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Exploring the plant-microbe interface by profiling the surface-associated proteins of barley grains Abida Sultan, Birgit Andersen, Birte Svensson, and Christine Finnie J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b01042 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 4, 2016

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Journal of Proteome Research

Exploring the plant-microbe interface by profiling the surface-associated proteins of barley grains Abida Sultan1, Birgit Andersen2, Birte Svensson1, Christine Finnie2*

1. Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Elektrovej, Building 375, DK-2800 Kgs. Lyngby, Denmark. 2. Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

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ABSTRACT Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4xylanases that are in turn inhibited by plant xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, a combined gel-based (2-DE coupled with MALDI-TOF-TOF MS) and gel-free (LCMS/MS) proteomics approach complemented with enzyme activity assays was used to profile the surface-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell wall and polysaccharide degradation, and included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment.

KEYWORDS: Cereal proteins, mass spectrometry, proteome analysis, xylanase activity, metaproteome, microbial communities, environmental proteomics


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INTRODUCTION Cereals are major crops for feed, food, and malting, by-products of which also represent an important biomass resource. All of these applications are dependent on efficient enzymatic degradation of complex polysaccharides, proteins, and/or other bioactive components. The mature barley grain contains a collection of hydrolytic enzymes that are synthesized during grain development and found in active and inactive forms in the grain. The full complement of these enzymes (also referred to as first wave enzymes) needs to be unraveled, but includes β-amylases, α-glucosidases, phytases, proteases and cell wall degrading enzymes, which are of fundamental biological importance due to their roles in the germination.

1, 2

The first wave enzyme activities are considered to be major quality

determinants for food and feed, since they may be activated during grain processing or in the digestive tract. Cereals are naturally populated by a diverse and complex microbial community, including fungi, bacteria and yeast. The amounts and species of these microbes strongly depend on the species, the stage of development, as well as environmental and postharvest storage conditions. 3 The colonizing microbial community actively interacts with the grains by various means, including production of enzymes, plant stimulating hormones and metabolites required for breakdown of cell wall components, nutrient acquisition, host colonization and virulence. 4 Fungi are the major colonizers after anthesis of the cereal grains and are reported to interfere with grain respiration, reduce grain germination/viability and produce mycotoxins. 3, 5 Available complete fungal genome sequences and advances in genomics, transcriptomics and proteomics have contributed significantly to a better understanding of plant-fungus interactions, fungal pathogenicity and plant defense mechanisms. 6 There is a constant demand by the industry for new enzymes with diverse properties and 3 ACS Paragon Plus Environment


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activities. For feed applications, several strategies such as liquid feed (pre-soaking of cereal grains) 7 and supplementation of enzymes or amino acids have been employed 8 with the aim of enhancing nutrient availability. Xylanases are routinely used to increase the nutritional value of animal feed by depolymerizing the arabinoxylan (AX) found in high abundance in cell walls by drastically changing their molecular mass, solubility and other physico-chemical properties. 9 Synergistic actions of degradative enzymes such as glucanases, glucosidases, and xylanases serve to modulate and depolymerize cell walls in grain development and germination. In barley grains, endo-1,4-β-xylanases are synthesized and accumulated as inactive precursors in aleurone cells and are released by programmed cell death after germination. 10 Approximately 85% of the xylanase activity in mature wheat grains has been attributed to the microorganisms populating the outer grain layers. 11 Xylanase activity associated with the surface of cereal grains has been largely disregarded because most of the microbial xylanases are inactivated by proteinaceous xylanase inhibitors of varying specificities, found in grain endosperm, nucellar tissue and outer grain layers. 12 These include the xylanase-inhibiting protein (XIP), Triticum aestivum xylanase inhibitor (TAXI) and thaumatin-like xylanase inhibitor (TLXI). 13, 14 Considerable variability in xylanases and xylanase inhibitors is found among cereal species and cultivars, due to genetic, climatic and agricultural factors. 15 Hitherto, no reports describe the composition of the surface-associated proteins on barley grains and their enzymatic activities. To gain better understanding of the importance of the colonizing microbial community and their enzymatic potential, a combination of a gel-based (2D-gel electrophoresis coupled with MALDI-TOF-TOF MS) and a gel-free (LC-MS/MS) proteomics approach was applied to profile the proteins found on the surface of the grains of two barley cultivars (Barke and Cabaret). We used activity assays


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in parallel to measure xylanase activities associated with the surface of barley grains. In addition, xylanase inhibitor activities in the corresponding grain extracts were determined to explore correlations between the surface-associated barley proteomes and this important cell wall polysaccharide degrading system. The combined strategy allows identification and characterization of proteins and enzymes derived from a complex and dynamic microbial community, as well as the host plant. This provides an insight into the molecular mechanisms in the plant-microbe interface, but also the untapped potential of the residing microbial community and their ability to produce novel enzymes and metabolites with potential biotechnological applications.

MATERIALS AND METHODS Barley grains, growing sites and harvest Grains from two spring barley cultivars, Barke and Cabaret, harvested in 2009 and 2011, were from Sejet Plant Breeding (Horsens, Denmark). Each cultivar was grown in three plots in the same field in a fully randomized block design and grains from different plots were mixed to eliminate location effects. The grains were selected in a systematic manner to ensure that they were intact and undamaged, as well to eliminate straw and other residues.

Washing procedure to separate the surface-associated and grain proteins A washing procedure 11 was implemented that effectively separates the surfaceassociated (including microbial) and endogenous grain proteins. Preliminary experiments showed most of the surface-associated xylanase activity to be extracted by 25 mM sodium acetate pH 5.0 containing 0.02% (w/v) sodium azide. No further increase in extractable xylanase activity was observed after washing for 8 h (data not shown), but for



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convenience, grains were washed for 15 h or overnight. In a subsequent wash by water and/or buffer to remove remaining surface-associated proteins, xylanase activity levels were close to zero (data not shown). Protein extracts prepared from the washed grains were therefore considered to contain negligible amounts of surface microbial xylanase and were used for determination of the endogenous barley grain xylanase and xylanase inhibitory activity.

Grain extract preparation Unwashed or washed grains were freeze-dried and ground (IKA Labortechnik A10 laboratory mill, Janke & Kunkel, Staufen, Germany) and flour (0.5 g) was extracted in 25 mM sodium acetate, pH 5.0 (5.0 mL) for 45 min at room temperature under agitation. After centrifugation (4,000 x g, 20 min), supernatants containing extracted proteins were filtered through MN 615 filter paper (Macherey-Nagel, Dueren, Germany) and kept at 20°C until use. Freezing at -20°C did not significantly affect the levels of xylanase activity measured in the grain extracts (data not shown). Duplicate measurements of two independent washing liquid preparations were performed.

Protein determination The protein content of washing liquids and grain extracts was estimated by the amido black method 16 using a standard curve of bovine serum albumin.

Xylanase activity assay Xylanase activity was determined using the colorimetric Xylazyme-AX method (Megazyme, Ireland) based on quantification of released products from the azurine-cross linked wheat arabinoxylan (AZCL-AX). Volumes of 0.5 mL of either washing liquid 6 ACS Paragon Plus Environment

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(surface-associated proteins) or grain extract (endogenous proteins) in 25 mM sodium acetate pH 5.0 were pre-incubated for 10 min at 40°C prior to addition of AZCL-AX tablets (30% w/v). The mixture was incubated for 30 min at 40°C and then added 5 mL stop solution (2% (w/v) Tris base pH 9.0) and vigorously mixed. After 10 min at room temperature, the reaction mixtures were filtered and the absorbance was measured at 590 nm (Ultrospec II, Amersham Biosciences, Uppsala, Sweden) against a blank prepared by adding 5 mL stop solution to samples prior to addition of substrate. Correction was made for non-enzymatic color release from the AZCL-AX tablets. All measurements were performed in duplicate from two independent washing liquid preparations.

Xylanase inhibition assay Relative levels of xylanase inhibitory activity in barley grains were determined using a variant of the Xylazyme AX method. 17 Washing liquid prepared from cultivar Cabaret (harvest year 2009) was used as the source of microbial xylanase activity for these experiments. The grain extracts were diluted to ensure a linear response between the amount of inhibitors and the measured residual xylanase activity. Twenty-two µg protein from grain extracts (containing xylanase inhibitors) or 25 mM sodium acetate pH 5.0 (for the uninhibited reference sample) was added to 450 µL Cabaret 2009 washing liquid to compare the efficiency of the different barley cultivars in inhibiting the surfaceassociated xylanases. The reaction mixtures were pre-incubated (30 min, RT) to allow formation of enzyme-inhibitor complexes before an AZCL-AX tablet was added and incubated for 30 min at 40°C. The reaction was stopped by addition of stop solution and immediate vortex mixing, filtered after 10 min and the absorbance at 590 nm was measured against a blank containing grain extract incubated with buffer (without washing liquid). Controls allowed correction for non-enzymatic color release while the blanks 7 ACS Paragon Plus Environment


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corrected for any xylanase activity present in the added grain extract. All measurements were performed in duplicate from two independent washing liquid preparations. Inhibition was expressed as percentage reduction of the total xylanase activity measured in Cabaret 2009 washing liquid. The inhibition efficiency towards xylanases corresponds to the amount of protein (grain extract containing mixture of xylanase inhibitors) needed to reduce xylanase activity by 50% in the described assay.

Sample preparation and 1D SDS-PAGE Surface-associated proteins were isolated by washing the grains as described above followed by desalting (NAP-5 column, GE Healthcare) of the washing liquids against 10 mM sodium acetate pH 5.0. Ten µg protein was precipitated by adding four volumes of ice-cold acetone and analysed by SDS-PAGE using 4−12% BisTris NuPAGE gels and a vertical slab mini gel unit (NuPAGE Novex system, Invitrogen) according to the manufacturer’s instructions. The gels were stained with colloidal Coomassie Blue. 18 A broad-range molecular mass protein ladder (Mark 12TM, Invitrogen) was used.

Zymogram staining of xylanase activity In-gel xylanase activity was detected in a 10% acrylamide gel containing 0.1% (w/v) wheat arabinoxylan (Megazyme) essentially as described. 19 The 10% acrylamide resolving gel, 4.5% stacking gel and running buffer contained 0.4%, 0.4% and 0.1% SDS, respectively. Samples of 10 µg protein were prepared and the electrophoresis was run as above followed by removal of SDS by washing the gel once with 50 mM sodium phosphate, pH 7.2, 25% (v/v) 2-propanol (1 h, RT) and subsequently with the buffer (50 mM sodium phosphate, 1 h). Proteins were renatured by incubating the gel in 50 mM sodium phosphate buffer, pH 7.2, 5 mM β-mercaptoethanol, 1 mM EDTA (4°C, 8 ACS Paragon Plus Environment

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overnight). The gel was then incubated (50°C, 4 h), followed by staining in 0.1% (w/v) Congo Red (1 h, RT) and washed with 1 M NaCl (30 min) until excess dye was removed. The zymogram was developed by soaking in 0.5% (v/v) acetic acid and showed xylanolytic activity as clear zones on a dark blue background.

Native PAGE for xylanolytic activity detection Protein samples of 10 µg were separated using pre-cast NativePAGE Novex 4−16% BisTris gels (8 x 8 cm gel, Invitrogen) according to the manufacturer’s instructions. After electrophoresis, the native gel was overlaid with a substrate gel (0.5% (w/v) Remazol Brilliant Blue (RBB)-dyed wheat arabinoxylan (WAX), 1.5% (w/v) agarose, 0.2 M sodium citrate-HCl, pH 4.8) and incubated for 4 h at 50°C. The substrate gel was developed in 96% ethanol: 0.2 M sodium citrate-HCl, pH 4.8 (2:1) for 2 h.

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Xylanase

activity appeared as transparent zones in the blue gel.

Agarose plate assay for endo-1,4-xylanase activity Agarose gels were prepared containing dyed substrate (0.1% (w/v) RBB-WAX (Megazyme), 1% (w/v) agarose, 0.2 M sodium citrate-HCl pH 4.8). Five microliters of washing liquid was added to 2 mm diameter wells punched into the plates and incubated overnight at room temperature. Xylanase activity appeared as clearing zones around the wells.

2D-gel electrophoresis of barley grain surface-associated proteins After desalting of washing liquids as described above, 50 µg of protein was precipitated overnight at -20°C in 4 volumes of acetone and dissolved in 125 µL rehydration buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 200 mM destreak reagent (bis (2-hydroxyethyl) 9 ACS Paragon Plus Environment


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disulfide; GE Healthcare), 0.5% (v/v) pharmalytes pH range 3−10 (GE Healthcare), trace of bromophenol blue). The samples were applied to 7 cm pH 3−10 IPG strips (GE Healthcare) for isoelectric focusing (IEF) (Ettan™ IPGphor; GE Healthcare) after rehydration (12 h at 50 mA/strip at 20°C), performed to reach a total of 20 kVh (1 h at 150 V, 1 h at 300 V, 1 h at 1000 V, gradient to 8000 V, held at 8000 V until a total of 20 kVh). Subsequently, the strips were equilibrated (2 × 15 min) in 5 mL equilibration buffer (6 M urea, 30% (v/v) glycerol, 50 mM Tris HCl, pH 8.8, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) supplemented with 1% (w/v) DTT and 2.5% (w/v) iodoacetamide in first and second equilibration step, respectively. The strips and molecular weight markers (Mark 12, Invitrogen) were placed on 4−12% Bis-Tris Zoom gels (NuPAGE, Novex system, Invitrogen) for SDS-PAGE (XCell SureLock mini-cell system; NuPAGE Novex System, Invitrogen) using NuPAGE MES running buffer (Invitrogen). Gels were fixed (50% (v/v) ethanol, 7% (v/v) acetic acid) for 1 h, rinsed 3 x 10 min in water, stained by Sypro Ruby (Invitrogen) overnight and washed (10% (v/v) ethanol, 7% (v/v) acetic acid) for 30 min and rinsed with water. Images of the Sypro Ruby-stained gels (three biological replicates of each cultivar, Supplementary Figure S1) were captured at excitation/emission wavelengths 532/580 nm, 100 microns (resolution), on a Typhoon scanner (9410 Variable Mode Imager, GE Healthcare, Uppsala, Sweden). Analysis of the gel images was performed using Progenesis Samespots software version 3.3 (Nonlinear Dynamics, Newcastle, UK). Gel images were aligned by automated calculation of alignment vectors after ten-fifteen manually assigned landmark vectors. A threshold of 1.5-fold spot volume ratio change and ANOVA p ≤ 0.05 was chosen to identify differentially abundant protein spots.

In-gel digestion and MALDI-TOF-TOF mass spectrometry


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Sypro Ruby-stained gels were post-stained with colloidal Coomassie Blue. 18 Spots were manually excised and subjected to in-gel tryptic digestion.

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Briefly, gel pieces were

washed (100 µL 40% (v/v) ethanol, 10 min), shrunk (50 µL 100% ACN) and soaked in 2 µL 12.5 ng/µL trypsin (Promega, porcine sequencing grade) in 25 mM (w/v) NH4HCO3 on ice (45 min). The gel pieces were rehydrated by addition of 10 µL 25 mM (w/v) NH4HCO3 followed by incubation at 37°C overnight. Tryptic peptides (1 µL) were loaded onto an AnchorChipTM target plate (Bruker-Daltonics, Bremen, Germany), covered by 1 µL matrix solution (0.5 µg/µL CHCA in 90% (v/v) ACN, 0.1% (v/v) TFA) and washed in 0.5% (v/v) TFA. Tryptic peptides were analyzed by Ultraflex II MALDITOF mass spectrometer (Bruker-Daltonics, Bremen, Germany) using Flex Control v3.0 and processed by Flex Analysis v3.0 (Bruker-Daltonics, Bremen, Germany). Peptide mass mapping was performed in positive ion reflector mode with 500 laser shots per spectrum. MS/MS data were acquired with an average of 1000−2000 laser shots for each spectrum. Spectra were externally calibrated using a tryptic digest of β-lactoglobulin (5 pmol/µL). Internal calibration was performed using trypsin autolysis products (m/z 842.5090, m/z 1045.5637 and m/z 2211.1040). Filtering of spectra was performed for known keratin peaks. Acquired MS and MS/MS spectra were analyzed using Biotools v3.1 (Bruker-Daltonics, Bremen, Germany). MASCOT 2.0 software (http://www.matrixscience.com) was used for database searches in the NCBInr (National Center for Biotechnology Information) bacteria, fungi and green plants (24712020, 2555264 and 1749148 entries, respectively), DFCI (Dana-Farber Cancer Institute) barley gene index Release 12.0 (http://compbio.dfci.harvard.edu/tgi) and MIPS/IBIS (Munich information center for protein sequence - Institute of Bioinformatics and Systems Biology) barley genome (79220 entries). The following search parameters were applied: monoisotopic peptide mass accuracy of 80 ppm; fragment mass accuracy to ± 0.7 Da; a



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maximum of one missed cleavage; carbamidomethylation of cysteine (fixed) and oxidation of methionine (partial). No restrictions with respect to protein Mw and pI were made. The signal to noise threshold ratio (S/N) was set to 1:6. A minimum of two matched peptides were considered for protein identification with a probability-based MOWSE score above the calculated threshold value corresponding to p < 0.05 (71, 86 and 77 for plants, bacteria and fungi, respectively). Identifications based on fewer than 5 matched peptides were confirmed by fragment ion spectra for at least one peptide, above the calculated threshold value corresponding to p < 0.05 (29, 34 and 35 for plants, bacteria and fungi, respectively).

Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis Tryptic digests of 1 µg of protein from washing liquids were prepared.

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The peptide

mixtures were separated on a nanoLC column (Dionex Acclaim PepMap RSLC C18 analytical column, 75 µm x 15 cm nanoviper, 2 µm particle size, 100 Å pore size combined with a Dionex Acclaim PepMap 100 pre-column, 75 µm x 2 cm nanoviper, 3 µm particle size, 100Å pore size (Thermo Fisher Scientific) using a 90 min gradient prepared from solvent A (0.1% formic acid (FA)) and B (80% ACN in 0.1% FA), at a flow rate of 300 nL min-1 on an EASY nLC 1000 coupled to a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Massachusetts, US). The gradient was ramped from 5 to 30% of solvent B in A for 70 min and then increased to 100% B for 10 min and held for 10 min. Each sample was run in triplicate. The Q Exactive was operated in data dependent mode with 10 MS/MS spectra for every full scan. The obtained MS and MS/MS spectra were processed using Xcalibur v.2.0 (Thermo Fisher Scientific), analyzed and searched using Mascot Daemon v.2.2.0 (Matrix Science) in the NCBInr bacteria, fungi and green plants protein database applying as search parameters: one 12 ACS Paragon Plus Environment

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missed trypsin cleavage; allowed modification: carbamidomethylated cysteine (fixed); oxidation of methionine (partial); peptide and fragment ion mass tolerance 5 ppm and 0.6 Da, respectively. Mass range for MS1 was 350-1850 m/z at resolution 35000, an AGC target of 3×106 or maximum injection time 120 ms. MS2 spectra were collected at 17500 resolution with fixed first mass 50 m/z, an isolation window of 3 m/z, intensity threshold 7.5×104 and exclusion of ions with a charge state 4)-beta-xylan endohydrolases. J. Cereal Sci. 2003, 37 (2), 111-127. (11) Dornez, E.; Gebruers, K.; Wiame, S.; Delcour, J. A.; Courtin, C. M. Insight into the distribution of arabinoxylans, xylanases and xylanases inhibitors in industrial wheat roller mill streams. J. Agric. Food Chem. 2006, 54 (22), 8521-8529. (12) Jerkovic, A.; Kriegel, A. M.; Bradner, J. R.; Atwell, B. J.; Roberts, T. H.; Willows, R. D. Strategic distribution of protective proteins within bran layers of wheat (Triticum aestivum L.) protects the nutrient-rich endosperm. Plant Physiol. 2010, 152 (3), 14591470. (13) Goesaert, H.; Elliott, G.; Kroon, P. A.; Gebruers, K.; Courtin, C. M.; Robben, J.; Delcour, J. A.; Juge, N. Occurrence of proteinaceous endoxylanase inhibitors in cereals. Biochimica Et Biophysica Acta. 2004, 1696 (2), 193-202. (14) Juge, N.; Svensson, B. Plant protein inhibitors of cell wall degrading enzymes. J. Sci. Food Agric. 2006, 86 (11), 1573-1586. (15) Gebruers, K.; Dornez, E.; Bedõ, Z.; Rakszegi, M.; Courtin, C. M.; Delcour, J. A. Xylanases and xylanase-inhibitors of wheat, barley and rye in the HEALTHGRAIN diversity screen. J. Agric. Food Chem. 2010, 58 (17), 9362-9371. (16) Popov, N.; Schmitt, M.; Schulzeck, S.; Matthies, H. Eine störungsfreie mikromethode zur bestimmung des proteingehaltes in gewbehomogenaten. Acta. Biol. Med. Ger. 1975, 34, 1441-1446. (17) Gebruers, K.; Brijs, K.; Courtin, C. M.; Goesaert, H.; Proost, P.; Van Damme, J.; Delcour, J. A. Affinity chromatography with immobilised endoxylanases separates TAXI- and XIP-type endoxylanase inhibitors from wheat (Triticum aestivum L.). J. Cereal Sci. 2002, 36 (3), 367-375. (18) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 2004, 25 (9), 13271333.


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(43) Yadav, S. K.; Singla-Pareek, S.; Ray, M.; Reddy, M. K.; Sopory, S. K. Methylglyoxal levels in plants under salinity stress are dependent on glyoxalase I and glutathione. Biochem. Biophys. Res. Commun. 2005, 337 (1), 61-67. (44) Maeda, K.; Finnie, C.; Ostergaard, O.; Svensson, B. Identification, cloning and characterization of two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, from the barley seed proteome. Eur. J. Biochem. 2003, 270 (12), 2633-2643. (45) Shahpiri, A.; Svensson, B.; Finnie, C. From proteomics to structural studies of cytosolic/mitochondrial-type thioredoxin systems in barley seeds. Mol. Plant. 2009, 2 (3), 378-389. (46) Shahpiri, A.; Svensson, B.; Finnie, C. The NADPH-dependent thioredoxin reductase/thioredoxin system in germinating barley seeds: gene expression, protein profiles, and interactions between isoforms of thioredoxin h and thioredoxin reductase. Plant Physiol. 2008, 2, 789-799. (47) Fan, S.; Guo-Jiang, W. Characteristics of plant proteinase inhibitors and their applications in combating phytophagous insects. Bot. Bull. Acad. Sin. 2005, 46, 273-292. (48) Pesquet, E. Plant proteases - from detection to function. Physiol. Plant. 2012, 145 (1), 1-4. (49) Dornez, E.; Croes, E.; Gebruers, K.; De Coninck, B.; Cammue, B. P. A.; Delcour, J. A.; Courtin, C. M. Accumulated evidence substantiates a role for three classes of wheat xylanase inhibitors in plant defense. Crit. Rev. Plant Sci. 2010, 29 (4), 244-264. (50) Gupta, A. K.; Kaur,N. Sugar signalling and gene expression in relation to carbohydrate metabolism under abiotic stresses in plants source. J.Biosci. 2005, 30 (5), 761-776. (51) Park, J. S.; Lee, W. C.; Yeo, K. J.; Ryu, K. S.; Kumarasiri, M.; HHaesek, D.; Lee, M.; Mobashery, S.; Song, J. H.; Kim, S. I.; Lee, J. C.; Cheong, C.; Jeon, Y. H.; Kim, H. Y. Mechanism of anchoring of OmpA protein to the cell wall peptidoglycan of the gramnegative bacterial outer membrane. FASEB J. 2012, 1, 219-228. (52) Higgins, C. F. ABC transporters: Physiology, structure and mechanism - an overview. Res. Microbiol. 2001, 152 (3-4), 205-210. (53) Rodriguez-Herva, J.; Ramos-Gonzalez, M.; Ramos, J. L. The Pseudomonas putida peptidoglycan-associated outer membrane lipoprotein is involved in maintenance of the integrity of the cell envelope. J. Bacteriol. 1996, 178 (6), 1699-1706. (54) Chandramouli, K.; Qian, P. Proteomics: Challenges, techniques and possibilities to overcome biological sample complexity. HGP. 2009, 1 (1), 239204, 1-22. (55) Praveen Rao, J.; Subramanyam, C. Calmodulin mediated activation of acetyl-CoA carboxylase during aflatoxin production by Aspergillus parasiticus. Lett. Appl. Microbiol. 2000, 4, 277-281. 33 ACS Paragon Plus Environment


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(56) Lorca, G. L.; Font de Valdez, G.; Ljungh, A. Characterization of the proteinsynthesis dependent adaptive acid tolerance response in Lactobacillus acidophilus. J. Mol. Microbiol. Biotechnol. 2002, 6, 525-532. (57) Prajapati, S. C.; Chauhan, S. S. Dipeptidyl peptidase III: a multifaceted oligopeptide N-end cutter. FEBS Journal 2011, 18, 3256-3276. (58) Waller, F.; Achatz, B.; Baltruschat, H.; Fodor, J.; Becker, K.; Fischer, M.; Heier, T.; Huckelhoven, R.; Neumann, C.; von Wettstein, D.; Franken, P.; Kogel, K. H. The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc. Natl. Acad. Sci. U. S. A. 2005, 38, 1338613391. (59) Makovec, T.; Breskvar, K. J. Catalytic and immunochemical properties of NADPHcytochrome P450 reductase from fungus Rhizopus nigricans. Steroid Biochem. Mol. Biol. 2002, 1, 89-96. (60) van den Brink, Hans (J.) M.; van Gorcom, R. F. M.; van den Hondel, C. A. M. J. J.; Punt, P. J. Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans. Fungal Genetics and Biology 1998, 1, 1-17. (61) Chakrabarty, A. M. Nucleoside diphosphate kinase: Role in bacterial growth, virulence, cell signaling and polysaccharide synthesis. Mol. Microbiol. 1998, 5, 875-882. (62) Tonukari, N. J. Enzymes and fungal virulence. J. Appl. Sci. Environ. Manag. 2003, 7 (1), 5-8. (63) Wilson, M. A.; St Amour, C. V.; Collins, J. L.; Ringe, D.; Petsko, G. A. The 1.8-A resolution crystal structure of YDR533Cp from Saccharomyces cerevisiae: a member of the DJ-1/ThiJ/PfpI superfamily. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (6), 1531-1536. (64) Bolwell, G. P.; Wojtaszek, P. Mechanisms for the generation of reactive oxygen species in plant defence - a broad perspective. Physiol. Mol. Plant Pathol. 1997, 51 (6), 347-366. (65) Pegg, G.F., Brady, B.L. Pathogenesis. In: Verticillium wilts, Pegg, G. F.; Brady, B. L., Eds.; CABI Publishing; Wallingford, 2002, pp 126. (66) Manamgoda, D.; Cai, L.; Bahkali, A.; Chukeatirote, E.; Hyde, K. Cochliobolus: an overview and current status of species. Fungal Divers 2011, 1, 3-42. (67) Ciuffetti, L. M.; Manning, V. A.; Pandelova, I.; Betts, M. F.; Martinez, J. P. Hostselective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis-wheat interaction. New Phytol. 2010, 4, 911-919. (68) Collins, T.; Gerday, C.; Feller, G. Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol. Rev. 2005, 1, 3-23.


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(69) Beaugrand, J.; Cronier, D.; Thiebeau, P.; Schreiber, L.; Debeire, P.; Chabbert, B. Structure, chemical composition, and xylanase degradation of external layers isolated from developing wheat grain. J. Agric. Food Chem. 2004, 23, 7108-7117.



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Page 36 of 51

Table legends Table 1. Identification of proteins in spots resolved by 2-DE of the washing liquids of barley cultivar Barke and Cabaret by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. The spot numbers correspond to the gel image shown in Figure 3. Table 2. Identification of proteins in washing liquids of barley cultivar Barke and Cabaret of year 2009 and 2011 by nanoLC-MS/MS. Abbreviation: ID: identification.


Page 37 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. Identification of proteins in spots resolved by 2-DE of the washing liquids of barley cultivar Barke and Cabaret by MALDI-TOF-TOF MS and MS/MS. Probabilitybased MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. Spot numbers correspond to Figure 3. Spot no.

Locus

Organism

Protein name

PMF score

E-value

Sequence coverage %

Functional a) group

CABARET 2009 1

WP_002893595

Streptococcus sanguinis

Acetyl-CoA carboxylase subunit beta

87

0.046

41

FP

6

CCA66671

Piriformospora indica

Probable dipeptidylpeptidase III

79

0.031

17

CIM; DSM

9

ENI03940

Bipolaris maydis C5

77

0.05

30

HP; EM; CIM

10

P15737

Hypothetical protein - cytochrome P450 oxidoreductase Endo-1,3-betaglucosidase GII GH17 Beta-(1-3)-glucanase

240

1.10E-18

54

CD; CTM

121

8.60E-07

17

CD; CTM

91

0.00062

12

CD; CE; DSM

120

1.10E-06

27

CD; CE; DSM

95

0.00022

12

CD; CE; DSM

11

AAA32939

Hordeum vulgare subsp. vulgare Hordeum vulgare

12

P15326

Coix lacryma-job

13

BAJ89873

14

P15326

Hordeum vulgare subsp. vulgare Coix lacryma-jobi

15

P23951

Hordeum vulgare

Alpha-amylase inhibitor/endochitinase 26 kDa Endochitinase

93

5.30E-04

13

CD; CE; DSM

16

P23951

Hordeum vulgare

26 kDa Endochitinase

88

1.70E-03

13

CD; CE; DSM

17

BAJ89873

Predicted protein - Chitinase GH19

72

7.30E-02

13

CD; CE; DSM

19

AAM96690

The nucleus-encoded protein MOC1

82

0.011

32

EM; PPNN

20

P23951

Hordeum vulgare subsp. vulgare Chlamydomonas reinhardtii Hordeum vulgare


89

1.40E-03

15

CD; CE; DSM

21

P15326

Coix lacryma-jobi

106

1.80E-05

12

CD; CE; DSM

22

P23951

Hordeum vulgare

Alpha-amylase inhibitor/endochitinase 26 kDa Endochitinase

114

4.30E-06

15

CE; DSM

23

ABX09990

1.30E-02

14

OS; EM

P15326

89

0.0008

12

DSM; CD; CE

25

EMS54278

Triticum urartu

NADPH-dependent thioredoxin reductase isoform 2 Alpha-amylase inhibitor/endochitinase Hypothetical protein TRIUR3_13181

79

24

Hordeum vulgare subsp. vulgare Coix lacryma-jobi

80

0.02

36

HP

26

CAA55345

Hordeum vulgare

Chitinase

80

0.0078

40

CE; DSM

29

CAB99486

Chitinase II

122

6.80E-07

20

CE; DSM

30

IAAB_HORVU

Amylase/trypsin inhibitor CMb

89

0.00096

40

DSM; CD; CE

33

AAQ08998

Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Phaseolus vulgaris

36

XP_002531643

Ricinus communis

37

1GHS_A

41

CAB99486

42

AAA56787

Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare

44

AEE81086

45

XP_001782939

Pseudomonas abietaniphila Physcomitrella patens subsp. patens

Alpha-amylase inhibitor/endochitinase Predicted protein - Chitinase - GH19

Polyubiquitin 1, partial

93

0.00033

63

CP; EM

Disease resistance protein RPS2, putative Beta-1,3-glucanase II

84

0.0025

8

DSM

81

7.80E-03

13

CD; CTM

Chitinase II

167

2.20E-11

20

CE; DSM

Chitinase

140

1.10E-08

9

CE; DSM

OprF, partial

112

1.20E-04

17

CE

Predicted protein - Glyoxalase I

77

0.013

55

OS

BARKE 2009 58

AK377081

Oryza sativa

Metallo-beta-lactamase-like

72

0.041

24

DSM; CP; UF

59

BAK06170

Hordeum vulgare subsp.

Predicted protein - Alginate_lyase2

83

9.70E-02

15

CTM



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 38 of 51

vulgare 60

1GHS_A

Beta-1,3-glucanase II

114

2.80E-09

56

CD; CTM

61

1GHS_A

62

1GHS_A

Hordeum vulgare


119

1.40E-06

25

CD; CTM

Hordeum vulgare


289

1.40E-23

36

63

CHI2_HORVU

CD; CTM

Hordeum vulgare


112

6.80E-06

16

CE; DSM

64 65

ABG01419

Arabidopsis thaliana

Disease resistance protein, partial

78

0.01

51

DSM

BAE44506

Thottea tomentosa

Phytochrome A, partial

77

0.014

39

EM; CP; RF

66

CAA33407

Chitinase GH19

102

6.80E-05

22

CE; DSM

67

BAJ89873


109

1.40E-05

13

CE; DSM

69

BAJ89873


101

8.60E-05

13

CE; DSM

70

1GHS_A

Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare

71

CAB99486

72

BAJ89873

73

AF355455_1

75

CAB99486

Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare

78

AAA56787

Hordeum vulgare

79

BAJ89873

80

CAB99486

81

CAA55345

82

CAA55345

86

AAA56787

Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare

88

CAB99486

a)

Hordeum vulgare

Hordeum vulgare subsp. vulgare


105

2.20E-05

37

CD; CTM

Chitinase II

73

0.035

27

CE; DSM


99

1.40E-04

16

CE; DSM

Thaumatin-like protein TLP4

83

0.0037

45

DSM

Chitinase II

347

20

CE; DSM

Chitinase

82

6.70E-03

9

CE; DSM


99

1.30E-04

13

CE; DSM

Chitinase II

158

1.70E-10

24

CE; DSM

Chitinase

437

2.20E-38

37

CE; DSM

Chitinase

334

4.30E-28

35

CE; DSM

Chitinase

117

2.20E-06

9

CE; DSM

Chitinase II

302

6.80E-25

26

CE; DSM

Functional role: CD: Carbohydrate degradation; CE: Cell envelope; CIM: Central intermediary metabolism; CP: Cellular processes; CTM: Carbohydrate transport and metabolism; DM: DNA metabolism; DSM: Defense and stress-related mechanism; EM: Energy metabolism; FP: Fatty acid and phospholipid metabolism; HP: Hypothetical proteins; OS: Oxidative stress; PF: Protein fate; PPNN: Purines, pyrimidines, nucleosides and nucleotides;

PS: Protein synthesis; RF:

Regulatory functions; SP: Storage protein/deposition; ST: Signal transduction; T: Transcription; TR: Transport; UF: Unknown functions


Page 39 of 51

Table 2. Identification of proteins in washing liquids of barley cultivar Barke and Cabaret of year 2009 and 2011 by nLC-MS/MS. Abbreviation: ID: Identification.

Barke 2009 Cabaret 2009 Barke 2011 Cabaret 2011

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ID no. PLANT 1 x 2 3 4

Protein name

Organism

Target Functional a) b) group P

Hordeum vulgare Triticum aestivum Triticum aestivum Triticum aestivum

S S S S

DSM DSM DSM DSM

Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare

S _

CD; CTM UF DSM DSM DSM

AAZ94265

Putative protease inhibitor Wali6 Wali3 Putative proteinase inhibitorrelated protein Beta-glucosidase Baker's asthma allergen Protein synthesis inhibitor I Protein synthesis inhibitor II Chain C, Amy2BASI proteinprotein complex from barley seed Bifunctional alphaamylase/subtilisin inhibitor Antifungal protein S Antifungal protein R Thaumatin-like protein TLP4 Thaumatin-like protein TLP7 Thaumatin-like protein TLP8 Pathogenesis-related protein PRB1-3 Pathogenesis-related protein 1 PR-1a pathogenesis related protein (Hv-1a) Pathogenesis-related protein 1, partial Pathogenesis-related 1a

x x

x x

CAA88619 AAC37417 AAA50848 AAS49905

5 6 7 8 9

x x x x x

x

AAA87339 AAB34366 RIP1_HORVU RIP2_HORVU 1AVA_C

10

x

x

CAA78305

11 12 13 14 15 16 17 18

x x x x

Locus

x

x x x

x x x x x x

x x x x x x

x x x x

THHS_HORVU THHR_HORVU AF355455_1 AF355457_1 AF355458_1 PR13_HORVU

x x

x x

x x

x x

PR1_HORVU CAA52893

19

x

20

AAF62171 x

21 22

x

23

_

Hordeum vulgare

DSM

Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare

S S S

DSM DSM DSM DSM DSM DSM

Hordeum vulgare Hordeum vulgare

S S

DSM DSM DSM

Betula pendula S

DSM

x x

x

CAA29331 CAA66232

Protein Z (180 AA), partial Protein z-type serpin

Triticum monococcum Hordeum vulgare Hordeum vulgare

x

x

BSZ7_HORVU

Serpin-Z7

Hordeum vulgare

x

CAA55344 BAG12896 CAA55345

Chitinase Chitinase Chitinase

S S

2007234A AAA56787 1CNS_A

Hordeum vulgare Bromus inermis Hordeum vulgare subsp. vulgare Secale cereale Hordeum vulgare Hordeum vulgare Hordeum vulgare Oryza sativa Japonica Triticum aestivum

S

CE; DSM CE; DSM

S

CE; DSM

24 25 26

x x x

x x x

x x x

27 28 29

x x

x x x

x x x

x

30 31

x

CHI1_HORVU CAA39535

Chitinase a Chitinase Chain A, Crystal structure of chitinase at 1.91a resolution 26 kDa Endochitinase 1 Chitinase, partial

32

x

AAR11388

Class I chitinase

x

x

S

DSM DSM; CP; UF DSM; CP; UF CE; DSM CE; DSM CE; DSM CE; DSM CE; DSM CE; DSM



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

33 34 35 36

x

x x

x x x

THN5_HORVU THNA_HORVU THNB_HORVU CAA35188

x

37

x

x

x

IAAB_HORVU

38

x

x

39 40

x x

x x

41 42

x x

x x

x

IAA2_HORVU IAA1_HORVU

43

x

x

x

1208404A

IAAD_HORVU

x

IAA_HORVU IAAA_HORVU

44 45 46 47

x x x x

CAA42453 CAA11029 ABI18856 ABW71749

48

x

ICI2_HORVU

49 50

x x

1602257A ICIB_HORVU

51 52 53

x x x

ABV22584 AAC49288 XP_002467027

54 55 56 57

x x

CAA74594 CAA71774 BARW_HORVU CAJ40963

x x

BAE16420 1MID_A

58 59

x

x x

x x x x

x

x

x x

60

x

61 62

x

63

x

64

x

65 66

x

x x

67

x

x

68

x

AAL71854

Subtilisin-chymotrypsin inhibitor-2A Chymotrypsin inhibitor 1A Subtilisin-chymotrypsin inhibitor CI-1B PR17d precursor Unknown Hypothetical protein SORBIDRAFT_01g018490 Hypothetical protein Pathogenesis-related protein 4 Barwin Putative vacuolar defense protein PR-4 homolog, partial Chain A, Non-specific lipid transfer protein 1 from barley in complex with L-alfalysophosphatidylcholine, laudoyl. Non-specific lipid transfer protein 6 Lipid transfer protein 7a2b Probable non-specific lipidtransfer protein Endosperm transfer cell specific PR60 precursor Putative lipid transfer protein precursor Cystatin Hv-CPI8 GSH-dependent dehydroascorbate Reductase 1 Dehydroascorbate reductase

AAW52718

Peroxidase 4

AAV49759 CAA65680 NLTP2_HORVU

x x

x

ACA04813 ACN54189

x

x

Leaf-specific thionin Alpha-hordothionin Beta-hordothionin Trypsin inhibitor CMe precursor Alpha-amylase/trypsin inhibitor CMb Alpha-amylase/trypsin inhibitor CMd Alpha-amylase/trypsin inhibitor Alpha-amylase/trypsin inhibitor CMa Alpha-amylase inhibitor BDAI-1 Alpha-amylase inhibitor BMAI1 Trypsin/amylase inhibitor pUP13 CM 17 protein precursor BTI-CMe1 Hordoindoline b, partial Chymotrypsin inhibitor-2

CAG38129 BAA90672

Page 40 of 51

Hordeum vulgare Hordeum vulgare Hordeum vulgare Hordeum vulgare

S S S

DSM DSM DSM DSM

Hordeum vulgare

S

DSM

Hordeum vulgare

S

DSM


S S

DSM DSM


S

DSM DSM DSM

Hordeum vulgare Triticum aestivum Hordeum vulgare Hordeum vulgare Hordeum vulgare subsp. spontaneum Hordeum vulgare

S S S _

DSM DSM DSM DSM

_

DSM


_ _

DSM DSM

Hordeum vulgare Triticum aestivum Sorghum bicolor

S S S

DSM UF HP

Hordeum vulgare Hordeum vulgare Hordeum vulgare Triticum aestivum

S

HP DSM DSM DSM DSM FP; DSM

Solanum melongena Hordeum vulgare

Hordeum vulgare

S

FP; DSM


S S

FP; DSM FP; DSM

Triticum aestivum

S

TR

Triticum durum

S

FP; DSM

Hordeum vulgare Oryza sativa Japonica Triticum aestivum

_

Triticum

S

DSM; PF OS; EM; DSM OS; EM; DSM OS; EM;


Page 41 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


69 70 71

x

AAA32973

Peroxidase BP 1

x

CAC12881 AAB35914

AAA62698

Cold-regulated protein 32 kDa Antifreeze protein/endo-beta-1,3glucanase homolog, {Nterminal} [Peptide partial, 20 aa]. Beta-amylase Beta-amylase Tissue-ubiquitous beta-amylase 2 Ubiquitin

x

72 73 74 75

x

x x x

x x

x

AMYB_WHEAT CAC16789 AAX37358

76

x

AF240445_1

Polyubiquitin

77

x

AAC67551

Tetra-ubiquitin

78

x

1PKU_A

x

ENO1_HEVBR AAC49173

Chain A, Crystal structure of nucleoside diphosphate kinase from rice Hypothetical protein LOC_Os12g07340 Enolase 1 Enolase

82 83

x x

AMY2_HORVU CAX51373

Alpha-amylase type B isozyme Alpha-amylase

84 85

x x

ACN34260 CAA45903

Unknown Alpha-amylase

E13B_HORVU

Glucan endo-1,3-betaglucosidase GII Endo-1,3-beta-glucanase

79

x

80 81

86 87

x

x

ABA96563

x

x x

88

x x

89 90

1607157A 1GHS_A

x x

AAP33176 CAJ58513

91 92

x x

AAK38482 AAC49170

93

x

1EX1_A

94 95

x x

ALDR_HORVU TPIS_HORVU

96

x

T06212

97

x

NP_001105603

monococcum Hordeum vulgare

S

Hordeum vulgare Secale cereale

_

Triticum aestivum Hordeum vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Populus tremula x Populus tremuloides Saccharum hybrid cultivar H32-8560 Oryza sativa

Oryza sativa Japonica Group Hevea brasiliensis Oryza sativa Japonica Group Hordeum vulgare Hordeum vulgare subsp. vulgare Zea mays Oryza sativa Indica Group Hordeum vulgare

Hordeum vulgare subsp. vulgare Chain A, The three-dimensional Hordeum vulgare structures of two plant betaglucan endohydrolases with distinct substrate specificities 1,3-Beta glucanase Avena sativa Glucan endo-1,3-beta-DSecale cereale glucosidase precursor Beta-D-xylosidase Hordeum vulgare Beta-D-glucan exohydrolase, Hordeum vulgare isoenzyme ExoII subsp. vulgare Chain A, Beta-D-glucan Hordeum vulgare exohydrolase from barley Aldose reductase Hordeum vulgare Triosephosphate isomerase, Hordeum vulgare cytosolic Glucose and ribitol Hordeum vulgare dehydrogenase homolog barley Malate dehydrogenase, Zea mays] cytoplasmic

DSM OS; EM; DSM DSM DSM; CD; CTM

CD; CTM CD; CTM CD; CTM _

PF; CP

_

PF; CP

_

PF; CP

_

EM

HP EM EM S S

CD; CTM CD; CTM

S S

UF CD; CTM

S

CD; CTM

_

CD; CTM

_

CD; CTM

S _

CD; CTM CD; CTM

S

CD; CTM CD; CTM

_

CD; CTM EM EM

_

EM

EM



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

98 99

x x

CYSP1_HORVU ABO72663

100

x

G3PX_HORVU

101

x

G3PC_HORVU

Cysteine proteinase EP-B 1 Glyceraldehyde 3-phosphate dehydrogenase, partial Glyceraldehyde-3-phosphate dehydrogenase, cytosolic Glyceraldehyde-3-phosphate dehydrogenase, cytosolic Glyceraldehyde-3-phosphate dehydrogenase

102

x

ABA03227

103

x

BAC80257

104

x

CAC80383

105 106

x x

CBP1_HORVU 1314177A

107

x

CAA70815

108

x

ABC55717

Serine carboxypeptidase II, CPMII Beta-mannosidase 2

109

x

BAB71741

Glyoxalase I

110 111

x

ABK22263 CAA59104

Unknown D-Hordein, partial

x

Glyceraldehyde-3-phosphate dehydrogenase, partial Glyceraldehyde-3-phosphate dehydrogenase, partial Serine carboxypeptidase 1 CPase I A

112

x

BAA11642

D-Hordein

113

x

AAV37977

Grain softness protein

ABI18927 CAA25509

Grain softness protein, partial Unnamed protein product, partial Histone H3 variant, partial

114 115

x x

116

x

XP_001699985

x

PDI_HORVU LE19A_HORVU

x

LE19B_HORVU

120

x

LE194_HORVU

123

x

117 118

x

119

x

124 126

x

CAN68695

x

BAD15703 x

CAN63684

Protein disulfide-isomerase Late embryogenesis abundant protein B19.1A Late embryogenesis abundant protein B19.1B Late embryogenesis abundant protein B19.4 Hypothetical protein VITISV_035922 Hypothetical protein Hypothetical protein VITISV_020448

Page 42 of 51

Hordeum vulgare Prosopis pubescens

S

EM

Hordeum vulgare Hordeum vulgare Populus maximowiczii x Populus nigra Houttuynia cordata Sphagnum cuspidatum Hordeum vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Oncidium Gower Ramsey Oryza sativa Japonica Picea sitchensis Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare subsp. vulgare Hordeum vulgare Hordeum vulgare Chlamydomonas reinhardtii Hordeum vulgare Hordeum vulgare

PF; DSM EM

_

EM EM

_

EM EM

_

PF PF PF CD; CTM

_

OS

S

UF SP

S

SP

S

SP

S

SP UF PPNN; PF

S

CTM; PF DSM; SP

Hordeum vulgare

DSM; SP

Hordeum vulgare

DSM; SP

Vitis vinifera Oryza sativa Japonica Vitis vinifera

_

HP HP

_

HP


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ID # FUNGI 1

Barke 2009 Cabaret 2009 Barke 2011 Cabaret 2011

Page 43 of 51

Locus

Protein name

Organism

Target Functional P a) group b)

x

x

ABE02800

Xylanase

Verticillium dahliae

S

x

CAA06151

Beta-1,4-xylanase precursor

Cochliobolus sativus

S

XP_001939205

Endo-1,4-beta-xylanase I precursor Glucan 1,3-beta-glucosidase precursor Hypothetical protein

Pyrenophora triticirepentis Pt-1C-BFP Pyrenophora triticirepentis Pt-1C-BFP Podospora anserina S mat+ Phaeosphaeria nodorum SN15

S

2 3

x

5

x

6

x

XP_001941009 x

XP_001905591

x

XP_001799969

8

x

XP_001598620

9

x

XP_002628332

7

x

10

x

11

XP_001805825

x

XP_001935291

Hypothetical protein SNOG_15684 G3P_COLGL Glyceraldehyde 3phosphate dehydrogenase (GAPDH) NADP-specific glutamate dehydrogenase Hypothetical protein CIMG_06611 Ceramide glucosyltransferase YALI0F09229p Nucleoside diphosphate kinase ThiJ/PfpI family protein

x

AAQ81899

Actin, partial

BAC55128

Actin, partial

x

EEH49656

Predicted protein

x

XP_002562870

Pc20g03180 Pre-rRNA processing protein IPI3

x

XP_386433

12

x

XP_001934415

13

x

XP_001242715

14

x

17 18

x

19

x

22

x

23

Hypothetical protein SNOG_09682 Transaldolase Hypothetical protein SS1G_00709 Transaldolase Transaldolase

x

XP_505197

_

CD; CE; DSM CD; CE; DSM CD; CE; DSM CD; CTM; DSM HP

_

CTM

Sclerotinia sclerotiorum 1980 UF-70 Ajellomyces _ dermatitidis SLH14081 Phaeosphaeria nodorum SN15 Gibberella zeae PH-1

CTM

EM

_

EM

_

FP

_

EM; DM; PS

Pyrenophora triticirepentis Pt-1C-BFP Coccidioides immitis RS Yarrowia lipolytica (Yeast) Pyrenophora triticirepentis Pt-1C-BFP Sphaerophorus globosus (Lichen) Debaryomyces hansenii (Yeast) Paracoccidioides brasiliensis Pb18 Penicillium chrysogenum Wisconsin 54-1255

S

CTM

HP

DSM; PF _

RF

_

RF UF PS


ID # BACTERIA

Cabaret 2011

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Barke 2009 Cabaret 2009 Barke 2011


Page 44 of 51

Locus

Protein name

Organism

Target a) P

Functiona b) l group

1

x

WP_010255028

Membrane protein

Pantoea

S

CE; TR

2

x

YP_004115247

Pantoea sp. At-9b

S

CE; TR; DSM

3

x x

YP_004212300

Rahnella sp. Y9602

S

4

x

WP_017802501

OmpA domain-containing protein transmembrane region-containing protein OmpA/MotB domaincontaining protein Membrane protein

Erwinia toletana

S

CE; TR; DSM CE; TR

_

S S

5

x

ADC44455

OmpA, partial

6

x

YP_003530744

Candidatus Erwinia dacicola Erwinia amylovora CFBP1430 Pantoea sp. Sc1

7

x

WP_009091389

Hypothetical protein EAMY_1386 Translocation protein TolB

8

x

WP_017802169

Murein lipoprotein

Erwinia toletana

9

x

LPP_SERMA

Major outer membrane lipoprotein Hypothetical protein YPN_1848 Major outer membrane lipoprotein

Serratia marcescens

10

x

YP_647777

11

x

YP_049963

12

x

WP_009088983

13

x

WP_009087536

14

x

YP_002870995

15

x

YP_003931167

16

x

WP_010247363

17

x

YP_004212788

18

x

NP_669476

19

x

WP_017810955

Amino acid ABC transporter substrate-binding protein Amino acid ABC transporter Substrate-binding protein Branched amino acid ABC transporter substrate-binding protein Periplasmic oligopeptidebinding protein precursor Sugar ABC transporter substrate-binding protein Glyceraldehyde-3-phosphate dehydrogenase, type I Glyceraldehyde-3-phosphate dehydrogenase Hypothetical protein

20

x

WP_003848945

21

x

22

x

23

x

WP_010208010

a)

S

CE; TR; DSM HP TR; CP; DSM CE; DSM CE; DSM

S

HP

S

TR; CP; DSM

S

CTM; TR

Pantoea sp. Sc1

S

CTM; TR

Pseudomonas fluorescens SBW25

S

CTM; TR

Yersinia pestis Nepal516 Pectobacterium atrosepticum SCRI1043 Pantoea sp. Sc1

Pantoea vagans C9- S 1 Pantoea sp. SL1_M5 S

TR CTM; TR

Rahnella sp. Y9602

EM

Yersinia pestis KIM10+ Paenibacillus sp. A9

EM _

HP

Hydroperoxidase II

Pantoea

_

OS

WP_010245500

Hydroperoxidase II

Pantoea

_

YP_001908129

DNA starvation/stationary Erwinia _ phase protection protein Dps tasmaniensis Et1/99 Molecular chaperone GroEL Pseudomonas _

OS DM; DSM PF

Prediction of localization: C: Chloroplast; M: Mitochondrion; S: Secretory pathway; –: any other location.


Page 45 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


b)

Functional role: CD: Carbohydrate degradation; CE: Cell envelope; CIM: Central intermediary metabolism; CP: Cellular processes; CTM: Carbohydrate transport and metabolism; DM: DNA metabolism; DSM: Defense and stress-related mechanism; EM: Energy metabolism; FP: Fatty acid and phospholipid metabolism; HP: Hypothetical proteins; OS: Oxidative stress; PF: Protein fate; PPNN: Purines, pyrimidines, nucleosides and nucleotides; PS: Protein synthesis; RF: Regulatory functions; SP: Storage protein/deposition; ST: Signal transduction; T: Transcription; TR: Transport; UF: Unknown functions.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 46 of 51

Figure legends Figure 1. Xylanolytic activities of barley cultivars Barke and Cabaret of harvest year 2009 and 2011. (A) The xylanase activities (endogenous) in the grain extracts. (B) Plate zymograms (Rimazol Brilliant Blue (RBB)-dyed wheat arabinoxylan containing agarose) showing xylanase activity in the washing liquids. (C) The xylanase activities (microbial) determined in the washing liquids. (D) Inhibition of surface-associated xylanases in washing liquids of Cabaret from 2011 harvest by grain sxtracts of Barke and Cabaret. Figure 2. (A) 1D SDS-PAGE of the washing liquids of barley cultivars Barke and Cabaret of harvest year 2009. Each lane was loaded with equal amount of protein approximately 10 µg, where lane (1) Barke 2009 and (2) Cabaret 2009. (B) Zymogram developed with Congo Red dye, where samples were run in a SDSPAGE incorporated with 0.1% (w/w) WAX. (C) 4−16% Native PAGE gel and substrate gel (0.5% Remazol Brilliant Blue-dyed WAX and 1.5% agarose). Molecular markers are as indicated. The detected protein bands on the zymogram are indicated with an arrow. Cleared zones/xylanolytic activity is indicated with brackets. Figure 3. Two-dimensional gel electrophoresis Sypro Ruby-stained gels loaded with 50 µg protein of washing liquids of barley cultivar Cabaret (A) and Barke (A) of harvest year 2009 (greyscale, 16 Bit). After post-staining with Coomassie Blue, visible spots were excised and subjected to MALDI-TOF-TOF MS and MS/MS. Identified proteins are given in Table 1. Molecular marker and pI range are as indicated. Figure 4. Overview of the plant-microbe interactions and insight into the identified proteins/enzymes at the plant-microbe interface.


Page 47 of 51

(A) Absorbance 590 nm

Endogenous xylanase activity

0.150 0.100

2009 2011

0.050 0.000

(B) Barke

Cabaret

2009

2011

(C) Absorbance 590 nm

Surface-associated xylanase activity

3.00 2.00

2009 2011

1.00 0.00

(D) Xylanase inhibition levels 100 % Inhibition

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80 60

2009

40

2011

20 0

Figure 1.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a) kDa 200 116,3 97,4 66,3 55,4 -

1

(b) 2

(c) Protein gel

1

Page 48 of 51

2

kDa

1

2

Substrate gel

1

2

1048720480-

36,5 31,0 -

242-

21,5 14,4 -

14666-

]

6.0 3,5 2.5 -

20-

Figure 2.


Page 49 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

kDa 200-

pI

3

10

116.397.466.355.4-

36.531.0-

21.514.4-

6.0-

(B)

kDa 200-

pI

3

10

116.397.466.355.4-

36.531.021.514.4-

6.0-

Figure 3.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 50 of 51

Figure 4.


Page 51 of 51

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC only 190x190mm (96 x 96 DPI)

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