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Zeitschrift für Pflanzenkrankheiten und Pflanzenschutz Journal of Plant Diseases and Protection 110 (5), 419–431, 2003, ISSN 0340-8159 © Eugen Ulmer GmbH & Co., Stuttgart

Induction of systemic resistance to Xanthomonas oryzae pv. oryzae by salicylic acid in Oryza sativa (L.) Induktion einer systemischen Resistenz gegen Xanthomonas oryzae pv. oryzae durch Salicylsäure in Oryza sativa (L.) R. Mohan Babu1, A. Sajeena2, A. Vijaya Samundeeswari2, A. Sreedhar3, P. Vidhyasekaran2, K. Seetharaman2, M. S. Reddy4 1 Department of Botany, University of Toronto at Mississauga, Mississauga, Canada L5L 1C6, Fax: 905-828-3792, Email: [email protected] 2 Department of Plant Pathology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India 3 Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive Saskatoon, Saskatchewan S7N 5A8, Canada 4 Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, USA Received 15 January 2003, accepted 17 June 2003

Summary The effect of salicylic acid (SA) is hypothesized to be a natural signal that triggers the systemic induction of phenolics, pathogenesis-related proteins and disease resistance in rice against the bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Rice plants pretreated with 1000 μmol/l SA showed resistance to challenge inoculation with Xanthomonas oryzae pv. oryzae and the effectiveness persisted in the susceptible cv. ‘IR 50’ for at least 3 days prior to inoculation with Xoo. To investigate the role of SA in rice disease resistance, we examined the endogenous levels of SA in the SA-pretreated rice plants with Xoo inoculation. A three-fold increase in the endogenous SA levels was observed in the rice tissues pretreated with 1000 μmol/l SA and the resistance persisted for at least 3 days after SA treatment prior to inoculation with Xoo. Increasing the endogenous level of SA in rice leaves to those naturally observed during systemic acquired resistance resulted in increased resistance to Xanthomonas oryzae pv. oryzae, expressed as a reduction in leaf blight lesion length. Immunoblot analysis revealed an induction of a 25 kDa protein cross-reacting with rice thaumatin-like protein (TLP) antiserum in response to SA-pretreated and SA non-pretreated rice plants followed by pathogen inoculation. A significant increase in the induction of TLPs 3 days after Xoo inoculation in the tissues pretreated with SA was observed when compared with the 2 days and 1 day after Xoo inoculation in SA-treated plants. Increased phenolics content and enhanced activities of some pathogenesis-related (PR) proteins, viz., TLP, chitinase and β-1,3-glucanase were observed in rice plants treated with SA. Based on these experiments, it was investigated that the defense responses are induced locally at the infection site only after pathogen attack and are augumented when the rice tissue has been pretreated with SA. These data further support the hypothesis that the defense responses in rice can be rapidly triggered and induced in a genetically susceptible cultivar after treatment with SA. Key words:

Bacterial leaf blight; Xanthomonas oryzae pv. oryzae; chitinases; β-1,3-glucanases; induced resistance; Oryza sativa; pathogenesis-related protein; salicylic acid; thaumatinlike proteins (TLPs)

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Zusammenfassung Salicylsäure (SA) soll ein natürliches Signal sein, das die systemische Induktion von phenolischen Verbindungen, PR-Proteinen und Krankheitsresistenz in Reis gegen Xanthomonas oryzae pv. oryzae (Xoo), dem Erreger der Blattbräune, auslöst. Reispflanzen, die mit 1000 μmol/l SA vorbehandelt worden waren, zeigten Resistenz nach Inokulation mit X. oryzae pv. oryzae. Die Wirkung hielt in der anfälligen Sorte ‘IR 50’ wenigstens 3 Tage vor der Inokulation mit Xoo an. Um die Rolle von SA in der Krankheitsresistenz zu untersuchen, wurde der endogene Gehalt an SA in den Geweben der mit SA vorbehandelten Pflanzen nach Inokulation mit Xoo bestimmt. Eine dreifache Zunahme des endogenen SA-Gehaltes in den mit 1000 μmol/l behandelten Reispflanzen wurde nachgewiesen. Die Resistenz dauerte wenigstens 3 Tage lang an vor Inokulation mit Xoo. Eine Erhöhung des endogenen SAGehaltes auf den unter natürlichen Bedingungen in Reisblättern beobachteten Gehalt während der systemisch induzierten Resistenz führte zu einer erhöhten Resistenz gegen X. oryzae pv. oryzae, gemessen an der Reduktion der Länge der Blattflecken. Durch Immunoblot-Analyse konnte die Induktion eines 25-kDa Proteins nachgewiesen warden, welches mit dem Thaumatin-ähnlichen rotein (TLP)Antiserum von Reis kreuzreagierte als Reaktion auf Reispflanzen, die mit SA vorbehandelt oder unbehandelt waren mit nachfolgender Xoo-Inokulation. Ein signifikanter Anstieg der Induktion von TLPs 3 Tage nach Inokulation mit Xoo in den mit SA vorbehandelten Geweben konnte im Vergleich zu den 2 Tage oder 1 Tag später inokulierten Pflanzen nachgewiesen warden. Erhöhte Gehalte an phenolischen Verbindungen und vermehrte Aktivitäten einiger PR-Proteine, nämlich TLP, Chitinase und β-1,3-Glucanase wurden nach Behandlung von Reispflanzen mit SA festgestellt. Auf der Basis dieser Ergebnisse kann gesagt werden, dass die Abwehrreaktionen lokal am Ort der Infektion nur nach Angriff des Pathogens induziert warden und dass sie verstärkt warden, wenn das Reisgewebe mit SA vorbehandelt wird. Die Hypothese, dass Abwehrreaktionen in genetisch anfälligen Reissorten durch Behandlung mit SA schnell ausgelöst und induziert warden können, wird durch die vorliegenden Daten unterstützt. Stichwörter: Bakterielle Blattbräune; Xanthomonas oryzae pv. oryzae; Chitinase; β-1,3-Glucanase; indu-

zierte Resistenz; Oryza sativa; PR-Proteine; Salicylsäure; Thaumatin-ähnliche Proteine; TLP 1

Introduction

Bacterial leaf blight (BLB) of rice (Oryza sativa L.) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important destructive diseases of rice throughout the world (Mew 1987). In some areas of Asia, rice yield losses caused by BLB can be as high as 50 % (Adhikari et al. 1995). Effective chemical control measures against the disease are lacking but development of rice cultivars with durable resistance is ideal. However, success in this regard is limited. The phenomenon of a promising environment-friendly strategy, nowadays known as induced systemic resistance (ISR; Kuc 1995) or, synonymously, systemic acquired resistance (SAR; reviewed by Ryals et al. 1996; Sticher et al. 1997) can be exploited using an exogenous application of salicylic acid (SA) to induce systemic resistance in rice against Xoo. It is well known that plants possess a range of active defense responses, which contribute to induced resistance to pathogens (Akinwunmi et al. 2001). The induced resistance occurs not only at the site of the initial treatment but also in distal, untreated plant parts. The various induced resistance phenomena are all associated with an enhanced capacity for the rapid and effective activation of cellular defense responses, which are induced only after contact with a pathogen (Zimmerli et al. 2000; Jakab et al. 2001; Conrath et al. 2001; Kohler et al. 2002). The systemic resistance response activated upon infection of plants with necrotizing pathogens is called SAR (Ryals et al. 1996; Sticher et al. 1997), but SAR can also be induced by exogenous application of salicylic acid. Establishment of SAR requires an endogenous increase in salicylic acid levels (Ryals et al. 1996; Sticher et al. 1997; Kuc 2001) and its onset is associated with the expression of SAR genes (Ryals et al. 1996), some of which encode pathogenesis-related (PR) proteins (Ryals et al. 1996; Sticher et al. 1997; Hoffmann-Sommergruber et al. 1999; Dempsey et al. 1999). Some PR proteins display antimicrobial activity in vivo (van Loon et al. 1999), but their actual role in SAR

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remains uncertain. Subsequently, the resistance spreads systemically and develops in distal, untreated parts of the plant and thus confers an elevated level of protection (Siegrist et al. 1997). In SAR, a number of defense pathways are stimulated and diverse defense products are synthesized which includes lignin, PR proteins, phytoalexins, thionins and defensins (Kessmann et al. 1994; Terras et al. 1995; Epple et al. 1997; Thomma et al. 2002). The PR proteins are constitutively expressed in plants at low levels, but the expression of most of the PR proteins is turned on in response to pathogen attack. It has been proposed that the induction of PR proteins is a consequence of the activation of plant defensive pathways, which limit the entry, or the further spread of the pathogen (Baker et al. 1997; Conrath et al. 2002). The systemic response involves the de novo production, in some cases, of phytoalexins and of PR proteins (van Loon 1999; Dempsey et al. 1999; van Loon and van Strien 1999). Originally, PR proteins were detected and defined as being absent in healthy plants but accumulating in large amounts after infection. During the past few decades, there has been increasing evidence for augmentation of locally induced defense responses upon pathogen infection of systemically protected plants (for review, see Sticher et al. 1997). Although this conditioning phenomenon has been known for quite a while, not much attention was paid to it when studying SAR and, therefore, little is known so far about the molecular and biochemical mechanism(s) that mediate(s) conditioning. The broad-spectrum activity of SA, conferring protection against bacterial, fungal and viral diseases, strongly suggests an indirect mode of action via activation of plant defense mechanisms. Salicylic acid (SA) has an important role in the signalling pathway leading to ISR (Mauch-Mani and Métraux 1998; Thulke and Conrath 1998; Métraux 2002). The role of SA as a defense signal in plants has been well established in cucumber (Smith-Becker et al. 1998), tobacco (Seskar et al. 1998), pea (Frey and Carver, 1998), grape (Kassemeyer and Busam 1997), arabidopsis (Cameron et al. 1999; Pieterse et al. 1998 ) and tomato (M’Piga et al. 1997). Studies on the basal levels of SA and effects of exogenously applied SA in different plant species suggest that substantial differences in SA responsiveness exist among plants. Although SA response in dictotyledonous plants is well documented, there is little information on the response of monocotyledonous plants to exogenously applied SA to induce resistance against bacterial pathogens of the Xanthomonas genus. More over, the involvement of SA in the activation of defense responses in rice plant against Xoo is also unclear. To ascertain the possibility of using SA as a SAR inducer in rice crops, its efficacy in controlling BLB disease caused by Xoo was investigated. In the present study, we explored the possibility of inducing systemic resistance in rice against Xoo by application of SA. Following SA treatment, the normally susceptible interaction became resistant, with a rapid increase in defense-related enzymes, phenolics and the accumulation of PR proteins. These results support the idea that the induction of systemic resistance by exogenous application of SA may be an important component in integrated plant disease management strategies.

2

Materials and Methods

2.1

Plant material

Rice (Oryza sativa L.) seeds of the susceptible cultivar ‘IR 50’ were obtained from the Paddy Breeding Station, Coimbatore, Tamil Nadu, India. This cultivar was chosen for the current study because of its proven susceptibility to BLB disease. Twenty-five seeds of the ‘IR 50’ rice cultivar were soaked in water overnight and germinated on wet filter paper in the dark. The sprouted seeds were placed on wire meshes. Five hundred ml of Hoagland solution (Hoagland and Arnon 1938) were taken in a 500-ml beaker and the wire mesh was placed over the beaker so that Hoagland solution touches the wire mesh. The sprouted seeds were allowed to grow for 25 days on the Hoagland solution. Plants were grown in an environmentally controlled growth chamber at 25 °C, 75 % RH, with a 16 h photoperiod (500 μmol m–2 s–1) that was provided by a combination of incandescent and cool-white fluorescent lights. 2.2

Pathogen isolation and maintenance

Xanthomonas oryzae pv. oryzae (Xoo) was isolated from bacterial leaf blight-infected rice plants (cv. ‘IR 50’) and maintained on Wakimoto’s semi-synthetic potato sucrose agar (PSA) medium (Wakimoto

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1960). After 48 h of incubation at 28 ± 2 °C, the yellowish bacterial growth on the medium was subcultured in slants containing the above medium and purified by the dilution plate technique (Waksman 1952). Pure culture was maintained on agar slants of PSA medium at –10 °C for further studies. To prepare inocula, bacteria were grown on PSA at 28 ± 2 °C for 48 h. The bacterial cells were collected by centrifugation at 1500 × g for 5 min, re-suspended in deionized water and the optical density measured at A600 = 0.3, corresponding to a concentration of ∼109 colony-forming units (cfu) ml–1. The virulence of the isolate was confirmed further by subjecting rice (cv. ‘IR 50’) leaves to pathogenicity test by clip inoculation. 2.3

Hydroponic experiments with salicylic acid (SA)

Different solutions of SA with final concentrations of 50, 100 and 1000 μmol/l were added into the Hoagland solution (500 ml / beaker) containing 25-day-old rice seedlings. To standardize the optimum incubation period, the SA with the above final concentrations was added to the Hoagland solution at 1, 2 and 3 days prior to inoculation with Xoo. After the addition of the inducer with a incubation period for 1, 2 and 3 days after the pretreatment, leaves of the rice plants were clipinoculated with the bacterial (Xoo) suspension containing ∼109 cfu ml–1. The BLB disease development was recorded for 7 days after each inoculation schedule and expressed in terms of lesion length. The Xoo inoculation of SA non-pretreated plants with a waiting period for 1, 2 and 3 days were maintained for comparison purposes. However, the plants treated with sterile distilled water without Xoo inoculation and SA treatment served as another internal control. The tissue samples were also analyzed for SA levels in the susceptible rice cultivar ‘IR 50’ in the Xoo inoculation of SA-pretreated and SA non-pretreated rice plants as per the protocol described by Enyedi et al. (1992). All data shown are the mean values of 25 leaves per treatment. All experiments were performed three times with similar results and the data presented are from one representative experiment. 2.4

Induction of pathogenesis-related (PR) proteins

Induction of PR proteins were assessed in Xoo-inoculated rice plants pretreated with SA and nonpretreated with SA for 1, 2 and 3 days (1000 μmol/l). Proteins were extracted from the leaf samples (1 g) in 2 ml of 0.1 mol/l potassium phosphate buffer (pH 6.5) using a pre-chilled pestle and mortar at 4 °C. The homogenate was centrifuged at 10,000 × g for 1.5 min at 4 °C. Protein content of the supernatant was determined according to the method described by Bradford (1976) using bovine serum albumin as a standard. Approximately 100 μg of protein per sample was added to 40 μl of a sample buffer containing 0.0625 mol/l Tris, 2 % sodium dodecylsulphate (SDS), 10 % glycerol, 5 % 2-mercaptoethanol and 0.01 % bromophenol blue at pH 6.8 by vigorous vortexing and then boiled for 5 min. SDS-PAGE was carried out using Hoefer Electrophoresis Unit according to the procedure of Laemmli (1970) with a 12 % separating gel and 4 % stacking gel. Electrophoresis was performed at 40 V until the dye reached the separating gel and then increased to 100 V and continued until the blue dye reached the bottom of the gel. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R250 (Bio-Rad) overnight. A protein marker with known molecular weight (Sigma St. Louis, USA) was co-electrophoresed to estimate the molecular weights of the proteins. 2.5

Western blotting analysis

After electrophoresis, the proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA) using Semidry “Transblot” apparatus (Bio-Rad, Richmond, CA, USA). The membrane was then blocked in Tris-buffered saline (TBS) (10 mmol/l Tris-HCl, 150 mmol/l NaCl, pH 8.0) containing 0.05 % Tween-20 supplemented with 2.5 % gelatin. Antiserum raised against rice thaumatin like protein (TLP) (a gift from Prof. S. Muthukrishnan, Kansas State University, USA) was used as primary antibody at 1 : 1500 dilution. Detection of TLP on the membrane was performed according to Winston et al. (1987) using a 1 : 1500 dilution of horseradish peroxidase conjugated goat-anti rabbit IgG (Bio-Rad). The protein bands were visualized using 4-chloro-1-napthol (Bio-Rad). Molecular weight of the proteins was determined using prestained kaleidoscope protein markers (Bio-Rad, USA).

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Estimation of phenolics content

Phenolics content of rice leaf tissue was estimated by the procedure described by Zieslin and BenZaken (1993). One g of SA-pretreated (1000 μmol/l) and SA non-pretreated rice leaf tissues collected at 0, 12, 24, 48 and 72 h, respectively, after pathogen inoculation were homogenized in 10 ml of 80 % methanol and agitated for 15 min at 70 °C. One ml of the methanolic extract was added to 5 ml of distilled water and 250 μl of Folin-Ciocalteau reagent (1 N) and the solution was kept at 25 °C for 3 min. One ml of a saturated solution of Na2CO3 and 1 ml of distilled water were added and the reaction mixture incubated for 1 h at 25 °C. The absorption of the developed blue color was measured using a Beckman DU64 spectrophotometer at 725 nm. The relative content of the total soluble phenolics was calculated according to a standard curve obtained from a Folin-Ciocalteau reaction with phenol and expressed as phenol equivalents in μg g–1 fresh weight. 2.7

Chitinase assay

One gram of SA-pretreated (1000 μmol/l) and SA non-pretreated rice leaf tissues collected at 0, 12, 24, 48 and 72 h respectively, after pathogen inoculation were immediately extracted with 10 ml of 0.1 mol/l sodium citrate buffer (pH 5.0). The homogenate was centrifuged for 10 min at 10,000 × g at 4 °C and the supernatant was used as the enzyme source. Colloidal chitin was prepared from crab shell chitin (Sigma) according to Berger and Reynolds (1958). The commercial lyophilized snail gut enzyme (Helicase, obtained from Sepracor, France) was desalted as described by Boller and Mauch (1988). For the colorimetric assay of chitinase, 10 μl of 1 mol/l sodium acetate buffer (pH 4.0), 0.4 ml of enzyme extract and 0.1 ml colloidal chitin (1 mg) were pipetted into a 1.5-ml Eppendorf tube. After 2 h at 37 °C, the reaction was stopped by centrifugation at 1,000 × g for 3 min. An aliquot of the supernatant (0.3 ml) was pipetted into a glass reagent tube containing 30 μl of 1 mol/l potassium phosphate buffer (pH 7.1) and incubated with 20 μl desalted snail gut enzyme for 1 h. The resulting monomeric N-acetylglucosamine (GlcNAc) was determined according to Reissig et al. (1959) using internal standards of GlcNAc in the assay mixtures for calculations. Enzyme activity was expressed as nmol GlcNAc equivalents min–1 g–1 fresh weight. 2.8

β-1,3-glucanase assay

β-1,3-glucanase activity was assayed colorimetrically by the laminarin-dinitrosalicylate method (Pan et al. 1991). One gram of SA-pretreated (1000 μmol/l) and SA non-pretreated rice leaf tissues collected at 0, 12, 24, 48 and 72 h, respectively, after inoculation with Xoo were extracted with 5 ml of 0.05 mol/l sodium acetate buffer (pH 5.0) by grinding at 4 °C using a pestle and mortar. The extract was then centrifuged at 10,000 × g for 15 min at 4 °C and the supernatant was used in the enzyme assay. The reaction mixture consisted of 62.5 μl of 4 % laminarin and 62.5 μl of enzyme extract. The reaction was carried out at 40 °C for 10 min. The reaction was then stopped by adding 375 μl of dinitrosalicylic reagent and heating for 5 min in a boiling water bath. The resulting colored solution was diluted with 4.5 ml of distilled water, vortexed and its absorbance at 500 nm was determined. Enzyme activity was expressed as nmol glucose min–1 g–1 fresh weight. 2.9

Statistical analysis

All experiments were laid out in a completely randomized design (CRD) with 25 plants maintained for each treatment with three replications. The data were statistically analyzed using IRRI STAT version 92-1 developed by the International Rice Research Institute Biometrics Unit, Philippines. 3

Results

All the test concentrations of SA, viz., 50, 100 and 1000 μmol/l were effective in inducing resistance to BLB pathogen in terms of reducing the lesion length. A significant reduction in BLB development in rice was observed due to pretreatment with SA. Of the various concentrations of SA tested, the best

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protection (20.34 % reduction in BLB lesion length) was observed at 1000 μmol/l and the effectiveness persisted in the susceptible cv. ‘IR 50’ for at least 3 days prior to inoculation with Xoo when compared to the SA non-pretreated rice plants. However, no significant symptom reduction in BLB lesion length was observed in control (plants non-pretreated with SA and uninoculated with Xoo) rice plants treated with sterile distilled water (Table 1). In the SA-pretreated rice plants with Xoo inoculation, there was a significant amount of increase observed in the endogenous SA levels in the rice tissues (Table 2). When the leaves of the rice plants were pretreated with 1000 μmol/l SA followed by Xoo inoculation, a three-fold increase in the levels of SA was observed at 3 days after SA treatment when compared to the rice plants non-pretreated with SA and uninoculated with Xoo pathogen. At the time of SA non-pretreated rice plants with Xoo inoculation, the level of SA was 54.67 μg g–1 fresh weight (FW) at 3 days after treatment, which is two-fold higher than the rice plants non-pretreated with SA and uninoculated with Xoo (Table 2). However, there is a significant increase of 44.35 % in the levels of SA in the Xoo-inoculated rice plants pretreated with 1000 μmol/l SA when compared to the SA non-pretreated rice plants at 3 days after treatment (Table 2). Regardless of SA, the addition of 1000 μmol/l SA to the hydroponic solution caused induced resistance to BLB in reducing the lesion length (as shown in Table 1). No symptoms of phytotoxicity were observed when SA was applied at these concentrations during the course of the experiment (Table 2).

Table 1.

Efficacy of SA pretreatment in the control of bacterial leaf blight in susceptible (‘IR 50’) rice cultivar at different days prior to inoculation with Xanthomonas oryzae pv. oryzae Bacterial leaf blight lesion length (cm)y Days after treatment with SA

SA concentration (μmol/l)

Control (non -pretreated with SA and uninoculated with Xoo) SA non-pretreated rice plants 50 μ mol/l 100 μ mol/l 1000 μ mol/l

1

2

3

7.9 ± 0.13 6.2 ± 0.21 3.8 ± 0.34 3.0 ± 0.15 2.1 ± 0.20

8.0 ± 0.11 6.0 ± 0.29 3.5 ± 0.14 2.9 ± 0.09 1.7 ± 0.11

8.1 ± 0.06 5.9 ± 0.30 3.3 ± 0.19 2.7 ± 0.12 1.2 ± 0.06

y Disease development was recorded 7 days after pathogen inoculation.

Data are expressed as means ± SE of three replications of bacterial leaf blight lesion length.

Table 2.

Levels of endogenous salicylic acid (μg/g fresh weight) determination in 3-week-old rice seedlings (cv. ‘IR 50’) after Xanthomonas oryzae pv. oryzae inoculation of SA-pretreated and SA non-pretreated rice plants

Treatments

Control (Plants non-pretreated with SA and uninoculated with Xoo) Xoo inoculation of rice plants non-pretreated with SA Xoo inoculation of rice plants pretreated with 50 μ mol/l SA Xoo inoculation of rice plants pretreated with 100 μ mol/l SA Xoo inoculation of rice plants pretreated with 1000 μ mol/l SA Values are means ± SE of three replications

Leaf SA (μg/g fresh weight) Days after treatment 1

2

3

18.35 ± 1.01

21.54 ± 1.06

27.96 ± 0.52

45.18 ± 0.09

49.37 ± 1.24

54.67 ± 0.98

59.02 ± 1.24

66.25 ± 1.36

70.25 ± 0.42

74.24 ± 0.59

79.59 ± 1.57

82.51 ± 1.02

88.65 ± 0.47

90.06 ± 0.68

98.24 ± 1.11

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Western blot analysis indicated that a 25 kDa protein cross-reacting with rice TLP antiserum was induced in Xoo inoculation of SA-pretreated and SA non-pretreated plants (Fig. 1). The induction of the TLPs was more pronounced when the plants were pretreated with 1000 μmol/l SA followed by pathogen inoculation when compared to the SA non-pretreated Xoo-inoculated rice plants (Fig. 1). A significant increase in the induction of TLPs 3 days after Xoo inoculation in the tissues pretreated with 1000 μmol/l SA was observed when compared with the 2 days and 1 day after Xoo inoculation in SA treated plants. Similar induction was observed but at a lower level with no significant differences on the third day, second and first day in the SA non-pretreated Xoo-inoculated rice plants. However, in the SA non-pretreated and uninoculated rice plants, the induction of TLP antibody cross-reacting protein appeared to be only in trace (Fig. 1). Activity of PR proteins and accumulation of phenolics in SA pre-treated and in SA non-pretreated rice leaf tissues in response to inoculation with Xoo were examined. A significant increase in phenolic content, chitinase and β-1,3-glucanase activity were observed in the rice plants pretreated with 1000 μmol/l of SA 24 h after Xoo inoculation. The increase was sustained throughout the experimental period of 72 h (Fig. 2). As shown in Figure 2, the mock plants treated with sterile distilled water alone, did not elicit increases in phenolic content, chitinase and β-1,3-glucanase activity. An almost 76 % increase in the levels of phenolics content and β-1,3-glucanase activity and 78 % increase in the levels of chitinase activity were recorded 72 h after pathogen inoculation in rice tissues pretreated with 1000 μmol/l SA in comparison with the SA non-pretreated rice tissues with Xoo inoculation (Fig. 2).

4

Discussion

Biological, chemical and physical induction of resistance to pathogens has been described in cereals (Schweizer et al. 1999; Gorlach et al. 1996; Silverman et al. 1995). However, the molecular events associated with the establishment of acquired resistance in cereals are less well understood, and information about signals involved in acquired resistance or existence of SAR in cereals is scarce (Ryals et al. 1996; Tamas and Huttova 1998). It is well known that plants are endowed with various defense

Fig. 1. Induction of 25 kDa TLP antibody cross-reacting protein in rice in response to Xanthomonas oryzae pv. oryzae inoculation of SA-pretreated and SA nonpretreated plants. a – Control (rice plants non-pretreated with SA and uninoculated with Xoo); b – 1 day after Xoo inoculation in SA non-pretreated plants; c – 2 day after Xoo inoculation in SA non-pretreated plants; d – 3 day after Xoo inoculation in SA non-pretreated plants; e – 1 day after Xoo inoculation in 1000 μ mol/l SA pretreated plants ; f – 2 days after Xoo inoculation in 1000 μ mol/l SA pretreated plants; g – 3 days after Xoo inoculation in 1000 μ mol/l SA pretreated plants. Aliquots (100 μg) of proteins were analyzed by western blotting analysis after SDS-PAGE, using rice TLP antiserum. Sizes of the marker protein are indicated on the left.

KDa 2191246741302519-

a b c d e f g

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Changes in phenolics content in SA prereated and SA non-pretreated rice plants in response to inoculation with Xanthomonas oryzae pv. oryzae . μ g g -1 fresh weight

1000 800 600 400 200 0 0

12

A

24 48 Hours after inoculation

72

Changes in chitinase activity in SA prereated and SA non-pretreated rice plants plants in response to inoculation with Xanthomonas oryzae pv. oryzae.

n molGlcNAc min- 1 g-1

80 60 40 20 0 0

B

12

24

48

72

Hours after inoculation Changes in b-1,3-glucanase activity in SA prereated and SA nonpretreated rice plants in response to inoculation with Xanthomonas oryzae pv. oryzae .

n mol glucose min -1 g-1

1000 800 600 400 200 0

0

C

12

24

48

72

Hours after inoculation

Fig. 2. Changes in phenolics (A), chitinase activity (B) and β-1,3-glucanase (C) activity in SA-pretreated and SAnonpretreated rice plants in response to inoculation with Xanthomonas oryzae pv. oryzae. Data are means from three independent experiments. Xoo inoculation in 1000 μ mol/l SA pretreated plants (■); Xoo inoculation in SA nonpretreated plants (▲); Control (non-pretreated with SA and uninoculated with Xoo) (◆).

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mechanisms against pathogens. However, in the susceptible plants, the defense mechanisms are not induced. For the induction of defense mechanisms, signals are needed. The defense mechanisms can be triggered even in the susceptible cultivars by manipulating the signal transduction system (Vidhyasekaran 1997; Lucas 1999). Many defense mechanisms are triggered in plants in response to infection by pathogens (M’Piga et al. 1997), including the deposition of phenolics (Vidhyasekaran et al. 2001), phytoalexins, callose, PR proteins and hydroxyproline-rich glycoproteins (HRGPs) (Vidhyasekeran 1997). The genes encoding PR proteins are expressed not only in the infected leaves, but to certain extent also in the non-infected leaves of the same plant (Sticher et al. 1997). This phenomenon, SAR, plays a major role in disease resistance of plants (Schweizer et al. 1999). Naturally induced SAR is not predictable in timing and level of expression and therefore would not be useful for agricultural practice. Thus, it is obvious that, from a practical point of view, only chemical means of plant activation would have definite advantages in plant disease control. Many studies of SA action have been predicted on its functions in SAR and hence involved experiments designed to monitor the induction of defense-related responses in the economically important cereal crop rice against Xoo in the SA pretreated and SA non-pretreated rice plants. Signal potentiation was observed in all the test concentrations of SA, viz., 50, 100 and 1000 μmol/l and was effective in inducing resistance to BLB in terms of reducing the lesion length (Table 1). However, the best protection was observed with 1000 μmol/l SA and the effectiveness persisted in the susceptible cv. ‘IR 50’ for at least 3 days prior to inoculation with Xoo. The results of the present study indicated clearly that plants treated with SA were effective in inducing systemic resistance in rice plants against Xoo. Feeding SA prior to Xoo inoculation in the ‘IR 50’ rice plants grown hydroponically resulted in an increase in the levels of SA (Table 2). The SA levels in the rice plants pretreated with 1000 μmol/l SA with Xoo inoculation increased significantly for at least 3 days of treatment when compared to those observed during SAR in SA non-pretreated rice plants with Xoo inoculation. The data presented in the study supported that when rice plants were pretreated with 1000 μmol/l SA or at a lesser concentration, following pathogen inoculation, the pathogen could not colonize from the site of inoculation. However, in the SA non-pretreated rice plants with Xoo inoculation the plants dried completely within 3 days. These results have proved that SA induced systemic resistance against the BLB in rice. Artificially increasing the endogenous SA to levels typically observed during SAR increased resistance to BLB as demonstrated by a reduction in lesion length (Table 1). These observations indicate that rice plants pretreated with 1000 μmol/l SA following Xoo inoculation of ‘IR 50’ rice leaves was found to prime the rice plants for enhanced activation of defense responses against BLB. Similar induction of SAR has been reported in various host/pathogen systems (Oostendorp et al. 1996; Cohen et al. 1999; Hong et al. 1999; Narusaka et al. 1999; Toal et al. 1999; Métraux et al. 2002). The induction of SAR in response to inducers is often correlated with the systemic accumulation of β-1,3-glucanase, chitinase and thaumatin-like-protein (Velazhahan et al. 1998; Xu and Reddy 1997). In the present study, western blot analysis revealed the induction of a new protein that crossreacted with a TLP antibody and with a molecular mass of 25 kDa in rice leaves in response to pretreatment with SA followed by pathogen inoculation. The inoculation with Xoo rapidly induced the 25 kDa protein with higher induction in plants pretreated with SA with an incubation period of 3 days. Similar induction was observed, but at a lower level with no significant differences on the third, second and first day in the SA non-pretreated Xoo-inoculated rice plants. However, in the SA nonpretreated and uninoculated rice plants, the induction of TLP antibody cross-reacting protein appeared to be only in trace (Fig. 1). PR proteins accumulate abundantly at the site of infection, but some also accumulate in the uninoculated parts of an infected plant, although to a lesser degree than in inoculated parts (Sticher et al. 1997). It can be concluded from the study that the 25 kDa protein induced in SA-treated plants may be a TLP antibody cross-reacting protein. Further investigations are on the way to clone the gene and sequence the protein to justify it to be a TLP protein. The systemic resistance is associated with the co-ordinate expression of a complex set of so-called ‘SAR’ genes (Ryals et al. 1996; Conrath et al. 2001), which include genes for some of the PR proteins (van Loon and van Strien 1999). The enzymatic activities of several PR proteins have been identified and include β-1,3-glucanases (PR 2), chitinases (PR 3) and TLPs (PR 5) which can hydrolyze microbial cell wall components. Furthermore, over-expression of several PR genes in transgenic plants has been shown to enhance their resistance to certain fungal pathogens (Lin et al. 1995; Chen et al. 1999; Datta et al.

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1999; Datta et al. 2001). Therefore, the expression of PR genes and the associated accumulation of the encoded PR proteins have often been considered as the molecular basis of ISR. However, this assumption proves the fact that ISR is often effective against a variety of fungal and viral pathogens as well as against bacteria (Kuc 1995; Ryals et al. 1996; Sticher et al. 1997; Conrath et al. 2001; Mohan Babu et al. 2003). Antibacterial effects have not yet been shown for any PR protein, nor has enhanced resistance to Xoo pathogen been reported anywhere in the world with respect to the over-expression of the PR genes. In the present study, a concomitant increase in the levels of β-1,3-glucanase and chitinase was observed in rice plants pretreated with SA following Xoo inoculation. Increase in phenolics content in plants has been correlated with resistance to pathogens in both dicots and monocots (Vidhyasekeran 1997). It is evident that resistant plants contain more phenols or produce polyphenols more rapidly than susceptible ones (Vidhyasekeran 1997; Chong et al. 1999; von Ropenack et al. 1998). The increased phenolics content and accumulation of PR proteins following application of SA might be involved in the resistance of rice against Xoo. However, in the rice BLB pathogen interactions, induction of phenolics synthesis was not suppressed in the susceptible interactions. It suggests that the defensive mechanism can be activated even in the susceptible cultivar but early induction may be necessary to induce resistance. Hence, it appears that only delayed induction in the defense mechanisms has resulted in susceptibility. Furthermore, the SA itself is a phenolics compound and hence application of SA could have increased the phenolics content of the plant (Vidhyasekeran 1997). Since the SA has been reported to be a signal molecule, the enzymes involved in the phenolics synthesis like PAL and 4CL have been studied resulting in higher synthesis of phenolics (Thulke and Conrath 1998; Vidhyasekeran et al. 2001). However, this possibility has not yet been studied. The data presented in this study further support the hypothesis that pretreatment of SA following pathogen inoculation is the probable inducer of PR proteins. Nevertheless, the data presented in this paper further elucidate a major regulatory role of defense responses that are induced locally at the infection site and only after pathogen attack, the accumulation of PR proteins are augumented when the rice tissues has been pretreated with SA. Recently, Conrath et al. (2002) showed that SA could potentiate the expression of defense genes after pathogen attack or wounding. The important role of defense response potentiation in SAR is also supported by Siegrist et al. (1994), who reported on increased deposition of various cell wall phenolics and augmented chitinase activity upon Colletotrichum lagenarium infection of cucumber seedlings exhibiting enhanced disease resistance upon pretreatment with SA. Such chemicals might provide interesting tools to gain insights into the pathway leading to resistance. Since conditioning of cells or whole plants does not lead to changes that are immediately apparent but manifests itself only after an elicitor treatment or a challenge infection. Interestingly, the induction of resistance in plant parts remote from the site of inoculation is postulated to result from the translocation of a hitherto unknown systemic signal produced at the site of infection (Sticher et al. 1997). This signal primes the plant against further pathogen attacks, probably by triggering a complex array of defense responses. Hence, in the rice system, studying cell priming and the resulting defense response potentiation have opened a new avenue for protecting rice plants against BLB by SA.

Acknowledgements The author thanks Dr. S. Muthukrishnan, Kansas State University, USA for providing the rice TLP antiserum.

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