Regulation of reactive oxygen species production by a14-3-3 protein in elicited tobacco cells*

TALINE ELMAYAN1†, JÉRÔME FROMENTIN1, CHRISTOPHE RIONDET2‡,GÉRARD ALCARAZ2, JEAN-PIERRE BLEIN1 & FRANÇOISE SIMON-PLAS1

1Unité Mixte de Recherche Plante-Microbe-Environnement INRA 1088/CNRS 5184/Université de Bourgogne, BP 86510,21065 Dijon Cedex and 2UPSP PROXISS, ENESAD, 26 Bd Petitjean, 21079 Dijon Cedex, France

ABSTRACT

The regulation of the system responsible for the productionof reactive oxygen species (ROS) during plant–micro-organism interaction is still largely unknown. The proteinNtrbohD has been recently demonstrated as the plasmamembrane oxidase responsible for ROS production in elic-ited tobacco cells. Here, its C-terminus part was used as abait in a two-hybrid screen in order to identify putativeregulators of this system. This led to the isolation of acDNA coding for a member of the 14-3-3 protein family.The corresponding transcript was induced after infiltrationof tobacco leaves with the fungal elicitor cryptogein.Tobacco cells transformed with an antisense construct ofthis 14-3-3 no longer accumulated ROS, which constitutes afunctional validation of the two-hybrid screen. This workprovides new insights to the understanding of the regulationof ROS production in a signalling context and gives a newlight to the possible role of 14-3-3 proteins in plant–micro-organisms interactions.

Key-words: cryptogein; NADPH oxidase; signalling.

INTRODUCTION

Plants are exposed to a great number of pathogenic micro-organisms, although only a relatively small proportion ofthem are able to cause diseases. Indeed, plants defendthemselves against pathogens by triggering a wide range ofmechanisms, including the rapid and intense accumulationof reactive oxygen species (ROS), mainly superoxide anion(O2

-·) and hydrogen peroxide (H2O2). The generation ofthese ROS has been long believed to contribute to severaldisease resistance strategies (for reviews, see Bolwell &

Wojtaszek 1997; Lamb & Dixon 1997; Wojtaszek 1997;Bolwell 1999). More recently, the complicated role of theseROS in the regulation of the cell death accompanying thehypersensitive response (HR) and the establishment of thesystemic acquired resistance has been underlined (Apel &Hirt 2004; Torres, Jones & Dangl 2005, 2006). Because ofthe key role played by these ROS in the orchestration ofplant defence, the mechanism of their biosynthesis has beenextensively studied using whole plants or suspension-cultured cells treated with various elicitor molecules. Cellwall peroxidases and oxalate oxidases have been proved tobe responsible for apoplastic ROS production (Apel & Hirt2004; Mittler et al. 2004). But studies performed on differentplant species indicated that enzymes similar to the respira-tory burst oxidase of mammalian neutrophil cells act as themain ROS-producing system during plant–micro-organisminteraction (Desikan et al. 1996; Groom et al. 1996; Kelleret al. 1998; Torres et al. 1998; Amicucci, Gaschler & Ward1999; Simon-Plas, Elmayan & Blein 2002; Yoshioka et al.2003).

The mammalian NADPH oxidase is a proteic complexcomposed of a membrane-bound heteroflavocytochromeb558 and cytosolic regulatory proteins (p47phox, p67phox,p40phox and the small GTP-binding protein Rac2) (Babior2004; Cross & Segal 2004). The flavocytochrome is formedof a transmembrane protein, gp91phox, which contains theNADPH binding site and the electron transport chainleading to the reduction of molecular oxygen into super-oxide anion, and of the p22phox subunit. The plant oxidasehas been so far described as a single protein which exhibitssome characteristic features of gp91phox, that is, the numberand position of the putative membrane spanning helixes,part of the C-terminus domain that contains the motifsresponsible for the binding of FAD and NADPH cofactors,and in particular the four histidines postulated to bind thetwo haemes (Davis et al. 1998), but also has a hydrophilicN-terminal extension with a Ca2+-binding motif, typical ofplant oxidases (Keller et al. 1998; Torres et al. 1998;Amicucci et al. 1999).

Published work of several groups first suggested a pos-sible similarity of regulation at the molecular level betweenplant and animal oxidases (Desikan et al. 1996; Xing,Higgins & Blumwald 1997). However, several lines of evi-dence may question the conclusions raised from these

Correspondence: F. Simon-Plas. Fax: +33 0 3 80 69 32 65; e-mail:simon@epoisses.inra.fr

*Upon request, all novel materials described in this publication willbe made available in a timely manner for non-commercial researchpurposes.†Present address: Laboratoire de Biologie Cellulaire INRA Routede Saint Cyr, 78026 Versailles Cedex, France.‡Present address: Laboratoire Génome et Développement desPlantes, UMR 5096 CNRS/Université de Perpignan, 52 Avenue deVilleneuve, 66860 Perpignan, France.

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studies: (1) to date, no evidence for sequences presentingsignificant homology with p22phox, p47phox or p67phox exists inthe entire Arabidopsis genome database, (2) attempts todetect clones of putative subunits of a plant NADPHoxidase by expression screening using antibodies directedagainst subunits of the neutrophil oxidase led to the isola-tion of unrelated proteins (Tenhaken & Rubel 1998; Kieffer& Elmayan, unpublished results), (3) the sites known inneutrophil gp91phox to interact with p47phox (indicated inFig. 1) are not conserved in plant oxidases and (4) the use ofp47phox and p67phox as baits in a heterologous two-hybridscreen with a tobacco cDNA library did not lead to theisolation of any tobacco clones able to interact with thesesubunits of neutrophil NADPH oxidase (Elmayan, unpub-lished results).

It has been demonstrated that plant rboh (respiratoryburst oxidase homolog) proteins are stimulated directly byCa2+, and can produce O2·- in the absence of additionalcytosolic components (Sagi & Fluhr 2001). Several studieshave also underlined the role of small GTP-binding pro-teins of the Rac family in the regulation of plant oxidases(Ono et al. 2001; Morel et al. 2004; Moeder, Yoshioka &Klessig 2005) and the involvement of the a-subunit of aheterotrimeric G protein upstream ROS production

(Suharsono et al. 2002). As each of these studies has beenfocused on one particular aspect of the regulation of rboh,and performed on different biological materials, the pictureof the global regulatory mechanism involved in the contextof plant defence is still unclear.

Cryptogein, is a protein secreted by the oomycete Phy-tophthora cryptogea, able to induce a hypersensitive-likeresponse and an acquired resistance in tobacco (Ricci1997). We identified a tobacco homolog of gp91phox,NtrbohD, responsible for ROS production triggered ontobacco cells by this elicitor (Simon-Plas et al. 2002) anddemonstrated that this oxidase was regulated by a small Gprotein of the Rac family, without direct interactionbetween the two proteins (Morel et al. 2004).

Here, in order to further characterize the regulation ofthis enzyme, we used a two-hybrid screen in yeast to findproteins able to specifically interact with NtrbohD. Theseexperiments led to the isolation of a cDNA encoding aprotein belonging to the family of 14-3-3 proteins. Theexpression of the corresponding mRNA was examined invarious tissues elicited or not with cryptogein, and theactual involvement of the protein in the regulation of ROSproduction was assessed using suspension cells transformedwith antisense constructs of this cDNA.

Figure 1. Part of the tobacco oxidase NtrbohD used as a bait in the two-hybrid screen. The upper part of the Figure is a schematicrepresentation of the structure of the tobacco oxidase NtrbohD with its hydrophilic N-terminus part comprising an EF-hand domain, sixtransmembrane helixes and the hydrophilic C-terminus part. The lower part of the Figure shows an alignment of the C-terminus parts ofthe proteic sequences of oxidases from Arabidopsis thaliana (AtrbohD), rice (OsrbohA), neutrophil cells (human gp91) and tobacco(NtrbohD). It corresponds to AA 602 to 939 for NtrbohD. The part of the C-terminus region of NtrbohD (AA 753 to 939) which wasused as a bait in the two-hybrid screen is indicated in bold. Regions underlined 1, 2 and 3 correspond to the sites of fixation of p47phox onthe human gp91phox oxidase.

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MATERIALS AND METHODS

Plant material

Tobacco plants (Nicotiana tabacum cv. Xanthi) and BY2cells (N. tabacum cv. Bright Yellow 2) were grown as previ-ously described (Milat et al. 1991; Simon-Plas et al. 1997).Cells were maintained by weekly dilution (2:80) into freshmedium. Cryptogein was purified and treatments were per-formed as previously described (Milat et al. 1991; Simon-Plas et al. 1997).

Two-hybrid cDNA library construction

RNA was extracted from young leaves (nearest to theapex) from 55-day-old tobacco plants (N. tabacum cv.Xanthi). cDNA library was prepared as described in Kiefferet al. (2000). Approximately 3 ¥ 106 colonies contain-ing the primary cDNAs were obtained, before libraryamplification.

Screening of the tobacco cDNA library usingthe two-hybrid system

In the system used here, one hybrid is a fusion between theLexA DNA-binding protein and one protein of interest, thetobacco NtrbohD (in pLex vector), and the other hybridis a fusion between the activation domain of Gal4 andproteins encoded by the tobacco cDNA library (inpGAD3S2X). Two hydrophilic C-terminal coding region ofNtrbohD (AA753 to AA939 and AA602 to 939) wereamplified by PCR before insertion in frame with the LexADNA-binding protein into the BamHI and SalI sites ofpLex10 vector. Transactivation of the HIS3 and lacZreporter genes occurs only if both proteins of interest inter-act when co-expressed in the appropriate Saccharomycescerevisiae strain. Interaction can therefore be monitored byb-galactosidase activity or by prototrophy for histidine. Thetwo-hybrid system used was obtained from Clontech (yeastMatchmaker kit; Mountain View, CA, USA). The L40 yeastreporter strain used has the genotype: MATa, trp1, leu2,his3, LYS2::lexA-HIS3, URA3::lexA-LacZ.Yeast was grownin synthetic minimal medium consisting of 2% glucose(w/v), 0.67% nitrogen base without amino acids (w/v)(Difco, San Jose, CA, USA) with the appropriate supple-ments: amino acids, uracil and adenine of cell culture grade(Sigma, St Louis, MO, USA).

To identify proteins interacting with the C-terminal ofNtrbohD, the S. cerevisiae L40 reporter strain containingLexA/NtrbohD fragment was transformed with the tobaccolibrary fused to the activation domain of Gal4 inpGAD3S2X vector. Approximately 5.106 transformantswere plated onto selective medium. His+ colonies appeared3 to 5 d after plating and were tested for b-galactosidaseactivity by the colour filter assay using the substrate5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (Xgal)as previously described (Kieffer et al. 2000). A fraction ofthe yeast plasmid extracted from 28 His+/LacZ+ clones was

used to electroporate HB101 Escherichia coli. The libraryplasmid was rescued by complementing the leu- phenotypeof HB101 on minimal medium. This plasmid was trans-formed again into either the L40 strain carrying LexA/NtrbohD to confirm interaction or an L40 strain carrying anunrelated hybrid protein, LexA/Lamin to test for false posi-tive clones. Only 13 His+/LacZ+ clones were specific forLexA/NtrbohD and led to the isolation of two cDNAsencoding a homolog to known proteins, Nti14-3-3(AJ309008), further renamed Nt14-3-3h/omega1 andNtP2C (AJ309007). L40 yeasts were co-transformed withthe pGAD3S2X vector containing the Nti14-3-3 cDNA infusion with the activation domain of Gal4 and each of thefollowing sequences inserted in the pLex vector in fusionwith the lexA binding protein: the tobacco small G proteinNtRac5, the neutrophil small G protein Rac2, the neutro-phil proteins p47phox and p67phox, the neutrophil proteinRho-GDI, a human lamin and the yeast proteins SUT1(involved in sterol uptake), and ERG 19 (a cytosolicenzyme involved in sterol biosynthesis). Yeast were thenplated on a medium without leucine and tryptophan (DO-II) selective for the efficacy of the transformation or on amedium without leucine tryptophan and histidine (DO-III)selective for the interaction between the proteins whosesequences are inserted in the plasmids.The 786 bp fragmentof Nt14-3-3h/omega1 was used as a probe to screen atobacco cDNA library made from tobacco leaves elicitedwith Pseudomonas solanacearum (Czernic, Huang &Marco 1996) in the bacteriophage lambda zap Express(Stratagene, West Cedar Creek, TX, USA). The positiveclones inserted into pBK-CMV phagemid after excisionshared the same nucleotide sequence and the largest cloneof 1090 bp (AJ309008) encodes a full length protein of 258amino acids.

DNA sequencing and analysis

Both strands of cDNA were sequenced by the GenomeExpress corporation (Meylan, France). DNA sequence datawere analysed using the GCG package (University of Wis-consin Genetics Computer Group, Madison,WI, USA), andthe deduced amino acid sequences were compared withsequences in the current databases (GenBank, SwissProtein, EMBL . . .) using the Blast network service of theNational Center for Biotechnology Information (NCBI,Bethesda, MD, USA), and using the programs Bestfit, Clust-alw and Edtaln (for alignment and consensus).

RNA isolation and blot hybridization

Total RNA from BY2 tobacco cells and from differenttissues of mature tobacco plants and seedlings wasextracted and purified, followed by a CsCl gradient accord-ing to Sambrook, Fritsch & Maniatis (1989). Samples oftotal RNA (10 mg) were analysed by electrophoresis in8% formaldehyde, 1.5% agarose gels, and transferred toPall biodyne B membranes according to the manufac-turer’s instructions. A NotI fragment of 0.8 kb bearing the

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Nt14-3-3h/omega1 coding region (fragments purified fromthe pGAD3S2X vector) was labelled to 108 c.p.m. mg-1 usingrandom priming and 32P-dCTP (Amersham, Piscataway, NJ,USA). Labelling, hybridization and washes were performedessentially as described by Sambrook et al. (1989).

A single-stranded RNA probe (labelled with 32P-CTP)corresponding to antisense transcripts of Nt14-3-3h/omega1was obtained by transcription in vitro using the kit Ribo-probe (Promega, Madison, WI, USA). Nt14-3-3h/omega1cDNA was cloned into pGEMT-easy vector, allowing syn-thesis of antisense transcripts derived from the insertsequence only, using SP6 RNA polymerase promoters afterlinearization of the plasmid by NcoI.

Antisense gene construction andcell transformation

A SalI fragment of 786 bp from Nt14-3-3h/omega1 clonewas inserted in antisense into pKYLX71-35S2, a plant trans-formation vector allowing inserts to be cloned between a35S promoter with a duplicated enhancer and a rbcS 3′UTR (Elmayan & Tepfer 1994).

The resulting plasmid, introduced by triparental matinginto a disarmed strain of Agrobacterium tumefaciens,C58C1 (pMP90), was used to transform BY2 cells. Twomillilitres of a 3-day-old BY2 culture was co-cultivated with50 mL of each Agrobacterium culture (OD600 0.3) in Petridishes in the dark for 2–3 d at 26 °C. Cells were washedthree times by centrifugation in 10 mL of fresh medium at50 g, 3 min, and plated onto agar-MS medium containing100 mg/L kanamycin and 500 mg/L cefotaxime. Trans-formed micro-calli were propagated during four to five sub-cultures in 10 mL MS liquid medium containing theselection agent before maintaining by weekly dilution of2 mL in 80 mL of MS liquid medium.

ROS determination

Cells were harvested 6 d after subculture, filtered, resus-pended (1 g for 10 mL) in a 2 mM MES buffer pH 5.90,containing 175 nmM mannitol, 0.5 mM CaCl2 and 0.5 mMK2SO4.After 3 h equilibration on a rotary shaker (150 rpm)at 25 °C, cells were treated with cryptogein and/or chemi-cals as indicated in the legend of the Figures. The accumu-lation of ROS was determined by chemiluminescence usingluminol and a luminometer (LUMAT, LB 9501; BertholdTechnologies, Bad Wildbad, Germany). Every 10 min, a250 mL aliquot of the cell suspension was added to 50 mL of0.3 mM luminol and 300 mL of the assay buffer (175 mMmannitol, 0.5 mM CaCl2, 0.5 mM K2SO4 and 50 mM MESpH 6.5).

RESULTS

In search for proteins regulating NtrbohD:screening of a tobacco cDNA library usingNtrbohD as bait in the two-hybrid system

The hydrophilic C-terminal part of NtrbohD, was used asbait for two hybrid screen, as this method is not suitable

for the study of integral membrane proteins exhibitinghydrophobic transmembrane segments. This part of theprotein is indeed known to contain the sites of interactionwith the cytosolic components in the neutrophil gp91phox

(Heyworth, Curnutte & Badwey 1999). In the system usedhere,one hybrid is a fusion between the LexA DNA-bindingprotein and the hydrophilic C-terminal coding region ofNtrbohD (AA753 toAA939,Fig. 1) and the other hybrid is afusion between the activation domain of Gal4 and proteinsencoded by the tobacco cDNA library.A larger fragment ofthe C-terminal region of NtrbohD (AA 602 to AA 939,Fig. 1) was first used as bait but it acts as a transactivator ofthe HIS3 and lacZ reporter genes in yeast and was notsuitable for screening with the two-hybrid system.

Thirteen His+/LacZ+ clones were recovered in thisscreen interacting specifically with lexA/NtrbohD and ledto the isolation of two cDNAs encoding homologs to knownproteins, a protein belonging to the family of 14-3-3 proteins(AJ309008) whose characteristics are developed in thispaper, and a protein phosphatase 2C (AJ309007) whosefunction is still under investigation.

To further confirm the specificity of the interactionobserved between NtrbohD and Nt14-3-3, L40 yeastswere co-transformed with the Nt14-3-3 clone and each ofthe following sequences: the tobacco small G proteinNtRac5 (AJ250174), the neutrophil small G protein Rac2(AF498965), the neutrophil proteins p47phox (AF330627)and p67phox (AF527950), the neutrophil protein Rho-GDI(BC000317), and the yeast proteins SUT1 involved in steroluptake (X77766) and ERG 19, a cytosolic enzyme involvedin sterol biosynthesis (X97557). As indicated on Fig. 2,a specific interaction was observed only for the co-transformation of the C-terminus part of NtrbohD and theNt14-3-3 protein.

The strength of the interaction between the C-terminuspart of NtrbohD and the 14-3-3 protein was measuredwith a quantitative test using b-galactosidase activity(Miller 1972). This strength appeared to be similar towell-established interactions such as Raf/Ras (Vojtek,Hollenberg & Cooper 1993) and significantly higher thanthe value of negative controls already used in a previouslypublished paper (Kieffer et al. 2000) and repeated in thisstudy (Table 1).

The Nt14-3-3 cDNA of 786 bp isolated in this two-hybridscreen corresponds to an open reading frame which extendsfrom 25 amino acids upstream the ‘start codon’ to Ser 237located 20 amino acids before the end of the coding region(Fig. 3). Although it is not complete, the correspondingprotein expressed in yeast represents more than 90% of thesequence of the entire protein and is thus supposed to givea good representation of its interacting capacities.

This 786 bp fragment was used as a probe to screen atobacco cDNA library from tobacco leaves elicited with P.solanacearum (Czernic et al. 1996) (a gift from Dr Y.Marco). The positive clones shared the same nucleotidesequence and the largest clone of 1090 bp (AJ309008),encoded a full-length protein of 258 amino acids (Fig. 3).Asobserved in all yeast, plant and animal 14-3-3 sequences,

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nine a helices (a1 to a9) and five highly conserved blocks(B1 to B5) can be identified (Fig. 3), including those neces-sary for dimerization according to the crystal structure ofanimal 14-3-3 dimers (Piotrowski & Oecking 1998). Block 2contains a consensus sequence SSIEK, which is known to bephosphorylated by PKC in animal cells (Jones, Ley &Aitken 1995).

At the moment we isolated this 14-3-3 isoform, it was anew member of the tobacco 14-3-3 family and we called itNti14-3-3. Between depositing this clone in EMBL libraryand submission of the current paper, Konagaya et al. (2004)reported an inventory of 14-3-3 isoforms from N. tabacumvar. Samsun, among which, two are identical to Nti 14-3-3(h1/h2 isoforms), and Moriuchi et al. (2004) found the sameisoform (called omega 1) in N. tabacum cv. Petit Havana.Wewill thus name this isoform Nt14-3-3h/omega1 in the rest ofthe paper to be in agreement with the literature.

A phylogenetic analysis of all the sequences availablefrom different cultivars of N. tabacum indicates the prob-able presence of 17 isoforms according to the extensivedescription made in the Samsun cultivar by Konagaya et al.

(2004). All these sequences can be divided in the fourgroups (omega, kappa psi and epsilon) classically observedfrom dicotyledon sequence analysis (Piotrowski & Oecking1998), with Nt14-3-3h/omega1 isolated in this work belong-ing to the omega group (Konagaya et al. 2004). An align-ment of Nt14-3-3h/omega1 with two other 14-3-3 proteins,a1 and c1/omega2 isoforms of the omega group also knownto be induced during tobacco mosaic virus (TMV) infectionis shown on Fig. 3 (Konagaya et al. 2004).

Expression of Nt14-3-3h/omega1

The expression of the corresponding gene was examined indifferent tobacco plant tissues by Northern blot analysis.The Nt14-3-3 probe hybridized with a transcript of 1.1 kbpresent in pollen, apex, young leaves and roots, less abun-dant in flowers and seedlings, and absent from matureleaves (Fig. 4). Upon elicitation of excised leaves with 1 mgcryptogein, the level of expression of the transcript wassignificantly higher after 7 h of treatment and mRNA accu-mulated until 30 h (Fig. 4). This timing of induction is iden-tical to the one observed for NtrbohD transcripts on thesame material (Simon-Plas et al. 2002).

Involvement of Nt14-3-3h/omega1 in theregulation of ROS production during elicitation

In order to assess the role of the corresponding protein inthe production of ROS upon elicitation, transgenic BY2tobacco cells, transformed with antisense constructs of thiscDNA, were obtained. Molecular and physiological analy-ses were performed for four lines (14.1 to 4).

Upon elicitation with 50 nM cryptogein, ‘pky’ cells trans-formed with the pKY vector alone exhibited an intenseaccumulation of ROS (100 mM at the maximum rate)(Fig. 5a). On the other hand, when the 14-3-3 cell linestransformed with an antisense construct of the Nt14-3-3h/omega1 cDNA were elicited with the same concentration ofcryptogein, the amount of ROS detected was much lower(from 2 to 20% of the control, depending on the cell line)(Fig. 5a).

lexA-lamine

lexA-NtRac5

lexA-NtrbohDCterm

lexA-p47phox

lexA-p67phox

lexA-Rac2

lexA-Rho-GDI

DO-II DO-III

lexA-erg19

lexA-SUT1

Gal4-Nti14.3.3 Gal4-Nti14.3.3

Figure 2. Interaction of Nt14-3-3 with various proteins in yeast.L40 yeasts were co-transformed with the pGAD3S2X vectorcontaining the Nti14-3-3 cDNA in fusion with the activationdomain of Gal4 and each of the following sequences inserted inthe pLex vector in fusion with the lexA binding protein: thetobacco small G protein NtRac5, the neutrophil small G proteinRac2, the neutrophil proteins p47phox and p67phox, the neutrophilprotein Rho-GDI a human lamin and the yeast proteins SUT1(involved in sterol uptake) and ERG 19 (a cytosolic enzymeinvolved in sterol biosynthesis). Yeasts were then plated on amedium without leucine and tryptophan (DO-II) selective forthe efficacy of the transformation or on a medium withoutleucine tryptophan and histidine (DO-III) selective for theinteraction between the proteins whose sequences are inserted inthe plasmids.

Table 1. Quantitative assay of protein–protein interaction usingb-galactosidase activity

Protein interaction b-Galactosidase activitya

Lamine/NtRhoGDIb 0.06 � 0.01NtrbohD/NtRhoGDIb 0.07 � 0.01Ras/Rafc 3.45 � 0.65NtrbohD/Nt14-3-3 1.30 � 0.09

Mild-to-late exponential yeast cells were collected, resuspended ina Z-buffer, and assayed for b-galactosidase activity as described byMiller (1972) using O-nitrophenyl b-D-galactopyranoside (ONPG)as a substrate. The values indicated in the Table represent themeans and SD of three independent experiments.ab-Galactosidase activity unit = 1000 ¥ OD420/[OD600 ¥ reactiontime (min) ¥ volume of culture (mL)].bThis experiment and Kieffer et al. (2000).cVojtek et al. (1993).

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We checked for each transgenic line that the peroxidaseactivity of cells, necessary for the chemiluminescence reac-tion used for ROS detection, was not affected by the trans-formation, so that the discrepancies observed in ROSaccumulation are not due to a modification in their detec-tion (data not shown). So, the antisense Nt14-3-3 lines arestrongly affected in their ability to accumulate hydrogenperoxide.

We investigated the expression of the antisense transgenein these lines by Northern blot analysis using a single-stranded RNA probe (Fig. 5b). In the ‘pky’ control cells

Figure 3. Sequence of Nt14-3-3 protein and alignment with two other tobacco 14-3-3 proteins of the omega group. The sequence of theprotein deduced from the 14-3-3 cDNA isolated in the two-hybrid screen, (Nti), identical to the isoform h/omega1, is aligned withisoforms a1 and c1/omega2 described by Konagaya et al. (2004) and Moriuchi et al. (2004), respectively. Residues that differ from theconsensus are boxed. The nine a-helixes (a1 to a9 are underlined and the five conserved blocked are in grey boxes. Ser 237corresponding to the end of the clone isolated in the two-hybrid screen is indicated with an asterisk.

2.8 kb1.6 kb

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H2O

L11 + cry

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erPo

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xL

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seed

ling

1.1 kb Nti14.3.3

Figure 4. Expression of the Nt14.3.3h/omega1 gene in differenttissues of tobacco and during elicitation by cryptogein. TotalRNAs (10 mg) were analysed by Northern blot, probed with a32P-labelled restriction fragment corresponding to theNth/omega114.3.3 cDNA. Total RNAs were extracted from theindicated tissues. L1, L11, L16: 1st, 11th, 16th leaves from the topof mature tobacco plants; flower: floral tissue without stamen,pistil, stigma, anther and pollen; L11 + cry: L11 leaves treated bycryptogein during 0, 7, 24, 28, 29 and 30 h (10 mL of 0.1 mg/mLcryptogein was applied to the petiole of excised tobacco leaves);L11 + H2O: control L11 leaves treated with water during 7 h,necroses of the HR type (hypersensitive-like) appeared after11 h. The autoradiograph of the hybridized blot is shown abovethe corresponding gel stained with ethidium bromide.

100 0.2 1.8 0.2 2 0.1 17 0.2 21

pkyT 14.1C 14.1T 14.2C 14.2T 14.3C 14.3T 14.4C 14.4T

pkyT 14.1C 14.1T 14.2C 14.2T 14.3C 14.3T 14.4C 14.4T

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(b)

Figure 5. Reactive oxygen species (ROS) accumulation andexpression of the transgene in tobacco cells transformed withantisense construct of Nt14.3.3h/omega1 cDNA. Transgenic linesof BY2 tobacco cells were prepared and treated with 50 nM ofcryptogein as described in Materials and Methods. pky: cell linetransformed with the empty binary vector pKY; 14.1 to 14.4:independent cell lines transformed with the antisense constructof Nt14.3.3 cDNA; C: control cells; T: cells treated by cryptogein.(a) Every 10 min, ROS accumulation was measured. Resultsrepresent the mean of three independent experiments. Total ROSmeasured during 100 min of cryptogein treatment were summedand expressed as percent taking ROS accumulation in ‘pkyT’ cellline as the 100%. (b) Total RNAs (10 mg) were extracted fromthe transgenic tobacco cell untreated (C cells) and treated (Tcells) by 50 nM cryptogein during 100 min up to the maximalrate of ROS accumulation and were analysed by Northern blot,probed with the 32P-labelled sense RNA synthesized in vitro,complementary to the antisense transcript of Nt14.3.3h/omega1transgene. The autoradiographs of the hybridized blots are shownabove the corresponding gel stained with ethidium bromide.

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(transformed with the empty vector), no antisense isdetected, whereas in the Nt14-3-3h/omega1 transgenic lines,antisense transcripts are accumulated in untreated or elic-ited cells (Fig. 5b). In particular lines (14.1, 14.3 and 14.4),this accumulation of antisense transcript seems to bedecreased following cryptogein treatment: this is probablydue to a post-transcriptional inactivation process related toRNA interference. This process known as co-suppressionfor sense gene and transgene occurs very often in transgenicplant tissues (Elmayan & Vaucheret 1996) and could betriggered after induction of the native gene by cryptogein.In the transgenic line 14.2 exhibiting a strong antisensetransgene expression before and after elicitation, the realantisense mechanism of pairing between the two comple-mentary mRNA without degradation of RNA is probablyinvolved. Thus, these results show that inhibition of ROSaccumulation is correlated to the expression of theNt14.3.3h/omega1 antisense transgene.

DISCUSSION

14-3-3 proteins and response to biotic stress

The use of NtrbohD as a bait, in a two-hybrid screen, led tothe isolation of a cDNA encoding a protein belonging to thefamily of 14-3-3 proteins. This result seemed quite interest-ing as these proteins represent good candidates for partici-pation to a signal transduction process. Studies over the past20 years have proven 14-3-3 to be ubiquitous, being found inmost eukaryotic organisms and tissues (DeLille, Sehnke &Ferl 2001). The degree of sequence conservation is higheven between plant and animal isoforms, with the C- andN-terminal regions being most variable. In animals, theseproteins play a central role in the regulation of many cellu-lar processes such as control of cell cycle, differentiation,apoptosis, targeting of proteins to different cellular loca-tions and coordination of multiple signal transduction path-ways (Palmgren, Fuglsgang & Jahn 1998; Finnie, Borch &Collinge 1999; Fu, Subramanian & Masters 2000; Roberts2000). These 14-3-3 proteins could achieve these functionsby directly regulating the activity of proteins involved in asignal transduction cascade, promoting the formation ofmulti-protein complexes, or modulating the expression ofparticular genes by regulating the activity or localizationof transcription factors.

There are many results showing that 14-3-3 proteinscould be involved in signalling pathways regulating plantgrowth and responses to environmental stress (Roberts,Salinas & Collinge 2002). Concerning more precisely theplant defence, a 14-3-3 gene was identified to be regulatedin the non-host HR between barley and Blumeria graminisF. sp. tritici (Brandt et al. 1992). An mRNA encoding a14-3-3 protein was reported to be differentially inducedduring the hypersensitive reaction of soybean after inocu-lation with Pseudomonas syringae pv. glycinea (Seehaus &Tenhaken 1998). Roberts & Bowles (1999) also describeddifferent subsets of 14-3-3 genes from tomato (TFT 1-10),specifically induced after treatment with the fungal toxin

fusicoccin and during a gene for gene resistance response.The induction of several genes of 14-3-3 has also been evi-denced during the incompatible interaction between riceand fungal pathogens (Chen et al. 2006) and betweentobacco and the TMV (Konagaya et al. 2004).All these datastrongly suggest the involvement of 14-3-3 proteins in plantdefence although there is a crucial lack for precise rolesplayed by these proteins in such physiological situations. Adifferent functional role for 14-3-3 proteins during plant–pathogen interactions was indicated by results concerningthe Arabidopsis AKR2 gene (Yan, Wang & Zhang 2002).The AKR2 protein was initially identified as a 14-3-3 inter-acting protein in a yeast two-hybrid system, and the anti-sense expression of the corresponding gene in plantsshowed that this protein was a negative regulator of theresistance response to pathogens, but no precise functioncould be hypothesized for this protein.

Nt14-3-3h/omega1 induction by cryptogein

In a given organism, 14-3-3 are usually present as a family ofisoforms with a molecular mass of 30 kDa. There are 15members of the 14-3-3 gene family in Arabidopsis thalianaamong which at least 12 are expressed (Sehnke, DeLille &Ferl 2002), and a phylogenetic analysis of the 14-3-3 genefamily of N. tabacum cv. Samsun made an inventory of 17isoforms (Konagaya et al. 2004).This relatively high numbermay be explained by the fact that tobacco possesses a dupli-cated genome of two ancestral species (Nicotiana tomen-tosiformis and Nicotiana sylvestris), which is confirmed bythe fact that two very closely related genes have been iden-tified for several of these isoforms (Konagaya et al. 2004).

The question of the specificity of expression is raised bythe presence of such a gene family. Different studies havedemonstrated that Arabidopsis isoforms present a high cell-and tissue-type specificity (Sehnke et al. 2002), and this hasbeen confirmed recently by similar studies performed onrice (Chen et al. 2006). Finally, recent data obtained inSolanum tuberosum indicated that the 14-3-3 gene expres-sion specificity in response to stress is promoter-dependent(Aksamit et al. 2005).

Analysis of Nt14-3-3h/omega1 expression by Northernblot on excised leaves indicates a very low expression incontrol plants and a strong induction after cryptogein treat-ment with a timing quite comparable to the induction ofNtrbohD (Simon-Plas et al. 2002): both mRNAs areinduced 7 h after treatment with the elicitor. These resultsare confirmed by the work of Konagaya et al. (2004)showing that this isoform was specifically induced duringthe incompatible interaction occurring upon inoculation oftobacco (NN) with the TMV. Furthermore, the clone TFT6,encoding a protein which is the tomato isoform exhibitingthe highest homology with Nt14-3-3h/omega1 (97% at theprotein level and 89% at the nucleotide level), is specificallyinduced in Cf9 and not in Cf0 tomato leaves, a few hoursafter infiltration with Avr9-containing intercellular fluid(Roberts & Bowles 1999). Nti 14-3-3 is also the tobaccoclosest homolog (100% at the protein level and 87% at the

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nucleotide level) of the S. tuberosum isoform 16R, whichpossesses a promoter containing elicitor-responsive ele-ments (EIRE) sequence required for plant defence signal-ling, and induced by salicylic acid and PVY virus (Aksamitet al. 2005).

Regulation of ROS production: a role for a14-3-3 protein during the set-up of plantdefence mechanisms

If many studies (more than 200) suggest a putative role for14-3-3 proteins in essential physiological process of plants,such as growth and development, or response to stress, veryfew of them assign to these proteins a precise function inthe corresponding signalling pathways. Indeed, functionalapproaches using modification of expression of these pro-teins have rarely been reported (Sehnke et al. 2001; Zuket al. 2003; Yan et al. 2004).

In this study, when BY2 cell lines are transformed withantisense constructs of Nt14-3-3h/omega1, the expression ofthe antisense transgene is correlated with a strong inhibi-tion of ROS accumulation following elicitation with cryp-togein. This demonstration of the involvement of a 14-3-3protein in the transduction process leading to ROS accu-mulation is a quite new and interesting point, even if it stillhas to be precisely defined. Firstly, it constitutes one of thefirst clearly established roles for these proteins in the sig-nalling accompanying the development of an incompatibleinteraction. Then, if it has been clearly established that aplasma membrane NADPH oxidase produces ROS inplanta (Torres, Dangl & Jones 2002; Yoshioka et al. 2003) orin elicited cells (Simon-Plas et al. 2002) during incompatibleinteraction, its particular regulation is at the moment quitepoorly known. Only general lines of signal transduction,such as calcium influx, phosphorylation, nitrate efflux orsmall G proteins, have been reported as regulating ROSproduction mediated by this oxidase. Furthermore, thisROS production occurs not only during plant–pathogensinteractions, but also in abscisstic acid (ABA) signallingduring stomatal closure, or in response to an abiotic stresssuch as ozone exposure (Torres & Dangl 2005). All theseprocesses use the same general signal transduction ele-ments listed here, but lead to a different physiologicalresponse. This renders all the more necessary the elucida-tion at the molecular level of the different steps of thesignalling cascades involved. To this respect, the involve-ment, upstream of ROS production induced by a fungalelicitor, of a 14-3-3 isoform which has been proved to beinduced during incompatible interactions, is not devoid ofinterest. Such a regulation of ROS production by a 14-3-3protein, quite new in plants, cannot either be discussed inthe context of animal literature. No evidence exists for theinvolvement of a 14-3-3 protein in the regulation of therespiratory burst oxidase from phagocytes, but, as devel-oped in the Introduction, there seems to be obvious differ-ences between the regulation of these proteins.

If the regulation of ROS accumulation by a 14-3-3protein seems functionally established in our system, the

questions are raised of (1) the direct or indirect nature ofthis regulation and (2) its isoform specificity.

Northern blots performed on RNA extracted from celllines transformed with antisense constructs of Nt14-3-3h/omega1 demonstrated that mRNAs corresponding toNtrbohD, were still induced following a cryptogein treat-ment, as observed in wild-type cells (Simon-Plas et al. 2002;and data not shown). Although indirect ways of regulationcould be hypothesized, the fact that a significant interactionbetween Nti4-3-3h/omega1 and the C-terminus part ofNtrbohD has been observed in a two-hybrid screen, makesplausible a direct regulation of ROS production by thisprotein–protein interaction. In an attempt to confirm thein vivo interaction of Nt14-3-3h/omega1 and NtrbohD,pull-down assays were performed using the antibody wegenerated against NtrbohD, but we never succeeded toimmunoprecipitate the oxidase, may be due to the antipep-tide origin of the antibody, or from the transmembranenature of the enzyme. Initially, a specific consensussequence for 14-3-3 binding was suggested but after identi-fication of a large number of 14-3-3 target sites, the assig-nation of a unique consensus motif proved to be impossible(Fu et al. 2000). The present agreement on this subject isthat three modes of interaction may occur between a givenprotein and a 14-3-3. Modes 1 and 2 are characterized by thepresence on the target protein of well-defined motifs (notpresent on the C-terminus of NtrbohD), RSXpS(pT)XPand RXFX(pS)XP, respectively (where pS and pT arephosphorylated serine and threonine residues, F an aro-matic or aliphatic amino acid, X any amino acid) (Coblitzet al. 2006). Mode 3 is characterized by a consensussequence derived from natural interactors which do notexactly match mode 1 and mode 2 ligands. The search forsuch a motif in the C-terminus part of NtrbohD, using theELM server (Eukaryotic Linear Motif resource for func-tional sites in proteins, http://elm.eu.org), led to the identi-fication of three sequences (KSGSAS, RVKSHF andKTSTKF) which correspond respectively to amino acids791-796, 877-882 and 927-932, and contain several threonineand serine residues potentially phosphorylated.

The C-terminus part of NtrbohD used as a bait, did notallow us to isolate any other 14-3-3 isoform but Nt14-3-3h/omega1. However, the question of the specificity of functionof this isoform remains and a possible interaction betweenNtrbohD and another member of the 14-3-3 family cannotbe ruled out. Studies on the binding of different isoforms of14-3-3 to different isoforms of H+-ATPase in Arabidopsis,indicated a relative and not absolute specificity: 12 isoformswere able to bind to the H+-ATPase in vitro, but the level ofexpression and relative affinity in a particular physiologicalsituation, seem to determine in vivo an isoform specificity(Alsterfjord et al. 2004). Thus, there is no biochemicalargument in favour of a specific interaction of Nt14-3-3h/omega1 with NtrbohD but rather a first line of argumentscoming from the induction of the corresponding mRNAs bythe elicitor in the same timing. In this context, an additionalinteresting result came from proteomic studies performedon microdomains. We isolated, from tobacco BY2 cells,

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plasma membrane domains enriched in sterols and sphin-golipids, similarly as previously known for animal rafts. Theanalysis of their proteic composition (Mongrand et al. 2004;Morel et al. 2006) showed the presence in these domains ofNtrbohD, and revealed their relative enrichment in proteinsinvolved in response to biotic and abiotic stress, suggestingthat they could play a role in the regulation of such pro-cesses, as already known in yeasts and animal cells(Rajendran & Simons 2005). Complementary mass spec-trometry analysis (Morel et al., unpublished data) indicatedthe presence of 14-3-3 proteins in microdomains isolatedform BY2 cells elicited with cryptogein. The identifiedpeptides could not allow to discriminate between thesethree close isoforms, Nt14-3-3h/omega1, a and c/omega2(sequences shown in Fig. 3) all induced during incompatibleinteraction between tobacco and TMV (Konagaya et al.2004). A great variety of sub-cellular localization (chloro-plast, cytoplasm, nucleus, mitochondria and plasma mem-brane region) has been evidenced for plant 14-3-3 proteins(Finnie et al. 1999), and this colocalization in specializeddomains is an additional argument in favour of the biologi-cal relevance of the regulation of NtrbohD by Nt14-3-3h/omega1.All the future investigations aiming to identify stepby step the protein–protein interactions involved in thesignal transduction cascade triggered by the elicitor shouldbe performed, keeping in mind, the role of plasma mem-brane microdomains in the regulation of the spatio-temporal distribution on the membrane, of proteinsinvolved in this signalling.

ACKNOWLEDGMENTS

We are grateful to Michel Ponchet for the gift of cryptogein,Marie-Claire Dagher for thep47phox, p67phox and Rac2cDNA clones, and Thierry Berges for the SUT1 and ERG19cDNA clones. We thank Sylvain Morel and CarolineAndrieux for technical assistance, and David Wendehennefor helpful discussions. We would like to acknowledge theConseil Régional de Bourgogne for his financial support.

REFERENCES

Aksamit A., Korobczak A., Skala J., Lukaszewicz M. & Szopa J.(2005) The 14-3-3 gene expression specificity in response tostress is promoter-dependent. Plant and Cell Physiology 46,1635–1645.

Alsterfjord M., Sehnke P.C., Arkell A., Larsson H., Svennelid F.,Rosenquist M., Ferl R.J., Sommarin M. & Larsson C. (2004)Plasma membrane H+-ATPase and 14-3-3 isoforms of Arabi-dopsis leaves: evidence for isoform specificity in the 14-3-3/H+-ATPase interaction. Plant and Cell Physiology 45, 1202–1210.

Amicucci E., Gaschler K. & Ward J.M. (1999) NADPH oxidasegenes from tomato (Lycopersicon esculentum) and curly-leafpondweed (Potamogeton crispus). Plant Biology 1, 524–528.

Apel K. & Hirt H. (2004) Reactive oxygen species: metabolism,oxidative stress, and signal transduction. Annual Review of PlantBiology 55, 373–399.

Babior B.M. (2004) NADPH oxidase. Current Opinion in Immu-nology 16, 42–47.

Bolwell G.P. (1999) Role of active oygen species and NO in plantdefence responses. Current Opinion in Plant Biology 2, 287–294.

Bolwell G.P. & Wojtaszek P. (1997) Mechanisms for the generationof reactive oxygen species in plant defence – a broad perspective.Physiological and Molecular Plant Pathology 51, 347–366.

Brandt J., Thordal-Christensen H., Vad D., Gregersen P.L. &Collinge D.B. (1992) A pathogen-induced gene of barleyencodes a protein showind high similarity to a protein kinaseregulator. The Plant Journal: For Cell and Molecular Biology 2,815–820.

Chen F., Li Q., Sun L. & He Z. (2006) The rice 14-3-3 gene familyand its involvement in responses to biotic and abiotic stress.DNA Research 13, 53–63.

Coblitz B., Wu M., Shikano S. & Li M. (2006) C-terminal binding:an expanded repertoire and function of 14-3-3 proteins. FEBSLetters 580, 1531–1535.

Cross A.R. & Segal A.W. (2004) The NADPH oxidase of profes-sional phagocytes-prototype of the NOX electron transportchain system. Biochimica et Biophysica Acta 1657, 1–22.

Czernic P., Huang H.C. & Marco Y. (1996) Characterization ofhsr201 and hsr515, two tobacco genes preferentially expressedduring the hypersensitive reaction provoked by phytopathogenicbacteria. Plant Molecular Biology 31, 255–265.

Davis A.R., Mascolo P.L., Bunger P.L., Sipes K.M. & Quinn M.T.(1998) Cloning and sequencing of the bovine flavocytochrome bsubunit proteins, gp91-phox and p22-phox: comparison withother known flavocytochrome b sequences. Journal of LeukocyteBiology 64, 114–123.

DeLille J.M., Sehnke P.C. & Ferl R.J. (2001) The Arabidopsis 14-3-3family of signaling regulators. Plant Physiology 126, 35–38.

Desikan R., Hancock J.T., Coffey M.J. & Neill S.J. (1996) Genera-tion of active oxygen in elicited cells of Arabidopsis thaliana ismediated by a NADPH oxidase-like enzyme. FEBS Letters 382,213–217.

Elmayan T. & Tepfer M. (1994) Synthesis of a bifunctionalmetallothionein/b-glucuronidase fusion protein in transgenictobacco plants as a mean of reducing leaf cadmium levels. ThePlant Journal: For Cell and Molecular Biology 6, 433–440.

Elmayan T. & Vaucheret H. (1996) Expression of single copiesof a strongly expressed 35s-transgene can be silenced post-transcriptionally. The Plant Journal: For Cell and MolecularBiology 9, 787–797.

Finnie C., Borch J. & Collinge D.B. (1999) 14-3-3s: eukaryotic regu-latory proteins with many functions. Plant Molecular Biology 40,545–554.

Fu H., Subramanian R.R. & Masters S.C. (2000) 14-3-3 proteins:structure, function, and regulation. Annual Review of Pharma-cology and Toxicology 40, 617–647.

Groom Q.J., Torres M.A., Fordham-Sketon A.P., Hammond-Kosack K.E., Robinsion N.J. & Jones J.D.G. (1996) rbohA, a ricehomologue of the mamalian gp91phox respiratory burst oxidasegene. The Plant Journal: For Cell and Molecular Biology 10,515–522.

Heyworth P.G., Curnutte J.T. & Badwey J.A. (1999) Structure andregulation of NADPH oxidase of phagocytic leukocytes. InMolecular and Cellular Basis of Inflammation (ed. P.A. CNaWSerhan), pp. 165–190. Humana Press Inc, Totowa, NJ, USA.

Jones D.H.A., Ley S. & Aitken A. (1995) Isoforms of 14-3-3 proteincan form homo- and heterodimers in vivo and in vitro: implica-tions for function as adapter proteins. FEBS Letters 368, 55–58.

Keller T., Damude H.G., Werner D., Doerner P., Dixon R.A. &Lamb C. (1998) A plant homolog of the neutrophil NADPHoxidase gp91(phox) subunit gene encodes a plasma membraneprotein with Ca2+ binding motifs. Plant Cell 10, 255–266.

Kieffer F., Elmayan T., Simon-Plas F., Dagher M.C. & Blein J.-P.(2000) A tobacco cDNA encoding a Rac-like protein cloned

730 T. Elmayan et al.

Journal compilation © 2007 Blackwell Publishing LtdNo claim to original French government works, Plant, Cell and Environment, 30, 722–732

using the two-hybrid system in an heterologous screen. Bio-chimie 82, 1099–1105.

Konagaya K., Matsuhita Y., Kasahara M. & Nyunoya H. (2004)Members of 14-3-3 protein isoforms interacting with the resis-tance gene product N and the elicitor of Tobacco mosaic virus.Journal of General Plant Pathology 70, 221–231.

Lamb C.J. & Dixon R.A. (1997) The oxidative burst in plantdisease resistance. Annual Review of Plant Physiology and PlantMolecular Biology 48, 251–275.

Milat M.-L., Ducruet J.-M., Ricci P., Marty F. & Blein J.-P. (1991)Physiological and structural changes in tobacco leaves treatedwith cryptogein, a proteinaceous elicitor from Phytophthoracryptogea. Phytopathology 81, 1364–1368.

Miller J.H. (1972) Experiments in Molecular Genetics. Cold SpringHarbor Laboratory, Cold Spring Harbor, NY.

Mittler R., Vanderauwera S., Gollery M. & Van Breusegem F.(2004) Reactive oxygen gene network of plants. Trends in PlantScience 9, 490–498.

Moeder W., Yoshioka H. & Klessig D.F. (2005) Involvement of thesmall GTPase Rac in the defense responses of tobacco to patho-gens. Molecular Plant Microbe Interactions 18, 116–124.

Mongrand S., Morel J., Laroche J., Claverol S., Carde J.P., HartmannM.A.,Bonneu M.,Simon-Plas F.,Lessire R. & Bessoule J.J. (2004)Lipid rafts in higher plant cells: purification and characterizationof TX-100 insoluble microdomains from tobacco plasma mem-brane. Journal of Biological Chemistry 279, 36277–36286.

Morel J., Fromentin J., Blein J.-P., Simon-Plas F. & Elmayan T.(2004) Rac regulation of NtrbohD, the oxidase responsible forthe oxidative burst in elicited tobacco cell. The Plant Journal: ForCell and Molecular Biology 37, 282–293.

Morel J., Claverol S., Mongrand S., Furt F., Fromentin J., BessouleJ.J., Blein J.-P. & Simon-Plas F. (2006) Proteomics of plantdetergent-resistant membranes. Molecular and Cellular Pro-teomics 5, 1396–1411.

Moriuchi H., Okamoto C., Nishihama R., Yamashita I., Machida Y.& Tanaka N. (2004) Nuclear localization and interaction of RolBwith plat 14-3-3 proteins correlates with induction of adventi-tious roots by the oncogene rolB. The Plant Journal: For Cell andMolecular Biology 38, 260–275.

Ono E., Wong H.L., Kawasaki T., Hasegawa M., Kodama O. &Shimamoto K. (2001) Essential role of the small GTPase Rac indisease resistance of rice. Proceedings of National Academy ofScience USA 98, 759–764.

Palmgren M.G., Fuglsgang A.T. & Jahn T. (1998) Deciphering therole of 14-3-3s. Experimental Biology Online 3, 1–17.

Piotrowski M. & Oecking C. (1998) Five new 14-3-3 isoforms fromNicotiana tabacum L.: implications for the phylogeny of plant14-3-3 proteins. Planta 204, 127–130.

Rajendran L. & Simons K. (2005) Lipid rafts and membranedynamics. Journal of Cell Science 118, 1099–1102.

Ricci P. (1997) Induction of the hypersensitive response and sys-temic acquired resistance by fungal proteins: the case of elicitins.In Plant-Microbe Interactions (eds G. Stacey & N.T. Keen),Vol 3, pp. 53–75. Chapman & Hall, New York NY, USA.

Roberts M.R. (2000) Regulatory 14-3-3 protein-protein interac-tions in plant cells. Current Opinion in Plant Biology 3, 400–405.

Roberts M.R. & Bowles D.J. (1999) Fusicoccin, 14-3-3 proteins, anddefense responses in tomato plants. Plant Physiology 119, 1243–1250.

Roberts M.R., Salinas J. & Collinge D.B. (2002) 14-3-3 proteins andthe response to abiotic and biotic stress. Plant Molecular Biology1031, 1031–1039.

Sagi M. & Fluhr R. (2001) Superoxide production by plant homo-logues of the gp91phoxNADPH oxidase. Modulation of activity bycalcium and by tobacco mosaic virus infection. Plant Physiology126, 1281–1290.

Sambrook J., Fritsch E.F. & Maniatis T. (1989) Molecular Cloning:A Laboratory Manual. Cold Spring Harbor Laboratory, ColdSpring Harbor, NY, USA.

Seehaus K. & Tenhaken R. (1998) Cloning of genes by mRNAdifferential display induced during the hypersensitive reaction ofsoybean after inoculation with Pseudomonas syringae pv. gly-cinea. Plant Molecular Biology 38, 1225–1234.

Sehnke P.C., Chung H.J., Wu K. & Ferl R.J. (2001) Regulation ofstarch accumulation by granule-associated plant 14-3-3 proteins.Proceedings of National Academy of Science USA 98,765–770.

Sehnke P.C., DeLille J.M. & Ferl R.J. (2002) Consummating signaltransduction: the role of 14-3-3 proteins in the completion ofsignal-induced transitions in proteins activity. Plant Cell 14(Suppl.), S339–S354.

Simon-Plas F., Rustérucci C., Milat M.-L., Humbert C., MontilletJ.-L. & Blein J.-P. (1997) Active oxygen species production intobacco cells elicited by cryptogein. Plant Cell and Environment20, 1573–1579.

Simon-Plas F., Elmayan T. & Blein J.-P. (2002) The plasma mem-brane oxidase NtrbohD is responsible for AOS production inelicited tobacco cells. The Plant Journal: For Cell and MolecularBiology 31, 137–147.

Suharsono U., Fujisawa Y., Kawasaki T., Satoh H. & Shimamoto K.(2002) The heterotrimeric G protein alpha subunit acts upstreamof the small GTPase rac in disease resistance of rice. Proceedingsof National Academy of Science USA 99, 13307–13312.

Tenhaken R. & Rubel C. (1998) Cloning of putative subunits of thesoybean plasma membrane NADPH oxidase involved in theoxidative burst by antibody expression screening. Protoplasma205, 21–28.

Torres M.A. & Dangl J. (2005) Functions of the respiratory burstoxidase in biotic interactions, abiotic stress and development.Current Opinion in Plant Biology 8, 397–403.

Torres M.A., Onuchi H., Hamada S., Machida G., Hammond-KosakK.E., Robinsion N.J. & Jones J.D.G. (1998) Six Arabidopsisthaliana homologues of the human respiratory burst oxidase(gp91-phox). The Plant Journal: For Cell and Molecular Biology 14,365–370.

Torres M.A., Dangl J. & Jones J.D.G. (2002) Arabidopsis gp91phox

homologues AtrbohD and AtrbohF are required for accumula-tion of reactive oxygen intermediates in the plant defenseresponse. Proceedings of National Academy of Science USA 99,517–522.

Torres M.A., Jones J.D.G. & Dangl J.L. (2005) Pathogen-induced,NADPH oxidase-derived reactive oxygen intermediates sup-press spread of cell death in Arabidopsis thaliana. NatureGenetics 37, 1130–1134.

Torres M.A., Jones J.D.G. & Dangl J.L. (2006) Reactive oxygenspecies signaling in response to pathogens. Plant Physiology 141,373–378.

Vojtek A.B., Hollenberg S.M. & Cooper J.A. (1993) MammalianRas interacts directly with the serine threonine kinase Raf. Cell74, 205–214.

Wojtaszek P. (1997) Oxidative burst: an early plant response topathogen infection. The Biochemical Journal 322, 681–692.

Xing T., Higgins V.J. & Blumwald E. (1997) Race-specific elicitorsof Cladosporium fulvum promote translocation of cytosoliccomponents of NADPH oxidase to the plasma membrane oftomato cells. Plant Cell 9, 249–259.

Yan J., Wang J. & Zhang H. (2002) An ankyrin repeat-containingprotein plays a role in both disease resistance and antioxidationmetabolism. The Plant Journal: For Cell and Molecular Biology29, 193–202.

Yoshioka H., Numata N., Nakajima K., Katou S., KawakitaK., Rowland O., Jones J.D.G. & Doke N. (2003) Nicotiana

A 14-3-3 protein regulates ROS production in tobacco cells 731

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benthamiana gp91phox homologs NbrbohA and NbrbohB partici-pate in H2O2 accumulation and resistance to Phytophthorainfestans. Plant Cell 15, 706–718.

Zuk M., Prescha A., Kepczynski J. & Szopa J. (2003) ADP ribosy-lation factor regulates metabolism and antioxydant capacity of

transgenic potato tubers. Journal of Agriculture and FoodChemistry 51, 288–294.

Received 25 October 2006; received in revised form 16 February2007; accepted for publication 18 February 2007

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