Induction of Pathogenesis-Related Genes, PR-17A and N-Methyltransferase, in Barley Infested by the Aphid Rhopalosiphum Padi

Naima Grönberg Master thesis

Induction of pathogenesis-related genes, PR-17a and N-methyltransferase, in barley infested by the aphid Rhopalosiphum padi

Master thesis by Naima Grönberg 2006

Supervisor: Gabriele Delp, Ph.D. Examiner: Professor Lisbeth Jonsson, Ph.D

Södertörns högskola, University College

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Table of contents

List of abbreviations…………………………………………………………………..3

Abstract………………………………………………………………………………...4

Introduction…………………………………………………………………………… 5 Barley (Hordeum vulgare L.)...... 5 Bird cherry-oat aphid (Rhopalosiphum padi)…………………………………. 5 Pathogenesis-related proteins (PR-17)………………………………………… 6 Gramine and N-methyltransferase……………………………………………. 8 Aim of the project………………………………………………………………. 10

Materials and methods……………………………………………………………….. 10 Plants and growth conditions………………………………………………….. 10 Aphids infestation………………………………………………………………. 10 Protein extraction………………………………………………………………. 11 Protein determination………………………………………………………….. 11 SDS-PAGE………………………………………………………………………. 11 Staining SDS-polyacrylamid gel……………………………………………….. 11 Western blot and detection…………………………………………………….. 12 Primary antibodies………………………………………………………………12 Ponceau S staining……………………………………………………………… 12 Total RNA isolation…………………………………………………………….. 13 Integrity of the total RNA……………………………………………………… 13 Preparation of the probe for Northern blot detection……………………… 13 Northern blot……………………………………………………………………. 13 Reverse transcription-PCR (RT-PCR)………………………………………... 14

Results…………………………………………………………………………………. 14 Purity and integrity of the RNA……………………………………………….. 15 Northern blot analysis………………………………………………………….. 16 Gene expression analyses of PR-17a…………………………………………... 16 Accumulation pattern of PR-17a and PR-17b in barley seedlings upon R. padi infestation……………………………………………………………….. 16 Gene expression analyses of NMT……………………………………………. 18 Accumulation pattern of NMT in barley seedlings upon R. padi infestation.. 19

Discussion……………………………………………………………………………... 22 Gene expression analyses of PR-17a ………………………………………….. 22 The N-methyltransferase …………………………………………………….. 23 Conclusion ……………………………………………………………………… 24

Acknowledgements…………………………………………………………………… 24

References……………………………………………………………………………... 25

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List of abbreviations

Abs Absorbance AMI 3-aminomethylindole AP Alkaline phosphatase BCIP/NBT Bromochloroindolyl phosphate/nitro blue tetrazolium BSA Bovine serum albumine COMT caffeic acid-O-methyltransferase DTT 1,4-dithiothreitol ECL Enhanced chemiluminescence EDTA Ethylenediaminetetraacetic acid Gap Glycerol aldehyde 3-P dehydrogenase HRP Horseradish peroxidase MAMI N-methyl-3-aminomethylindole MOPS 3-[N-morpholino] propanesulfonic acid NMT N-methyltransferase OD Optical density OMT O-methyltransferase PBS Phosphate buffered saline PCR polymerase chain reaction PR proteins Pathogenesis-related proteins PVDF Polyvinyl difluoride PVP Polyvinylpyrrolidone PVPP Polyvinyl poly-pyrrolidone RT Room temperature RT-PCR reverse transcription-polymerase chain reaction SAM S-adenosyl-L-methione SB Sample buffer SDS Sodium dodecyl sulphate SDS-PAGE SDS Poly acrylamide gel electrophoresis SSPE Sodium chloride sodium phosphate + ethyleneaminetetraacetic acid TBE Tris-Borate-EDTA TBS Tris-buffered saline TE Tris-EDTA

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Abstract

Plants produce a large diverse array of organic compounds that may function in protection against pathogens. Diverse antifungal compounds were reported to exist in barley (Hordeum vulgare L.); the indole alkaloid, gramine, and the pathogenesis-related proteins are some of them. Both the N-methyltransferase that is involved in gramine biosynthesis and PR-17a were studied in barley upon infestation by the bird cherry-oat aphid (Rhopalosiphum padi). The effect of infestation by R. padi on induction of PR-17a and N-methyltransferase was investigated in different barley lines, susceptible and resistant. The gene expression of PR-17a was down-regulated in the susceptible cv. Golf and to some extent up-regulated at the first days in var. Lina and then down-regulated. The PR-17a was induced by the aphid infestation in the resistant line CI16145; the gene expression was stronger in the infested plants than in the controls. The different responses in resistant and susceptible lines indicate that the induced PR-17a may play a role in the resistance against aphid infestation. PR-17a was up-regulated systemically in the base in barley after infestation by R. padi. In the susceptible varieties Lina and Golf, the accumulation of N-methyltransferase did not increase with time from 1 day to 7 days after infestation, as determined by western blots with antibody raised against NMT from barley. The NMT-gene was down-regulated after 7 days infestation in both variety Lina and Golf both locally in the first leaf and in the base. Barley line CI16145 had no accumulation of NMT as was seen by western blotting. There was no induction of NMT in barley upon aphid infestation.

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INTRODUCTION

Plants have developed complex signalling and defence mechanisms to protect themselves against invading pathogens. Recognition of the pathogen by the plant stimulates multiple signalling pathways leading to the activation of defence mechanisms, production of reactive oxygen species (ROS) (Taiz and Zeiger, 2002), cell wall modification such as callose formation and lignification (Barker and al., 1989; Lozovaya et al., 1998), and accumulation of phytoalexins (Hahlbrock and Scheel, 1989), induction of pathogenesis-related (PR) proteins (Schlumbaum et al., 1996; van Loon et al., 1994), as well as systemic acquired resistance and hypersensitive response (HR) (Park et al., 2002;Taiz and Zeiger, 2002). A common feature of plant-aphid interactions is that plants activate genes known to increase defences against bacterial and fungal pathogens (Fidantsef et al., 1999; Moran and Thompson, 2001). Such a response is generally not stimulated by chewing insects (Stotz et al., 2000). Besides pathogen attacks, plants protect themselves from various stresses as wounding, application of chemicals and heavy metals, air pollutants like ozone, ultraviolet rays, and harsh growing conditions. These protective reactions are known as defence responses of higher plants, and the proteins actively synthesized in accordance with these reactions are called “defence-related proteins” (Bowles, 1990).

Barley (Hordeum vulgare L.)

Barley was one of the first domesticated cereals, dating back to 8000 BC. It is an important crop for direct human consumption and for animal feed. It is a source of malt for beer and other products. Cultivated barley is one of 31 Hordeum species, belonging to the tribe Triticeae, family Poaceae. It is an annual diploid species. The genetic system is relatively simple, while the species is genetically diverse, making it an ideal study organism (http://oregonstate.edu). Diverse antifungal compounds and proteins were reported to exist in barley; gramine (Wippich and Wink, 1985), and pathogenesis-related proteins such as peroxidases (Thordal-Christensen et al., 1992) are some of them.

Bird cherry-oat aphid (Rhopalosiphum padi)

The bird cherry-oat aphid (Rhopalosiphum padi L.) is holocyclic in Sweden, carrying out alternations of parthenogenic and sexual generations. Bird cherry (Prunus padus L.) is the main primary host, while Gramineae, especially maize, barley, oats and wheat are secondary hosts (www.inra.fr). The bird cherry-oat aphid overwinters in the egg stage on bird cherry. The first fundatrices of R. padi hatch from the winter eggs in the end of April. These morphs, wingless aphids, start to produce parthenogenetic offspring and feed on the opening buds and then on the undersides of young leaves. The bird cherry-oat aphid population reaches a maximum on the winter host in the middle of May. An increasing number of individuals develops wings (alatae) and usually after 2 to 3 generations they migrate from their primary (winter) to their secondary (summer) host, colonizing the cereals and various grasses (Dixon, 1971). When the emigrants arrive at the cereal fields the crop is at a growth stage that provides an excellent source of nutrition. As the lower leaves export more of their assimilates downwards than upwards (Patrik, 1972), the stem-base becomes a highly suitable feeding area for the aphids. The aphid population growth becomes huge. This crowding disturbs feeding and decreasing food quality gradually induces the development of winged individuals. These winged individuals leave the crop in search for other grasses. The third migration from mid- August is the return to the primary host to lay their eggs (Dixon, 1971).

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Aphids penetrate plant tissues by probing intercellularly through epidermal and mesophyll cell layers with their stylet-like mouthparts to establish feeding sites in veins of the phloem (Miles, 1999) inflicting considerable fitness costs in many crop plants (www.ipm.ucdavis. edu). Aphids secrete watery saliva containing peroxidases, β-glucosidases, and other potential signal-generating enzymes into phloem sieve elements (Miles, 1999). The aphid´s stylet is in continuous contact with plant cells and causes limited tissue damage. Plant responses to phloem-feeding insects are distinct from that of chewing insects and tissue damaging cell- content feeders (Walling, 2000). And some piercing/sucking insects induce the defense- signaling pathways most commonly activated by bacterial, fungal, and viral pathogens (Moran and Thompson, 2001; Forslund et al., 2000; Walling, 2000; Fidantsef et al, 1999).

Aphids are important vectors of viral plant pathogens (www.ipm.ucdavis.edu.). R. padi also transmits several virus diseases, being the principal vector of barley yellow dwarf virus (BYDV). In spring, alatae aphids leave their overwintering hosts, migrate into spring-sown cereals and, if carrying BYDV, may infect susceptible plants (Mann et al., 1997).

Pathogenesis-related proteins, with special reference to (PR-17)

Protective plant proteins specifically induced in pathological or related situations have been intensively studied from an agricultural perspective and are called “pathogenesis-related proteins”. PR proteins, which have been found in many plant species to date, are classified into 17 families (Table 1), regardless of the original plant species. The sequence similarities, serologic or immunologic relationships, and enzymatic properties are the basis of this classification (Van Loon et al., 1994 and 1999).

PR proteins are also induced and upregulated by phytohormones including jasmonic acid (Wasternack and Hause, 2002), salicylic acid (Zeier et al., 2004; Ryals et al., 1996) as well as by chemicals or osmotic stress (Liu et al., 2003; Kitajima and Sato, 1999). In addition some PR proteins are constitutively expressed at distinct developmental stages in roots, in senescent leaves (Tamás et al., 1998) or during the flowering stage (Cote et al., 1991).

Not all families of PR proteins have been identified in each plant species examined. Plant species differ in the types of PR proteins present or, at least expressed upon infection (van Loon and Strien, 1999). In tobacco, the major PR proteins are acidic, located extracellularly, and expressed upon infection, whereas basic vacuolar counterparts with different stress expression patterns are also present in healthy plants where their expression is temporally and spatially controlled in a cell-type and organ-dependent manner. The presence of PR-type proteins in healthy plant tissues, such as glucanases and chitinases in dicotyledons and thionins (PR-13) and lipid-transfer proteins (LTPs) (PR-14) in monocotyledons, appears to be fairly common (van Loon and Strien, 1999).

PR-proteins may be considered as stress proteins (van Loon and Gerritsen, 1989) produced in response to, particularly necrotizing, infections by viruses (Park et al., 2002), viroids, fungi and bacteria, and thought to function in the acquired resistance against further infection (van Loon, 1989). However, in contrast to most other types of stress proteins, they accumulate in plant tissues to levels that are easily detectable on gels by general protein stains (van Loon, 1997). The role of PR proteins and other herbivore-induced gene products on plant resistance to herbivores that use piercing/sucking mode of feeding is unknown (Walling, 2000).

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Families Type member Properties Gene symbol Reference PR-1 Tobacco PR-1a antifungal Ypr1 Antoniw et al., 1980 Ypr2, [Gns2 PR-2 Tobacco PR-2 ß-1,3-glucanase Antoniw et al., 1980 ('Glb')] chitinase type I,II, PR-3 Tobacco P, Q Ypr3, Chia Van Loon, 1982 IV,V,VI,VII PR-4 Tobacco 'R' chitinase type I,II Ypr4, Chid Van Loon, 1982 PR-5 Tobacco S thaumatin-like Ypr5 Van Loon, 1982 Green and Ryan, PR-6 Tomato Inhibitor I proteinase-inhibitor Ypr6, Pis ('Pin') 1972 Vera and Conejero, PR-7 Tomato P endoproteinase Ypr7 69 1988 PR-8 Cucumber chitinase chitinase type III Ypr8, Chib Métraux et al., 1988 Tobacco 'lignin-forming Lagrimini et al., PR-9 peroxidase Ypr9, Prx peroxidase' 1987 Somssich et al., PR-10 Parsley 'PR1' 'ribonuclease-like' Ypr10 1986 Melchers et al., PR-11 Tobacco 'class V' chitinase chitinase, type I Ypr11, Chic 1994 PR-12 Radish Rs-AFP3 defensin Ypr12 Terras et al., 1992 PR-13 Arabidopsis THI2.1 thionin Ypr13, Thi Epple et al., 1995 García-Olmedo et PR-14 Barley LTP4 lipid-transfer protein Ypr14, Ltp al., 1995 PR-15 Barley OxOa (germin) oxalate oxidase Ypr15 Zhang et al., 1995 PR-16 Barley OxOLP 'oxalate oxidase-like' Yrp16 Wei et al., 1998 Okushima et al., PR-17 Tobacco PRp27 unknown Yrp17 2000

Table 1. PR-proteins classification (source: http://dmd.nihs.go.jp/latex/defense-e.html)

Several PR proteins have direct antimicrobial activity, and as such represent a conceptually simple form of defence against the invading microbe. In the barley-powdery mildew interaction, the peroxidases (PR-9) are enzymes with possible implications in the oxidative cross-linking of plant cell wall components to prevent the pathogen from penetrating (Thordal-Christensen et al., 1992). Another defensive response to infection is the formation of hydrolytic enzymes that attack the cell wall of the pathogen. An assortment of glucanases, chitinases, and other hydrolases are induced by fungal invasion.

Aphid infestation triggers an accumulation of the PR-proteins, β-1,3-glucanase (PR-2a) and chitinase (PR-3a) in barley (Forslund et al., 2000). When barley leaves are attacked by the powdery mildew fungus, the majority of the established families are represented in the response (Gregersen et al., 1997; Christensen et al., 2002). The PR-protein oxalate oxidase (PR-15) is accumulated in barley leaves in response to the powdery mildew fungus Blumeria graminis f. sp. hordei and may play a role in a signal transduction pathway for regulation of the hypersensitive response (Zhou et al., 1998).

The encoded proteins from barley (designed Hv-PRs), HvPR-17a and HvPR-17b belong to the plant pathogenesis-related proteins PR-17 (Christensen et al., 2002). The family includes also NtPRp27 from tobacco (Okushima et al., 2000) and WCI-5 from wheat (Görlach et al., 1996) responsive to viral and fungal infection, respectively. The HvPR-17a and HvPR-17b

7 Naima Grönberg Master thesis protein sequences were found to be members of a small family of plant proteins. HvPR-17b exhibits the highest overall identity of 83% to WAS-2, an abscisic acid-induced secreted protein from wheat (Kuwabara et al., 1999). HvPR-17a exhibits the highest overall identity of 62% to a putative rice protein. Additionally, HvPR-17a and HvPR-17b are 61% and 66% identical, respectively, to a protein encoded by the wheat cDNA clone, WCI-5 (Christensen et al., 2002). This clone represents an mRNA that accumulates in response to treatment with benzothiadiazole, a chemical inducer of systemic acquired resistance. Importantly, the WCI-5 transcript was also found to accumulate in wheat leaves infected by the wheat powdery mildew fungus (Görlach et al., 1996). HvPR-17a and HvPR-17b have molecular weights of 26 and 24 kDa respectively. They accumulate in the mesophyll apoplast following Bgh- inoculation, as well as in the leaf epidermis, the only tissue to be invaded by the fungus (Christensen and al, 2002). HvPR-17a and HvPR-17b proteins are monomeric polypeptides. Both proteins were found in the stem but only HvPR-17a was seen in roots at a high level, while HvPR-17b had a high level in the stem in barley, and in the leaves at low levels (Christensen et al., 2002).

Gramine and N-methyltransferase

Secondary metabolites protect plants against being eaten by herbivores and against being infected by microbial pathogens (Taiz and Zeiger, 2002) and many of them are found in only one plant species or related group of species. Each species contains only a subset of genes for secondary metabolism and the enzymes in plant secondary metabolism are specific for a given substrate and produce a single product (Pichersky and Gang, 2000). There are three different chemical groups belonging to the secondary metabolites; nitrogen-containing compounds is one of them. Most nitrogenous secondary metabolites are biosynthesized from common amino acids. Alkaloids are usually synthesized from one of a few common amino acids, in particular, lysine, tyrosine and tryptophan.

The indole alkaloid, gramine, is a constitutive compound of barley and is synthesized from tryptophan. Two methylation reactions are needed for the formation of gramine. Both methylation reactions need a methyl donor, in the case of barley the methyl donor is S- adenosyl-L-methione (SAM). The biosynthetic pathway of gramine is considered as follows, 3-aminomethylindole (AMI) is synthesized from tryptophan through unknown metabolic steps. AMI is catalysed into N-methyl-3-aminomethylindole (MAMI) by an N- methyltransferase (NMT) (Leland et al., 1985). The NMT also converts MAMI into gramine (Fig. 1).

Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) (Matsuo et al., 2001). Gramine is mainly located in parenchymal cells and on the leaf surface in barley (Yoshida et al., 1993).

Gramine is the most toxic secondary metabolite against aphids (Corcuera, 1984 and 1993). Gramine contained in artificial diets decreased fecundity and longevity of Schizaphis graminum (Kawada and Lohar, 1989). These observations suggest gramine may be one of the resistance factors against cereal aphids.

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Figure 1. The synthetic pathway of gramine (source: www.hort.purdue.edu)

Kanehisa et al. (1990) and Rustamani et al. (1992) found that resistant lines of barley contained higher concentrations of gramine than susceptible lines and a negative effect of gramine on aphid survival on artificial diets. However, the abundance of this alkaloid varies significantly among different barley cultivars (Hanson et al., 1981). Forslund et al. (1998) found no correlation between gramine concentration and preference of the aphid in settling behaviour. This means that high gramine concentrations did not confer resistance to aphids.

An O-methyltransferase (OMT) (Genbank accession number U43498) showing similarity to caffeic acid O-methyltransferase was isolated as being regulated when screening a cDNA library from aphid-infested barley (Delp et al., 2002). Lee et al. (1997a, 1997b) had isolated the same cDNA earlier as methyljasmonate-induced O-methyltransferase. The gene was mapped to chromosome 5 and was absent in the barley cultivar Morex that did not contain gramine either. The transcript for OMT was detected only in the leaf sheath of barley and accumulated in leaf segments after jasmonate application (Lee et al., 1997b). One type of O-methyltransferase (OMT) plays an important role in methylating caffeic acid or 5-hydroxylferulic acid leading to lignin precursors (Whetten and Sederoff, 1995) or the methylation of various classes of flavonoid compounds leading to phytoalexins (Gustine et al., 1978). In addition to its role in lignification during vascular tissue development, OMT has been widely described to be induced during pathogen attack or elicitor treatment (Pelligrini et al., 1993). OMT catalyses the methylation of caffeic acid to ferulic acid and the enzyme is called caffeic acid O-methyltransferase (COMT). Larsson et al. (2006, under publication) demonstrated that the OMT gene identified earlier by Lee et al. (1997a) was in fact an N-methyltransferase (NMT) gene. The purified protein, NMT, expressed enzyme activity with AMI and MAMI, intermediates of gramine synthesis, but not with caffeic acid. Not all barley cultivars had the methyltransferase gene in their genome. The methyltransferase gene was detected in eight of thirteen barley cultivars tested. In all barley varieties missing the gene, the indole alkaloid gramine is present only in trace amounts.

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Aim of the project

K. Forslund identified a peptide sequence for PR-17b from a protein spot that was induced by aphids on two-dimensional gel electrophoresis (Personal communication). Therefore, the aim of this study was to confirm these results by studying the regulation of the expression of PR- 17 gene at different times of infestation of barley with aphids. The N-methyltransferase was originally isolated as aphid-induced sequence, but later studies using Northern blot gave inconclusive results. Therefore the aim for this project was to study the regulation of the expression of N-methyltransferase at RNA level and at protein level at different times of infestation of barley using both resistant and susceptible varieties.

MATERIALS AND METHODS

Plant materials and growth conditions

Seeds of barley (Hordeum vulgare L.) of cv. Golf, Lina, double haploid line 5175-50:20 and CI 16145 were surface sterilized with ethanol 70% for 1 min, then washed with tap water for 15 min, and with sodium hypochlorite 20% for 5 min and washed for 15 min with tap water. The seeds were kept at 4°C for 2 days and then planted in individual pots filled with soil. They were kept in a growth chamber at 20°C and 16-hr-light period at 190 µmol m-2 s-1/8-hr- dark period, and watered with tap water.

Aphid infestation of plants

All the aphids are female aphids. When the plants were 7 days old, when the primary leaf was fully developed, each primary leaf was infested with 10 aphids Rhopalosiphum padi and covered with a cage. Control plants carried an empty clipcage on the primary leaf without adding aphids. After 1, 2, 3, 4 and 7 days of infestation, all aphids were removed and the plants were divided into local tissues, which consist of the primary leaf, that had carried the cage, the top of the second leaf and the base of the plant (Fig. 2). The plant samples were flash-frozen in liquid nitrogen, weighed and stored at -80°C until protein extraction or RNA extraction.

Figure 2. Selected tissues for extraction of the total RNA and total proteins. The tissues used were those from the first leaf located in the cage, the top of the second leaf and the base.

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Protein extraction

Total protein was prepared from barley tissues by homogenizing frozen samples in liquid nitrogen using a mortar and pestle. The frozen tissue powder was extracted with 3 x (w/v) of ice-cold 0.1 M phosphate buffer (0,1 M K2HPO4, 0,1 M KH2PO4), pH 7.1, 16 mM 2- mercaptoethanol and 50 g l-1 PVPP. The homogenate was vortexed and centrifuged at 12 000 g for 10 min at 4°C. The supernatant was kept and the pellet was washed with 1 volume of ice-cold 0.1 M phosphate buffer (see above) with 2-mercaptoethanol and PVPP (see above) and centrifuged under the same conditions. The supernatants were combined to form the total protein extract. All protein samples were stored at -20°C.

Protein determination

Protein was quantified in triplicate by spectrophotometry (595 nm) using an UV-2501 PC UV-VIS Recording Spectrophotometer (Shimadzu) according to the method of Bradford with bovine serum albumin as standard (Bradford, 1976).

SDS-PAGE

Before loading the gel, equal amounts of total protein (10 µg) were mixed with 4x SDS loading buffer (1,25 M Tris-HCl pH 6,8, 20% SDS, 0,05% Bromophenol blue, 20% Glycerol) and 1M DTT and then heated for 5 minutes at 100οC. Then equal amounts of protein (10 µg) were separated on 4-20% Tris-glycine gels (Cambrex PAGE). After loading the samples and the molecular weight protein marker (PageRulerTMPrestained Protein Ladder) the electrophoresis was run for about 90 minutes using an EI 9001-X Cell IITM Mini Cell (Novel Experimental Technology, USA) and 1 x SDS running buffer (19,2 mM Glycine, 2,5 mM Tris-base pH 8.8, 0,01% SDS) at room temperature. A voltage of 125 volt and 40 mA per gel were used. The gel was run until the bromophenol blue reached the bottom of the resolving gel.

Staining SDS-polyacrylamide gel

Proteins separated by SDS-PAGE were detected by staining with Coomassie Brillant Blue as described by Sambrook and Russel (2001). Coomassie Brillant Blue binds nonspecifically to proteins but not to the gel, thereby allowing visualization of the proteins as discreet blue bands within the transluscent matrix of the gel. Coomassie Brillant Blue is an aminotriarylmethane dye that forms strong but not covalent complexes with proteins, most probably by a combination of van der Waals forces and + electrostatic interactions with NH3 groups. Coomassie Brillant Blue is used to stain proteins after electrophoresis through polyacrylamide gels. The gel must be stained at least 4 hours in Coomassie Brillant Blue (0,25 g Coomassie Brillant blue R-250, 90 ml of methanol:H2O (1:1), 10 ml glacial acetic acid) and with shaking. The gel is then destained in methanol:acetic acid solution without the dye on a slowly rocking platform for more than 8 hours changing the destaining solution 3 or 4 times. After destaining the gels were stored in H2O in a sealed plastic bag.

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Western blot and detection

The proteins were blotted onto a Hybond-P membrane (PVDF-membrane, Amersham Biosciences) by using a Semi-Phor Hoefer Scientific Instruments (San Franscisco) for 60 minutes at 0.8 mA/cm2. Before transfer, the PVDF-membrane was wetted with methanol 100% for one minute and then transferred to water for 15 minutes and at last to transfer buffer or Towbin buffer (96mM Glycine, 12 mM Tris-base pH 8.3, 10% methanol) for 15 minutes. After transfer, the membrane was blocked with blocking buffer (1 x TBS (20 mM Tris/HCl, pH 7.5, 150 mM NaCl), 5% low-fat dried milk powder, 0.05% Tween-20) at RT during 2 hr with gentle shaking. After blocking, the membrane was washed briefly twice, then 15 min and 4 x 5 min at RT in washing buffer (1 x TBS, 0.05% Tween-20). The membrane was then incubated with the primary antibody (see below) in antibody buffer (1 x TBS, 2% milk powder, 0.05% Tween-20) at a dilution of 1:10 000 (anti-NMT) or 1:2000 (anti-PR17) over night at 4 °C. After the incubation, the membrane was washed briefly twice with washing buffer, then 1 x 15 min and 4 x 5 min in washing buffer (see above). The membrane was then incubated with secondary antibody using alkaline phosphatase-conjugated secondary antibodies (Anti-Chicken IgY (IgG) Alkaline Phosphatase diluted in antibody buffer at 1:50 000 (to detect NMT) or Anti-Rabbit IgG alkaline phosphatase diluted in antibody buffer at 1:30 000 (to detect PR17)) during 2 h at RT and with gentle shaking. Afterwards, the membrane was washed with washing buffer, 4 x 5 min and then with distilled water 1 x 15 min. For detection, the membrane was rinsed in AP buffer (100 mM Tris-Cl pH 9.5, 100 mM NaCl, 5 mM MgCl2) and then the membrane was incubated with BCIP/NBT Blue liquid substrate for membranes (Sigma). The reaction was stopped by briefly rinsing the membrane with distilled water and drying it between two Whatman papers. The BCIP/NBT substrate generates an intense black-purple precipitate at the site of enzyme binding. The BCIP/NBT substrate characteristically produces sharp bands with very little background coloring the membrane (Sambrook and Russel, 2001).

Primary antibodies

The NMT antibodies were produced by immunizing chicken with the purified recombinant protein (Larsson et al., 2006). Antibody production and purification were performed by AgriSera (Sweden). The antibodies against PR-17 were a polyclonal antiserum produced by injecting rabbits with the in vitro expressed protein. The antisera were kindly provided by Professor D. Collinge (Royal Veterinary and Agricultural University, Copenhagen).

Ponceau staining

To confirm that transfer of sample proteins to the membrane occurred, a total protein stain such as Ponceau S is most commonly used (Salinovich and Montelano, 1986). After blotting the membrane was washed for a few seconds in 100% methanol, then immersed in sufficient Ponceau S staining solution for 5 min to just cover the membrane. After that, the membrane was immersed in 100% methanol until the bands appeared, then the membrane was washed with distilled water until the background was clear and the bands were visible. Before proceeding with the immuno-detection, the membrane was destained first in water and then in 0,1 M NaOH until the protein bands had disappeared. Then the membrane was rinsed again with distilled water.

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Total RNA isolation

Total RNA was extracted from barley samples. Each frozen sample (approximately 0,14-0,58 g fresh weight) was ground to a fine powder in liquid nitrogen and stored at -80°C prior to extraction with plant RNA reagent. RNA extraction was carried out using ConcertTM Plant RNA Reagent according to the protocol for Large-Scale RNA isolation (Invitrogen). Three replicates were used to determine the purity and concentration of RNA using NanoDrop ND-1000 Spectrophotometer. The RNA samples were stored at -80°C until they were used for Northern Blot or RT-PCR.

Integrity of the total RNA

To determine the quality and integrity of the isolated RNA, aliquots of the RNA samples were run on a 1% agarose gel with 17% formaldehyde in 1 x running buffer (0,2 M MOPS (pH 7.0), 20 mM sodium acetate and 10 mM EDTA (pH 8.0)). The RNA samples were mixed with equal volume of 2 x RNA loading buffer (Fermentas), incubated at 70°C for 10 minutes, then put on ice before loading. The gel was run at 80 volt about 1h.

Preparation of the probe for Northern blot detection

A plasmid containing the cDNA for PR-17 (obtained from prof. D. Collinge, Royal Veterinary and Agricultural University, Copenhagen) was used as template (1 µl of plasmid solution) and amplified using specific primers for PR17 (primer sequence, see below) and one unit of Taq DNA polymerase (Fermentas). PCR conditions were as follows: 1cycle of 94°C, 4 min; 40 cycles of 94°C, 30 sec, 58°C, 30 sec, 72°C, 1 min; and 1 cycle of 72°C, 5 min. PCR products were electrophoretically analyzed on a 1,5% agarose gel containing ethidium bromide 0,2 µg/ml in 1xTBE (0,089M Tris base, 0,089 M Boric acid, 0,002M EDTA pH 8,3). The gel was run at 80 volt. The DNA fragments in the gel were cut out on a UV-light transilluminator with a scalpel and transferred to an eppendorf tube. The fragment-containing gel pieces were purified with the Geneclean®Turbo Kit (Q biogene). The purified DNA could be used immediately for radio-labeling. After isolation the purified DNA fragments were kept at -20°C until use.

Northern blot

The RNA samples (15 µg/sample) were loaded on agarose/formaldehyde gel as described above. The gel was run at 80 volt during 1h and 30 min. After running the gel, the RNA was transferred from the gel to a nylon filter (Hybond N+ membrane, Amersham Biosciences) by capillary action with a high salt solution transfer buffer 20 x SSPE (3 M NaCl, 0,2 M * NaH2PO4 H2O, 19,8 mM EDTA, pH 7,4) overnight. After desalting the membrane in 2 x SSPE for a few minutes, the membrane was baked during 2h at 80ºC. The membranes were pre-hybridized and blocked in (5 x SSPE, 5 x Denhart´s (1 mg/ml BSA, 1 mg/ml Ficoll, 1 mg/ml PVP), 0,5% SDS, 0,2 mg/ml herring sperm DNA) at 65ºC during 4- 8h in a hybridisation oven. 25 ng DNA probe (MT-specific probe or PR-17) was radioactively labelled with 50 µCi of α-32P dCTP using the Amersham Pharmacia Rediview kit. The membranes were hybridised over night with the probe at 65ºC. The membrane was washed twice in 0,5 x SSPE + 0,1% SDS during 20 min at 65ºC.

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The membrane was exposed over night in a Fuji Imaging Plate and then analysed with a Fluorescent Image analyser FLA-3000 (Fujifilm). The reading was done with an Image Reader FLA-3000/3000G ver.1.0 and an Image Gauge Program ver.3.12 (Fujifilm).

Reverse transcription-PCR (RT-PCR)

RT-PCR is a very sensitive method for detection of gene expression. One Step RT-PCR can replace methods for detection and quantifying gene expression such as Northern blots. Using RobusTTM II RT-PCR kit (Finnzymes) or SuperScript III One Step RT-PCR system with Platinum® Taq DNA polymerase (Invitrogen), RT-PCR was accomplished with 50 ng of total RNA (see Total RNA extraction) following the supplier´s protocol and using suitable primers. All primers were designed based on internal sequences of the full-length cDNA. (All primers used are listed in Table 2). cDNA synthesis and PCR amplification of cDNA were performed successively in a single tube during a continuous thermal-cycling program using a PTC-100TM Programmable Thermal Controller (MJ Research, Inc.).

Primer Sequence Amplic Annealing PCR on temperature amplification GapHvF 5´-TTC ACT GAC AAG GAC AAG GC-3´ 292 bp 55ºC 30 cycles GapHvR 5´-CCA CCT CTC CAG TCC TTG CT-3´ PR17aF 5´-GGC CAG CGA TTC GAC AGG GA-3´ 590 bp 58ºC 40 cycles PR17aR 5´-ACG GAT CAG CCC TGG GAG TAC-3´ NMT-F 5´-ATA TAG CAG AGG CGG TGA CT-3´ 348 bp 55ºC 35 cycles NMT-R 5´-AAG AGA ACC GCA TCT CCA GT-3 Table 2. List of primers used.

Many different PCR reactions were made to optimise the reaction. The final PCR conditions used were as below: The reverse transcription at 45ºC 30 min, was followed by an inactivation of reverse transcriptase at 94ºC 2 min, the PCR amplification was reached by x cycles (Table 2) of 94ºC 30s for the template denaturation, followed by the primer annealing at xºC (Table 2) 30s, the primer elongation at 72ºC 1 min, and by one cycle for the final extension at 72ºC 5 min. Negative controls included water samples (without the template) subjected to RT-PCR. RT-PCR products were electrophoretically analyzed on a 1,5% agarose gel as described above.

RESULTS

The study included 2 susceptible varieties of barley, Golf and Lina, and 2 resistant varieties, CI16145 and double haploid line 5175-50:20. Both Golf and Lina contain gramine and the NMT-gene but Lina has medium high content of gramine and Golf has very low gramine content; CI16145 has hardly detectable gramine and lacks the NMT-gene (Larsson, 2006), and the double haploid line 5175-50:20 has the NMT-gene and contains gramine (G. Delp, personal communication). Unfortunately, we could not do comparisons between resistant and susceptible varieties, as the only resistant variety containing NMT that was used, the double haploid line 5175-50:20 gave after seven days very small seedlings that were not usable for our experiments.

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Ten aphids infested the first leaf (primary “caged” leaf) of each variety. Primary “caged” leaf, the second leaf and the base of the same seedling from infested and uninfested plants (controls) were harvested at different time points after infestation.

Purity and integrity of the RNA

The purity of the total RNA isolated with ConcertTM Plant Reagent (Invitrogen) was high as measured by UV absorbance ratios. Pure preparations of RNA have an A260/280 ratio of approximately 2.0. If there were protein contamination in the sample, this ratio would be significantly lower (Sambrook and Russel, 2001). As when analysed on agarose-formaldehyde denaturing gels (Figure 3), the total RNA (2µg) showed sharp and clear 28S and 18S rRNA bands. The total RNA isolated showed no serious signs of degradations.

Figure 3. Agarose-formaldehyde gel electrophoresis on total RNA isolated from barley. Total RNA was extracted from primary “caged” leaves and bases at 1, 2, 3, 4 and 7 days after infestation by R. padi. Numbers above panel indicate days after infestation. (-): non-infested (control) (+): infested by R. padi

Northern blot analysis

Accumulation of the gene transcripts corresponding to Gap, NMT and PR-17 were studied by hybridisation to Northern blots of total RNA (10 to 15 µg) isolated from barley leaves at different time points after infestation by aphids. The RNA was not degraded before blotting as verified by visualizing on formaldehyde- agarose gel (not shown). After transfer on the nylon membrane, the gel was visualized to determine the transfer of the RNA (not shown).

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No hybridisation signals were observed with RNA from uninfested and infested plant material due to technical failure. To remedy the situation, we used higher salt to reduce stringency. We proceeded also in removing the unincorporated (free) nucleotides and short oligomers using a ProbeQuant G-50 (Amersham Biosciences). This removal technique showed that the labelling of the probe was very poor, which might explain the failure to obtain a signal. Therefore we used RT-PCR, which is a very sensitive method for detection and quantifying gene expression and can replace Northern blot.

Gene expression analyses of PR-17a

Microarray studies have identified PR-17a and PR-17b as being up-regulated after 5 days infestation in both the resistant var. CI16145 and the susceptible var. Lina (G. Delp, personal communication). To confirm these results, a time course experiment was performed. Gene expression studies with RT-PCR were carried out on total RNA extracted from barley samples of the first leaf, where the aphids had been feeding, and the base, where no aphids had been sitting. 50 ng of total RNA was reverse transcribed and amplified using RobustTM II RT-PCR kit (Finnzymes) or SuperScriptTM One-Step RT-PCR with Platinum® Taq (Invitrogen).

As a control we investigated the expression of Gap, the gene coding for glycerol aldehyde 3-P dehydrogenase. We assumed that this gene, which is expressed in all tissues, is not regulated to a significant degree by aphid attack and can then serve as a loading control for the RT- PCR. As can be seen in the lower panels of Fig. 4, 5 and 6, equal amounts of template RNA in the samples gave rise to equal amount of product when using Gap-specific primers.

We did many attempts with the primers PR-17b but we did not obtain any expression of the gene. The gene coding for PR-17a (Fig. 4) presented different pattern of expressions depending on the barley variety. In the resistant line CI16145, the gene coding for PR-17a was expressed constitutively in the base but was still induced when the plants were infested by aphids (up- regulation) (Fig. 4A). In the susceptible barley var. Golf, the gene coding for PR-17a was only expressed in non-infested plants. The infested seedlings showed no expression of the gene indicating a down-regulation of the gene after infestation by aphids (Fig. 4B). Variety Lina had a lower level of constitutive expression than CI16145 for the very first days. One and 2 days after infestation, the gene was up-regulated but less than in CI16145. The gene was down-regulated after 3 days infestation (Fig. 4C).

We could not obtain any results in the primary leaf, where the aphids were feeding, even with so many attempts. I did many amplifications with many different PCR machines without any result. I think the problem was due to that the primers PR-17a were not functioning any more, as I used new dilutions of RNA that were working with the primers Gap but not with the primers PR-17a.

Accumulation pattern of PR-17a and PR-17b in barley seedlings upon R. padi infestation

In order to investigate the accumulation of the proteins PR-17a and PR-17b in barley seedlings when infested by R. padi we used polyclonal antisera against PR-17a and PR-17b. The immunostained bands observed in Western immunoblot analysis of SDS-PAGE were likely to be composed of many different polypeptides with different molecular masses but the

16 Naima Grönberg Master thesis bands that we were looking for of 26 and 24 kDa were missing (not shown). One possible explanation is that the antisera had been degraded during storage.

Figure 4. Gene expression analyses of barley seedlings var. CI16145 (A), Golf (B) and Lina (C) of uninfested plants (-) and plants infested with aphids (+) for one to 7 days. RT-PCR one step analysis of PR-17a gene expression with 50 ng of total RNA as template. The lower panels of A, B and C indicate Gap, amplicon size 292 bp, used as a control for the RT-PCR. The samples used are from the base (where no aphids had been sitting). (N): negative control, without the template.

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Gene expression analyses of NMT

The expression of NMT-gene was clearly detected in the base and the first leaf (were the aphids were feeding) for both barley var. Lina and var. Golf (Figures 5 and 6), indicating that NMT is constitutively expressed in barley plants. The base of var. Lina showed a constant expression of NMT, both for the controls and the infested seedlings (Fig. 5A), but the primary leaf (where aphids were sitting) of the same seedling showed a down-regulation after 3 days infestation and an up-regulation after 4 days infestation; and a new down-regulation after 7 days infestation (Fig. 5B).

A B B

Figure 5. Gene expression analyses of barley seedlings var. Lina of uninfested plants (-) and plants infested with aphids (+) for one to 7 days. RT-PCR one step analysis of N-methyltransferase (NMT) gene expression with 50 ng of total RNA. The lower panels of A and B indicate Gap, amplicon size 292 bp, used as a control for RT-PCR. A: base and B: primary leaf (where aphids were feeding) (N): negative control, without the template.

A B

Figure 6. Gene expression analyses of barley seedlings var. Golf of uninfested plants (-) and plants infested with aphids (+) from one to 7 days. RT-PCR one step analysis of NMT-gene expression with 50 ng of total RNA used as template. The lower panels of A and B indicate Gap, amplicon size 292 bp, used as a control for RT-PCR. A: base and B: primary leaf (where aphids were feeding) (N): negative control, without the template.

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Barley var. Golf had an expression of the gene in the base similar to var. Lina but after 7 days infestation the NMT-gene was down-regulated and the same pattern was seen in the first leaf (where the aphids were sitting) after 7 days infestation (Fig. 6A, 6B). Both the susceptible varieties Lina and Golf had a stronger expression of NMT in the base than in the primary leaf.

Accumulation pattern of NMT in barley seedlings upon R. padi infestation

In order to investigate the accumulation of the protein N-methyltransferase (NMT) in barley seedlings when infested by R. padi we used polyclonal antisera against NMT. Total protein extracts from non-infested and infested barley seedlings were immunoblotted after SDS- PAGE and probed with antibodies raised against NMT. The results are shown in figures 7 and 8. The antibodies recognized a 42 kDa band of NMT in the first leaf (where the aphids were sitting), the second leaf and the base of both barley var. Lina and Golf (Fig. 7 and 8), in infested seedlings and the non-infested seedlings (controls). The band has the same size as the purified protein that was run on the same gel as a control.

The accumulation of the protein NMT as a band of 42 kDa was seen in barley var. Lina primary “caged” leaf in both the controls and the infested seedlings (Fig. 7B). The lanes corresponding to 4 days showed weak bands due to an unequal loading of the proteins in the gel, which was revealed by Ponceau staining of the membrane. In the base, the NMT was detected in both the controls and the infested plants (Fig. 7A) and these accumulations seemed to be equally strong in the controls and the infested plants. The lanes corresponding to one-day had also less protein due to an unequal loading of the proteins in the gel. The second leaf showed an unequal loading of the proteins in lanes corresponding to one-day and 2-days. The second leaf showed the same accumulation of the protein NMT as for the first leaf and the base (Fig. 7C). Furthermore, the antibodies recognized other protein bands of ca 50 kDa in the first leaf and the second leaf (Fig. 7B and 7C) and a band of ca 100 kDa in the base (Fig. 7A).

The antibody recognized the 42 kDa NMT band in the first leaf, the second leaf and the base of barley var. Golf (Fig. 8). In the primary “caged” leaf, the bands of 42 kDa were very weak both for the controls and the infested plants (Fig.8B). In the base after one-day infestation, a very faint band of 42 kDa due to unequal loading was seen and the accumulation was the same for both the infested plants and the controls (Fig.8A). In the second leaf, the bands of 42 kDa were as strong as in both the controls and the infested seedlings at 2, 3 and 4 days. After 1day infestation, both the control and the infested samples showed very weak bands due to unequal loading of the proteins (Fig. 8C). However, the antibodies recognized bands of ca 100 kDa in the base (Fig. 8, A). Other bands of ca 50 kDa were recognized in the first leaf and the second leaf (Fig. 8B and 8C).

The antibody did not recognize any band of 42 kDa corresponding to the NMT in the resistant breeding line CI16145 (not shown). This line had been shown to lack the NMT-gene (K. Larsson, personal communication).

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Figure 7. Expression of the NMT protein in response to the infestation of barley var. Lina by R. padi (+) and in non-infested controls (-). Total proteins extracts (10 µg) were separated by SDS- PAGE and detected with antibodies raised against NMT after transfer onto a PVDF-membrane. A: the base. B: the primary leaf where aphids where sitting. C: the top of the second leaf. Numbers above panel indicate days after infestation. Purified NMT was used as positive control (C).

Barley var. Golf showed very low levels of NMT protein accumulation both for the controls and the infested seedlings as the detection time was prolonged twice comparing with barley var. Lina. For both varieties, the accumulation patterns were remained stable when we prolonged the exposing time. The aphid infestations did not induce NMT accumulation within the time period studied (from one day to 7 days; not shown for 7 days).

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When we compare the different parts of variety Golf and variety Lina, the bands of 42 kDa were strongest in the base and weakest in the first leaf (Fig.7A, 7B, 8A and 8B) and this was seen also with the gene expression (Fig. 5 and 6). The NMT was present in both the controls and the infested plants for both var. Lina and Golf showing that NMT is constitutively expressed in barley and not induced by aphid infestation.

Figure 8. Expression of the NMT protein in response to the infestation of barley var. Golf by R. padi (+) and non-infested controls (-). Total proteins extracts (10 µg) were separated by SDS-PAGE and detected with antibodies raised against NMT after transfer onto a PVDF-membrane. A: the base. B: the primary leaf where aphids where sitting. C: the top of the second leaf. Numbers above panel indicate days after infestation. Purified NMT was used as positive control (C).

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DISCUSSION

The bird cherry-oat aphid (Rhopalosiphum padi) like other aphids, inserts its stylet into phloem sieve elements and consumes phloem sap as its food source (Miles, 1999). In contrary to the bird cherry-oat aphid, the greenbug (Schizaphis graminum) causes necrotic spots (Burd, 2002) on crops such as sorghum and wheat, ultimately followed by general necrosis and plant death (Porter et al., 1997; Miles, 1999). In this study no symptoms were visible after 7 days infestation with bird cherry-oat aphids on the two susceptible lines Lina and Golf and the resistant line CI16145. Maybe more morphological symptoms could be seen if aphids were much longer time in contact with the leaves and especially with the susceptible lines. It has been proposed that phloem-feeding insects are perceived as pathogens due to similarities in the manner of penetration of plant tissues by fungal hyphae and aphid stylets (Forslund et al., 2000), and to some extent, by the similar hydrolytic enzymes released during fungal growth and insect feeding (Fidantsef et al, 1999; Walling, 2000). It may be caused by pectinases and other hydrolytic enzymes that via the aphid saliva may cause the release of oligosaccharide fragments which trigger defence reactions (Forslund et al., 2000).

Gene expression analyses of PR-17a

The expression of PR-17a was constitutive in barley non-infested plants. Christensen et al. (2002) found that in their study of the barley-Blumeria graminis f.sp hordei interaction in inoculated and non-inoculated leaves, the HvPR-17a antiserum strongly recognized a band as a 26 kDa protein in SDS-PAGE, but in non-inoculated leaves low levels of protein expression could be detected following prolonged development of the western blot. While there was an obvious up-regulation at 1 and 2 days after infestation in the resistant line CI16145, the variety Golf showed a down-regulation of the gene PR-17a. Variety Lina seemed less up-regulated than CI16145. These transcript accumulation patterns resembled those seen for PR17 when barley leaves were attacked by the powdery mildew fungus (Christensen et al., 2002). It also fits with studies on the expression pattern of several other pathogenesis related proteins in barley (Forslund, 2000), which also showed stronger up- regulation upon aphid infestation of both β-glucanase and chitinase in CI16145 than in Golf (Forslund, 2000). Immunoblot experiments of M. Kielkiewicz (unpublished) showed that PR-17a was constitutive in the first leaf (where the aphids were feeding) of both barley var. Lina and var. CI16145 and that the protein was more expressed upon aphid infestation (up-regulation) in the resistant line CI16145 than in the susceptible var. Lina. The PR-17a did not only accumulate locally in the primary leaf as seen in the case of the powdery mildew (Christensen et al., 1002), but the PR-17a was also induced systemically as shown in our case, in the base. These results are very important as it is the first time that PR-17a is shown to be up-regulated in barley and in systemic manner upon aphid infestation, and need to be confirmed.

Systemic acquired resistance is dependent on the accumulation of salicylic acid (van Loon and Strien, 1999). Salicylic acid was shown to be effective in barley for the induction of systemic resistance against the powdery mildew (Walters et al., 1993). Salicylic acid activates genes encoding pathogenesis-related proteins, which could include enzymes capable of degrading microbial cell walls, as chitinases, and other proteins that block microbial enzymes (Taiz and Zeiger, 2002). Furthermore, Muthukrishnan et al (2001) postulated that many of the PR proteins were encoded by more than one gene and because the PR proteins often acted co-

22 Naima Grönberg Master thesis ordinately, no studies have shown definitively that inactivation of a specific PR-protein results in enhanced susceptibility to a pathogen or insect. PR-17a gene was activated in both resistant (CI16145) and to lesser extent the susceptible variety Lina in response to aphid infestation. Aphids’ saliva contains various hydrolytic enzymes (Miles, 1999) that may function as elicitors (Zhu-Salzman et al., 2004), which allow the plant to perceive aphid invasion. This recognition resulted in induction of defence gene expression compared with the controls. The different responses in resistant and susceptible lines indicate that the induced proteins (PR-17a) may play a role in the resistance against aphid infestation. Although up-regulation of PR genes by several aphid species has been reported, no direct role has been established for PR-proteins in defense against phloem- feeding insects (Zhu-Salzman et al., 2004).

The N-methyltransferase

The accumulation patterns of NMT were investigated both at protein and RNA level in time- course experiments. As in previous studies, no clear regulation (either up- or down- regulation) of NMT expression could be observed at both RNA and protein level, except for the down-regulation seen on RNA level after 7 days infestation.

It was intended to include the resistant barley line 5175-50:20, which is resistant and has the NMT-gene, in our experiments but the seeds of this line gave after seven days very small seedlings that were not usable for our experiments. It would have been interesting to use a resistant barley variety that had the NMT gene in its genome to study how is the accumulation pattern and the gene expression of NMT behaving when infested by aphids; and to be able to do comparisons with the susceptible varieties.

During the immunoblotting experiments, other bands than those corresponding to the NMT, cross-reacted with the antiserum raised against NMT in chicken. The antiserum used was a polyclonal serum; therefore, the bands of 50 kDa and 100 kDa that were observed in Lina, CI16145 and Golf may result from the recognition of proteins related, but not identical to NMT (Christensen et al., 1998) by the antibodies directed against NMT, or may be derived from other unrelated antibodies present in the serum. This could be improved by affinity purification of the antiserum (L. Jonsson, oral communication).

Antibodies against NMT detected proteins that did not increase with time in infested leaves, either locally or systemically. The accumulation patterns of the proteins resembled those of the gene expression of NMT. The expression of the NMT-gene in response to aphid infestation was characterized by a late down-regulation; both variety Lina and variety Golf had the NMT-gene down-regulated after 7 days infestation. This down-regulation was only observed at RNA level, while in the immunodetection there was no difference between aphid- infested and control plants after 7 days infestation. This is in line with results from K. Larsson (personal communicaion) who observed that the NMT gene was down-regulated in barley varieties Lina and Golf after 5 days infestation when the aphids were sitting on the base of the seedlings and no clear conclusion could be drawn from the expression of NMT-gene at earlier time points in Lina when infested. The variation in expression could be caused by some abiotic disorders as light or water stress. K. Larsson (personal communication) showed that the NMT-enzyme activity suggested transient increase in Lina and Golf after 2 days but no difference was shown after 5 days infestation by aphids.

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As seen by our results, barley var. Golf had very low levels of NMT protein accumulation both for the controls and the infested seedlings when compared to var. Lina. The finding by Larsson et al. (2006) that var. Lina had high gramine content and var. Golf only trace amounts of gramine, indicates a relation between NMT protein amount and gramine amount. Gramine is a constitutive compound in many barley cultivars (H. vulgarae L.) and wild barley lines (Hanson et al., 1981). Matsuo et al. (2001) suggested that the constitutively produced gramine scarcely affects the infection but that only newly produced or transported gramine is able to inhibit the infection by a pathogen. Forslund et al. (1998) showed that a high gramine concentration did not affect the settling preference of R. padi. Gramine has long been in focus in its associated role in the resistance in barley (Matsuo et al., 2001; Velozo et al., 1999). In previous laboratory studies, R. padi was fed either artificial diets, plants with gramine added at varying concentrations, or plants with inherently different gramine contents (Zuñiga and Corcuera, 1986; Lohar, 1989). Results from those studies suggest that gramine acts as a feeding deterrent and can be toxic if ingested. Kanehisa et al. (1990) suggested that the aphid distribution pattern that they found in the fields might have been due to aphids moving away from high gramine accessions. However, Åhman et al. (2000) observed that high gramine concentrations do not confer resistance to R. padi. Based on gramine content, it was expected to find higher expression levels of NMT protein in Lina than in Golf. This was also observed in this study, where the protein band detected by the NMT antibodies was weaker in Golf as compared to Lina. The resistant barley line CI16145 does not have N-methyltransferase, which is in accordance with the observations of Larsson et al. (2006), that var. CI16145 does not have the NMT gene, and contains only trace amounts of gramine. Furthermore, both var. Lina and var. Golf showed a stronger expression of the protein NMT in the base than in the primary leaf; the same pattern was also seen when the gene was expressed. These results are in concordance with those of Lee et al. (1997b) who observed that the leaf sheath was the only tissue where the gene was expressed.

Conclusions

Primers for PR-17b did not work well in our experiments. New primers could be designed to get results even for this PR-protein. The results obtained with the PR-17a specific primers in RT-PCR were very interesting and it is the first time that it was shown that aphids induce PR-proteins systemically in barley seedlings after infestation by R. padi. These experiments should be done again to ascertain these results. The accumulation patterns of NMT at protein level changed with time neither locally nor systemically. The same pattern was seen at RNA level except that there was a late down- regulation after 7 days infestation. The time course experiment has to be performed and to study the regulation after 7 days infestation.

Acknowledgement

I am grateful to Professor in Botany and PhD Lisbeth Jonsson for letting me join her group to do my master theses. I would like to thank for all expertise during my work and for the experienced reflection and many discussions throughout this period. I want to thank Dr Gabriele Delp for all guidance, assistance and expertise during my work and for the critical reading of the manuscript.

24 Naima Grönberg Master thesis

I want to thank Therese Gradin for the introduction and support at the lab in the beginning of the project and for her kindness during all my projekt; to PhD student Kristina Larsson for her help with the northern blot; to Andrea Didon for her kindness and her happiness.

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