A benzothiadiazole derivative induces systemic ...

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A benzothiadiazole derivative induces systemic acquired resistance in tobacco Article in The Plant Journal · July 1996 DOI: 10.1046/j.1365-313X.1996.10010061.x

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Jean-Pierre Metraux

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The Plant Journal (1996) 10(1), 61-70

A benzothiadiazole derivative induces systemic acquired resistance in tobacco Leslie Friedrich 1, Kay Lawton 1, Wilhelm Ruess z, Peter Masner 2, Nicole Specker2, Manuela Gut Rella 2, Beatrice Meier 2, Sandra Dincher 1, Theodor Staub 2, Scott Uknes 1, Jean-Pierre Metraux 3, Helmut Kessmann 2 and John Ryals 1,* lCiba-Geigy Agricultural Biotechnology, P.O. Box 12257, Research Triangle Park, NC 27709-2257, USA, 2Plant Protection Division, Ciba-Geigy Ltd, CH-4002 Base/, Switzerland, and 31nstitut de Bio/ogie V~g~tale, Universit~ de Fribourg, CH- 1700 Fribourg, Switzerland Summary Systemic acquired resistance (SAR) is a pathogen-induced disease resistance response in plants that is characterized by broad spectrum disease control and an associated coordinate expression of a set of SAR genes. Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a novel synthetic chemical capable of inducing disease resistance in a number of dicotyledenous and monocotyledenous plant species. In this report, the response of tobacco plants to BTH treatment is characterized and the fact that it controls disease by activating SAR is demonstrated. BTH does not cause an accumulation of salicylic acid (SA), an intermediate in the SAR signal transduction pathway. As BTH also induces disease resistance and gene expression in transgenic plants expressing the nahG gene, it appears to activate the SAR signal transduction pathway at the site of or downstream of SA accumulation. BTH, SA and TMV induce the PR-la promoter using similar cis-acting elements and gene expression is blocked by cycloheximide treatment. Thus, BTH induces SAR based on all of the physiological and biochemical criteria that define SAR in tobacco. Introduction Systemic acquired resistance (SAR) is an inducible defense mechanism that plays a central role in disease resistance (Delaney et aL, 1994; Ross, 1961). SAR is induced by most pathogens that cause tissue necrosis ranging from a hypersensitive response (HR) (Delaney et al., 1994; Ross, 1961) to a disease lesion (Cruikshank and Mandryk, 1960; Hecht and Bateman, 1964; Jenns and Kuc, 1979) and is characterized by a long-lasting, systemic resistance against

Received15December1995;revised22 March1996;accepted17April 1996. *For correspondence(fax+1 919 541 8557;[email protected]).

a broad spectrum of pathogens (Kuc, 1982; Mclntyre et aL, 1981; Ryals et al., 1994). It has been proposed that SAR could serve as the basis for novel disease control strategies which include genetically engineered plants with enhanced disease resistance and novel plant protection chemicals (Chester, 1933; Kuc, 1982; Ryals et al., 1994). Tobacco is one of the best characterized systems for the study of SAR. Tobacco mosaic virus (TMV) infection of local lesion hosts leads to the development of resistance against subsequent TMV infections as well as resistance against other viruses (Ross, 1961), fungi and bacteria (Mclntyre et aL, 1981; Vernooij et aL, 1995). However, SAR is not effective against all tobacco pathogens (Vernooij et al., 1995); there is a defined spectrum of pathogen resistance that serves to distinguish the SAR response from other inducible disease resistance responses. SAR is also characterized by the associated, coordinate accumulation of mRNAs encoded by a set of SAR genes (Ward et al., 1991). Expression of this set of nine gene families, which comprise many of the genes encoding pathogenesisrelated (PR) proteins, also serves as a criterium that can distinguish SAR from other resistance responses. Thus, in tobacco SAR can be defined as an inducible, systemic disease resistance response that provides protection against a specific group of pathogens and is associated with the expression of a particular set of genes. Little is known about the signal transduction process that leads to SAR in tobacco. It is assumed that at some point during the formation of a necrotic lesion, a signal is released that travels throughout the plant to trigger SAR. This unknown signal is perceived in target cells and is transduced by a signal transduction pathway that is dependent on the accumulation of salicylic acid (SA) (Malamy et al., 1990; M~traux et al., 1990; Rasmussen et al., 1991; Vernooij et al., 1994). Following SA accumulation, SAR genes are induced and the establishment of the resistance state is tightly correlated with this gene expression (Vernooij et al., 1994; Ward et aL, 1991). This is particularly true for the expression of PR-la, a protein with unknown function in tobacco. Interestingly, transgenic plants expressing PR-la have decreased susceptibility to Peronospora tabacina and Phytophthora parasitica, suggesting that SAR gene expression may play an active role in the maintenance of resistance (Alexander et aL, 1993). Certain chemicals have been shown to induce SAR in tobacco. For example, the exogenous application of either SA or 2,6-dichloroisonicotinic acid (INA) to leaves results in an induction of resistance against the same spectrum of pathogens and induction of the same SAR genes as 61

62

Leslie Friedrich et al.

with TMV (Vernooij et al., 1995; Ward et al., 1991). Thus, both of these chemicals are capable of inducing SAR. However, SA only induces SAR in the leaf tissues that have been treated with the compound. This is not surprising because SA is quickly converted to a glucoside which is likely immobile in the plant (Enyedi et al., 1992; Malamy et al., 1992). Because SA appears to function in signal transduction and not as a signal itself (Beffa et aL, 1995; Rasmussen et al., 1991; Vernooij et al., 1994), it would have to be translocated in order to induce the SAR response systemically. On the other hand, INA has been shown to induce SAR both in treated and untreated tissues. Because INA does not cause SA accumulation and INA induces SAR in transgenic tobacco plants unable to accumulate SA, INA most likely functions as a mimic of SA, inducing SAR at the same site or downstream of SA accumulation (Vernooij et al., 1995). However, INA is efficiently translocated in plants and thus its systemic activity is most likely due to its own translocation and not the release of a systemic signal. Presently, two compounds, SA and INA, have been clearly demonstrated to induce SAR. Other compounds have been shown to induce either PR-1 gene expression or resistance against one or two pathogens, but they have not been shown to induce all of the characteristics of SAR (Asselin et al., 1985; Cohen, 1994; Iwata et al., 1980). While both SA and INA are potent inducers, both have problems with crop tolerance (e.g. phytotoxicity) which has prevented their development as plant protection compounds. Here we describe the mode-of-action of a novel compound, benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (i.e. BTH, CGA 245704; Schurter et al., 1987). We show that, based on all of the criteria that define SAR in tobacco, BTH works by activating SAR. BTH is translocated throughout the plant and most likely activates SAR signal transduction at a step downstream or at the same site of SA accumulation. BTH is currently being developed as a novel disease control compound that acts by inducing an inherent disease resistance response in the plant. Results

Spectrum of activity of BTH in tobacco Benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) was identified in a biological screen as a compound that caused disease resistance in tobacco and cucumber but had no antifungal activity. The chemical structure for BTH is shown in Figure 1; details of the synthesis and the field activity will be published elsewhere (manuscript in preparation). Because BTH treatment led to disease resistance with no direct antifungal activity, it was possible that the compound acted by inducing SAR. Chemical activators of SAR have been proposed to exhibit three characteristics:

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Figure 1. Structureof BTH.

(i) the chemical should induce resistance againstthe same spectrum of pathogens as in the biological model; (ii) neither the compound nor its significant metabolites should have antimicrobial activity; (iii) chemical treatment should induce expression of the same biochemical markers as in the biological model (Kessmann et al., 1994). To determine whether BTH acted as an activator of SAR in tobacco, we evaluated the ability of the chemical to fulfill these criteria. In tobacco, SAR can be induced by tobacco mosaic virus (TMV) inoculation of local lesion hosts (e.g. Xanthi.nc). Resistance is established in the uninoculated leaves 5-7 days following this inducing inoculation. As shown in Figure 2, both TMV inoculation and BTH treatment induced resistance against Cercospora nicotianae (frog eye leaf spot), Erwinia carotovora, Phytophthora parasitica (black shank), Pseudomonas syringae pv. tabaci (bacterial wild fire) and TMV. BTH also induced resistance to Peronospora tabacina (blue mold); TNV (tobacco necrosis virus) inoculation was not included in this particular experiment, but has been included as a control in other experiments and TNV-induced resistance to P. tabacina has been reported (Vernooij et al., 1995). Neither the biological inducers nor BTH treatment induced resistance against Alternaria alternata or Botrytis cinerea (data not shown). Thus, for the eight pathogens tested, BTH induced resistance against the same spectrum of fungal, bacterial and viral pathogens as observed with TMV and failed to protect against the same two fungal pathogens as with TMV induction. Given the broad spectrum activity of BTH against several classes of fungi, two different bacteria and a virus, it would be unusual if this compound acted as an antibiotic. However, to test directly the antimicrobial activity of BTH, we carried out standard in vitro evaluations on the 18 fungi listed in Table 1. No antifungal activity of BTH was detected, even at concentrations higher than those present in treated plant tissues. Similar results were obtained with the major metabotite of BTH (the free carboxylic acid) as well as with several minor metabolites (data not shown). Thus, BTH

BTH induces SAR in tobacco 150

100

Table 1. In vitro assays for BTH activity against different fungi

Alternaria brassicae Alternaria brassicicola Botrytis cinerea Ceratocystis ulmi Cladosporium cucumerinum Fusarium culmorum Helminthosporium oryzae Helminthosporium teres Mucor hiemalis

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Penicilfium digitatum Penicillium expansum Penicillium italicum Phytophthora infestans Pyricularia oryzae Rhizoctonia solani Septoria nodorum Ustilago maydis Verticiflium dahliae

BTH was tested at 1.4 mM against the fungi listed for antibiotic activity. The in vitro assays were standard plate assays as described in Experimental procedures.

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mco,ro, ) . . v D.T. Figure 2. BTH provides protection against a broad spectrum of pathogens. The levels of pathogen protection provided by pretreatment of Xanthi.nc tobacco plants with 1.2 mM BTH relative to untreated controls or TMVinduced plants were compared. Plants were treated and disease symptoms were evaluated as described in Experimental procedures. Each bar in the histograms represents the average disease level of six plants. The solid bars represent values from untreated controls, the hatched bars represent values of plants following an inducing inoculation of TMV and the open bars represent values following BTH treatment.

appears to effect disease control in tobacco through an indirect mechanism.

BTH protects tobacco from systemic infection of TMV

SAR has not been reported to protect TMV-susceptible tobacco (i.e. lacking the N or N' TMV resistance gene) from TMV infection. Because BTH appeared to be a very strong resistance inducer, we tested its ability to provide TMV resistance in a tobacco cultivar without N gene-mediated resistance (Xanthi 'nn'). Figure 3(a) shows leaves from TMV-inoculated plants that were pretreated with either water (left) or 1.2 BTH (right). TMV-inoculated control leaves exhibited mosaic symptoms typical of TMV infection while leaves from BTH-treated plants did not show disease symptoms. Furthermore, as shown in the RNA gel blot in Figure 3(b), viral RNA accumulation in BTH-treated plants was reduced in both the TMV-inoculated leaves and the

Figure 3. BTH acts as an antiviral compound against TMV in genetically susceptible tobacco. Xanthi 'nn' plants were pretreated with H20 or 1.2 mM BTH 7 days prior to TMV infection. Twelve days after TMV inoculation, leaves were harvested and photographed or frozen for RNA extractions. (a) Upper, uninoculated leaves from control (left) and BTH-treated plants (right). (b) Northern blot of total RNA extracted from inoculated (I) and uninoculated (U) leaves; the blot was hybridized to a 32p-labeled probe derived from reverse-transcribed TMV RNA.

64


uninoculated leaves. Based on Phosphorlmage analysis of the RNA blots, TMV levels were reduced by 95% in the inoculated leaves of BTH-treated plants relative to TMVinfected control leaves and by 98% in the uninoculated BTH-treated leaves relative to uninoculated control leaves (data not shown). Interestingly, in BTH-treated plants there was no sign of lesions associated with TMV infection, even upon close inspection of the inoculated leaves. Further microscopic inspection or inspection of the leaves illuminated by UV light also did not reveal the presence of lesions. Thus, substantial resistance was induced in these plants by BTH without the occurrence of an associated HR.

(b) 1.5

BTH induces PR-1 mRNA accumulation and TMV protection in a dose-dependent manner

If the mode-of-action of BTH is to induce SAR in tobacco, then both TMV resistance and PR-I mRNA accumulation would be expected to increase in a dose-dependent fashion following BTH application. Accumulation of PR-1 mRNA provides a useful molecular marker for SAR as its expression is tightly correlated with the establishment of the resistant state (Vernooij et al., 1995; Ward et aL, 1991). The Northern blot in Figure 4(a) shows that an increase in PR-1 mRNA accumulation was observed at BTH concentrations as low as 1.2 pM and reached a maximum level of expression at about 36 tJM. As shown in Figure 4(b), the size of TMV lesions decreased as a function of BTH concentration. In addition, the size of TMV lesions correlated inversely with PR-1 mRNA accumulation.

BTH induces SAR mRNA accumulation

A number of genes are coordinately expressed in tobacco leaves during the induction and maintenance of SAR (Ward et al., 1991). If BTH acts by inducing SAR, then the accumulation of the same set of mRNAs should result. Tobacco plants were treated with 1.2 mM BTH and leaves were harvested for RNA extraction and analysis at various times after treatment. As shown in Figure 5, BTH treatment results in the coordinate induction of the same set of mRNAs that are induced by TMV. These same mRNAs are also induced by application of SA which is an intermediate involved in the SAR signal transduction cascade. The accumulation of these SAR mRNAs was observed within 4-12 h after BTH treatment, consistent with the induction by SA. Similarly, the basic glucanase mRNA was slightly induced by BTH treatment from an already high constitutive level, while the basic chitinase did not seem to be BTHinduced. In this particular experiment the acidic peroxidase mRNA was induced slightly at 1 and 3 days, but this was not a reproducible response.

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BTH (mM) Figure 4. PR-1 mRNA accumulation and TMV resistance in Xanthi.nc plants treated with increasing concentrations of BTH. (a) Total RNA was extracted from leaves harvested 7 days following BTH treatment. The Northern blot was hybridized with a 32p-labeled probe derived from a cDNA clone of PR-I (Ward et al., 1991). (b) Additional BTH-treated leaves were infected with TMV and, after 7 days, at least 60 lesions per treatment were measured. The average lesion sizes + standard deviations were plotted as a function of BTH concentration.

BTH does not induce the accumulation of either free or total SA

One step in the signal transduction pathway of biologically induced SAR leading to the resistant state is the accumulation of free and glucosylated SA. To determine whether BTH induced SA accumulation, plants were treated with 1.2 mM BTH, samples were harvested at various times following BTH application and the levels of free and total SA were measured. It is clear from the results shown in Figure 6 that BTH does not induce a significant increase in SA concentration. Following TMV infection of Xanthi.nc plants, the levels of free and total SA typically increase to 3.6 tJg gfw -1 and 25 pg gfw -1, respectively (Enyedi et al., 1992; Friedrich etal., 1995), which is two to three orders of magnitude higher than measured following BTH treatment. Thus, it is unlikely that BTH induces resistance or SAR mRNA accumulation through SA.

BTH induces SAR in tobacco

65

BTH induces PR-1 mRNA accumulation and disease resistance in NahG plants Transgenic tobacco plants that express a bacterial salicylate hydroxylase gene (nahG) cannot accumulate significant amounts of SA (Gaffney et al., 1993). If BTH induces SAR independently of SA, then both PR-1 mRNA accumulation and disease resistance should be induced in NahG plants following BTH application. To test the dependence of BTH activity on SA accumulation, NahG tobacco plants were sprayed with water or 1.2 mM BTH and plants were assayed for accumulation of PR-1 mRNA as well as resistance to P. tabacina (blue mold) and TMV. As shown in the Northern blot in Figure 7, PR-1 mRNA accumulated in NahG plants with essentially the same time course as wild-type Xanthi.nc (see Figure 5). The development of blue mold disease following water or BTH treatment of Xanthi.nc, NahG or NahG-9, a transgenic tobacco line that harbors the nahG gene but neither accumulates nahG mRNA nor inhibits SA accumulation (Gaffney et aL, 1993), is shown in Figure 8. All three tobacco lines show resistance to blue mold disease upon application of BTH. These results demonstrate that BTH treatment can induce resistance against P. tabacina in a dose-dependent manner that is independent of the expression of the nahG gene. Furthermore, experiments with TMV shown in Table 2 show that lesion size is reduced in NahG plants pretreated with BTH. Taken together, the results in Figures 6, 7 and 8 and Table 2, indicate that BTH induces resistance in an SAindependent manner.

BTH activates the PR la promoter using cis-acting sequences similar to SA and TMV We have previously shown that regions of the PR-Ia promoter upstream of position -661 are sufficient for maximal induction of GUS activity by either SA or TMV (Uknes et aL, 1993). To determine whether BTH activated the PR-la promoter using these same sequences, tobacco plants transformed with 5' deletions of the PR-la promoter fused to GUS (Uknes et al., 1993) were treated with BTH. Expression of the GUS reporter gene following BTH treatment was assayed in 11-20 independent transformants for each deletion construct. The results shown in Figure 9 indicate that GUS activity was maximally induced by BTH in plants containing promoter fragments longer than 661 bp, while plants with constructs containing 600 bp or less were not strongly induced. This result is consistent with our Figure 5. Timecourseof the accumulationof mRNAencodedby SARgenes in BTH-treatedXanthi.ncplants. Tissueswere harvestedat the indicatedhoursor days following treatment and total RNA was extracted from these tissues. Individual blots were hybridizedwith 32p-labeledprobes derived from the indicatedcDNAs.The origins of the cDNAsare listed in Table 2 from Ward eta/. (1991),


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Figure 6. Accumulation of free and total SA in response to BTH treatment. Tobacco leaves from wild-type Xanthi.nc (Xanthi) and NahG plants were harvested at various times after BTH treatment and SA levels were measured (see Experimental procedures). Total SA equals free SA plus glucosylated SA (SAG) levels. Values are shown as the average z: standard deviation of triplicate assays. For reference, free SA levels reach 3.6 pg gfw -~ in TMV-inoculated plants while total SA can routinely reach levels of 25 ~tg gfw -1 (Enyedi et aL, 1992; Friedrich et al., 1995).

previous studies (Uknes et al., 1993) and those of Van de Rhee (Van de Rhee et al., 1990) showing that the cis-acting elements sufficient for PR-1a induction by SA and TMV are located upstream of position -643 in the PR-la promoter. Also, because BTH induces GUS accumulation, at least part of the mRNA accumulation seen following BTH treatment is due to transcriptional activation. The tissue specificity of GUS induction following BTH treatment of a number of independent lines containing the -903 bp PR-la promoter/GUS construct was also analyzed. The results shown in Table 3 demonstrate that GUS activity was expressed primarily in green leaf tissues. While some expression was observed in other tissues of the plant, the highest levels were in leaf tissue. This result is consistent with our previous findings with TMV-infected and SA-treated plants (Uknes et al., 1993). Therefore, BTH has the same effect on PR-1a gene expression as TMV and SA based on both the promoter elements involved and the tissue specificity of PR-Ia expression.

BTH induction of PR-1 is dependent on protein synthesis We have previously demonstrated that SA-induced PR-1a mRNA accumulation is blocked by cycloheximide (CHX) which suggests that ongoing protein synthesis is required for PR-1a gene expression (Uknes et al., 1993). To understand further where BTH might act in the SAR signal

Figure 7. PR-1 mRNA accumulation in NahG tobacco plants following BTH treatment. Material was obtained from NahG plants as described in Figure 5, The Northern blot was hybridized to a 32p-labeled probe for PR-1 as in Figure 4,

transduction pathway, we asked if BTH-induced PR-Ia mRNA accumulation was affected by CHX. Xanthi.nc or NahG tobacco leaves were injected with water, 1.2 mM BTH or a 1 mg m1-1 CHX + 1.2 mM BTH solution (the latter after a pretreatment with 1 mg m1-1 CHX). In previous experiments, treatment of tobacco leaves with 1 mg m1-1 CHX reduced protein synthesis by 99% (Uknes et al., 1993). Treated tissues were harvested 24 h post-treatment and analyzed for PR-1 mRNA accumulation. As shown in Figure 10, PR-1 mRNA was not detectable in tissues treated with the CHX + BTH solution (lanes 4 and 5), while plants treated with BTH alone accumulated substantial levels of PR-1 mRNA (lanes 2 and 3). Thus, BTH-induced PR-1 mRNA

BTH induces SAR in tobacco • Xanthi.nc [ ] NahG E~ NahG-9

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67

Table 2. TMV lesion measurements in control and induced tobacco leaves Water

BTH

Experiment

Lesion size (mm)

Lesionsize % of water (mm) control

Xanthi.nc

1 2

0.98 -+ 0.22 1.07 + 0.18

0.22 _+0.04 0.31 _+0.07

22 29

NahG

1 2

2.62 +_0.63 1.74 +_0.35

0.27 +_0.06 0.34 _+0.08

10 20

80

Plants were treated and lesions measured in two different experimentsas described in Experimentalprocedures.The relative reduction in lesion sizes induced by BTH pretreatment is given as % of water control. 0 CON

0

\ 0.03

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1.0

BTH (mM) Figure 8. Infection of Xanthi.nc and NahG tobacco plants by P. tabacina following treatment with increasing concentrations of BTH. Xanthi.nc, NahG (expressing the nahG gene) and NahG-9 (carrying but not expressing the nahG gene) plants were treated with a wettable powder solution containing no active BTH (0 mM) or increasing concentrations of BTH. These plants were infected with P. tabacina and disease symptoms measured as described in Experimental procedures. Untreated control plants (Con) were also inoculated as a check for disease development.

accumulation also appears to depend upon de novo protein synthesis, suggesting that the gene may be regulated, at least partially, by a factor that requires de novo synthesis prior to gene activation.

Discussion

BTH induces the SAR response in tobacco In 1961, Ross reported that TMV infection of local-lesionhost tobacco plants (e.g. Xanthi.nc) resulted in resistance to subsequent TMV infection in the uninfected leaves of these plants. This response was called systemic acquired resistance (SAR). Based on many years of study, SAR is now characterized by the spectrum of pathogen resistance induced, distinct biochemical changes associated with this resistance and the systemic nature of the resistance (Ryals et al., 1994). BTH treatment of tobacco leads to the same broadspectrum pathogen resistance as induced by TMV inoculation. Resistance is conferred against three fungi, two bacteria and a virus. Furthermore, neither BTH nor TMV protect tobacco against Alternaria alternata or Botrytis cinerea. Thus, for the eight pathogens tested, BTH and TMV treatment provide the same spectrum of pathogen control. Importantly, BTH-dependent resistance does not appear to be due to an antibiotic effect of the compound.

Experiments with a diverse set of fungal species showed that neither BTH nor its major metabolites exhibit in vitro activity, even at concentrations that exceed levels shown to be efficacious in plants. It cannot be excluded that BTH might have a very active minor metabolite that in some way acts synergistically with a plant component to form a potent antibiotic mixture. However, if this were the case then the antibiotic would possess an unprecedented activity against viral, fungal and bacterial pathogens. We find this possibility unlikely. Gene expression associated with SAR in tobacco is reasonably well characterized. Coordinate accumulation of mRNA from at least nine gene families is observed in tobacco tissues manifesting SAR after induction by TMV, SA and INA (Ward etal., 1991). BTH induces the accumulation of this same set of mRNAs. Moreover, TMV inoculation, SA and BTH treatment all induce PR-1 gene expression using the same cis-acting promoter elements and in the same tissue types (Uknes et al., 1993). In addition, PR-1 mRNA accumulation by both SA and BTH are blocked by CHX treatment. Thus, based on all of the biochemical criteria that characterize the SAR response and based on the spectrum of pathogen protection, BTH induces SAR in tobacco.

Does BTH induce a systemic signal? Another characteristic of SAR is that it is a systemic resistance. Because BTH is highly mobile in tobacco (data not shown), it is unclear how to determine whether it is capable of inducing a systemic signal. However, BTH appears to stimulate the SAR signal transduction pathway by acting at a step downstream or at the same site of SA accumulation. This is based on the observations that BTH does not induce SA accumulation (Figure 6) and that BTH induces both SAR gene expression and pathogen resistance in NahG plants (Figures 7 and 8, Table 2). It is possible that BTH could induce the responses following


68

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Figure 9. Induction of GUS activity in transgenic tobaccoplants. Plantscontainingconstructswith deletionsof the PR-la promoter from -903 to -73 bp fused to a GUS reportergenewere previously described (Uknes et aL, 1993). Leaves from plantscontainingthe indicatedconstructwere treatedwith 1.2 mM BTH and fluorometrically assayed for GUS activity. Each point representsthe levelof GUS activity in an individual primary transformant after BTH treatment divided by the GUS activityof the sameclone after treatmentwith H20.

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aData derived from Uknes et al. (1993). b_ No detectable GUS activity. c÷ to + + + Increasing degrees of GUS staining. dExpressed predominantly in mesophyll cells. eExpressed predominantly in the cortex.

BTH inhibits mosaic disease without inducing an associated HR Because BTH was such a potent inducer, we tested whether it could inhibit TMV in susceptible (i.e. non-local lesion) hosts. BTH application resulted in substantial protection against both mosaic disease and viral replication (Figure 3). The level of TMV inhibition is close to that seen with N gene-dependent genetic resistance. However, there was no sign of HR lesions on the BTH-treated, TMV-infected leaves, even upon microscopic examination. This result leads to the question of what role the HR plays in TMV resistance. While an HR is associated with some, but not all pathogen resistance, it is clear that there is live virus in green tissues surrounding TMV lesions (Israel and Ross, 1967). Thus, the role of the HR in limiting TMV spread and in genetically determined disease resistance mechanisms is not clear.

Importance of BTH as a plant activator Figure 10. Requirementof protein synthesisfor BTH-inducedPR-1mRNA accumulation. Xanthi.nc or NahG plants were treated as described in Experimental proceduresand RNA was extractedfrom treatedtissues.The Northern blot was hybridized to a 32p-labeledprobe for PR-1 as in Figure 4. Lane 1, Xanthi.nc treated with H20; lane 2, Xanthi.nc treated with BTH; lane 3, NahGtreatedwith BTH; lane4, Xanthi.nctreatedwith CHX + BTH; lane 5, NahG treatedwith CHX - BTH. an independent signaling pathway. However, we find this to be unlikely because BTH induction of both resistance and SAR gene expression is blocked in the Arabidopsis n i m l mutant (Lawton et al., 1996) which has been shown to have a suppressed SAR signaling pathway (Delaney et aL, 1995). Therefore, since SA has been shown to be involved in the transduction of the systemic signal and BTH appears to stimulate the pathway at or downstream of SA accumulation, it appears that BTH induces SAR by stimulating signal transduction and not by signal release.

As early as 1933, Chester had recognized the potential impact on agriculture that could result from the manipulation of a plant's inherent 'immune' system (Chester, 1933). Because of the broad-spectrum, systemic and long-lasting resistance characteristics of SAR, it has been suggested as a basis for disease control (Kuc, 1982; Ryals et al., 1994). Following BTH treatment, plants as diverse as tobacco, tomato, cucumber, wheat and rice are protected in the field against various diseases (our unpublished results). Plants treated with appropriate concentrations of BTH show no negative affects on yield or crop appearance. Thus, BTH is currently under development as a novel disease control technology. Along with conventional fungicides, biocontrol organisms and improved seed varieties, BTH will provide the farmer with a new option for disease control. We believe that the future use of BTH and related plant activators will have a profound impact on the control of plant disease.

BTH induces SAR in tobacco

Experimental procedures Plant material and treatments The following tobacco genotypes were used in these experiments: Xanthi.nc, a local lesion host for TMV; Xanthi 'nn', a systemic susceptible host for TMV; NahG, a transgenic Xanthi.nc line (also called NahG-10) that expresses the bacterial enzyme salicylate hydroxylase which metabolizes salicylic acid to catechol (Friedrich et al., 1995; Gaffney et aL, 1993); and NahG-9, a transgenic Xanthi.nc line that contains but does not express the nahG gene (Gaffney et al., 1993). Tobacco plants were grown and TMV inoculations were performed as previously described (Payne et aL, 1990). Water or BTH treatments were applied as a fine mist to leaves; a 1.2 mM BTH formulation as a 25% active ingredient in wettable powder was used unless specified otherwise. The wettable powder solution used in the BTH formulation does not induce SAR gene expression or resistance (Ryals, unpublished results). For each experiment, at least three different plants of each genotype were treated and tissues from like-treated leaves were pooled upon harvesting for analysis.

Pathogen assays For each of the pathogen assays shown in Figures 2 and 8, tobacco plants were pretreated by spraying with BTH or inoculating with TMV, or no pretreatment was given (controls). Seven days later, plants were challenged with the pathogen. TMV, Cercospora nicotianae, Peronospora tabacina, Phytophthora parasitica var. nicotianae and Pseudomonas syringae pv. tabaci assays were performed as described (Vernooij et al., 1994, 1995). Erwina carotovora assays were performed as follows: E. carotovora (EC12312 ATCC) was cultured for 24 h on YDC agar then used to make a bacterial suspension (5x105 m1-1) in distilled water. Stems from the top of each plant were cut with scissors dipped in this solution and plants were incubated at 22-24°C under 100% humidity. Four to five days later, the stem was cut (vertically) and the length of the lesion was measured. At least 10 plants were used per treatment. For TMV lesion measurements in Figure 4(b) and Table 2, plants were pretreated with water or BTH and challenged with TMV 7 days later. Lesions were measured (mm) 7 days following the TMV inoculation. A minimum of 10 lesions per leaf on nine leaves were measured for each treatment.

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RNA analysis RNA was isolated from frozen tissue samples by phenol-chloroform extraction followed by lithium chloride precipitation (Lagrimini et al., 1987). Total RNA samples (10 i~g) were electrophoretically separated through formaldehyde-agarose gels and blotted to hybridization membrane (GeneScreen Plus; DuPontNew England Nuclear Research Products) as described (Ausubel, et aL, 1987). Equal loading of samples was confirmed by including 40 ~tg m1-1 ethidium bromide in the sample loading buffer allowing visualization of RNA by photography under UV light. 32p-labeled cDNA probes were synthesized by random priming of isolated insert DNA using the random primers DNA labeling system (Gibco BRL). The cDNA probes were described by Ward et aL (1991). Hybridization and washing conditions were as described (Church and Gilbert, 1984). Relative amounts of transcript were determined using a Phosphorlmager (Molecular Dynamics) according to the manufacturer's instructions.

Deletion constructs and GUS analysis Tobacco plants transformed with constructs containing 5' deletions of the PR-la promoter fused to a GUS reporter gene were have been described (Uknes et al., 1993). Eleven to twenty independent primary transformants for each construct were treated with water or BTH and fluorometrically assayed for GUS activity as described (Uknes et aL, 1993). Fold induction by BTH was determined by dividing the GUS activity after BTH treatment by the GUS activity of the same clone (prepared by vegetatively splitting each plantlet at the five-leaf stage) after treatment with water. Tissue-specific expression of GUS activity following water, SA, TMV or BTH treatments of the -903 PR-Ia/GUS plants was determined by in situ assays performed as described (Uknes et aL, 1993).

Cycloheximide treatments Xanthi.nc or NahG leaves were injected with water, 1.2 mM BTH or a 1.2 mM BTH + 1 mg m1-1 CHX solution by pricking fully expanded leaves with a needle and forcing liquid into the leaves with a 10 ml syringe. Water-soaked areas were marked and harvested 1 day later for RNA extraction and analysis. Leaves treated with BTH + CHX were pretreated with 1 mg m1-1 CHX 1 h prior to injection of the BTH + CHX solution.

Acknowledgments In vitro assays Media for the in vitro assays were prepared as follows: a 2% filtered rye solution was added to 1% yeast extract, 0.5% Dglucose, 1% PDA and 1% agar and autoclaved. BTH was added to the warm media at a final concentration of 1.4 mM. Mycelia discs were placed on to the middle of solidified agar plates and radial growth determined over several days.

Salicylic acid extractions and analysis Free and total SA analysis was conducted on water- and BTHtreated leaves at various times after treament with 1.2 mM BTH. SA levels of triplicate samples were determined as described (Gaffney et al., 1993; Uknes et al., 1993). SA levels were not corrected for recovery.

We thank Dr Bernard Vernooij for assistance with SA assays and helpful discussions, Jay Johnson for excel lent technical assistance, Maggie Blair for plant care and Drs Bernard Vernooij, Henry-York Steiner and Dieter Nordmeier for critical reading of the manuscript.

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