<Beta>-Aminobutyric Acid-Mediated Enhancement of Resistance In

Physiological and Molecular Plant Pathology (2000) 56, 95–106 doi:10.1006\pmpp.1999.0255, available online at http:\\www.idealibrary.com on

β-Aminobutyric acid-mediated enhancement of resistance in tobacco to tobacco mosaic virus depends on the accumulation of salicylic acid

J. SIEGRIST*, M. OROBER and H. BUCHENAUER Institute of Phytomedicine, UniŠersity of Hohenheim, D-70593 Stuttgart, Germany

(Accepted for publication December 1999)

A number of chemical and biological agents are known as inducers of systemic acquired resistance (SAR) to tobacco mosaic virus (TMV) in tobacco plants. In the present study, a local spray application of the non-protein amino acid DL-β-aminobutyric acid (BABA) was effective in enhancing resistance to TMV in tobacco plants containing the N gene. In contrast, the isomer α-aminobutyric acid (AABA) showed a much lower activity whereas γ-aminobutyric acid (GABA) was completely inactive, indicating a strong isomer specificity of aminobutyric acid in triggering enhanced virus resistance. Rapid cell death was detected in tobacco leaf tissues after foliar application of BABA, subsequently resulting in the development of macroscopically visible, necrotic lesions. BABA-induced cell death was associated with the rapid generation of superoxide and hydrogen peroxide. As further consequences, the occurrence of lipid peroxidation in treated tissues, a local and systemic increase of salicylic acid (SA) levels and the expression of PR-1a, a molecular marker of SAR in tobacco, could be observed. None of these responses was detectable after treatment with GABA. Enhancement of virus resistance by BABA was found to be strictly dependent on SA-mediated signal transduction since it could not be detected in salicylate hydroxylase (nahG) expressing transgenic tobacco plants. These findings suggest that in tobacco, primary processes triggered by foliar application of BABA, resemble those initiated by microbes during a hypersensitive response (HR) that result in SAR activation. # 2000 Academic Press

Keywords: β-Aminobutyric acid; BTH; hypersensitive response; reactive oxygen species; systemic acquired resistance; tobacco; tobacco mosaic virus.

INTRODUCTION In this relation, scientific activities were mainly focused on the phenomenon of induced disease resistance. In Natural disease resistance of plants is based on preformed general, induced resistance can be defined as an activation and inducible mechanisms. The latter are activated in the of mechanisms that allow plants to defend themselves case of pathogen contact as an active response of plant against a broad spectrum of pathogens without any cells after recognition of the invader. Defense mechanisms changes of the plant genome [51]. Depending on the can be expressed locally, at the site of pathogen attacked activating stimulus, induced resistance can be devided in cells as well as systemically, in uninfected tissues. They pathogen-induced systemic acquired resistance (SAR) include changes like papilla formation, cell wall ligni- and rhizobacteria-mediated induced systemic resistance fication, production of reactive oxygen species (ROS), (ISR). In the case of SAR, SA-dependent signal trans- initiation of a hypersensitive reaction (HR), expression of duction was found to be imperative for triggering defense pathogenesis-related proteins (PRs) and accumulation of responses [27, 34]. In contrast, ISR seems to operate in an antimicrobial metabolites [28]. In recent years, the battery SA-independent manner [39]. Essential elements of the of the plants’ own defense reactions has attracted a lot of SAR and the ISR pathway have already been charac- attention both in applied agriculture and basic research. terized [15, 52]. Several natural and synthetic agents have been decribed * To whom all correspondence should be adressed. E-mail: as activators of defense-related processes when applied to jsieg!uni-hohenheim.de Abbreviations used in the text: AABA, DL-α-aminobutyric plants and some of these may have potential application acid; BABA, DL-β-aminobutyric acid; BTH, in agriculture [26, 58]. However, in only a few cases have benzo[1,2,3]thiadiazole-7-carbothioic acid-S-methyl ester; the criteria definded by Kessmann et al.[26] been clearly DAB, diaminobenzidine; GABA, γ-aminobutyric acid; HR, demonstrated. Thus, besides an effective disease control, hypersensitive response; NBT, nitroblue tetrazolium; ROS, re- chemical agents should be shown to induce plant specific active oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; TBARS, thiobarbituric acid reactive sub- defense responses. At the moment, the best characterized stances; TMV, tobacco mosaic virus. compounds within the group of chemical activators of 0885–5765\00\030095j12 $35.00\0 # 2000 Academic Press 96 J. Siegrist et al. disease resistance are 2,6-dichloroisonicotinic acid (INA) was provided by L. Friedrich (Novartis Ltd., Research and benzo[1, 2, 3]thiadiazole-7-carbothioic acid-S-methyl Triangle Park, U.S.A.). ester (BTH) which are both synthetic substances. These inducers were used in a number of recently published studies dealing with mechanisms involved in SAR [45]. Plants With the help of transgenic tobacco and Arabidopsis Seeds of transgenic PR-1a:uidA tobacco plants [10] were plants that constitutively express the bacterial nahG gene provided by D. F. Klessig (Rutgers University, NJ, encoding a salicylate hydroxylase, it was demonstrated U.S.A.), seeds of transgenic NahG-10 tobacco [18] came that both chemicals enter the SAR signalling pathway from K. Lawton (Novartis Ltd., Research Triangle Park, downstream of SA [17, 53]. Meanwhile, BTH was U.S.A.). Non-transgenic tobacco (Nicotiana tabacum cv. 2 introduced as a commercial product (Bion , Novartis Xanthi-nc), bearing the N gene derived from Nicotiana Ltd., Basel, Switzerland) in Europe for the protection of glutinosa for hypersensistive resistance to TMV, was used wheat against powdery mildew by means of SAR [19]. for inducer verification. Tobacco plants were grown 4 Other inducing agents like probenazole [55] or Milsana , under controlled conditions in a 14 h\10 h day\night an extract from the giant knotweed Reynoutria sachalinensis cycle at 24mC and 18mC, respectively. [12], were exclusively active in only a few selected pathosystems and can therefore not be considered as effective inducers in general. Inducer treatment Besides BTH and INA, the non-protein amino acid Chemical treatment was performed by spraying aqueous DL-β-aminobutyric acid (BABA) was described as SAR solutions of the aminobutyric acids (10 m) on leaves 3 inducer in several plant species belonging to the Solanaceae. and 4 of tobacco plants with six fully developed leaves. BABA treatment was effective in activating plants for a BTH (0.5 m) was applied in the form of the commercial sucessful defense against foliar diseases [7–9, 20, 47, 50] plant activator Bion2 (WP 50, Novartis Ltd., Frankfurt, and also against soilborne pathogens [4, 30]. In addition, Germany). For biological induction of resistance, cor- the inducing efficiency of BABA was recently demon- responding leaves of tobacco plants were inoculated with strated in cultured parsley cells, a model system for the TMV (10 µgml−" in 25 m sodium phosphate buffer pH testing of SAR-inducing agents [44]. In contrast to 7.0) using celite (0.1 mg ml−") as abrasive. BABA, the isomers DL-α-aminobutyric acid (AABA) and γ-aminobutyric acid (GABA) showed only weak or no activity in parsley cell cultures [44] and in all plant– Challenge inoculation with TMV pathogen interactions tested so far [7–9, 20, 47, 50]. This indicates a high isomer specificity of aminobutyric acid in Control plants and inducer-treated tobacco plants were µ −" defense activation. challenge inoculated on leaf 5 with TMV (10 gml in  In tomato, Cohen and coworkers were able to correlate 25 m phosphate buffer pH 7.0). Since N gene tobacco BABA-induced resistance to Phytophthora infestans with the is genetically resistant to TMV by local lesion formation, accumulation of several PR proteins in treated and in evaluation of inducer effects on these plants was performed non-treated parts of the plants [8]. When tested in Šitro, 7 days after inoculation by counting the number of local BABA exhibited no direct activity against various lesions as well as by measuring the diameter of 20 lesions pathogens [7–9, 20, 47, 50] which is a further important on each leaf. In this system, effective inducers mediate an prerequisite for abiotic inducers of disease resistance [26]. enhancement of resistance by reducing the symptom However, except for these biochemically based studies on severity [17]. BABA-activated mechanisms, the mode of action of this resistance-inducing chemical was not further examined in Quantification of SA detail. In the present study the classical SAR test system tobacco-tobacco mosaic virus (TMV) was used to gain an Total contents of SA (the sum of free and conjugated SA) insight into primary BABA-induced processes in plants. in tobacco leaves were determined according to Mo$ lders et al.[37] with some modifications. For SA extraction, tobacco leaf material (1 g) was grinded in liquid nitrogen MATERIALS AND METHODS and subsequently homogenized in a mortar after addition of 5 ml MeOH (90% v\v). The homogenate was Materials centrifuged for 15 min at 4mC with 15000 rpm and the BTH was supplied by K.-L. Nau (Novartis Ltd., resulting supernatant carefully removed. After resus- Frankfurt, Germany). BABA was purchased from Fluka penion of the pellet in 5 ml MeOH (90% v\v) and a (Buchs, Switzerland), all other chemicals came from further centrifugation, both methanolic supernatants were Sigma (Deisenhofen, Germany). Tobacco PR-1a c-DNA combined and vacuum-reduced to 1\10 of the original β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus 97 volume. Extracts were directly hydrolysed for 2 h at 80mC GUS activity is given in nanomoles MU mg−" protein h−" in the presence of 10  HCl to release glucosidically at 37mC. Protein content of samples was determined bound SA. After acidic hydrolysis, pH of the extract was according to Bradford [3]. adjusted to 6.5 with 1  NaOH. Total SA-contents were analysed by HPLC (Pharmacia, Uppsala, Sweden) using EŠaluation of cell death, callose formation and lipid a fluorescence detector (LC 304, Linear Instruments, peroxidation Reno, U.S.A.). Recovery rates were determined by adding SA standards to blank samples. SA levels of test samples Histochemical monitoring of cell death was carried out by were respectively corrected for recovery. infiltrating Evans blue in tobacco leaf discs according to Schraudner et al.[42]. As a second method, leaf discs were stained with trypan blue to visualize cell death as described Analysis of PR-1a gene expression previously [25]. For further microscopical evaluation of Expression of PR-1a was checked in wild-type tobacco BABA- and TMV-mediated lesion formation, leaf discs (Nicotiana tabacum cv. Xanthi-nc) by Northern gel blot were heated for 3 min in a mixture of lactic acid, phenol analysis. Total RNA was extracted from tobacco leaf discs and water (1:1:1, v\v\v). After cooling, the discs were (100 mg FW) which were collected from inducer-treated destained for 2 days in the same mixture [42]. and non-treated leaves according to Ka$ stner et al.[23]. Formation of callose was determined by staining leaf RNA (10 µg) was separated on 1.1% (w\v) agarose gels discs with aniline blue according to Dietrich et al.[14]. which contained 6% (v\v) formaldehyde as described Lipid peroxidation was estimated by measuring the previously [43]. Equal loading of samples was verified by accumulation of thiobarbituric acid reactive substances adding ethidium bromide (0.01 mg ml−") in the sample (TBARS) spectrophotometrically as described by buffer. RNA was blotted overnight onto a positively Rusterucci et al.[40]. The content of lipid peroxides was charged nylon membrane (Hybond-Nj, Amersham Life determined by using the molar extinction coefficient for & −" −" Science, Buckinghamshire, U.K.) by capillary transfer malondialdehyde (1.56i10  cm ). using 10iSSC (1iSSC l 0.15 m sodium chloride, 0.015  sodium citrate pH 7). After transfer, RNA was Histochemical detection of ROS covalently bound to the membrane by using an u.v. cross- − linker (Amersham Life Science). Generation of superoxide anions (O# ) was verified by Prehybridisation was carried out for 4 h at 47mCin staining leaf discs with nitroblue tetrazolium (NBT) as 2iSSC, 5iDenhard’s solution [1iDenhard’s l 1m described by Schraudner et al.[42]. Histochemical EDTA (pH 8.0)], 0.02% (v\v) each of polyvinyl- detection of hydrogen peroxide (H#O#) was performed by h pyrrolidone, Ficoll and bovine serum albumin, 1% (v\v) infiltrating 3,3 -diaminobenzidine 4 HCl (DAB) accord- formamide, sonicated salmon sperm DNA (100 µgml−") ing to Thordal-Christensen et al.[49]. Infiltrated leaf discs and 1% (w\v) SDS. $#P-labelled cDNA probes were were destained in ethanol:chloroform (4:1) containing \ $ synthesized by PCR amplification of insert DNA present 0.15% (w v) TCA according to Huckelhoven and Kogel in a pBluecript vector (Stratagene, La Jolla, U.S.A.) by [21]. using the primer OL 7 (5h-GGT GGC GGC TCT AG-3h) RESULTS and OL 53 (5h-GTA AAA CGA CGG CCA GTG AGC- BABA enhances resistance to TMV in tobacco 3h). A highly labelled probe was obtained by running PCR with dNTP concentration of 65 µ except dATP The aminobutyric acid isomers AABA, BABA and GABA which was used at 6.5 µ in each sample. In addition 1 µl were tested for their potential to enhance resistance to [α-$#P] dATP (10 mCi ml−"; Amersham Pharmacia TMV in tobacco plants that contain the N gene. As a Biotec, Freiburg, Germany) was included in each PCR positive control, the chemical activator BTH was included sample. Hybridization to labelled PR-1a cDNA was which was already demonstrated as a highly effective carried out for 16 h at 47mC. The blots were then washed inducer in tobacco [17]. Compared to the untreated 3i for 15 min at 65mC with 1iSSC containing 1% control, plants which were sprayed on lower leaves either (w\v) SDS . After air-drying, blots were exposed to X-ray with 10 m BABA or 0.5 m BTH, exhibited significantly film at k80mC. smaller lesions on upper leaves after challenge inoculation As a second approach to demonstrate PR-1a gene with TMV (Table 1). Furthermore, the number of lesions activation after inducer treatment, the expression of a was considerably reduced in treated plants (data not chimeric PR-1a:uidA reporter gene in transgenic tobacco shown). Foliar application of BABA at concentrations plants was tested. Activity of β-glucuronidase (GUS) was below 5 m resulted in a markedly reduced efficacy, at determined according to Conrath et al.[10]. The 0.5 m BABA was completely inactive (data not shown). fluorescence of 4-methylumbelliferone (MU) was In contrast to BABA, a treatment of tobacco plants with measured with a spectrofluorometer (Hitachi F 2000). 10 m GABA did not enhance resistance to TMV (Table 98 J. Siegrist et al.

TT1.EffectofatreatmentwithBTH,AABA,BABAor 120 GABA on the expression of resistance in tobacco plants (Xanthi- Treated Non-treated nc) to TMV. Resistance was assessed by inoculating an upper leaf 100 * (leaf 5) with TMV (10 µgmlk"), 7 days after application of the mg protein) × inducers to leaf 3 and 4. 80 –1

a Treatment TMV lesion size (mm) 60

None (control) 1.55p0.24 * 40 BTH (0.5 m) 0.25p0.11 AABA (10 m) 1.10p0.16 BABA (10 m) 0.53p0.30 20 * GABA (10 m) 1.47p0.18 *

GUS activity (nmol MU h 0 aValues are the meanspSD of five experiments with five Control BTH BABA GABA plants per treatment F. 2. Expression of a chimeric PR-1a:uidA reporter gene in treated (leaf 4) and non-treated (leaf 5) parts of transgenic tobacco plants. GUS activity was determined 7 days after application of 0.5 m BTH, 10 m BABA or 10 m GABA to leaf 3 and 4. *Means from four experiments (five plants per treatment) significantly different from control (P ! 0.05).

shown in Fig. 2, maximal GUS activity was detected in leaves sprayed with BTH which was used as a positive control. Lower but significantly induced activities were determined in leaves treated with BABA. In contrast,  F . 1. Northern Blot analysis of total RNA extracted from almost no GUS activity was measured in control plants tobacco (Xanthi-nc) leaves sprayed with 10 m BABA, 10 m GABA or 0.5 m BTH. Seven days after inducer application, and in plants sprayed with GABA. However, increased RNA from treated leaves (lanes 1, 3, 5 and 7) as well as from GUS activities were also evident in non-treated leaves of non-treated leaves (lanes 2, 4, 6 and 8) was separated by tobacco plants induced by a local application of BTH or electrophoresis and probed with $#P-labelled tobacco PR-1a BABA, indicating a systemic activation of the PR-1a cDNA. promotor by both chemicals (Fig. 2).

1). GABA was ineffective up to 50 m (data not shown). BABA mediates local cell death The application of AABA (10 m) resulted in slightly reduced TMV lesion sizes after challenge inoculation Foliar application of BABA to tobacco plants resulted in (Table 1). Due to its weak activity, AABA was not the appearance of HR-like lesions on the treated leaves. applied in the further experiments. Lesions were macroscopically visible 24 h after inducer treatment, especially on the adaxial leaf surface. Lesions subsequently increased in size within the next 24 h (not BABA induces local and systemic PR-1a gene expression shown). Compared to the formation of local lesions after Induction and maintenance of resistance in tobacco is TMV inoculation of tobacco plants bearing the N gene, a closely associated with the coordinated activation of SAR greater number of smaller lesions appeared in BABA- genes [54]. Genes coding for proteins of the PR-1 group treated tissues (Fig. 3). In contrast to BABA, no visible are highly expressed after treatments with chemical and signs of cell death were detectable after GABA application biological inducers [54]. In tobacco plants, foliar ap- (not shown). plication of 10 m BABA resulted in the induction of PR- Staining of tobacco leaf material with trypan bue and 1a mRNA in the treated as well as in non-treated Evans blue indicated a rapid initiation of cell death by (systemic) leaves (Fig. 1). As expected, BTH (0.5 m) BABA. Starting 14–16 h after inducer application, also induced local and systemic PR-1a expression. No localized blue spots, indicating irreversible uptake of accumulation of PR-1a mRNA was detectable after Evans blue and therefore cell death, were detected in application of 10 m GABA (Fig. 1). BABA-treated tissues [Fig. 4(a)]. Blue coloured regions As an additional experimental approach, transgenic expanded up to 48 h after inducer treatment [Fig. 4(b)]. tobacco plants containing a chimeric PR-1a:uidA reporter Microscopical evaluation of leaf discs stained with trypan gene construct were used to demonstrate the activation of blue, revealed clusters of dead cells surrounded by the PR-1a promotor by aminobutyric acid derivatives. As apparently unaffected cells [Fig. 4(c)]. Besides cell death, β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus 99

(a)

(b)

F. 3. Lesion phenotype on tobacco leaves provoked by various inducer treatments. (a) Phenotype of necrotic lesions, 6 days after application of BABA (10 m) or TMV (10 µgml−") inoculation. (b) Microscopical appearance of lesions in destained tobacco leaf tissue, 6 days after application of BABA (10 m) or TMV (10 µgml−") inoculation. 100 J. Siegrist et al. a staining of leaf discs with aniline blue showed a rapid deposition of callose around dying cells [Fig. 4(d)]. SA accumulation as prerequisite for BABA-mediated enhancement of Širus resistance Activation of SAR by HR-inducing microbes depends on Histochemical localization of BABA-mediated generation of the accumulation of SA [18] whereas chemical activators ROS like BTH and INA induce SAR in an SA-independent manner [17, 53]. Transgenic tobacco that constitutively Occurrence of cell death is expected to be closely related expresses a salicylate hydroxylase gene (nahG) from to the generation of ROS [35]. For this reason, histo- Pseudomonas putida cannot accumulate significant amounts − chemical staining methods for the detection of O# and of SA [18]. This transgene is a helpful tool to demonstrate H#O# were performed. Infiltration of NBT revealed a the involvement of SA in SAR expression. Since BABA − short period of O# generation at 6–8 h after BABA was found to induce SA accumulation in wild type application [Fig. 5(a)]. Subsequently, production of H#O# tobacco (Fig. 7), transgenic NahG plants were used to test was detectable by DAB staining by 12–14 h after inducer the relationship between BABA-induced enhancement of treatment. Staining continued increasing up to 48 h after TMV resistance and SA accumulation. For comparison, BABA treatment [Fig. 5(b) and (c)]. The spatial non-transgenic tobacco plants and the well characterized − distribution of O# production was more or less diffuse inducers BTH and TMV were included in this set of [Fig. 5(a)], whereas H#O# accumulation was distinctively experiments. As shown in Fig. 8 and already known from localized in regions where cell death could be detected by the literature [17], BTH enhanced resistance in wild type staining with Evans blue [Figs 4(b) and 5(c)]. Xanthi-nc as well as in the transgenic line NahG-10, whereas TMV was exclusively active as inducer in Xanthi- nc and not in NahG-10. Interestingly, BABA treatment BABA induces lipid peroxidation resulted in the same phenotype of resistance as seen in the case of the biotic induction by TMV. BABA as an Since cell death resembling an HR was detected as a effective inducer in non-trangenic tobacco, exhibited no consequence of foliar BABA application, lipid peroxi- activity in transgenic NahG plants (Fig. 8). A quantitative dation in tobacco leaf tissues was verified as a further evaluation of the plants revealed a significant reduction of typical marker related to cell death. The extent of disease symptoms in wild type and transgenic tobacco by membrane damage in control leaves and BABA-treated BTH compared to the control, but no enhanced resistance leaves was estimated with the thiobarbituric acid assay. in TMV-preinoculated or BABA-treated NahG tobacco BABA-induced lipid peroxidation was evident by plants (Fig. 9). These results indicate that BABA-mediated increased TBARS contents, measurable 48 h after inducer enhanced resistance to TMV in tobacco is SA-dependent. application. Compared to control plants, a three-fold In addition, it is interesting to note that BABA-mediated higher TBARS level was determined as maximal response lesion formation was still evident in NahG tobacco (not at 96 h after BABA treatment (Fig. 6). shown).

DISCUSSION BABA mediates local and systemic accumulation of SA It is expected that chemical inducers of disease resistance SA is known to play an important role in the process of will play the key role for the integration of the SAR SAR activation by HR-inducing microbes [27]. To concept in modern plant protection strategies [31]. For estimate whether the occurrence of BABA-mediated cell this reason, the search for new inducing agents as well as death could be correlated with an accumulation of SA, the elucidation of the mode of action of already available the content of total SA in tobacco plants was determined. inducers has intensified in recent years [58]. Indeed, only Increased SA levels were found in the leaves 7 days after a limited number of inorganic, natural organic or synthetic spraying with BABA [Fig. 7(a)]. As previously described compounds with a generally accepted resistance-inducing in the literature [17, 33], an inoculation with TMV activity are known so far [26, 45]. However, even in case caused a massive accumulation of SA whereas SA contents of the best studied inducers, INA and BTH, little did not increase in tobacco plants treated with the plant knowledge exists about their mode of action and the activator BTH [Fig. 7(a)]. Similarly, SA accumulated in cellular receptors or target molecules with which they the upper (systemic) leaves of BABA-treated tobacco interact [41]. The resistance inducing potency of BABA plants but at a lower level than in directly treated leaves. has also been documented in a number of studies Again, SA contents measured in systemic leaves of BABA- [7–9, 20, 47, 50]. The highest efficacy of the compound treated and TMV-preinoculated plants were much higher was demonstrated in several members of the Solanaceae. than in control and BTH-treated plants [Fig. 7(b)]. Therefore, tobacco was used in the present study to β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus 101

CON BABA (a) (a)

(b) (b)

(c) (c)

F. 5. Histochemical localization of ROS in tobacco leaf tissue  − following application of 10 m BABA. For the detection of O# , (d) leaf discs were vaccum-infiltrated with NBT. H#O# accumulation was demonstrated by DAB infiltation. (a) NBT staining, 8 h after BABA application. (b) DAB staining, 12 h after BABA application. (c) DAB staining, 48 h after BABA application.

examine biochemical and molecular mechanisms involved in BABA-induced resistance. In the pathosystem tobacco- TMV, BABA-mediated enhancement of virus resistance could be demonstrated. In contrast, the aminobutyric acid derivative GABA was completely inactive, the isomer AABA had a much lower activity indicating a high isomer specificity of aminobutyric acid in triggering enhanced virus resistance. Similar results have been obtained in tomato, pepper and sunflower [8, 20, 47, 50]. In a more F. 4. Histochemical detection of cell death by Evans related study, using the system tobacco-Peronospora blue\trypan blue staining and of callose formation by anilin blue tabacina, only BABA but not AABA and GABA was found staining in BABA-treated tobacco leaves. Trypan blue-stained to induce resistance against blue mold [7]. Thus, data leaf discs were examined by light microscopy, anilin blue-stained discs by fluorescence microscopy. (a) Control (left); Evans blue- available at the moment suggests a very specific interaction stained leaf disc, 16 h after application of 10 m BABA (right). of BABA with unknown cellular components in treated (b) Control (left); Evans blue-stained leaf disc, 48 h after plant tissues. As a further characteristic of plant activation  application of 10 m BABA (right). (c) Trypan blue staining, by BABA, relatively high concentrations of the chemical 48 h after application of 10 m BABA. (d) Anilin Blue staining, 24 h after application of 10 m BABA. were needed to trigger resistance in almost all systems 102 J. Siegrist et al.

20 12 (a) Control BABA * 10 15 *

FW) * –1 8 10 FW) –1 6 g TBARS (nmol g TBARS 5 µ SA ( 4

0 0 24 48 72 96 Time after application (h) 2 F. 6. Effect of a treatment with BABA (10 m) on the content of TBARS in tobacco leaves, indicating lipid peroxidation. *Means from three experiments (five plants per treatment), 0 significantly different from control (P ! 0.05). Control BTH TMV BABA

800 tested so far. Foliar applied concentrations of BABA in the (b) millimolar range (1–20 m) were usually utilized to achieve plant protection [8, 20, 47]. In addition, effective 700 BABA concentrations used for inducing resistance in  plants by root-drenching were 5–40 m [4, 20, 30, 50]. 600 Compared to BABA, a much lower concentration (0.5 m) of the plant activator BTH was sufficient for the enhancement of resistance in tobacco to TMV, indicating 500 \ differences in the uptake and or in the mode of action of FW) both inducers. –1 400 Activation of certain marker genes encoding PR proteins is a common feature associated with resistance SA (ng g 300 induction. Thus, in tobacco, PR 1-a gene expression could be demonstrated locally and systemically after foliar BABA application. This is in agreement with the results 200 obtained by immunoblot analysis of extracts from tobacco plants sprayed with BABA, where PR-1 protein ac- 100 cumulation was already shown [7]. In addition, BABA- induced resistance in tomato and pepper was also found to be correlated with the induction of PR-proteins [8, 22]. 0 Control BTH TMV BABA However, no PR protein accumulation could be detected in tobacco after stem injection of BABA although the F. 7. Local and systemic accumulation of SA in tobacco plants   plants were strongly protected against Peronospora tabacina treated with BABA (10 m ), BTH (0.5 m ) or preinoculated µ −" [7]. This indicates that different processes may be with TMV (10 gml ). Total SA contents were determined in leaf 4 (a) and leaf 5 (b) of tobacco plants, 7 days after activated by BABA in tobacco depending on the location application of the inducing agents to leaf 3 and 4. Values are of inducer application. A foliar spray treatment with meanspSD from six experiments with five plants per treatment. BABA triggers PR gene expression and virus resistance, whereas a stem application seems to activate yet unknown mechanisms which mediate resistance against the blue enhance virus resistance in tobacco (Siegrist et al., mold pathogen but were not related to PR protein unpublished results). accumulation. This view is further supported by the The occurrence of cell death as a rapid reaction observation that a stem application of BABA did not following application of BABA was an unexpected result β-Aminobutyric acid-mediated enhancement of resistance to tobacco mosaic Širus 103

F. 8. Phenotype of TMV lesions in wild type (Xanthi-nc) tobacco (upper row) and in transgenic NahG tobacco (lower row), 1 week after challenge inoculation with TMV (10 µgml−") on leaf 5. Inducers [0.5 m BTH, TMV (10 µgml−"), 10 m BABA] were applied to lower leaves (leaf 3 and 4), 7 days before challenge inoculation.

plant defense activation [7, 8, 47]. Moreover, it was also Xanthi-nc 3 NahG-10 observed that AABA, which exhibited a slight but reproducible activity in the tobacco\TMV system (Table 1), induced cell death when applied to tobacco leaves. However, compared to BABA, lesion formation occurring 2 after AABA treatment was markedly less pronounced (Siegrist et al., unpublished results). Further studies from our lab provide additional Lesion size (mm) 1 * evidence for BABA-mediated cell death in plants. In * * * tomato, foliar application of the inducer resulted in SAR expression against Phytophthora infestans [29]. In this system, 0 the plant phenotype observed after BABA treatment was Control BTH TMV BABA nearly identical to that appearing in the case of a biotic F. 9. Effect of various treatments [0.5 m BTH, TMV (10 µg SAR induction mediated by local lesion forming −"  ml ), 10 m BABA] on the expression of resistance in wild-type pathogens like tobacco necrosis virus [2, 29]. Furthermore, tobacco plants (Xanthi-nc) and in transgenic NahG tobacco it was recently demonstrated that BABA also causes cell against TMV. Resistance was assessed by inoculating an upper leaf (leaf 5) with TMV (10 µgml−"), 8 days after application of death in the roots of tomato plants after a soil drench the inducers to leaf 3 and 4. *Means from three experiments application leading to resistance against Fusarium oxysporum (5 plants per treatment) significantly different from control f.sp. lycopersici [4]. This kind of plant response prompted ! (P 0.05). us to examine how BABA-induced cell death could be linked to the enhancement of virus resistance in tobacco and the processes subsequently observed. As the first obtained in the present study. Indeed, a critical review of detectable reaction, a generation of ROS was evident, the literature dealing with BABA-induced resistance indicating an initial interaction of BABA with a cellular revealed that this HR-resembling reaction of plant tissues target which directly or indirectly provokes an oxidative to foliar application of the chemical was already noticed burst. The nature of this interacting component is still by other authors, but was not discussed in relation to unknown and awaits further studies. It could be localized 104 J. Siegrist et al. in the apoplast or in cell wall-associated regions, since a bacterio-opsin proton pump displayed necrotic lesions, small portion of radiolabelled BABA remained in the cell elevated SA levels, increased PR gene expression and wall of treated tomato leaves whereas no binding to enhanced resistance against pathogens [1, 36]. Further cytoplasmatic proteins could be detected [6]. transgenic, lesion-forming plants with similar properties Besides ROS generation, lipid peroxidation was have been described [13]. detected in tobacco tissues, providing further evidence for Other chemicals and natural compounds, besides typical cell death-aligned responses initiated by BABA. BABA, are known to induce resistance in plants by However, increased TBARS contents were not obvious triggering HR-like responses. In potato, a spray treatment until 2 days after inducer treatment when necrotic lesion with arachidonic acid generated massive local lesion formation was already macroscopically visible. The reason formation and subsequently SAR against Phytophthora could be the low sensitivity of the thiobarbituric acid infestans [5, 11] and Alternaria solani [11]. In this system, a assay that does not allow detection of small changes in local but not systemic accumulation of SA was demon- already damaged membranes at earlier stages. strated [11]. Nevertheless, by using NahG potato plants, In the case of a biotic induction of disease resistance, SA was found to play an essential role in arachidonic acid- localized cell death and the formation of HR-lesions mediated SAR [59]. resulting in defense gene activation are characteristically Other examples of cell death-inducing agents are triggered by viruses and microbes [45]. For this reason, chemicals like the herbicide Paraquat or the dye Rose we compared typical responses that appeared after Bengal, that act by producing ROS when applied to treatment of tobacco plants with the biotic inducer TMV plants as foliar sprays. Both chemicals have been and the chemical inducer BABA. In agreement with demonstrated as effective SAR inducers in tobacco already published data [33], local and systemic ac- [16, 46]. Furthermore, inorganic phosphate salts which cumulation of SA was evident in tobacco after TMV induce SAR in different plants were recently described to inoculation. Interestingly, we could also find increased SA operate by causing ROS generation, cell death and SA levels in treated and in non-treated parts of tobacco plants accumulation [38]. Similar results have been obtained sprayed with BABA, indicating an involvement of SA in after application of elicitins, proteins excreted by Phyto- BABA-mediated enhancement of TMV resistance. This phthora spp. [24]. Cell death-associated activation of assumption was further supported in experiments with defense genes and SA accumulation is also known from NahG plants. As expected, BTH was found to operate tobacco plants treated with ozone or exposed to u.v.-light independently of SA. Whereas, enhancement of resistance [57]. was blocked in plants induced by TMV and BABA. From In the case of BABA-mediated lesion formation, as well these results, it can be concluded that BABA triggers as in most of the above mentioned examples, a complex mechanisms in tobacco leaf tissues which are typical for regulation seems to be involved that link cell death biotic inducers of resistance acting by local lesion initiation and the succeeding processes leading to disease formation. Therefore, under our experimental conditions, resistance. In contrast, cell death caused by unspecific BABA seems to imitate a pathogen attack. However, the damage of plant tissues, e.g. dry ice treatment, did not formation of necrotic lesions in BABA-treated NahG activate SA synthesis and plant defense against pathogens tobacco indicates that primary processes initated by this [32; Siegrist et al., unpublished results]. inducer are in no way affected by the low SA level of the In conclusion, our data provide evidence that foliar transgenic plants. application of the chemical inducer BABA to tobacco Apart from microbial initiation of cell death, known plants results in the activation of mechanisms resembling lesion-mimic mutants of certain plants display necrotic those initiated by necrotizing microbes and viruses that lesions that resemble those of an HR, and exhibit activated trigger SAR. defense mechanisms as well as SAR. Intensively studied examples are cell death mutants from Arabidopsis thaliana which express spontaneous lesion formation in the absence of pathogens [14]. These mutants showed elevated We thank Margit Schmidtke, Carmen Kolle and Gisela concentrations of SA, constitutive expression of PR Moll for excellent technical assistance. We are grateful to proteins and enhanced resistance against virulent Prof. Dan Klessig (Rutgers University, NJ, U.S.A.) and pathogens [14, 56]. More recently, lesion-mimic mutants Dr Kay Lawton (Novartis Ltd., Research Triangle Park, of rice have been described which were resistant against U.S.A.) for providing seeds of transgenic tobacco. We also the blast fungus Magnaporthe grisea [48]. A further line of thank Dr Leslie Friedrich (Novartis Ltd., Research evidence for cell death-associated activation of SAR Triangle Park, U.S.A.) for providing tobacco PR 1a c- without any microbial stimulus can be deduced from DNA and Dr Jan Hinrichs-Berger for reviewing the studies with lesion-mimic transgenic plants. For example, manuscript. 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