Identification and characterisation of barley (Hordeum vulgare)respiratory burst oxidase homologue family members

Damien J. LightfootA, Annette BoettcherA, Alan LittleA, Neil ShirleyB and Amanda J. AbleA,C

ASchool of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB1, Glen Osmond,SA 5064, Australia.

BAustralian Centre for Plant Functional Genomics, The University of Adelaide, Waite Campus, PMB 1,Glen Osmond, SA 5064, Australia.

CCorresponding author. Email: amanda.able@adelaide.edu.au

Abstract. Respiratoryburst oxidasehomologues (RBOHs) of thehumanphagocytegp91phoxgenehavebeen isolated fromseveral plant species and the proteins that they encode have been shown to play important roles in the cellular response tobiotic stress via theproductionof superoxide. In this studywehave identifiedandpreliminarily characterised sixRBOHs frombarley (HordeumvulgareL.). Conservation of the genomic structure and conceptual protein sequencewas observedbetweenall six barley RBOH genes when compared with Arabidopsis and rice RBOH gene family members. Four of the six barleyRBOH transcripts hadwide-spread constitutive spatial expression patterns. The inducible expression profiles ofHvRBOHF1and HvRBOHF2 in response to infection by the necrotrophic fungal pathogens Pyrenophora teres f. teres Drechsler andRhynchosporium secalis (Oudem) J. Davis were further characterised by quantitative real-time PCR (qPCR). Increasedexpression of both transcripts was observed in leaf epidermal tissue in response to infection, which is in keeping with asuggested role for both transcripts in the early oxidative burst during the plant response to pathogen invasion. This researchprovides a basis for further analysis and establishment of the roles of this RBOH family in various reactive oxygen speciesdependent processes in barley.

Additional keywords: NADPH oxidase, necrotroph, plant--pathogen interaction, reactive oxygen species.

Introduction

Reactive oxygen species (ROS) are involved in many importantcellular processes in both plants and animals (reviewed byMittleret al. 2004).At low levels,ROScan function in cell signalling thatmediates responses to stress, infection, programmed cell deathand developmental cues. At higher levels, ROS may damagecellular macromolecules, such as lipids, proteins and DNA.A localised oxidative burst, which is associated with the planthypersensitive response (HR), is produced by plants undergoingan incompatible plant--pathogen interaction and can lead to bothplant cell and pathogen death (reviewed by Torres and Dangl2005). Although this response may form part of a successfuldefence against invasion from biotrophic pathogens, whichrequire living cells for infection, it is ineffective in protectingthe plant against necrotrophic pathogens, which utilise theresources made available by cell death.

The major source of ROS in plants is the NADPH oxidase-catalysed conversion of dioxygen (O2) to the superoxide radical(O2

�), and its protonated form, the perhydroxyl radical (HO2*

),which is followed by subsequent reactions that lead to theproduction of hydroxyl radicals (

*

OH) and hydrogen peroxide(H2O2).MammalianNADPHoxidase, alsoknownasRespiratoryBurst Oxidase (RBO), has been well characterised for itsrole in superoxide production in phagocytes (reviewed byEl-Benna et al. 2005). The mammalian RBO enzyme complex

is composed of the membrane-bound flavocytochrome b558and cytosolic regulatory proteins p40phox, p47phox and p67phox

and either Rac1 (in monocytes) or Rac2 (in neutrophils). Theflavocytochrome b558 complex is a heterodimer composed ofmembrane-bound proteins gp91phox and p22phox. Afterphagocyte stimulation, the cytosolic components translocateto, and associate with, the membrane-bound components toactivate the NADPH-oxidase enzyme complex (reviewed byBedard and Krause 2007).

Plant homologues of the mammalian gp91phox respiratoryburst NADPH-oxidase subunit have been identified andpartially characterised in several plant species, includingOsRBOHA, OsRBOHB, OsRBOHD and OsRBOHE fromOryza sativa (rice) (Groom et al. 1996; Yoshie et al. 2005),AtRBOHA--AtRBOHJ from Arabidopsis thaliana (Keller et al.1998;Torres et al. 1998;Torres andDangl 2005),LeRBOH1 fromLycopersicon esculentum (tomato) (Amicucci et al. 1999),StRBOHA and StRBOHB from Solanum tuberosum (potato)(Yoshioka et al. 2001), NbRBOHA, NbRBOHB and NtRBOHDfromNicotiana spp. (tobacco) (Simon-Plas et al. 2002; Yoshiokaet al. 2003), andHvRBOHA frombarley (Trujillo et al. 2006). Themammalian gp91phox and corresponding plant respiratory burstoxidase homologues (RBOHs) share many structural andfunctional similarities, but differ by the inclusion of anextended N-terminal region in the plant RBOHs. This plant

CSIRO PUBLISHING

www.publish.csiro.au/journals/fpb Functional Plant Biology, 2008, 35, 347--359

� CSIRO 2008 10.1071/FP08109 1445-4408/08/050347

RBOH-specific region contains two EF-hand motifs that bindCa2+, suggesting that calcium ions play a role in regulating thefunction of these oxidases (Keller et al. 1998; Torres and Dangl2005).

The RBOH gene family is best characterised in Arabidopsisthaliana, where studies have shown distinct roles for severalRBOHproteins in processes such asROSproduction, theHR androot cell expansion. Torres et al. (2002) demonstrated thatAtRBOHD is responsible for most of the extracellular ROSproduced in response to avirulent pathogens, while AtRBOHFis themajorArabidopsisRBOH involved in regulation of theHR,potentially in a pathogen-dependant manner. AtRBOHD andAtRBOHF have also been shown to be involved in abscisicacid (ABA) signal transduction (Kwak et al. 2003). AnotherArabidopsis RBOH, AtRBOHC (also known as ROOT HAIRDEFECTIVE 2), has been shown to play a role in regulating cellexpansionduring root-hair formation, viaROSproduction,whichactivates hyperpolarisation-activated Ca2+ channels to facilitateCa2+ influx for growth processes (Foreman et al. 2003).

To date, one RBOH, HvRBOHA, has been identified andpartially characterised in barley (Trujillo et al. 2006). Trujilloet al. (2006) found higher levels of resistance to plant cellpenetration by the biotrophic Blumeria graminis (powderymildew fungus) in plants that had undergone RNA-interference-mediated gene silencing of HvRBOHA. Theauthors suggested that superoxide (and associated hydroxylradicals) produced by HvRBOHA in response to Blumeriagraminis infection could induce cell-wall softening, thereforesupportinghaustoria establishment in compatible reactions,whilealso sensitising cells for cell death in incompatible interactions(Trujillo et al. 2006).

In the present study we report the identification of six RBOHgenes from barley and the characterisation of their constitutiveexpression patterns. Based on their evolutionary relationshipswith other plant RBOHs, the differential expression of two ofthese RBOHs (HvRBOHF1 and HvRBOHF2) in response toinfection by the necrotrophic fungal pathogens Pyrenophorateres f. teres and Rhynchosporium secalis has also beenanalysed. This is the first study to report on the geneticinteractions between RBOHs and necrotrophic pathogens inbarley. Further understanding of the interactions between theRBOH-mediated oxidative burst and fungal pathogens couldallow for future modulation of barley resistance to bothbiotrophic and necrotrophic pathogens.

Materials and methodsPlant and fungal material

Barley (Hordeum vulgare L.) cultivars (cvv.) Sloop and Turkand the breeding line CI9214 were grown in growth roomsat a constant 18�C with a 12 h day/night regime.Rhynchosporium secalis isolates 6 and 332a andPyrenophora teres f. teres isolate NB50 (kindly provided bythe South Australian Research and Development Institute,Urrbrae, South Australia, Australia) were used to study theinteraction of barley with necrotrophic fungal pathogens.A compatible interaction occurs when cv. Turk is inoculatedwith R. secalis isolate 332a or when cv. Sloop is inoculated with

the P. teres isolate or R. secalis isolates 6 and 332a. Anincompatible interaction occurs when cv. Turk is inoculatedwith R. secalis isolate 6 or when breeding line CI9214 isinoculated with P. teres f. teres NB50. Fungal growthconditions, manipulations and inoculations were as previouslydescribed (Able 2003; Sarpeleh et al. 2007).

Nucleic acid isolationGenomic DNA was isolated by grinding tissue samples in liquidnitrogen and mixing with an equal volume of extraction buffercontaining 1% sarcosyl, 100mM Tris--Cl, 100mM NaCl, 10mM

EDTA and 1% polyvinylpolypyrrolidone followed by phenolchloroform extraction, precipitationwith 0.1 volumes 3 M sodiumacetate (pH 5.2) and 2 volumes 100% isopropanol andresuspension in an appropriate volume of sterile distilledwater. Total RNA was extracted using TRIZOL Reagent(Invitrogen, Carlsbad, CA, USA) as per the manufacturer’sinstructions.

Barley RBOH gene isolationThe initial barley RBOH sequences were obtained by PCRof genomic DNA from barley cv. Sloop using forward (50-CCCTTTGAATGGCATCCCTTCTCAAT-30) and reverse (50-CCATTCTTGGCATGGTTGAGAGCTTG-30) oligonucleotidesdesigned for the rice OsRBOHA sequence (accession numberX93301; Groom et al. 1996) and a partial barley EST (accessionnumber AJ251717; Hückelhoven et al. 2001). To obtain moresequence information, genomicDNA-walkingwith theUniversalGenomeWalker Kit (BD Biosciences Clontech, Palo Alto, CA,USA)wascarriedoutonadaptor-ligatedgenomicDNAfragmentsderived from 10-day-old Sloop seedlings following themanufacturer’s instructions. Using the sequence derived fromthe genomic-walking experiments, probes specific to thepresumptive 30-untranslated regions of the barley RBOHs weredesigned and used to screen a bacterial artificial chromosome(BAC) library constructed from the barley cv. Haruna Nijo(Saisho et al. 2007).

Sequence analysisSequences were analysed using Vector NTI version 10(Invitrogen), Genedoc (http://www.nrbsc.org/gfx/genedoc) andBiomanager from the Australian National Genomic InformationService (http://www.angis.org.au). Subcellular localisations ofthe RBOH proteins were predicted using PSORT (Nakai andKanehisa 1992) and WoLF PSORT (Horton et al. 2007).The sequences of the genes reported in this paper have beensubmitted to the GenBank database under accession numbersEU566852 (HvRBOHB1), EU566853 (HvRBOHB2),EU566854 (HvRBOHE), EU566855 (HvRBOHF1),EU566856 (HvRBOHF2) and EU566857 (HvRBOHJ).

Exon identification

Exons were defined by analysis of the genomic DNAsequences with FGENESH (http://www.softberry.com,accessed 5 September 2007) and GENSCANW (http://genscanw.biosino.org, accessed 3 September 2007) software.Where the results of the two programs were in disagreement

348 Functional Plant Biology D. J. Lightfoot et al.

the conflicted area was analysed for stop codons and TBLASTXsearches were carried out to identify similar sequences within theavailable databases. The outputs of these programs were checkedby obtainingmRNA sequences forHvRBOHF1 andHvRBOHF2by performing 50- and 30-rapid amplication of cDNA ends(RACE) on cDNA prepared from 10-day-old Sloop seedlingsusing the GeneRacer Kit (Invitrogen) as per the manufacturer’sinstructions. These sequences have been submitted to theGenBank database under the accession numbers EU566858(HvRBOHF1) and EU566859 (HvRBOHF2).

Reverse transcription PCR

RNA was extracted from leaf, root and coleoptile tissues from10-day-old Sloop seedlings and from flag leaf and immatureheads of 12-week-old Sloop plants. Reverse transcription PCR(RT--PCR) was conducted using the Superscript One-StepRT--PCR Kit (Invitrogen) as per the manufacturer’s instructions.Amplification of products corresponding to barley 18S rRNAwascarried out as an internal control. The oligonucleotides usedwere as follows: HvRBOHB1-forward (50-CCTGCCTTTCTATGGTCTGCGTCATC-30), HvRBOHB1-reverse (50-CATGTACAGGCGGCATCTGCAC-30), HvRBOHB2-forward (50-ATTGGCGAAGCGTCTTCAAGCG-30), HvRBOHB2-reverse (50-ACACAGGACTAGGCAAAACCACTCCC-30), HvRBOHE-forward (50-TGATGGGAAGAAGCATTGATTCTTCTG-30),HvRBOHE-reverse (50-CCAACTCTTTACCCTGCCTCTTTCCTT-30), HvRBOHF1-forward (50-TTACAACATGGACCTGCGTCCCTACA-30), HvRBOHF1-reverse (50-TGCCTTGGTCAGACACTCAGCTGCAT-30), HvRBOHF2-forward (50-TATGCGGAGTCCCGCAGAAAGATG-30), HvRBOHF2-reverse(50-TGTACTGTACTCCCCCTGCCTGTGT-30), HvRBOHJ-forward (50-CATTGAAAGCTAGTGTCACCAGCATGC-30),HvRBOHJ-reverse (50-GGCAGGCCTCGAAAGGATAGAGC-30), 18S-forward (50-CACGGGGAGGTAACAATAAATAACAA-30) and 18S-reverse (50-GGTTGAGACTAGGACGGTATCTGATCGT-30).

Evolutionary analysis

The full-length amino acid sequences of 32 plant RBOHs andhuman gp91phox were aligned with ClustalW (Thompson et al.1994) using the BLOSUM 30 protein weight matrix with a gap-opening penalty of 10 and a gap-extension penalty of 0.05.Protein distance trees were then constructed using theneighbour-joining method (Saitou and Nei 1987) inBiomanager using the Protdist, Neighbour, Seqboot, Consenseand Retree programs (Felsenstein 1989) with gp91phox set as theoutgroup. The accession numbers of the protein sequences used(except where indicated) are as follows: from Arabidopsisthaliana: AtRBOHA (NP_196356), AtRBOHB (NP_973799),AtRBOHC (AAS15724), AtRBOHD (NP_199602), AtRBOHE(AAC39478), AtRBOHF (NP_564821), AtRBOHG(NP_194239), AtRBOHH (NP_200809), AtRBOHI(NP_192862) and AtRBOHJ (NP_190167); from Hordeumvulgare: HvRBOHB1 (EU566852), HvRBOHB2 (EU566853),HvRBOHE (EU566854), HvRBOHF1 (EU566855),HvRBOHF2 (EU566856) and HvRBOHJ (EU566857); fromOryza sativa: OsRBOHA (NP_916447), OsRBOHB (DNA:

AK120739), OsRBOHD (DNA: AK120905), OsRBOHE(XP_482730), OsRBOH (AAT35117), OsRBOH(AAT58779), OsRBOH (ABA94089), OsRBOH (ABA99453)and OsRBOH (BAB89740); from Solanum tuberosum:StRBOHA (BAB70750) and StRBOHB (BAB70751); fromNicotiana benthamiana; NbRBOHA (BAC56864) andNbRBOHB (BAC56865); from Nicotiana tabacum:NtRBOHD (CAC84140) and NtRBOHF (CAC87256); fromLycopersicon esculentum: LeRBOH1 (AAD25300) and fromHomo sapiens: gp91phox (NP_000388).

Chromosome localisation of HvRBOHF1and HvRBOHF2Seven barley : wheat addition lines (Islam et al. 1981) werescreened to determine the chromosome location of HvRBOHF1and HvRBOHF2. Genomic DNA was extracted (as previouslydescribed) from young leaf tissue of plants (grown in growthroomsat a constant 18�Cwith a 12 h day/night regime) from eachline, as well as from the barley (cv. Betzes) positive control andthe wheat (cv. Chinese Spring) negative control. This genomicDNA was screened using PCR with oligonucleotides targetedto the 30-untranslated regions of HvRBOHF1 and HvRBOHF2.The HvRBOHF1-forward, HvRBOHF1-reverse, HvRBOHF2-forward and HvRBOHF2-reverse oligonucleotides were used asoutlined above.

Quantitative real-time PCRTotal RNA was extracted from barley leaf epidermal peels andcDNA was synthesised using the Superscript III First-StrandSynthesis System (Invitrogen) according to the manufacturer’sprotocol. The HvRBOHF1-forward, HvRBOHF1-reverse,HvRBOHF2-forward and HvRBOHF2-reverse oligonucleotideswere used as outlined above. The quantitative real-time PCR(qPCR) analysis was carried out essentially as previouslydescribed (Burton et al. 2004) using the H. vulgareglyceraldehyde-3-phosphate dehydrogenase (HvGAPDH),cyclophilin (HvCycl), elongation factor-1a (HvEF-1a) and heatshock protein 70 (HvHSP70) genes as controls. The primers usedwere as follows: HvGAPDH-forward (50-GTGAGGCTGGTGCTGATTACG-30), HvGAPDH-reverse (50-TGGTGCAGCTAGCATTTGAGAC-30), HvCycl-forward (50-CCTGTCGTGTCGTCGGTCTAAA-30), HvCycl-reverse (50-ACGCAGATCCAGCAGCCTAAAG-30),HvEF-1a-forward (50-GGTACCTCCCAGGCTGACTGT-30), HvEF-1a-reverse (50-GTGGTGGCGTCCATCTTGTTA-30), HvHSP70-forward (50-CGACCAGGGCAACCGCACCAC-30) and HvHSP70-reverse (50-ACGGTGTTGATGGGGTTCATG-30). For each plant--pathogen interaction,the qPCR values were compared with uninfected controls for eachtime point and expressed as relative levels of expression.

Results

Isolation and characterisation of six barley RBOH genes

Six genes with similarity to other plant RBOH genes wereidentified from barley cv. Haruna Nijo. One of the identifiedgenes is very similar to a previously described barleyRBOHgene,showing 99.9% nucleotide identity within the coding region,while the other five appear to represent novel barley genes. Based

Barley respiratory burst oxidase homologues Functional Plant Biology 349

on evolutionary relationships with the Arabidopsis RBOHs, thebarleyRBOH genes have been namedHvRBOHB1,HvRBOHB2,HvRBOHE, HvRBOHF1, HvRBOHF2 (HvRBOHA) (Trujilloet al. 2006) and HvRBOHJ.

The barley RBOH genomic clones ranged from 5379 bp to10 183 bp with predicted open reading frames of between 2532and 2961 bp encoding proteins between 843 and 986 amino acidswith predicted molecular masses between 95 and 110 kDa(Table 1). The HvRBOHF2 genomic clone is very similar toHvRBOHA (Trujillo et al. 2006), showing 99.9% nucleotideidentity within the coding region and 99.7% amino acididentity. The differences may result from sequence variationbetween cultivars with HvRBOHA identified from the cv.Ingrid and HvRBOHF2 identified from the cv. Haruna Nijo.Comparison of the barley RBOH conceptual proteins withother plant RBOHs and human gp91phox allows for theidentification of several conserved features (Fig. 1). TheN-terminal regions of the six conceptual proteins arehydrophilic and contain two putative EF-hands, which arepredicted to bind Ca2+ in other RBOH proteins (Keller et al.1998; Torres et al. 1998; Yoshie et al. 2005). The barley RBOHproteins are predicted to contain six transmembrane domains thatcorrespond to those identified in plant RBOHs from Arabidopsisand rice, as well as in gp91phox (Keller et al. 1998; Yoshie et al.2005). The third and fifth transmembrane domains contain pairsof histidine residues that are required in gp91phox for hemebinding during the process of electron transfer across the cellmembrane (Finegold et al. 1996). The C-terminal regions of theconceptual proteins contain conserved binding sites for flavinadenine dinucleotide, NADPH-ribose and NADPH-adenine(Yoshida et al. 1998; Vignais 2002). The gp91phox residuesPro-415 and Asp-500, which are essential for catalytic activity(Segal et al. 1992), are also conserved in the barley RBOHs(Fig. 1).

The six barley RBOHs show between 49% and 91%identity with each other at the amino acid level, with theHvRBOHF1 and HvRBOHF2 and the HvRBOHB1 andHvRBOHB2 pairs being the most similar with 91% and 82%identity, respectively. Most of the barley RBOH conceptual

proteins showed higher sequence identity to members of theArabidopsis and rice RBOH protein families than to each other.The most similar pair-wise comparison between Arabidopsisand the barley RBOHs was between AtRBOHF (Torreset al. 1998) and HvRBOHF1 and HvRBOHF2, with 70%and 68% identity, respectively. HvRBOHF1 and HvRBOHF2were also the most similar of the barley RBOHs to OsRBOHA(Groom et al. 1996), showing 89% and 84% identity,respectively.

Analysis of intron/exon composition

The similarity of the barley RBOH genes to the previouslydescribed Arabidopsis and rice RBOH genes is furtherreflected in the intron/exon structure (Fig. 2). Withintheir coding regions AtRBOHF, OsRBOHA, HvRBOHF1 andHvRBOHJ contain 14 exons, while HvRBOHE and HvRBOHF2contain 13 exons and HvRBOHB1 and HvRBOHB2 contain12 exons (Fig. 2A). The reduction in exon number in severalof the barley genes appears to result from the loss of genomicintronic regions leading to longer individual exons, rather thanfrom the loss of genomic exonic regions. The order andapproximate size of exons among the barley RBOHs is wellconserved, while intron size is more variable, particularly atthe 50 end. Spacing between the third and fourth as well asbetween the fourth and fifth exons is particularly variableamong the barley RBOH genes ranging between 89 bp and774 bp for intron three and between 69 bp and 1769 bp forintron four.

Within the coding regions of barley, Arabidopsis and riceRBOH genes there are 14 possible intron locations (Fig. 2B), aswas suggested for the Arabidopsis RBOHs (Torres et al. 1998).The location of these intron positions, relative to the proteinsequence, is conserved across the three plant species (Fig. 1).Intron 9 is the least common intron within this set of genesequences, identified in nine of the 17 genes analysed, withthree of the six barley genes containing this intronic sequence(Fig. 2B). Introns 5, 11, 12 and 13 were also absent from severalgenes,with intron5missing from four genomic sequences and the

Fig. 1. Protein alignment of AtRBOHF, OsRBOHA and the six conceptual barley respiratory burst oxidase homologue (RBOH) proteins. The dark shadingindicates residues that are identical or chemically conserved across all sequences and the lighter shading represents positions where there is a lower level ofconservation. Putative transmembrane domains (TM), EF-hands (EF) and conserved binding sites for flavin adenine dinucleotide (FAD), NADPH-ribose andNADPH-adenine are indicatedwithbrackets below the alignment.Histidine residues involved in hemebindingand thegp91phox residuesPro-415andAsp-500areindicated with asterisks below the alignment. Intron positions (1--14) are indicated with labelled arrows above the alignment.

Table 1. Details of the six respiratory burst oxidase homologue (RBOH) genes isolated from Hordeum vulgare cv. Haruna NijoThe length of each genomic clone, its accession number, predicted open reading frame, protein size (amino acid number) and predicted

molecular mass are shown

Genomic clone (accession number) Length of gDNA (bp) Open reading frame (bp)A Predicted protein size (kDa)

HvRBOHB1 (EU566852) 8026 2538 (845) 95HvRBOHB2 (EU566853) 8630 2718 (905) 102HvRBOHE (EU566854) 10183 2961 (986) 110HvRBOHF1 (EU566855) 8376 2841 (946) 107HvRBOHF2 (EU566856) 7938 2892 (963) 108HvRBOHJ (EU566857) 5379 2532 (843) 95

AAmino acid number is shown in parentheses.

350 Functional Plant Biology D. J. Lightfoot et al.

Barley respiratory burst oxidase homologues Functional Plant Biology 351

others missing from seven. Introns 3, 7 and 10 were absent fromtwo genes each, while introns 1 and 8 were absent from one geneeach. Introns 2, 4, 6 and 14 were identified within all of theplant RBOH genes characterised, with the exception of intron14 within the AtRBOHH gene, which encoded a protein thattruncated before the genomic location of this intron. Thereare several similarities among the set of plant RBOHgenes shown in Fig. 2, most notably five Arabidopsis genes(AtRBOHA--D and AtRBOHG) and two barley genes(HvRBOHB1 and HvRBOHB2) lack introns 11, 12 and 13,while four Arabidopsis genes (AtRBOHD--F and AtRBOHI),three barley genes (HvRBOHE, HvRBOHF1 and HvRBOHF2)and one rice gene (OsRBOHA) lack intron 9. In addition, fourgenes lack intron 5 (AtRBOHD, AtRBOHH, AtRBOHJ andHvRBOHJ), two genes lack intron 7 (AtRBOHC andAtRBOHG) and two genes lack intron 10 (AtRBOHDand HvRBOHE). The intron/exon organisation of the barleyRBOHs is similar to that of the Arabidopsis RBOHs, withintrons 9, 11, 12 and 13 missing from multiple genes in bothspecies. An exception to this similarity is intron 1, which wasonly absent from one barley gene and introns 3, 7 and 8, whichwere absent from Arabidopsis RBOH genes, but were identifiedin all barley RBOH genes.

Constitutive spatial expression patterns

The levels of constitutive expression of the six barley RBOHgenes in various barley tissues was initially investigated usingnorthern blot analysis (data not shown). However, the expressionlevels were too low for detection by this method, as has beenpreviously reported for RBOHs in other species (Groom et al.1996; Torres et al. 1998; Yoshioka et al. 2003; Trujillo et al.2006). Therefore,RT--PCRwasperformedonRNA isolated fromyoung leaf, flag leaf, immature head, root and coleoptile tissuesand revealed differential expression patterns for the sixbarley RBOHs (Fig. 3). HvRBOHB2, HvRBOHE, HvRBOHF1and HvRBOHF2 mRNA was detected in all tissues tested.HvRBOHF1 was detected strongly in root tissue, whileHvRBOHF2 was most abundant in young and flag leaf tissues.

HvRBOHB2 was highly abundant in head, root and coleoptiletissues with minor levels of transcript in young leaf and flag leaftissues. HvRBOHE mRNA was also highly expressed in head,root and coleoptile tissues; however, although the transcript levelsin young leaf and flag leaf tissues were reduced, HvRBOHEmRNAwas still abundant.HvRBOHB1wasdetected in head, rootand coleoptile tissues, with no transcript detected in young leafand flag leaf tissues. HvRBOHJ was not detected by RT--PCR inany of the tissues tested.However,when the sameprimer pairwas

HvRBOHF2HvRBOHF1

HvRBOHJ

HvRBOHE

HvRBOHB1HvRBOHB2

AtRBOHAAtRBOHBAtRBOHCAtRBOHDAtRBOHEAtRBOHFAtRBOHGAtRBOHHAtRBOHIAtRBOHJ

01 02 03 04 05 06 07 08 09 10 1 12 1413

HvRBOHF2HvRBOHF1

HvRBOHJ

HvRBOHE

HvRBOHB1HvRBOHB2

AAtRBOHBAtRBOHCAtRBOHDAtRBOHEAtRBOHFAtRBOHGAtRBOHHAtRBOHIAtRBOHJ

HvRBOHF2HvRBOHF1

HvRBOHJ

HvRBOHE

HvRBOHB1HvRBOHB2

AAtRBOHBAtRBOHCAtRBOHDAtRBOHEAtRBOHFAtRBOHGAtRBOHHAtRBOHIAtRBOHJ

OsRBOHA

01 02 03 04 05 06 07 08 09 10 1 12 141301 02 03 04 05 06 07 08 09 10 11 12 1413(A) (B)

AtRBOHF

OsRBOHA

HvRBOHF2

HvRBOHF1

HvRBOHJ

HvRBOHE

HvRBOHB1

HvRBOHB2

1000 nt

1 32 4 65 12 15148 131179/10

Fig. 2. Intron/exon structures of Arabidopsis, rice and barley respiratory burst oxidase homologue (RBOH) genes. In (A), the exons are represented by boxesand the introns are represented by thin lines. The regions where introns are absent from individual genes are indicated with brackets above the ‘joined’ exonicregions. In (B), the intronspresent in theArabidopsis, rice andbarleyRBOHgenesarenumbered (andcorrespond to those indicated inFig. 1) andare indicatedbyavertical black line (adapted from Torres et al. 1998).

HvRBOHB1

HvRBOHB2

HvRBOHE

HvRBOHF1

HvRBOHF2

HvRBOHJ

18S RNA

Y HF R C

Fig. 3. The spatial expression patterns of the six barley respiratory burstoxidase homologue (RBOH) genes were determined by reverse transcriptionPCR (RT--PCR) of barley young leaf (Y), flag leaf (F), immature head (H),root (R) and coleoptile (C) tissues. Amplification of 18S rRNAwas used as aninternal control for the RT--PCR.

352 Functional Plant Biology D. J. Lightfoot et al.

used to amplify HvRBOHJ from genomic DNA a product wasobserved, indicating that HvRBOHJmRNA is either of very lowabundance or is not present in the tissues tested.

Phylogenetic analysis

The conceptual amino acid sequences of the barley RBOHproteins were compared with each other and with divergentRBOHs from Arabidopsis, rice, tobacco, tomato and potato toconstruct a phylogenetic tree to infer the evolutionaryrelationships among plant RBOHs (Fig. 4). The RBOH genefamily is best characterised in Arabidopsis, with 10 members ofthe family described to date. Most novel plant RBOHs are namedon the basis of their evolutionary relationships to theArabidopsismembers, AtRBOHA--J. As indicated in Fig. 4, the phylogeny of

plant RBOHs can currently be divided into six clades, namelyE, B, H/J, A/F, I andOther. As such, the barley RBOHs identifiedin this study have been named according to their phylogeneticgroupings with the Arabidopsis RBOH proteins.

The results of the phylogenetic analysis conducted in thepresent study are in agreement with previous evolutionaryanalyses of plant RBOHs (Yoshioka et al. 2001, 2003; Trujilloet al. 2006; Yamamizo et al. 2007). HvRBOHE grouped withAtRBOHE from Arabidopsis and with OsRBOHB andOsRBOHE from rice. HvRBOHB1 and HvRBOHB2 groupedwith AtRBOHB from Arabidopsis, with StRBOHB from potatoaswell aswith twouncharacterisedRBOHs from rice.HvRBOHJgrouped with AtRBOHH and AtRBOHJ from Arabidopsis aswell as with two unnamed RBOHs from rice. HvRBOHF1grouped most closely with rice OsRBOHA, while HvRBOHF2grouped most closely with rice OsRBOHD. HvRBOHF1 andHvRBOHF2also formedamonophyletic clade to the exclusionofother plant RBOHs with AtRBOHF from Arabidopsis,LeRBOH1 from tomato, NbRBOHA and NtRBOHF fromtobacco and StRBOHA from potato.

The highest level of divergence between RBOH proteins isfound within the plant-specific N-terminal region, whichoccupies approximately the first 300 amino acids. Todetermine to what degree the plant-specific N-terminal regioncontributes to the evolutionary relationships seen in Fig. 4,a phylogeny was constructed using conceptual proteinswithout the N-terminal extension (data not shown). No majordifferences were observed between the two phylogenies exceptfor aminor repositioning of theNbRBOHB/NtRBOHD subcladewithin the Other clade, indicating that the evolutionaryrelationships are robust and are not dependant on the highlydivergent N-terminal region.

Of particular interest to this study is the A/F clade, whosemembers have significant functional roles in ROS generationduring plant defence responses in Arabidopsis (Torres et al.2002), rice (Yoshie et al. 2005) and tobacco (Yoshiokaet al. 2003) and are required for promoting cell death duringthe HR in Arabidopsis (Torres et al. 2002) and rice (Yoshie et al.2005). The grouping of the proteins encoded byHvRBOHF1 andHvRBOHF2 within this clade, therefore, suggests that they mayalso play an important role in the plant immune response topathogen challenge and so became the focus of our studies.

Chromosome localisation of HvRBOHF1and HvRBOHF2

Owing to the close evolutionary relationship of HvRBOHF1 andHvRBOHF2 with members of the A/F clade, the chromosomelocations of the corresponding genes were investigated using aPCR-based analysis of barley : wheat addition lines (Fig. 5). Eachof the sevenbarley : wheat addition lines contains one of the sevenbarley chromosomes. The exception to this is the long arm ofchromosome 1, which is not contained within the barley : wheataddition line containing barley chromosome 1 because ofgenomic instability (Islam and Shepherd 1990). PCR usingprimers specific to the 30-untranslated region of HvRBOHF1and HvRBOHF2 gave a negative result for the wheat cv.Chinese Spring control and a positive result for the barley cv.Betzes control as expected (Fig. 5). A PCRproductwas amplified

A/F clade

I clade

B clade

Other clade

H/J clade

E clade

*

98

98

*

99

*

*

*

*

*

*

*

97

97

90

84

52

94

*

*

*

50

*

*

*

*

**

98

51

HvRBOHF1

OsRBOHA

HvRBOHF2

OsRBOHD

AtRBOHF

NtRBOHF

NbRBOHA

StRBOHA

LeRBOH1

AtRBOHI

HvRBOHB1

HvRBOHB2

OsAAT35117

OsABA99453

StRBOHB

AtRBOHB

AtRBOHA

AtRBOHC

AtRBOHG

NbRBOHB

NtRBOHD

AtRBOHD

OsABA94089

HvRBOHJ

OsBAB89740

OsAAT58779

AtRBOHH

AtRBOHJ

HvRBOHE

OsRBOHB

OsRBOHE

AtRBOHE

HsGP91phox

Fig. 4. Phylogenetic tree of the plant respiratory burst oxidase homologue(RBOH) protein family using protein distance methods. The proteins areidentified to the right of the alignment,with the taxonof origin indicatedby thefirst two letters of the protein name. The six major clades identified to date areindicated and labelled to the right of the tree. The length of the horizontal linesconnecting the sequences is proportional to the genetic distance between thesequences. The numbers next to the nodes indicate bootstrap values (%) from1000 pseudoreplicates and asterisk symbols indicate a 100% value at therelevant node.

Barley respiratory burst oxidase homologues Functional Plant Biology 353

with the HvRBOHF1-specific primers in the 3H barley : wheataddition line, suggesting that HvRBOHF1 is present on barleychromosome 3H. PCR analysis of the seven barley : wheat lineswithHvRBOHF2-specific primers did not give a positive result inany of the lines, but did give a strong positive result in the barley(cv. Betzes) control (Fig. 5). The PCR product amplified from thepositive control reaction was confirmed to be HvRBOHF2 bysubsequent cloning and sequencing (data not shown). AsHvRBOHF2 was not amplified from any of the barley : wheataddition lines, but was able to be amplified from the barleypositive control, the HvRBOHF2 gene is predicted to be on thelong arm of chromosome 1 (1HL).

HvRBOHF1 and HvRBOHF2 pathogen-inducedexpression

To further investigate the expression of the HvRBOHF1 andHvRBOHF2 genes and their potential roles in pathogen responsein barley, qPCR was used to measure the levels of inducibleexpression in leaf epidermal cells in response to infection by thefungal necrotrophic pathogens R. secalis and P. teres f. teres.Resistance toR. secalis, which is responsible for barley leaf scalddisease, is mediated by classical gene-for-gene resistance (Roheet al. 1995) and to analyse the interaction ofR. secaliswith barley,two different R. secalis isolates were used. Isolate 6 contains theNIP1 avirulence gene, and when inoculated onto a barley cv.containing the virulence-resistance gene Rrs1 (cv. Turk) anincompatible interaction occurs and no disease symptoms areobserved.When this isolate is inoculatedonto a susceptible barleycv., which does not contain the Rrs1 virulence-resistance gene(cv. Sloop), a compatible interaction occurs and symptomsdevelop. Isolate 332a of R. secalis does not contain the NIP1avirulence gene and therefore a compatible interactionbetween R. secalis and both cvv. Turk and Sloop is observed.TheP. teres f. teres isolate NB50, which is responsible for the netform of the net blotch disease, was also used in this study.Resistance to P. teres is conferred by several resistance genesvia a complex non-host response, which can be described on aquantitative scale (Williams et al. 2003; Grewal et al. 2007). TheNB50 isolate shows differential symptom severity on the barleycv. Sloop (susceptible) and the breeding line CI9214 (resistant),with the resistant interaction still resulting in minor symptomswithin 72 h and plant recovery is observed in 2 weeks (datanot shown).

All of the plant--pathogen interactions tested resulted in anincrease in transcript levels during the first 3--6 h post inoculation(hpi), with a return to near basal levels after 24 h (Fig. 6).

The incompatible and compatible interactions of barley withP. teres f. teres showed differences in the timing and level ofincrease of HvRBOHF1 and HvRBOHF2 relative expression(Fig. 6A,C). The incompatible interaction with CI9214resulted in an earlier peak of both HvRBOHF1 andHvRBOHF2 expression at 3 hpi relative to that seen during thecompatible interaction with Sloop at 6 hpi. The peak expressionlevel of HvRBOHF1 in the compatible interaction was 2.5-foldmore than the uninoculated control, but lower than the 4.4-folddifference observed in the incompatible interaction (Fig. 6A).Regardless of the interaction, the peak expression level forHvRBOHF2 was 3.3-fold more than the uninoculated control(Fig. 6C).

The timing of the peak relative expression levels during theR. secalis interactions did not vary, with the highest expressionlevels seen for all interactions at 6 hpi (Fig. 6B,D). Several trendswere observed, with all interactions between the cvv. Turk andSloop and isolate 6 yielding higher relative expression levels ofboth HvRBOHF1 and HvRBOHF2 than the correspondinginteractions with isolate 332a. In addition, the levels ofHvRBOHF1 and HvRBOHF2 were higher in the interactionsbetween cv. Sloop and both isolates, 6 and 332a, than theywere inthe corresponding interactions with cv. Turk. No difference wasobserved between the incompatible interaction between isolate 6and cv. Turk compared with the compatible interaction betweenisolate 332a and cv. Turk.

Discussion

Identification of barley RBOHs

This study has identified six barleyRBOH genes, comprising fivenovel barley sequences and one that closely corresponds to apreviously reported barley RBOH gene (Trujillo et al. 2006).A comparison of the predicted protein sequences with other plantRBOHs revealed several well-conserved sequences andstructural similarities that suggest that the novel barleygenomic sequences encode plant RBOH proteins. In additionto conserved sequence elementswithin theRBOHprotein family,the conceptual barley RBOH proteins as well as those fromArabidopsis and rice are all predicted to localise to the plasmamembrane, further suggesting that these barley proteins mayfunction as gp91phox homologues.

Chromosome localisation of HvRBOHF1and HvRBOHF2

The OsRBOHA gene has been mapped by Keller et al. (1998) tothe long arm of rice chromosome 1 near the NPR1 gene, whichcontrols plant resistance to a broad spectrum of pathogens via theonset of systemic acquired resistance (Cao et al. 1997).Comparative mapping has shown a high level of syntenybetween rice chromosome 1 and barley chromosome 3H(Smilde et al. 2001). HvRBOHF1, which is closely related toOsRBOHA, is predicted tobeencodedbyagene locatedonbarleychromosome 3H. Likewise, the genes encoding the closelyrelated barley HvRBOHF2 and rice OsRBOHD proteins arepredicted to be located on chromosome regions that have a

HvRBOHF1

HvRBOHF2

B CS

B/W

1HS 2H 3H 4H 5H 6H 7H

Fig. 5. Chromosome localisation of theHvRBOHF1 andHvRBOHF2 genesdetermined by PCR using barley : wheat addition lines. The PCRwas carriedout on the barley cv. Betzes (B), the wheat cv. Chinese Spring (CS) and sevenbarley : wheat (B/W) addition lines containing the barley chromosomes ‘1HS’(short arm only) and ‘2H--7H’ (entire barley chromosome).

354 Functional Plant Biology D. J. Lightfoot et al.

high level of synteny (Korzun andKünzel 1996) withOsRBOHDand have been mapped to the bottom of rice chromosome 5(International Rice Genome Sequencing Project 2005; Ohyanagiet al. 2006) and HvRBOHF2 is predicted to be localised on themacro-syntenous 1HL. The localisation of HvRBOHF1 andHvRBOHF2 to chromosomal regions syntenous to those fortheir suggested rice homologues further confirms theevolutionary relationships suggested in Fig. 4.

Constitutive spatial expression patterns

Several similarities exist between the spatial expression patternsof the barley RBOH genes and RBOH genes from other plantspecies. The AtRBOHF, HvRBOHF1 and HvRBOHF2 proteinsare closely related, and this is reflected by their grouping withinthe A/F clade of RBOHs. AtRBOHF is one of the most stronglyexpressed Arabidopsis RBOH genes in leaf tissue (Torres et al.1998), which correlates withHvRBOHF1 andHvRBOHF2 being

strongly expressed in barley leaf tissue. Torres et al. (1998) andSagi and Fluhr (2006) reported that AtRBOHF was expressed inall tissues tested, and the highest level of expressionwas observedin root tissue, which is similar to the expression pattern observedfor HvRBOHF1. In addition to similarities in expression amongmembersof theA/FcladeofRBOHgenes,BandEclademembersalso appear to have similar expression patterns. AtRBOHB isexpressed preferentially in the root tissue (Sagi and Fluhr 2006),similar to HvRBOHB1 and HvRBOHB2, which are expressedstrongly in root, head and coleoptile tissues. AtRBOHE isexpressed preferentially in root and seed tissues of Arabidopsis(Sagi and Fluhr 2006), while the highly related HvRBOHEis strongly expressed in root, head (containing the seeds) andcoleoptile tissues.HvRBOHJ transcripts were not detected in anyof the tissues tested, which is indicative of the expression patternof other H/J clade members, with AtRBOHH and AtRBOHJ onlydetected in pollen grains (Potocký et al. 2007). Although there islittle functional data available for plant RBOHs, members of

(A)

(C) (D)

(B)

–

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

0 3 6 9 12 15 18 21 24

Time (hpi)

Rel

ativ

e ex

pres

sion

HvRBOHF2 CI9214+NB50HvRBOHF2 Sloop+NB50

–

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

5.00

0 3 6 9 12 15 18 21 24Time (hpi) Time (hpi)

Rel

ativ

e ex

pres

sion

HvRBOHF1 CI9214+NB50HvRBOHF1 Sloop+NB50

–

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

0 3 6 9 12 15 18 21 24

Time (hpi)

Rel

ativ

e ex

pres

sion

HvRBOHF2 Sloop+6HvRBOHF2 Turk+6HvRBOHF2 Sloop+332aHvRBOHF2 Turk+332a

–

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

0 3 6 9 12 15 18 21 24

Rel

ativ

e ex

pres

sion

HvRBOHF1 Sloop+6HvRBOHF1 Turk+6HvRBOHF1 Sloop+332aHvRBOHF1 Turk+332a

Fig. 6. Expression levels ofHvRBOHF1 (A, B) andHvRBOHF2 (C,D) in the barley cvv. Sloop and Turk and the breeding line CI9214 inresponse to fungal infection by Pyrenophora teres f. teres (A, C) and Rhynchosporium secalis (B,D). The levels of expression (� s.d.) arerelative to the corresponding uninoculated plants, as measured by quantitative real-time PCR. The scale on the y-axis represents theexpression of the target transcript relative to the transcript level of the internal controls and the scale on the x-axis represents the hours postinoculation (hpi).

Barley respiratory burst oxidase homologues Functional Plant Biology 355

individual RBOH clades appear to share similar expressionpatterns. If mRNA expression can be correlated with proteinabundance and function, the evolutionary relatedness and similarmRNA expression patterns might suggest related or sharedin vivo functional roles across species for RBOHs from withinthe same clades.

Functional roles of the A/F clade members

Loss-of-function mutation or targeted suppression affecting A/Fclade RBOH genes have been shown to alter the responses ofplants topathogenattack (Torreset al. 2002;Yoshioka et al. 2003;Yoshie et al. 2005). Given the role of RBOHs inROSproduction,this altered response is likely to result from an impact onsignalling processes during responses to pathogen attack.Torres et al. (2002) demonstrated that AtRBOHF contributesto the levels of ROS produced during the incompatibleinteractions with the bacterial pathogen Pseudomonas syringaeand the Oomycete pathogen Hyaloperonospora parasitica. Inresponse to the avirulent H. parasitica isolate Emco5, atrbohfmutants showed increased levels of H2O2 and an enhanced HR,relative to wild-type Arabidopsis plants. The increased HR leadsto improved resistance toH. parasitica. However, in response tothe avirulent P. syringae isolate DC3000 (avrRpm1), atrbohfmutants display reduced H2O2 expression and a decreased HR,with reduced cell death around the site of infection (Torres et al.2002). Yoshie et al. (2005) demonstrated that RNAi knockdownof OsRBOHA in cultured rice cells eliminated early H2O2

expression and reduced HR-related cell death during theincompatible reaction with the bacterial pathogen Acidovoraxavenae. Yoshioka et al. (2003) showed that in transgenic plantswith reducedNbRBOHA levels there was noH2O2 accumulation.These plants also exhibited a suppressed HR and becamesusceptible to the usually avirulent potato Oomycete pathogenPhytophthora infestans.

A possible mechanism to explain the link between reducedRBOH expression and altered HR, via H2O2, has been suggestedby Delledonne et al. (2001). These authors showed that insoybean cell suspensions the pathogen-induced HR is onlytriggered in response to an appropriate balance between ROSandnitric oxide (NO

*

) and, furthermore, thatHR-related cell deathis activated only after interaction between NO

*

and H2O2.Alterations in the levels of ROS, such as in the RBOH mutantsdescribed here and elsewhere (Torres et al. 2002; Yoshioka et al.2003; Yoshie et al. 2005), may alter the ratio of NO

*

to otherROS. This can, in turn, alter the HR or HR-related cell deathsignals in response to pathogendetection and therefore change theimmunological responses of the plant to pathogen attack. Theroles of RBOH proteins, and the networks that they belong to, inthe production of ROS and in the subsequent induction of the HRand HR-related cell death are varied, as can be seen with thediffering responses of the atrbohf mutant to H. parasitica andP. syringae (Torres et al. 2002) and by the increased levels ofresistance by barley cells to penetration by the biotrophicBlumeria graminis in hvrboha (hvrbohf2) mutants (Trujilloet al. 2006). This suggests distinct molecular responses todifferent pathogens, which could depend on factors such aspathogen-specific strategies of infection and the molecularresponses of the host plants. Therefore, the expression of the

barley A/F clade members HvRBOHF1 and HvRBOHF2 duringthe responses of barley to necrotrophic pathogen attack was thefocus of this study.

HvRBOHF1 and HvRBOHF2 expression in responseto infection by necrotrophic fungi

When challenged with P. teres f. teres, the levels ofHvRBOHF1and HvRBOHF2 mRNA were elevated in both the compatible(with cv. Sloop) and incompatible (with CI9214) interactions.However, the expression peaked earlier in the incompatibleinteraction. Interestingly, the peak level of HvRBOHF2transcript is similar in both interactions, yet the HvRBOHF1transcript is twice as abundant at its peak in the incompatibleinteraction, suggesting that HvRBOHF1 may play a greater rolein ROS production, particularly in that interaction.

Aspreviouslydescribed, the incompatible interactionbetweenP. teres f. teres and CI9214 results in minor disease symptomswithin 72 h of inoculation and plant recovery within 2weeks. It ispossible that the delayed accumulation of HvRBOHF1 andHvRBOHF2 in the compatible reaction, compared with theincompatible reaction, is important in determining theresponse to P. teres f. teres challenge and might play a role inthe level of symptom severity. The resistant CI9214 variety maybe sensing and responding to the pathogen challenge fasterthan the susceptible cv. Sloop, as evidenced by the more rapidelevation ofHvRBOHF1 andHvRBOHF2mRNA, and activatingdefence mechanisms early enough to avoid irreversible diseasesymptoms.Given that theHR is notwitnessed in the incompatibleinteraction to P. teres (Able 2003), the role of HvRBOHF1 andHvRBOHF2 is likely to be in ROS production specifically forsignalling processes. ROS have been shown to function assecondary messengers that modulate many processes, such asthe activation of calcium-permeable channels (reviewed byMoriandSchroeder 2004), the activation ofG-proteins (Baxter-Burrellet al. 2002), the activationofphospholipid signalling (Rentelet al.2004) and the induction of cellular protectant genes (Levine et al.1994). In addition, ROS can induce hormone-signallingpathways, such as the ethylene (Thomma et al. 1999), methyl-jasmonate (Thomma et al. 2000) and salicylic acid (Murphy et al.2000) pathways, which have been implicated in plant defencefrom necrotroph attack.

In contrast to the differential timing of peak HvRBOHF1 andHvRBOHF2 expression in the incompatible and compatibleinteractions between barley and P. teres f. teres, the peaklevels of mRNA accumulation for both HvRBOHF1 andHvRBOHF2 in response to R. secalis occurred at 6 hpi,regardless of the type of interaction. This lack of differentialexpression therefore suggests that HvRBOHF1 and HvRBOHF2play no significant role in the gene-for-gene resistance at leastduring the first 24 hpi. However, the increased expressionsuggests that they still play a role in signalling processesduring pathogen attack.

The similarity in the abundance and temporal regulation ofHvRBOHF1 and HvRBOHF2 in response to pathogen infectionsuggests that the two genes may have overlapping or redundantfunctions. Evolutionarily they are very closely related to eachother and are both most closely related to other members of theA/FcladeofplantRBOHs.Within this clade,HvRBOHF1 ismost

356 Functional Plant Biology D. J. Lightfoot et al.

similar to OsRBOHA and HvRBOHF2 is most similar toOsRBOHD. Yoshie et al. (2005) demonstrated that OsRBOHAand OsRBOHD are both upregulated during the incompatiblereaction between rice and the bacterial pathogen Acidovoraxavenae (isolate N1141) by approximately eightfold andfourfold, respectively, at 6 hpi. As was the case withHvRBOHF1 in barley, OsRBOHA was more upregulated inrice in response to pathogen detection than the closely relatedHvRBOHF2 andOsRBOHD, respectively, further confirming thefunctional similarities suggested by the evolutionary analysis.

ROS are produced in at least one oxidative burst by a plantduring an incompatible reactionwith a pathogen and are involvedin signalling to initiate the HR against biotrophic pathogens(Doke 1983; Sutherland 1991). The timing and nature of thesebursts are variable betweendifferent plant--pathogen interactions.Yoshioka et al. (2001) demonstrated two ROS bursts in potatoafter elicitation with P. infestans. The first burst was rapid andoccurred at 1 hpi,while the secondburst,which occurred between6 and 9 hpi, was more sustained and greater in magnitude.StRBOHA was shown to be important for the first oxidativeburst, while StRBOHB was important for the second oxidativeburst. Likewise, Yoshie et al. (2005) reported that theincompatible interaction between A. avenae and rice wascharacterised by two distinct ROS bursts; OsRBOHA wasessential for the first burst between 0 and 3 hpi and OsRBOHEwas required for the secondburst at 6 hpi.An early oxidative bursthas previously been observed in both the compatible andincompatible interactions between barley and P. teres f. teres/R. secalis (Able 2003). However, a compatible-specific andlimited second oxidative burst at ~29 hpi was only observedfor R. secalis (Able 2003). The upregulation of HvRBOHF1and HvRBOHF2 reported here corresponds temporally withthe first oxidative burst in barley previously reported by Able(2003), further suggesting the key involvement of HvRBOHF1and HvRBOHF2 in the early barley responses to pathogeninfection. The genes identified to date that are involved in theearly oxidative burst (StRBOHA and OsRBOHA) are membersof theA/F clade, while the genes involved in the second oxidativeburst (StRBOHB and OsRBOHE) fall outside of the A/F clade.Here, we report that two barley RBOHs from the A/F clade areupregulated in response to P. teres f. teres infection much earlierand to a greater extent in the incompatible interaction comparedwith the compatible interaction. Additionally, we have identifiedfour other RBOH genes in the barley genome (HvRBOHB1,HvRBOHB2, HvRBOHE and HvRBOHJ) that are outside ofthe A/F clade, with the possibility of more being identified inthe future given the higher number of RBOH genes in other plantspecies. Further study is required to establish the expression ofthese barley RBOH genes during the early stages of interactionsbetween barley and necrotrophic pathogens and of all barleyRBOH genes during the later stages of these interactions.

Although this study reports on the response of RBOHmRNAlevels to pathogen infection it is important to remember thatthe abundance of the corresponding proteins as well asinteractions with other proteins are crucial to determining thefunctional responses to pathogen attack. RBOH proteins aredirectly stimulated by Ca2+, most likely via interactionwith the EF-hand calcium-binding domains in the N-terminalplant-specific region of the protein (Sagi and Fluhr 2001).

Cytosolic Ca2+ increases are observed before RBOH activationand ROS increases during elicitor-induced defence responses(Nürnberger and Scheel 2001; Zhao et al. 2005). Other signallingevents that precede the oxidative burst include the involvementof a plant homologue of the human Rac gene (Kawasaki et al.1999; Ono et al. 2001; Morel et al. 2004), salicylic acidproduction (Kauss and Jeblick 1995; Yoshioka et al. 2003),mitogen-activated protein kinase cascades (Yoshioka et al.2003; Yamamizo et al. 2007) and changes in proteinphosphorylation (Chandra and Low 1995; Kauss and Jeblick1995).

The work described in the present study characterises the roleof twoRBOH genes in the early production of ROS in response tonecrotrophic pathogen infection. The identification of six barleyRBOH genes and the analysis of their expression contributes tothe growing body of knowledge about plant homologues of themammalian gp91phox gene. The evolutionary analysis carried outhere further refines the relationships between RBOH familymembers and suggests possible functional roles for the barleyRBOHs. The quantitative analysis of the expression levels ofHvRBOHF1 andHvRBOHF2 represents the first report of RBOHgene activity in response to necrotrophic fungal infection andimplicates the involvement of HvRBOHF1 and HvRBOHF2 inthe early oxidative burst in barley. Future work using transgenicunderexpression and overexpression lines of key genes of theRBOH-mediated oxidative bursts will help further characterisethe involvement of RBOH gene products in plant reactions topathogen attack.

Acknowledgements

We thank Dr Rafiqul Islam (School of Agriculture, Food and Wine, TheUniversity of Adelaide) for providing access to the barley : wheat additionlines, Dr Kazuhiro Sato (Research Institute for Bioresources, OkayamaUniversity) for supplying the BAC library filters, Margaret Pallotta(Australian Centre for Plant Functional Genomics, The University ofAdelaide) for access to, and screening of, the barley BAC library filters,Dr Hugh Wallwork (South Australian Research and Development Institute)for providing the fungal isolates, Andrew Craig for providing theleaf epidermal peel cDNA and Dr Jason Able and Dr William Bovill(School of Agriculture, Food and Wine, The University of Adelaide) andDrCatherineMcLeod (Salk Institute) for reviewing themanuscript.Thisworkwas supported by the Molecular Plant Breeding Cooperative ResearchCentre and funded by the Grains Research and Development Corporation(Project No. CMB00006).

References

Able AJ (2003) Role of reactive oxygen species in the response of barleyto necrotrophic pathogens. Protoplasma 221, 137--143. doi: 10.1007/s00709-002-0064-1

Amicucci E, Gaschler K, Ward JM (1999) NADPH oxidase genes fromtomato (Lycopersicon esculentum) and curly-leaf pondweed(Potamogeton crispus). Plant Biology 1, 524--528. doi: 10.1111/j.1438-8677.1999.tb00778.x

Baxter-Burrell A, Yang Z, Springer PS, Bailey-Serres J (2002) RopGAP4-dependent Rop GTPase rheostat control of Arabidopsis oxygendeprivation tolerance. Science 296, 2026--2028. doi: 10.1126/science.1071505

Bedard K, Krause KH (2007) The NOX family of ROS-generating NADPHoxidases: physiology and pathophysiology. Physiological Reviews 87,245--313. doi: 10.1152/physrev.00044.2005

Barley respiratory burst oxidase homologues Functional Plant Biology 357

Burton RA, Shirley NJ, King BJ, Harvey AJ, Fincher GB (2004) The CesAgene family of barley. Quantitative analysis of transcripts reveals twogroups of co-expressed genes. Plant Physiology 134, 224--236.doi: 10.1104/pp.103.032904

Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The ArabidopsisNPR1 gene that controls systemic acquired resistance encodes a novelprotein containing ankyrin repeats. Cell 88, 57--63. doi: 10.1016/S0092-8674(00)81858-9

Chandra S, Low PS (1995) Role of phosphorylation in elicitation of theoxidative burst in cultured soybean cells. Proceedings of the NationalAcademy of Sciences of the United States of America 92, 4120--4123.doi: 10.1073/pnas.92.10.4120

Delledonne M, Zeier J, Marocco A, Lamb C (2001) Signal interactionsbetween nitric oxide and reactive oxygen intermediates in the planthypersensitive disease resistance response. Proceedings of the NationalAcademy of Sciences of the United States of America 98, 13454--13459.doi: 10.1073/pnas.231178298

Doke N (1983) Involvement of superoxide anion generation in thehypersensitive response of potato tuber tissues to infection with anincompatible race of Phytophthora infestans and to the hyphal wallcomponents. Physiological Plant Pathology 23, 345--357.

El-Benna J, Dang PM, Gougerot-Pocidalo MA, Elbim C (2005)Phagocyte NADPH oxidase: a multicomponent enzyme essential forhost defenses. Archivum Immunologiae et Therapiae Experimentalis53, 199--206.

Felsenstein J (1989) PHYLIP -- Phylogeny inference package (version 3.2).Cladistics 5, 164--166.

Finegold AA, Shatwell KP, Segal AW, Klausner RD, Dancis A (1996)Intramembrane bis-heme motif for transmembrane electron transportconserved in a yeast iron reductase and the human NADPH oxidase.Journal of Biological Chemistry 271, 31021--31024. doi: 10.1074/jbc.271.49.31021

Foreman J, Demidchik V, Bothwell JH, Mylona P, Miedema H et al. (2003)Reactive oxygen species produced by NADPH oxidase regulateplant cell growth. Nature 422, 442--446. doi: 10.1038/nature01485

Grewal TS, Rossnagel BG, Pozniak CJ, Scoles GJ (2007) Mappingquantitative trait loci associated with barley net blotch resistance.Theoretical and Applied Genetics 116, 529--539. doi: 10.1007/s00122-007-0688-9

Groom QJ, Torres MA, Fordham-Skelton AP, Hammond-Kosack KE,Robinson NJ, Jones JD (1996) rbohA, a rice homologue of themammalian gp91phox respiratory burst oxidase gene. The Plant Journal10, 515--522. doi: 10.1046/j.1365-313X.1996.10030515.x

Horton P, Park KJ, Obayashi T, Fujita N, Harada H, Adams-Collier CJ,Nakai K (2007) WoLF PSORT: protein localization predictor. NucleicAcids Research 35, W585--W587. doi: 10.1093/nar/gkm259

Hückelhoven R, Dechert C, Trujillo M, Kogel KH (2001) Differentialexpression of putative cell death regulator genes in near-isogenic,resistant and susceptible barley lines during interaction with thepowdery mildew fungus. Plant Molecular Biology 47, 739--748.doi: 10.1023/A:1013635427949

International Rice Genome Sequencing Project (2005) The map-basedsequence of the rice genome. Nature 436, 793--800. doi: 10.1038/nature03895

Islam AKMR, Shepherd KW (1990) Incorporation of barley chromosomesinto wheat. In ‘Biotechnology in agriculture and forestry, Vol. 13Wheat’.(Ed. Y Bajaj) pp. 128--151. (Springer-Verlag: Berlin)

Islam AKMR, Shepherd KW, Sparrow DHB (1981) Isolation andcharacterization of euplasmic wheat--barley chromosome additionlines. Heredity 46, 161--174. doi: 10.1038/hdy.1981.24

Kauss H, Jeblick W (1995) Pretreatment of parsley suspension cultureswith salicylic acid enhances spontaneous and elicited production ofH2O2. Plant Physiology 108, 1171--1178.

Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H,Shimamoto K (1999) The small GTP-binding protein Rac is aregulator of cell death in plants. Proceedings of the National Academyof Sciences of the United States of America 96, 10922--10926.doi: 10.1073/pnas.96.19.10922

Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C (1998)Aplant homologof the neutrophilNADPHoxidase gp91phox subunit geneencodes a plasma membrane protein with Ca2+ binding motifs. The PlantCell 10, 255--266. doi: 10.2307/3870703

KorzunL,KünzelG (1996)Thephysical relationship of barley chromosome5(1H) to the linkage groups of rice chromosomes 5 and 10. Molecular &General Genetics 253, 225--231. doi: 10.1007/s004380050316

Kwak JM,Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE,Bodde S, Jones JD, Schroeder JI (2003) NADPH oxidase AtrbohD andAtrbohF genes function in ROS-dependent ABA signaling inArabidopsis. The EMBO Journal 22, 2623--2633. doi: 10.1093/emboj/cdg277

Levine A, Tenhaken R, Dixon R, Lamb C (1994) H2O2 from the oxidativeburst orchestrates the plant hypersensitive disease resistance response.Cell 79, 583--593. doi: 10.1016/0092-8674(94)90544-4

Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactiveoxygen gene network of plants. Trends in Plant Science 9, 490--498.doi: 10.1016/j.tplants.2004.08.009

Morel J, Fromentin J, Blein JP, Simon-Plas F, Elmayan T (2004) Racregulation of NtrbohD, the oxidase responsible for the oxidative burstin elicited tobacco cell. The Plant Journal 37, 282--293.

Mori IC, Schroeder JI (2004)Reactive oxygen species activationof plantCa2+

channels. A signalingmechanism in polar growth, hormone transduction,stress signaling, and hypothetically mechanotransduction. PlantPhysiology 135, 702--708. doi: 10.1104/pp.104.042069

Murphy AM, Holcombe LJ, Carr JP (2000) Characteristics of salicylic acid-induced delay in disease caused by a necrotrophic fungal pathogen intobacco. Physiological and Molecular Plant Pathology 57, 47--54.doi: 10.1006/pmpp.2000.0280

Nakai K, Kanehisa M (1992) A knowledge base for predicting proteinlocalization sites in eukaryotic cells. Genomics 14, 897--911.doi: 10.1016/S0888-7543(05)80111-9

Nürnberger T, Scheel D (2001) Signal transmission in the plant immuneresponse. Trends in Plant Science 6, 372--379. doi: 10.1016/S1360-1385(01)02019-2

OhyanagiH,TanakaT, SakaiH,ShigemotoY,YamaguchiK et al. (2006)TheRice Annotation Project Database (RAP-DB): hub for Oryza sativa ssp.japonica genome information. Nucleic Acids Research 34, D741--D744.doi: 10.1093/nar/gkj094

Ono E, Wong HL, Kawasaki T, Hasegawa M, Kodama O, Shimamoto K(2001) Essential role of the small GTPaseRac in disease resistance of rice.Proceedings of the National Academy of Sciences of the United Statesof America 98, 759--764. doi: 10.1073/pnas.021273498

Potocký M, Jones MA, Bezvoda R, Smirnoff N, Žárský V (2007) Reactiveoxygen species produced by NADPH oxidase are involved in pollentube growth. The New Phytologist 174, 742--751. doi: 10.1111/j.1469-8137.2007.02042.x

RentelMC,LecourieuxD,OuakedF,Usher SL, PetersenL et al. (2004)OXI1kinase is necessary for oxidativeburst-mediated signalling inArabidopsis.Nature 427, 858--861. doi: 10.1038/nature02353

Rohe M, Gierlich A, Hermann H, Hahn M, Schmidt B, Rosahl S, KnoggeW(1995) The race-specific elicitor, NIP1, from the barley pathogen,Rhynchosporium secalis, determines avirulence on host plants of theRrs1 resistance genotype. The EMBO Journal 14, 4168--4177.

Sagi M, Fluhr R (2001) Superoxide production by plant homologues of thegp91(phox) NADPH oxidase. Modulation of activity by calcium and bytobacco mosaic virus infection. Plant Physiology 126, 1281--1290.doi: 10.1104/pp.126.3.1281

358 Functional Plant Biology D. J. Lightfoot et al.

Sagi M, Fluhr R (2006) Production of reactive oxygen species by plantNADPH oxidases. Plant Physiology 141, 336--340. doi: 10.1104/pp.106.078089

SaishoD,MyorakuE,Kawasaki S, SatoK,TakedaK(2007)Construction andcharacterization of a bacterial artificial chromosome (BAC) library fromthe Japanese malting barley variety ‘Haruna Nijo’. Breeding Science 57,29--38. doi: 10.1270/jsbbs.57.29

Saitou N, Nei M (1987) The neighbor-joining method: a new method forreconstructing phylogenetic trees. Molecular Biology and Evolution 4,406--425.

Sarpeleh A, Wallwork H, Catcheside DEA, Tate ME, Able AJ (2007)Proteinaceous metabolites from Pyrenophora teres contribute tosymptom development of barley net blotch. Phytopathology 97,907--915. doi: 10.1094/PHYTO-97-8-0907

Segal AW, West I, Wientjes F, Nugent JH, Chavan AJ, Haley B, Garcia RC,Rosen H, Scrace G (1992) Cytochrome b245 is a flavocytochromecontaining FAD and the NADPH-binding site of the microbicidaloxidase of phagocytes. The Biochemical Journal 284, 781--788.

Simon-Plas F, Elmayan T, Blein JP (2002) The plasma membrane oxidaseNtrbohD is responsible for AOS production in elicited tobacco cells. ThePlant Journal 31, 137--147. doi: 10.1046/j.1365-313X.2002.01342.x

Smilde WD, Halukova J, Sasaki T, Graner A (2001) New evidence for thesynteny of rice chromosome 1 and barley chromosome 3H from riceexpressed sequence tags. Genome 44, 361--367. doi: 10.1139/gen-44-3-361

Sutherland MW (1991) The generation of oxygen radicals during host plantresponses to infection. Physiological and Molecular Plant Pathology 39,79--93. doi: 10.1016/0885-5765(91)90020-I

Thomma BP, Eggermont K, Tierens KF, Broekaert WF (1999) Requirementof functional ethylene-insensitive 2 gene for efficient resistance ofArabidopsis to infection by Botrytis cinerea. Plant Physiology 121,1093--1102. doi: 10.1104/pp.121.4.1093

Thomma BP, Eggermont K, Broekaert WF, Cammue BPA (2000) Diseasedevelopment of several fungi onArabidopsis can be reduced by treatmentwith methyl jasmonate. Plant Physiology and Biochemistry 38, 421--427.doi: 10.1016/S0981-9428(00)00756-7

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving thesensitivity of progressive multiple sequence alignment through sequenceweighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Research 22, 4673--4680. doi: 10.1093/nar/22.22.4673

Torres MA, Dangl JL (2005) Functions of the respiratory burst oxidase inbiotic interactions, abiotic stress and development. Current Opinion inPlant Biology 8, 397--403. doi: 10.1016/j.pbi.2005.05.014

Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE,Jones JDG (1998) Six Arabidopsis thaliana homologues of the humanrespiratory burst oxidase (gp91phox). The Plant Journal 14, 365--370.doi: 10.1046/j.1365-313X.1998.00136.x

Torres MA, Dangl JL, Jones JDG (2002) Arabidopsis gp91phox homologuesAtrbohD and AtrbohF are required for accumulation of reactive oxygenintermediates in the plant defense response. Proceedings of the NationalAcademy of Sciences of the United States of America 99, 517--522.doi: 10.1073/pnas.012452499

Trujillo M, Altschmied L, Schweizer P, Kogel KH, Hückelhoven R (2006)Respiratory burst oxidase homologue A of barley contributes topenetration by the powdery mildew fungus Blumeria graminis f. sp.hordei. Journal of Experimental Botany 57, 3781--3791. doi: 10.1093/jxb/erl191

Vignais PV (2002) The superoxide-generating NADPH oxidase: structuralaspects and activation mechanism. Cellular and Molecular Life Sciences59, 1428--1459. doi: 10.1007/s00018-002-8520-9

Williams KJ, Platz GJ, Barr AR, Cheong J, Willsmore K, Cakir M,Wallwork H (2003) A comparison of the genetics of seedling and adultplant resistance to the spot form of net blotch (Pyrenophora teres f.maculata). Australian Journal of Agricultural Research 54, 1387--1394.doi: 10.1071/AR03028

Yamamizo C, Doke N, Yoshioka H, Kawakita K (2007) Involvement ofmitogen-activated protein kinase in the induction of StrbohC andStrbohDgenes in response to pathogen signals in potato. Journal of General PlantPathology 73, 304--313. doi: 10.1007/s10327-007-0020-1

Yoshida LS, Saruta F, Yoshikawa K, Tatsuzawa O, Tsunawaki S (1998)Mutation at histidine 338of gp91phox depletes FADand affects expressionof cytochrome b558 of the human NADPH oxidase. Journal of BiologicalChemistry 273, 27879--27886. doi: 10.1074/jbc.273.43.27879

Yoshie Y, Goto K, Takai R, Iwano M, Takayama S, Isogai A, Che F (2005)Function of the rice gp91phox homologs OsrbohA and OsrbohE genes inROS-dependent plant immune responses. Plant Biotechnology Journal22, 127--135.

Yoshioka H, Sugie K, Park HJ, Maeda H, Tsuda N, Kawakita K, Doke N(2001) Induction of plant gp91phox homolog by fungal cell wall,arachidonic acid, and salicylic acid in potato. MolecularPlant--Microbe Interactions 14, 725--736. doi: 10.1094/MPMI.2001.14.6.725

Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O,Jones JDG, Doke N (2003) Nicotiana benthamiana gp91phox homologsNbrbohA and NbrbohB participate in H2O2 accumulation and resistanceto Phytophthora infestans. The Plant Cell 15, 706--718. doi: 10.1105/tpc.008680

Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading toproduction of plant secondary metabolites. Biotechnology Advances 23,283--333. doi: 10.1016/j.biotechadv.2005.01.003

Manuscript received 4 April 2008, accepted 29 May 2008

Barley respiratory burst oxidase homologues Functional Plant Biology 359

http://www.publish.csiro.au/journals/fpb