010), and CCS1 protein is required for chloroplast Cu/Zn SOD stability (Chu et al., 2005). This suggests the possibility that HvSOD1 is regulated in a similar manner.

Research 1409

Reduced accumulation of HvSOD1 in the Sultan 5-derived mutants appears to be under genetic control of the rar1-m100 mutation. Subsequent up-regulation of HvSOD1 accumulation was coupled to the restoration of Mla-specified resistance via rom1 (Mla12, rar1-m100, rom1-1). The rar1 rom1 double mutant both induced hvu-miR398 expression (Fig. 4b) and restored HvSod1 transcripts from the level in rar1-m100 mutants to a level equivalent to that of wild-type Sultan 5 (Table 1; Fig. 4c). Thus, we postulate that Rom1, along with Rar1 and Mla, is required to control the levels of HvSOD1. Analysis of large-scale expression data has suggested that microRNA-mediated regulatory loops, including coherent and incoherent feedforward loops, form a key part of gene regulatory networks (Alon, 2007; Tsang et al., 2007; Cohen & Herranz, 2010). However, few have been experimentally verified in plants (Gutierrez et al., 2009; Kim et al., 2009). The data presented here support a hypothesis wherein hvu-miR398-mediated coherent and incoherent feedforward loops are regulating HvSod1 expression (Fig. 8). In the coherent loop, the effector-triggered resistance gene Mla may up-regulate the expression of HvSod1 by inhibiting hvu-miR398 accumulation. By contrast, Rom1 appears to act as master regulator in the incoherent loop to inhibit the expression of both hvu-miR398 and its target HvSod1. Although Mla alleles confer race-specific resistance in barley, Mla1 also functions in the dicot Arabidopsis, illustrating that mechanisms underlying CC-NB-LRR-triggered immunity are conserved (Maekawa et al., 2012). Recent identification of the Mla homolog TmMla (Jordan et al., 2011) and the Mla ortholog Sr33 (Periyannan et al., 2013), specifying resistance to wheat powdery mildew (Blumeria graminis tritici) and Ug99 stem rust (Puccina gramimis tritici), respectively, presents the possibility that Mla-mediated control of Sod1 through miR398 may also be operating in these systems.

Acknowledgements

Fig. 8 Model for barley superoxide dismutase 1 (HvSod1) expression and function. The combined evidence suggests that HvSod1 expression is regulated at both transcription and post-transcription stages. This model postulates that barley miR398 is regulated by the disease resistance proteins MLA and ROM1. In turn, miRNA398 controls the chloroplast copper/zinc superoxide dismutase protein HvSOD1, which also influences H2O2 accumulation and the hypersensitive reaction (HR). (1) Mla, either directly or indirectly, appears to have a positive effect on HvSod1 expression through suppression of miR398. (2) Rom1 appears to both antagonize Mla12 resistance1 (Rar1)-mediated HvSod1 transcription and impact HvSOD1 accumulation through hvu-miR398-mediated feedforward loops. (3) Subsequently, HvSOD1-derived H2O2 may function as an intra- or intercellular signal in plant disease resistance (Gupta et al., 1993; Ogawa et al., 1995; Kliebenstein et al., 1999; Rizhsky et al., 2003; Sunkar et al., 2006). Down-regulated HvSod1 diminishes the barley H2O2-associated HR upon inoculation of incompatible Blumeria graminis f. sp. hordei (Bgh). In this model, however, the effect of Rar1 on miR398 remains unresolved; rar1 plants do not detectably influence hvumiR398 expression, but this may be masked by wild-type Mla.

No claim to original US Government works New Phytologist Ó 2013 New Phytologist Trust

We thank Liz Miller for mutant screening, Greg Fuerst for expert technical assistance, Matthew Moscou for initial microRNA data mining, and Priyanka Surana for statistical analysis. The research was supported by USDA-ARS CRIS project no. 3625-21000057-00D and National Science Foundation-Plant Genome grants 05-00461 and 09-22746.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 Alignment of the HvSod1 promoter and protein coding regions for 10 barley genotypes.

Fig. S2 Images of two-dimensional polyacrylamide gels for identification of differentially expressed proteins. Fig. S3 Mapping of hvu-miR398-mediated cleavages on HvSod1 mRNA. Fig. S4 Images representing autofluorescence and DAB staining in BSMV-induced HvSod1-silenced seedlings. Table S1 Primers used for genomic DNA PCR, inverse-PCR, RNA gel blot analysis, miR398 over-expression, and BSMVVIGS constructs Table S2 Differentially expressed proteins identified by twodimensional polyacrylamide gel electrophoresis coupled with MS/MS analysis and MASCOT search Table S3 Results of Tukey–Kramer ANOVA of autofluorescence (HR) Table S4 Results of Tukey–Kramer ANOVA for DAB staining Table S5 Results of Tukey–Kramer ANOVA for elongating secondary hyphae (ESH) Please note: Wiley Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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