Proc. Natl. Acad. Sci. USAVol. 91, pp. 10556-10560, October 1994Plant Biology

Expression of the gene for a small GTP binding protein intransgenic tobacco elevates endogenous cytokinin levels,abnormally induces salicylic acid in response to wounding,and increases resistance to tobacco mosaic virus infection

Jasnonic add/pathogenesis-related proteins/proteinase inhibitor li/rgpl/wound signal transduction)

H. SANO*t, S. SEOt, E. ORUDGEV*, S. YOUSSEFIAN*, K. ISHIZUKAt, AND Y. OHASHI§t*Laboratory of Molecular Genetics, Biotechnology Institute, Akita Prefectural College of Agriculture, Akita 010-04, Japan; tInstitute of Applied Biochemistry,University of Tsukuba, Tsukuba, Ibaraki 305, Japan; and §Department of Molecular Biology, National Institute of Agrobiological Resources, Tsukuba,Ibaraki 305, Japan

Communicated by Clarence A. Ryan, July 18, 1994

ABSTRACT Tobacco plants transformed with rgpl, a geneencoding a Ras-related small GTP binding protein, werepreviously shown to exhibit a distinct reduction in apicaldominance with increased tillering. These abnormal pheno-types were later found to be associated with elevated levels ofendogenous cytokinins (zeatin and zeatin riboside). Analysis ofthe expression of several genes known to be affected bycytokinins identified a clear increase in the mRNA levels ofgenes encoding acidic pathogenesis-related proteins in bothtransgenic plants and their progenies. This increase was di-rectly attributable to elevated levels of the acidic pathogenesis-related protein inducers, salicylic acid (SA) and salicylic acidf-glucoside, due to an abnormal and sensitive response of thetransgenic plants to wounding. In contrast, mRNA levels of thegene for proteinase Inhibitor II, which is normally induced bywounding, were generally suppressed in the same woundedplants, probably due to SA overproduction. The changes in SAand pathogenesis-related protein levels in the transgenic plantsresulted in a distinct increase in their resistance to tobaccomosaic virus infection. In normal plants, the wound andpathogen-induced signal transduction pathways are consideredto function independently. However, the wound induction ofSA in the trangenic plants suggests that overexpression of thissmallGTP binding protein somehow interferes with the normalsignal pathways, possibly by affecting cytokinin biosynthesis,and results in cross-sling between these two transductionsystems.

In earlier studies, we isolated a rab/ypt-related gene, rgpl,encoding a small GTP binding protein from rice. Expressionof rgpl was age-dependent and reduced in seedlings aftertreatment with 5-azacytidine, an inhibitor of DNA methyl-ation in vivo (1). This treatment also simultaneously induceddwarf plants and suggested that the rgpl product rgpl-p25was somehow involved in the regulation of plant growth anddevelopment, especially in view ofthe regulatory functions ofthe small GTP binding proteins. To investigate some of thephysiological functions ofrgpl-p25, we constructed (6) trans-genic tobacco plants with the rgpl gene (2) and found that theresulting transformants showed distinct phenotypic changes,most notably a reduction in apical dominance with increasedtillering and also dwarfism and/or abnormal flower struc-tures. The observed phenotypes implicated rgpl-p25 in thehormone metabolic or response pathways. Indeed, morerecently, we found that the endogenous cytokinin levels were6-fold higher in these transgenic plants than in control plants.We subsequently examined the expression ofgenes known to

be differentially regulated in response to cytokinins andfound that genes encoding the acidic pathogenesis-related(PR) proteins were severely affected in rgpl transformants.PR proteins, which were originally identified in plants upon

viral infection (3), consist of both acidic and basic isoformsthat differ in their tissue and organ localizations and in themechanisms by which they are induced (4-6). Among theinducers of PR proteins, salicylic acid (SA) has attractedparticular interest as its endogenous levels are dramaticallyincreased in tobacco mosaic virus (TMV)-infected plants (7,8), suggesting that SA functions as a natural transductionsignal (9). However, other stresses, such as wounding, do notusually induce SA (7), and it is generally considered thatplants have two distinct signal transduction pathways for thepathogen- and wound-induced responses (10).

In this paper we demonstrate that transgenic tobaccoplants expressing the rgpl gene produce abnormally highlevels of SA in response to wound stress and that this, withan associated increase in acidic PR proteins, confers a highlevel of resistance against TMV infection.

MATERIALS AND METHODSPlant Materials and Stress Treatments. Tobacco plants

(Nicotiana tabacum, cv. Xanthi nc), with or without the rgplgene (2), were grown in a controlled-environment chamber at200C under a 14-h light/10-h dark cycle. Transgenic plantsused were A2 (Ro, the original transformant with rgpl in thesense orientation) (2) and the R1_1 and R1.9 progenies ob-tained by manual self-pollination of the A2/Ro plant. Thesynthesis of rgpl-p25 in the A2 plant was confirmed byimmunoelectrophoresis using a polyclonal antibody againstrgpl-p25 (data not shown). To obtain young plants, smalltillers from the transformants were detached, cultured inwater for 1-2 weeks until adventitious roots developed, andthen transferred onto soil (such plants are distinguished fromthe original parent by reference to them as A2'). For SA andjasmonic acid methyl ester (MeJA) treatments, a younghealthy leaf was cut from the plant and floated on a solutioncontaining 50 ,.M sodium salicylate or MeJA (Wako Bio-chemicals, Osaka) at 200C under continuous light for 2-3days.

Extraction, Purification, and Quantitation of Cytokinins.Cytokinins [zeatin and zeatin riboside (Z and ZR)] wereextracted from 10 g of leaf samples and purified by HPLC

Abbreviations: JA, jasmonic acid; MeJA, jasmonic acid methylester; PI-II, proteinase inhibitor II; PR, pathogenesis-related; SA,salicylic acid; SAG, salicylic acid P-glucoside; TMV, tobacco mosaicvirus; Z, zeatin; ZR, zeatin riboside.tTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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(Wakosil-II-5C18) essentially as described (11). Quantitationwas performed by ELISA with a monoclonal antibody (IDE-TEC, San Bruno, CA) to trans-ZR. Alkaline phosphatase-trans-ZR conjugate was synthesized by periodate coupling(12) and used in the antigen capture assay (12).RNA Isolation and Northern Blot Hybridization. Total cel-

lular RNA was isolated by the guanidine thiocyanate methodand Northern blot hybridization analysis was performed asdescribed (13). A cDNA probe encoding proteinase inhibitorII (PI-II) was a generous gift from C. A. Ryan (WashingtonState University). The PR-la cDNA was prepared as de-scribed (4). The other cDNA probes were synthesized by thePCR using synthetic primers designed from known sequencedata.

Quantitative Determination of PR-i Proteins. The quanti-tation of PR-i proteins was performed by rocket immuno-electrophoresis with an antibody raised against purifiedPR-la protein as described (14).

Quantitation of SA and Salicylic Acid 3Glucoside (SAG)Content of Leaves. Fully expanded leaves of wild-type or A2'plants were wounded by gentle rubbing of the upper epider-mis ofthe leafwith wet carborundum. Quantitation offree SAand SAG was performed essentially as described (15, 16).Known amounts of authentic SA were added to the sampleduring extraction to determine losses during the extractionand purification procedures. The overall recovery was 55%,and hence, all data were corrected on this basis. In mostcases, 5-10 g of leaf material was divided into several lots,which were assayed simultaneously, and the final valueswere normalized on a per g (fresh weight) basis. The detec-tion limit was thus lowered to 5-10 ng/g (fresh weight).

RESULTSIncrease of Endogenous Cytokinin Levels in Transgenic

Tobacco. The tillering morphology of the rgpl-transgenicplants (2) strongly suggested altered levels of, or responsesto, phytohormones, particularly cytokinins. Subsequently,the endogenous levels of Z and ZR, which constitute themajority of native cytokinins in higher plants, were exam-ined. The Z and ZR content in wild-type and GUS (vectoralone)-transgenic plants was -5 pmol/g (fresh weight),whereas in transgenic tobacco plants expressing the sense-oriented rgpl gene (A2) and the R1 progeny, their levels wereincreased to 23 pmol/g (fresh weight) and to >13 pmol/g(fresh weight), respectively (Fig. 1).

Increase of PR-1 mRNA Levels by Wound Stress in Trans-genic Tobacco. To identify cytokinin-responding genes thatwere differentially expressed in the transgenic plants, North-ern blot analysis with various cDNA probes was performedusing total cellularRNA from healthy leaves ofwild-type andseveral transgenic tobacco plants expressing the sense-oriented rgpl gene (Al, A2, and A3a plants; see ref. 2). Ofthe14 genes examined, only those encoding acidic PR proteins(PR-1, PR-Q, and PR-S) showed high levels of transcripts inthese transgenic plants (data not shown). Subsequently, themRNA levels of PR-1 and also PI-II, which has a differentinduction mechanism to PR-1, were examined in detail (Fig.2). The rgpl gene was constitutively expressed in the A2plants, and actin was expressed at equal levels in all the RNAsamples loaded. In untreated leaves, PR-1 mRNA was de-tected in the original A2/Ro plant but was undetectable in thewild-type or A2/R1 progeny plants. In excision-stressedleaves floated on water, PR-i mRNA was not detected in thecontrol but accumulated to high levels in both the A2/Ro andA2/R1 plants. In leaves treated with SA, the PR-i mRNAlevels increased further in both control and A2 plants. Incontrast, PI-II mRNA was induced in both control and A2plants simply by cutting stress but was suppressed by SAtreatment. In general, however, the PI-II mRNA level was

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FIG. 1. Quantitation of endogenous cytokinins (Z+ZR) in to-bacco leaves. A 10-g sample of apical leaves from healthy plants wasused to extract endogenous cytokinins. Quantitation was performedby using monoclonal antibodies. Values are the means of threemeasurements and standard deviations are shown by error bars.Plant materials were wild type (bar WT) and transgenics with thef-D-glucuronidase gene (pBI121 vector alone) (bar C) and rgpl [barsA2(Ro) and A2(R1.1)].

lower in the A2 plants than in the control. These unusualresponses of the A2 plants to wounding were clearly theresult of rgpl overexpression, as demonstrated by the heri-table nature of the trait. The high level of PR-1 mRNA in the

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FIG. 2. Effects of wounding and SA treatment on the mRNAlevels of genes encoding PR proteins. A whole leaf from either thewild type (WT) or A2 plants, both Ro (A2/Ro) and R1 (A2/R1_. andA2/R1_9) generations, was detached, floated on water (lanes W) ora solution containing 50 pM SA (lanes S) at 20TC under continuouslight for 3 days, and then used for isolation oftotal RNA. As a control(lanes C), total RNA was immediately isolated from freshly har-vested leaves without any treatment. A 20-ag aliquot of RNA wasassayed for mRNA levels of genes encoding actin as an internalstandard, rgpl-p25 (rgpl), PR-1, and PI-1I by Northern blot hybrid-ization.

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untreated A2/Ro plant may have been due to woundingduring previous sampling.

Induction of SA by Wound Stress. As SA is a powerfulinducer of acidic PR proteins, its levels in young A2' leaveswere examined after injury of the upper epidermis of leaveswith carborundum. One day after injury the leaves beganproducing SA, and this reached a maximum level of =z180ng/g (fresh weight) on day 2 and was maintained for up to atleast 6 days (Fig. 3).

Induction of PI-II Transcripts by MeJA. To examinewhether the low PI-II transcript levels in the wounded A2plants resulted from defects fn the wound signal transductionsystem itselfor were due to the elevated levels ofSA (17), theeffect of MeJA on the PI-II transcript level was determinedby Northern blot hybridization (Fig. 4). The results clearlyshow that PI-II transcripts are equally induced by MeJA inboth wild-type and A2 plants, suggesting that in wounded A2plants overproduction of SA may inhibit the jasmonic acid(JA) biosynthetic pathways (17).Production ofAcidic PR-1 Proteins by Wound Stress. To test

whether the PR-1 mRNAs in transgenic plants were normallytranslated and the products were transported into the inter-cellular spaces (soluble fraction) as in normal plants, healthyyoung plants were wounded by detaching the lower leaves,and PR-1 proteins were isolated from lower, middle, andupper leaves and subjected to immunoelectrophoresis (Fig.5). Simultaneously, RNA was isolated from the same leavesand assayed for PR-1 mRNA levels. The results demonstratethat the PR-1 proteins were normally translated from theircorresponding mRNAs and transported to the soluble frac-tion as in normal plants (18). This observation was furtherconfirmed by the detection ofPR-1 proteins secreted into theculture medium from a suspension culture of A2 cells (datanot shown). In addition, the results demonstrate that the PR-1proteins were also synthesized in the middle and upperleaves, at levels that were in direct proportion to theirmRNAlevels and, therefore, decreased with distance from thedetached leaf, suggestive of a systemic response of the A2plants to wounding.

Systemic Induction of Acidic PR-1 Proteins, SA, and SAG inResponse to Wounding. The systemic induction of PR-1proteins by wounding was examined in further detail. Thefifth leaf (leaf 5) of healthy 3-month-old A2' plants waswounded every 2-4 days, and the amounts of PR-1 proteinsin leafS and also in the unwounded distal leaves 6 and 7 were

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FIG. 3. Induction ofSA by wound stress in the A2' plant. A fullyexpanded leaf from the wild-type or A2' plant was wounded withcarborundum, harvested after appropriate intervals as indicated, andthen used for quantitation of the SA content. The amount of free SAis expressed in ng/g (fresh weight). Values are the means of morethan three assays and the standard deviation is shown by bars.

WT A2/RorI I

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FIG. 4. Effects of wounding and MeJA on PI-II mRNA levels.Leafpieces from wild-type (WT) or A2 (A2/Ro) plants were floatedon water (lanes W) or a solution containing 50 ,tM MeJA (lanes J)at 20'C under continuous light for 3 days and used for RNAisolation. The control (lanes C) RNA was from untreated leaves.RNA (20 Ag) was assayed for mRNA levels of genes encodingactin, as an internal standard, and PI-II by Northern blot hybrid-ization.

measured. While the control plant showed no response towounding, there was a rapid production of PR-1 proteins inthe A2' plant after wounding (Fig. 6A). In addition, theunwounded distal leaves also produced PR-1 proteins after8-10 days (Fig. 6A). To determine whether the systemicresponse of PR-1 proteins to wounding was at all associatedwith a similar activation of SA and SAG, leaf S of a healthy3-month-old A2' plant was injured and assayed successivelyfor SA and SAG at appropriate intervals after injury. At thesame time, adjacent leaves (leaves 4 and 6) that had not beeninjured were sampled and similarly assayed for SA and SAG.The production of SA in both injured and uninjured leavesbegan as early as 6 h after injury (Fig. 6B). The induction ofSAG occurred much faster than that ofSA in both injured anduninjured leaves, reaching a maximum at 6 h after injury (Fig.6C). These experiments clearly demonstrate that the abnor-mal wound response of the A2 plants is systemically andrapidly transmitted to adjacent leaves.

Resistance of Transgenic Plants to TMV Infection. Asexogenously applied SA is known to induce resistance toviral infection (19), healthy leaves were detached from

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FIG. 5. Expression of acidic PR-i genes and products in the A2'plant. Leafsamples were obtained from a 3-month-old A2' plant fromwhich lower leaves had been repeatedly detached. Total RNA wasisolated from leaves taken from relatively basal positions (lanesLower), from the mid-region (lanes Middle), or from the distal (lanesUpper) positions ofthe plant and assayed for PR-i mRNA levels (A).Simultaneously, -4 mg of leaf tissue was assayed for PR-i proteinsin the soluble fraction by immunoblot analysis using a polyclonalantibody against highly purified PR-la protein. Known amounts ofPR-la proteins were subjected to coelectrophoresis for standardiza-tion (the four lanes on the right) (B).

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FIG. 6. Systemic induction of PR-1 proteins, free SA, and SAGby wounding of the A2' plant. Fully expanded fifth leaves (leaf 5) ofwild-type (Control) and Al' plants were wounded by punching outleaf discs. The wounded leaves (leaf 5) and the unwounded adjacentleaves (leaves 6 and 7) from individual plants were harvested at theindicated times from the first wounding and used for quantitation ofthe PR-1 protein (A). For quantitation of SA and SAG, similarexperiments were performed except that the leaves (leaf 5) ofthe Al'plants were wounded by gentle rubbing of the upper epidermis of theleaf with wet carborundum. The injured leaves (leafS) and adjacentleaves (leaves 4 and 6) from individual A2' plants were assayed forSA (B) and SAG (C). Values are the means ofmore than three assaysand the standard deviation is shown by bars. To simplify theillustration ofthe leafpositions, many lateral stems (tillers) ofthe Al'plant have been omitted.

wild-type and A2' plants and inoculated with TMV, and thesize of local lesions was then analyzed. The size of necroticlesions were considerably smaller in the A2 plants than inwild-type plants from about 2 days after inoculation (Fig.7A). As lesion size is almost proportional to the viralcontent (19), the relatively smaller lesions of the A2 plantsare clearly indicative of their increased resistance to TMVinfection (Fig. 7B). The necrotic lesions in the A2 plantswere visible within 24 h ofinoculation, suggesting that these

A2 TransgenicFiG. 7. Resistance of the A2 plant toTMV infection. Upper fully

expanded leaves of wild-type or A2' plants were detached, inocu-lated with TMV (10 /g/ml) by using carborundum (Mesh 600), andincubated at 20"C under continuous illumination. The size of locallesions, resulting from TMV infection of wild-type (o) and A2' (e)plants, was measured by counting 50 local lesions with a stereoscopicmicroscope and estimating a time course oflocal lesion development.Standard deviations are indicated by error bars (A). Lesion formationwas photographed 6 days after inoculation in wild-type (Control) andA2' (A2 Transgenic) plants (B).

plants have a hypersensitive response to pathogens incomparison with control plants that require at least 36 h todevelop visible local lesions.

DISCUSSIONTo date, =30 genes encoding small GTP binding proteinshave been isolated from higher plants, but little is knownabout their physiological functions (20). The notable excep-tions are the genes pra2 and pra3 from pea, which have beenshown to be negatively regulated by light and mediated byphytochrome (21), and rhal from Arabidopsis, which ispredominantly expressed in developing guard cells, suggest-ing that its product may function in vesicle transport (22).These observations are indicative of the diverse develop-mental processes in which these small GTP binding proteinsare involved and the various pleiotropic effects they mayhave in response to different environmental stimuli.A clear example of such involvement is our transgenic

tobacco plants containing rgpl, which contain 6-fold higherlevels of cytokinins than control plants. The small GTPbinding proteins are, therefore, also involved in phytohor-mone metabolism, and it is tempting to speculate that rgpl-p25 affects cytokinin biosynthesis by, for example, regulatingthe transport of enzymes of cytokinin biosynthesis or medi-ating the signals necessary for its induction.

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Wounding, or mechanical injury, usually induces the tran-scriptional activation of a specific set ofgenes encoding basicPR proteins (5, 6). In marked contrast, however, genesencoding acidic PR proteins are activated by necrotic lesionformation after pathogenic attack or by SA treatment (6, 23)but usually not by wounding (6, 24). The response of thetransgenic A2 plants to wounding in the present study is thusquite abnormal in comparison with normal tobacco plants, inthat there was a concomitant increase in the levels of acidicPR-1 transcripts and SA and SAG levels and a decrease inPI-II transcripts. Indeed, the endogenous SA levels increasedup to nearly 200 ng/g (fresh weight) within 2 days of injury ofthe A2 plant, which is comparable to that in leaves ofXanthitobacco plants 2 days after TMV infection (7). Since as littleas 54 ng ofSA perg (fresh weight) is sufficient to induce acidicPR proteins (16), we consider that the increased levels ofacidic PR-1 proteins and decreased levels of PI-II were adirect result of the increases in SA and SAG in response towounding. Thus, the mechanism of acidic PR protein induc-tion and PI-II suppression by SA appears to function nor-mally in the transgenic plants. It is, therefore, the inductionof SA by wound stress that constitutes the abnormal responseof these transgenic plants.Due to its diverse biological activities, it has been sug-

gested that SA functions as a native regulator and evenpossibly as a phytohormone (25). In particular, it is consid-ered to be involved in various signal transduction systems,especially those leading to systemic acquired resistanceagainst pathogen attack (10, 24). In contrast, SA is notinduced in response to wounding (7). Indeed, transduction ofthe wound signal is thought to follow a separate pathway fromthat ofSA (10) and to involve ethylene and/or JA, which mayact as direct intracellular signal intermediates (26, 27). Withregard to the distinct wound and pathogen-induced signalpathways, it should be noted that the biosynthesis ofethyleneand JA is inhibited by SA (17). Thus, in normal plants, thesignal cascade involving SA is regulated quite distinctly fromthat involving ethylene and/or JA.

In transgenic plants expressing rgpl, however, the nor-mally distinct pathogen- and wound-induced signal transduc-tion systems appear to be modified, possibly by an unusualcrossing of the relevant signals. As it is generally consideredthat some phytohormones, such as cytokinins, auxins, andabscisic acids, are involved in the pathogen and woundsignaling pathways (4, 28), and as the A2 transgenic plantsconstitutively produce high levels of cytokinins, it is possiblethat the hormonal imbalance induced by rgpl-p25 is theprimary cause of the observed effects. Although the woundsignal reception system may be highly sensitized by theelevated cytokinin levels in our transgenic plants, this stilldoes not explain the mechanism by which the wound signalinduces SA.An additional possibility for this induction of SA is that, in

addition to mediating abnormal cytokinin production, rgpl-p25 or its homologs are directly involved in, or disturb, themolecular switch system (20) of wound and pathogen signaltransduction. Based on the structural similarities between JAand prostaglandins, which serve as powerful signaling mol-ecules in animals, it has been proposed that a lipid-basedsignaling system may also function in the control of plantgene expression, particularly in the stress response (29).Since GTP binding proteins function as the direct receptorsof prostaglandins (30) and since it has been proposed thatsome ras-related small GTP binding proteins are involved inthis signaling pathway (31), the involvement of a set of smallGTP binding proteins in the wound signaling system in plantsseems highly probable. If so, a disturbance in such signalcascades may result in cross-signaling ofthe normally distincttransduction pathways. Since the A2 plants appear to re-

spond normally to MeJA in the production of PI-II, suchcross-signaling may occur further upstream of the JA signal-ing pathway, for example, in plasma membranes (17, 32),where the GTP binding proteins are supposed to function asmolecular switches (33).

We thank Ms. Y. Mimura, Ms. M. Kudo, Biotechnology Institute(BI), Ms. Y. Gotoh, and Ms. H. Ochiai, National Institute ofAgrobiological Resources (NIAR) for maintenance of the plantmaterials and technical assistance; Mr. M. Ohshima, Dr. M. Mat-suoka, and Dr. M. Ugaki (NIAR) for valuable discussions; and Ms.K. Futada (BI) for manuscript preparation.

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