Association of hydrogen peroxide with restriction of Septoria tritici in resistant wheat

Physiological and Molecular Plant Pathology 62 (2003) 333–346 www.elsevier.com/locate/pmpp

Association of hydrogen peroxide with restriction of Septoria tritici in resistant wheat N.P. Shettya,b,*, B.K. Kristensenc, M.-A. Newmana, K. Møllera, P.L. Gregersend, H.J.L. Jørgensena a

Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark b Downy Mildew Research Laboratory, Department of Applied Botany and Seed Pathology and Biotechnology, University of Mysore, Manasagangothri, Mysore-570006, Karnataka, India c Plant Products, Plant Research Department Risø National Laboratory, Frederiksborgvej 399, P.O. Box 49, DK-4000 Roskilde, Denmark d Department of Plant Biology, Molecular Genetics and Biotechnology, Danish Institute of Agricultural Sciences, Research Centre Flakkebjerg, DK-4200 Slagelse, Denmark Accepted 16 June 2003

Abstract Infection of wheat by Septoria tritici was studied in a compatible (cultivar ‘Sevin’-isolate IPO323) and an incompatible (cultivar ‘Stakado’-isolate IPO323) interaction. A second incompatible interaction (cultivar ‘Flame’-isolate IPO323) was included as a control of the most important observations made for Stakado. Quantitative studies of the initial stages of infection confirmed that penetration occurs through stomata. However, direct penetration attempts were also observed, indicated by papilla-formation. Pre-penetration growth and penetration frequency was not different between the interactions. Hyphal growth in Stakado was inhibited after penetration and no pycnidia formed whereas in Sevin, hyphal growth progressed and pycnidia formed 15 days after inoculation. Significantly higher amounts of H2O2 accumulated in Stakado than in Sevin until 11 days after inoculation. Timing and localization of H2O2 in Stakado correlated with arrest of pathogen growth, thus indicating a role for this molecule in resistance. H2O2 accumulation is known to arrest biotrophic pathogens and therefore also likely the hemibiotrophic pathogen S. tritici. More H2O2 accumulated in Sevin than Stakado 13 and 15 days after inoculation, coinciding with pycnidium formation and host cell collapse. This late accumulation in the compatible interaction is thought to be a stressrelated response. After inoculation with S. tritici, total peroxidase activity and gene transcript of an apoplastic peroxidase increased in Stakado. The peroxidase activity pattern and transcript accumulation profile suggest a role for peroxidase in resistance, probably in cell wall cross-linking. Accumulation patterns of the gene transcript of a catalase and the total catalase enzyme activity suggest roles for catalase synthesis and inactivation in regulating H2O2 accumulation. q 2003 Elsevier Ltd. All rights reserved. Keywords: Septoria tritici; Mycosphaerella graminicola; Defence responses; Infection biology; Wheat; Triticum aestivum; Hydrogen peroxide; Bipolaris oryzae; Puccinia striiformis f.sp. tritici

1. Introduction Septoria tritici Roberge in Desmaz. (teleomorph Mycosphaerella graminicola (Fuckel) J. Schro¨t. in Cohn) causes Abbreviations: ABTS, 2,20 -azino-bis-(3-ethylbenzothiazoline-6sulfonic acid); AOS, active oxygen species; dai, days after inoculation; DAB, 3,30 diaminobenzidine; hai, hours after inoculation; HR, hypersensitive response; 20 £ , SSC buffer (3 M NaCl and 0.3 M sodium citrate). * Corresponding author. Present address: Department of Plant Biology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Tel.: þ45-35-28-23-23; fax: þ 4535-28-33-10. E-mail address: [email protected] (N.P. Shetty). 0885-5765/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0885-5765(03)00079-1

speckled leaf blotch or septoria tritici blotch of wheat (Triticum aestivum L). The disease is economically significant in almost all wheat-growing areas and has become more serious in recent years [16]. The infection of wheat by S. tritici has been studied extensively [13,15,20, 27,44,53]. In incompatible interactions, hyphal proliferation is inhibited and pycnidia often do not develop [3,13,17,27]. However, very little is known of the mechanisms responsible for inhibiting growth of S. tritici in wheat plants. Previously, callose accumulation was reported in resistant wheat cultivars [13] but it was concluded that a possible role in resistance needed further investigation. Accumulation of autofluorescing substances in response to infection was also

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reported [13,15]. A potential role for these substances in resistance was suggested [13], but was not investigated further. There are no indications of compartmentalisation, hypersensitive responses (HR) or deposition of lignin or polyphenolic substances as defence responses [13,27]. Kema et al. [27] suggested that resistance was the result of production of compounds inhibiting fungal colonisation and pycnidium formation. Knowledge on the structural and biochemical basis of wheat resistance to S. tritici is important in order to understand how the wheat plant defends itself in this host – pathogen interaction. The oxidative burst is one of the very early and initial responses of plant tissues to pathogens and elicitors, resulting in a rapid generation and release of Active Oxygen Species (AOS) [34]. AOS include superoxide (O2 2 ), the hydroxyl radical (OHz) and hydrogen peroxide (H2O2), all of which potentially affect many cellular processes involved in plant – pathogen interactions [5,55]. Both O2 2 and OHz are highly reactive and easily undergo dismutation to yield H2O2, which is relatively stable and therefore easily detected [55]. AOS have been suggested to have direct antimicrobial effects and to play a role in other defence responses such as cell wall modifications [8,9,22,34,35,40,43,48], lipid peroxidation, phytoalexin production and HR [22,23,28,34,35,48] and activation of defence related genes [23,34,35,48]. The oxidative burst is tightly regulated by antioxidants. Peroxidases can be involved in production as well as scavenging of H2O2 [21,32,54], in reinforcement of cell walls [51], deposition of polyphenolics [33] and polymerisation of suberins [4]. In addition, plant phenolic compounds have shown strong antimicrobial activity when oxidised by peroxidase [29]. Catalases are one of the primary types of enzymes for removing H2O2 from plant cells [54], thus regulating the accumulation of H2O2 in the plant and preventing harmful effects of the H2O2. The present investigation was undertaken to study the infection biology of S. tritici in a susceptible and a resistant wheat cultivar, i.e. the mode of invasion and growth in the tissue, and to examine the role of H2O2 in restricting the pathogen development. A preliminary report of this work has been published [47]. This is the first report from a series of experiments to elucidate defence responses in wheat against S. tritici.

2. Materials and methods 2.1. Plants Two wheat cultivars were used throughout the experiments. Cv. ‘Sevin’ is susceptible to isolate IPO323 of S. tritici and cv. ‘Stakado’ resistant to this isolate. H2O2 accumulation studies and enzyme assays also included cv. ‘Flame’ (also resistant to isolate IPO323) as a control for the most important observations made for Stakado. The plants

were grown in the soil mix Weibulls Enhetsjord [25] in a growth chamber under cycles of 16 h light and 8 h darkness. Light was supplied by fluorescent tubes (Osram L 36W/11860 Lumilux plus Eco Daylight, Osram GmbH, Augsburg, Germany, 200 mE m22 s21). Temperature and RH were approximately 198C/50 – 60% RH and 168C/80 –90% RH in light and darkness, respectively. At the two-leaf stage (14 days after sowing), the second leaf on each plant was fixed in a horizontal position on bent plastic plates using unbleached cotton strings [24]. 2.2. Inoculum and inoculation S. tritici (isolate IPO323) was grown on Potato Dextrose Agar (Difco) at 15 – 208C under cycles of 16 h of near-UV light (Philips TLD 36W/08, Philips Lighting B.V., Roosendaal, The Netherlands) and 8 h of darkness for 4 days. Inoculum was harvested in glass-distilled water, the concentration adjusted to 106 spores ml21 and sprayed onto the fixed leaves until run-off, using a glass hand sprayer. The inoculated plants were sealed in plastic bags to secure 100% RH and subsequently placed in the dark in the same growth chamber as before. After 72 h, the bags were opened and light applied again. 2.3. Infection biology For studying the infection biology of S. tritici, leaves of Stakado, Flame and Sevin were harvested 1, 3, 5, 7, 9, 11, 13 and 15 days after inoculation (dai) and cleared on filter paper saturated with a mixture of absolute ethanol:glacial acetic acid (3:1, v/v) [37]. After clearing, the leaves were transferred to filter paper saturated with lactoglycerol (lactic acid/glycerol/water, 1:1:1, v/v) where they were stored until examination. At each sampling time, four leaves of each cultivar were harvested. For microscopy, leaves were stained with 0.1% Evans blue in lactoglycerol for localization of fungal structures. The initial stages of infection were studied 1, 3, 5 and 7 dai. The number of non-germinated spores were recorded on each leaf and subsequently, the development of 100 randomly chosen germinated spores were studied (a total of 400 spores per cultivar per time point), using normal light microscopy and epifluorescence microscopy (excitation maximum 330– 385 nm, dicroitic mirror DM 400 nm, barrier filter . 420 nm). For each germinated spore, the direction of the germ tubes were recorded, i.e. whether the germ tubes grew towards stomata (the tip of the germ tube grew towards the stomatal aperture and had reached the cells of the stomatal complex), whether the germ tubes stopped their growth over a stomatal aperture, whether the germ tubes passed the stomatal apertures or whether the germ tubes grew in other directions than towards stomata. The occurrence of appressorium-like hyphal swellings was also recorded, i.e. whether they occurred over stomatal apertures, over anticlinal or over periclinal cell walls. Finally, it

Odds ratio for comparison of Stakado and Sevin (Sevin used as a reference, odds ratio ¼ 1.00). The number of asterisks indicates the degree of significance. NS: non-significant difference, ***: significant at P # 0:001; **: significant at P # 0:01; *: significant at P # 0:05:

1.15NS 1.16NS 1.21NS 1.34NS 0.81NS 0.41NS 1.00NS 1.32NS 0.59NS 0.24** 37.07*** 52.2 3.3 11.0 1.5 84.3 4.8 0.0 8.8 5.8 65.5 0.3 55.6 3.8 13.0 2.0 81.3 2.0 0.0 11.3 3.5 31.3 8.5 1.22NS 0.90NS 0.80NS 0.74NS 1.27NS 0.94NS 1NS 0.77NS 0.42NS 0.16* 1*** 55.2 2.5 6.5 4.0 87.0 4.0 0.0 4.5 3.5 79.2 0.0 58.0 2.3 5.3 3.0 89.5 3.8 0.5 3.5 1.5 34.8 3.3 0.81NS 0.85NS 0.51* 0.61NS 1.70* 0.70NS 1NS 0.42NS 0.00NS 0.00NS 1.00NS 61.7 3.5 7.5 5.5 83.8 4.3 0.0 5.8 0.3 3.8 0.0 57.1 3.0 4.0 3.3 89.8 3.0 0.3 2.5 0.0 0.0 0.0 1.05NS 5.87*** 0.61NS 1.40NS 0.65NS 0.25*** 0.00*** 1.25NS 0.50NS 0.60NS 1.00NS 55.7 0.8 3.3 4.0 92.0 5.8 2.0 1.0 0.5 16.7 0.0 56.9 4.3 2.0 5.5 88.3 1.5 0.0 1.3 0.3 10.0 0.0 Pct germinated spores Pct spores with germ tubes growing towards a stoma Pct spores with germ tubes ending on a stoma Pct spores with germ tubes growing past a stoma Pct spores with germ tubes growing in other directions Pct spores with germ tubes having swellings anticlinal Pct spores with germ tubes having swellings periclinal Pct spores with germ tubes having swellings on stomata Pct spores causing penetration Pct swellings on stomata giving penetrations Pct spores causing fluorescence

Stakado Sevin Odds ratio Stakado Sevin Odds ratio Stakado

Sevin Odds ratio

Stakado Sevin Odds ratio

7 days 5 days 3 days 1 day

Table 1 Observed incidence of various developmental steps in the infection course of Septoria tritici in leaves of the two wheat cultivars Stakado (resistant) and Sevin (susceptible). Values given are percentages

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was recorded whether the spores caused penetration and autofluorescence in the tissue. Further development of S. tritici was studied qualitatively 9, 11, 13 and 15 dai. Results for Stakado and Sevin are presented in Table 1. 2.4. In vivo detection of H2O2 In vivo detection of H2O2 was carried out by staining with 3,30 diaminobenzidine (DAB, D-8001, Sigma) as described by Thordal-Christensen et al. [50]. Leaf samples were harvested from inoculated plants 3 and 6 h after inoculation (hai) and then 1, 3, 5, 7, 9, 11, 13 and 15 dai. At each time point, four leaves from each cultivar were harvested and at each leaf, 20 microscopic fields were studied (400 £ magnification, total area in field of vision approx. 0.22 mm2). The fields were selected randomly across the leaf and in each field, the total number of cells was counted as well as the number of cells with DABstaining. These numbers also comprised cells of which only a part was seen within the field of vision. The ability of cv. Sevin to produce H2O2 was checked by inoculating plants with the non-wheat pathogen Bipolaris oryzae (Breda de Haan) Shoemaker. 2.5. Cell death For localisation of cell death, living leaves on the plants were infiltrated with a 0.1% aqueous solution of Evans Blue using a syringe without needle. The leaves were harvested 1 h after infiltration and immediately studied in the microscope. Positive (dark blue) staining of whole cells indicated cell death. As control of the staining procedure, wheat leaves infected with Puccinia striiformis Westendorp f.sp. tritici Eriksson were also infiltrated with Evans blue. 2.6. Scanning electron microscopy Leaf pieces, 4 £ 7 mm2 were cut from inoculated leaves 1, 3, 5, 7, 9, 11, 13 and 15 dai and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) in vacuum. After 24 h, the leaf pieces were washed in buffer and dehydrated in a graded series of acetone. Subsequently, the tissue was dried in an EMS 850 Critical Point Dryer, coated with a 20 nm gold-palladium alloy in a SC 7640 Sputter Coater and examined in a JEOL JSM 840-A Scanning microscope. In order to study the progress of S. triticiin the interior of the leaf, the adaxial epidermis and parts of the mesophyll were ripped off some leaf pieces with double adhesive tape prior to sputter coating. Both the inner side of the epidermal layer as well as the remaining part of the leaf were examined. 2.7. Statistical analysis Data from the studies of infection biology as well as the DAB-staining data represent discrete variables since it was

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recorded whether or not a certain event had taken place (e.g. whether or not a spore germinated or whether or not an epidermal cell was stained with DAB). Consequently, these data were analysed by logistic regression, assuming a binomial distribution (corrected for overdispersion when present) [14]. For comparison of the variables (percentages), odds ratios [14] were calculated using cv. Sevin as a reference (odds ratio ¼ 1.00). For example, the odds ratio for percent spores causing penetration 1 dai is 0.50 (Table 1). This means that odds (P½1 2 P21 ; in which P is the probability of a spore giving penetration) in Sevin are two times higher than odds for Stakado. Hypotheses were rejected at P # 0:05: All data were analysed by PC-SAS (release 6.12; SAS Institute, Cary, NC). Experiments on infection biology and H2O2 accumulation were performed twice with similar results. Only results from one experiment are presented. 2.8. Protein extraction and enzyme assay Leaves were sampled for enzyme assays 1, 5, 7, 9, and 15 dai, immediately frozen in liquid nitrogen and stored at 2 808C until extraction. Total protein was prepared from the leaves by homogenizing the frozen samples in liquid nitrogen using a mortar and pestle. The frozen tissue powder was extracted with two volumes (v/w) ice-cold buffer (50 mM potassium acetate (pH 5.2), 100 mM KCl, 1 M NaCl, 1 mM CaCl2 and 1 mM ascorbic acid) followed by centrifugation at 15,000g, (48C) for 10 min. Protein samples were stored on ice until further analysis. Protein was quantified in duplicate according to Bradford [6] using coomassie reagent (Pierce, Ill, USA) with IgG as standard protein (Biorad Ltd). Specific peroxidase activities were measured spectrophotometrically at 208C (Powerwave X, Bio-Tek Instruments, Inc., Vermont, USA) using a 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate solution (100 mM sodium acetate (pH 5.5), 0.36 mM ABTS (Sigma) and 6 mM H2O2). The increase in absorbance (405 nm) was followed for 7 min with 10 s intervals. The rates were determined from the linear phase of the slope and the specific activities were calculated. A unit is defined as the amount of peroxidase decomposing 1 mmol ABTS min21, using the extinction coefficient of 36.0 mM21 cm21 [11]. For specific catalase activity, protein extraction was carried out in a mixture of 50 mM potassium phosphate buffer (pH 7.8), 1 mM dithiothreitol, 2.5 mM phenylmethylsulfonylfluoride, 8% (w/v) polyvinylpolypyrrolidone and 0.1% Triton X-100. The extract was then centrifuged at 13,000g for 10 min and the supernatant used for the assay. Catalase activity was determined spectophotometrically by measuring the rate of decrease in absorbance at 240 nm of a solution of 30 mM H2O2 in 50 mM potassium buffer (pH 7.0) at 258C. Activity was expressed in enzyme units mg protein21. A unit is defined as the amount of catalase

decomposing 1 mmol H 2O 2 min21, calculated from the extinction coefficient for H2O2 (39.4 mM21 cm21) at 240 nm [1]. Experiments on enzyme activities were performed at least twice with similar results. Only representative data are presented. 2.9. Northern blots Wheat leaves for total RNA extraction was sampled at 0, 0.5 (12 hai), 1, 3, 5, 7, 9, 11, 13, 15 dai and immediately frozen in liquid nitrogen. Approximately 100 mg ground leaf material (fresh weight) was used to extract total RNA using a Tri reagent kit (T-9424, Sigma Chemical Co., St Louis, MO, USA) according to the manufacturer’s protocol. Northern blots were made using the procedure of Sambrook et al. [46]. Fifteen micrograms of total RNA was separated in a 1.2% denaturing formaldehyde agarose gel. The RNA was then transferred to a Nylon membrane (Zeta Probe, Biorad Ltd) by capillary transfer overnight using 20 £ SSC buffer (3 M NaCl and 0.3 M sodium citrate). After the transfer, the membrane was washed with 2 £ SSC twice and then exposed to a UV-transilluminator for 5 min to fix the RNA to the membrane. The membrane was stained with 0.02% Methylene Blue dissolved in 0.3 M sodium acetate (pH 5.5) to check the equal loading of the RNA. Radioactive probes were prepared using the Megaprime DNA Labelling System (RPN 1607, Amersham Phamacia Biotech UK Ltd). The two cDNA probes used encoded a barley peroxidase (pBH6-301, [49]) and a barley catalase 2 (clone E-107, with sequence 100% identical to GenBank accession number U20778, P.L. Gregersen, unpublished). Hybridisation and washing were done at 688C using the protocol of Church and Gilbert [12], with a final wash at high stringency in 25 mM phosphate buffer containing 1 mM EDTA and 1% SDS. Finally, the filters were exposed for 48 h to X-ray film (Kodak Eastman, New York) before developing. All blots were made twice from independent experiments with similar results. Only representative data are presented.

3. Results 3.1. Infection biology S. tritici spores germinated by forming thin germ tubes (Figs. 1A – C, 2A and B), usually terminally or occasionally laterally from the central part of the spores. Percentage germination of spores was rather low (52 –62%) (Table 1), but was not significantly different between Stakado (resistant) and Sevin (susceptible) at any time point. The directions of germ tubes from germinating spores were divided into four categories. Most germ tubes (. 81%) grew away from stomata on both cultivars, with only a small fraction in the other categories (towards stomata, ending on stomata, growing past stomata). Generally, there were

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Fig. 1. Details of the infection biology of Septoria tritici, isolate IPO 323 in cv. Sevin (compatible interaction). (A) and (D)–(E) SEM micrographs. (B) and (C) light micrographs. (A) Penetration through stoma 5 dai; (B) stomatal penetration 5 dai. Arrow shows swelling of the hypha. Insert show appressorium-like swelling at higher magnification; (C) attempted direct penetration through the epidermis 5 dai. The penetration appears to be stopped by a papilla-like structure (arrow); (D) and (E) micrographs from the inside of the leaves, towards the adaxial epidermis 9 and 15 dai, respectively. In (D), a hypha has just entered the stoma and is growing towards the mesophyll cells. Another hypha is also seen passing the substomatal cavity. In (E), abundant hyphal growth is seen; (F) sporulating pycnidium exuding spores through a stoma 15 dai. Bars ¼ 20 mm.

no significant differences between the two interactions regarding the number of germ tubes in a particular category except that 1 dai, significantly more germ tubes grew towards a stoma in Stakado than in Sevin. Furthermore, 3 dai, Stakado had a significantly lower number of germ tubes ending on a stoma and a significantly higher number of germ tubes growing away from stomata than Sevin. Appressorium-like swellings were occasionally seen at the tips of germ tubes as round thickenings. These swellings were seen over anticlinal cell walls (Fig. 1C) and occasionally over periclinal cell walls (Fig. 2B). Swellings were also observed over stomata on germ tubes ending there (Fig. 1A and B). Only 1 dai, significant differences were observed between cultivars, Sevin having significantly more anticlinal as well as periclinal swellings than Stakado (Table 1).

Penetration by S. tritici into the host occurred exclusively through stomata, the germ tubes entering through the aperture and spreading intercellularly in the mesophyll (Figs. 1A, B and D, 2C, E and F). Slightly more penetrations occurred in Sevin than in Stakado, but the differences were not significant at any time point (Table 1). Penetration always took place from germ tubes that had produced a swelling over a stoma (Fig. 1A and B). A higher percentage of swellings over stomata in Sevin led to penetrations than in Stakado, but the difference was only significant 5 and 7 dai. In rare cases, S. tritici made attempts to penetrate the host directly, as indicated by formation of papilla-like structures in periclinal and anticlinal epidermal cell walls (Figs. 1C and 2B) as well as in guard cells. However, the papilla-like structures stopped penetration and hyphae could not be traced beyond the papilla-like structures.

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After successful stomatal penetration in Sevin, hyphae spread intercellularly (Figs. 1D and E, 2D – F). Initially, single hyphae spread for long distances longitudinally and transversely in the mesophyll. These hyphae branched and formed an increasingly denser network from 9 to 15 dai (Fig. 1D and E). From 11 dai, hyphae started to aggregate in the substomatal cavities to form pycnidial initials and 15 dai, fully developed pycnidia were seen from where sporulation took place (Figs. 1F and 2G). Hyphal growth after successful penetration was inhibited in Stakado compared to Sevin. The average length of penetrated hyphae until 7 dai were significantly shorter than in Sevin (data not shown) and only single and most often unbranched hyphae were seen in Stakado. Occasionally, such hyphae extended for long distances in the mesophyll, but most often, they did not grow beyond the substomatal cavities. No pycnidium formation took place. In Flame, fungal hyphae behaved similarly but progressed a little further than in Stakado. However, no pycnidia formed in Flame either. Autofluorescence was observed as a response to penetration and penetration attempts significantly more often in Stakado then in Sevin from 5 dai (Table 1). The autofluorescence was seen in the stomatal complexes 5 dai and in the mesophyll below the stomata after 7 dai, coinciding with attempted growth of S. tritici into the mesophyll. Occasionally, autofluorescence was also seen at a distance from penetrated substomatal cavities. Autofluorescence was seen to a similar extent in Flame as in Stakado. 3.2. H2O2 accumulation Accumulation of H2O2 appeared in Stakado and Flame (resistant) and Sevin (susceptible) 3 and 6 hai as numerous, small, discrete dots of DAB staining, scattered in the outer epidermal cell wall around spores on the leaf surface. Quantitative studies of accumulation of H 2O2 were performed from 1 dai in Stakado and Sevin. At 1 and 3 dai, in Stakado, several small and usually light spots were seen anticlinally or periclinally in the outer epidermal cell walls, most often at sites where the fungus was present (Fig. 2A). Only few staining reactions were observed in the mesophyll. From 5 dai, staining still took place in the epidermis (Fig. 2C). However, an increasing accumulation of H2O2 occurred in the mesophyll, particularly around substomatal cavities where penetrations occurred (Fig. 2C – E). Accumulation occurred in the cell walls, i.e. in the apoplast of the mesophyll. When S. tritici hyphae started

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to grow into the mesophyll from the substomatal cavities, H2O2 accumulated around them. This correlated with arrest of fungal growth, and hyphal growth beyond the H2O2 accumulation was not seen (Fig. 2E). Occasionally, DABpositive reactions were also seen in the outer epidermal cell walls in papilla-like structures below hyphae that had made an appressorium-like swelling on the leaf surface (Fig. 2B). A similar pattern of DAB accumulation as seen in Stakado was also seen in the incompatible interaction between Flame and isolate IPO323 and also here, H2O2 accumulation correlated with arrest of fungal growth. In Sevin, very little H2O2 accumulation was seen 1 – 11 dai, except for a few light staining reactions in epidermal and mesophyll cells. H2O2 was occasionally observed accumulating in and around the substomatal cavities (Fig. 2F), but only very rarely. However, from 13 dai, a massive H2O2 accumulation was seen throughout the mesophyll of the entire leaf (Fig. 2G and H), especially around the substomatal cavities where pycnidia formed. This H2O2 accumulation was also associated with cell collapse and coincided with appearance of macroscopic symptoms. A time course study was performed to quantify H2O2 accumulation in Stakado and Sevin (Table 2). The early (3 and 6 hai) accumulation of H2O2 was not quantified and is therefore not included in Table 2. Stakado had significantly more cells with accumulation of H2O2 than Sevin until 11 dai whereas the accumulation was significantly higher in Sevin than in Stakado 13 and 15 dai. In Stakado, accumulation of H2O2 increased with time, with a significant peak 5 dai. At 7 dai, accumulation decreased significantly but increased again 9– 13 dai to the same level as 5 dai. At 15 dai, accumulation decreased significantly to the same level as 7 dai. In Sevin, there was no significant difference in H2O2 accumulation from 1 to 11 dai, except for a slight but significant increase 5 dai, as in the resistant Stakado. At 9 and 11 dai, very little H2O2 accumulated whereas at 13 and 15 dai, a significant increase took place, resulting in a massive accumulation of H2O2 which coincided with sporulation of the pathogen. The increase in H2O2 accumulation in both cultivars 5 dai coincided with the start of pathogen penetration in the host (Table 1). Although there was no significant difference in penetration efficiency between Stakado and Sevin (Table 1), a higher percentage of spores causing penetration were associated with H2O2 accumulation in Stakado than in Sevin (Table 3). The differences were, however, only significant from 5 dai.

R Fig. 2. Accumulation of H2O2 as seen by accumulation of DAB (red-brown staining) in wheat after inoculation with Septoria tritici, isolate IPO 323. Fungal surface structures were stained with Evans blue (blue staining) (A)–(E) cv. Stakado (incompatible interaction), (F)–(H) cv. Sevin (compatible interaction). (A) accumulation of H2O2 beneath germinated spores 1 dai; (B) attempted direct penetration from an appressorium-like swelling, causing accumulation of H2O2 in the periclinal cell wall (arrow) 5 dai. Insert shows magnification of interaction site. 5 dai. (C) and (D) H2O2 accumulation 5 and 7 dai, respectively, in the apoplast underneath the epidermis (C) and in epidermis and mesophyll around penetrated substomatal cavity (D). (E) H2O2 accumulation in mesophyll and arrest of a progressing hypha (arrow), which has also accumulated H2O2; (F) accumulation of H2O2 in the mesophyll around a penetrated stoma in cv. Sevin 7 dai; (G) and (H) massive accumulation of H2O2 in cv. Sevin, coinciding with extensive tissue colonization and pycnidium formation 15 dai (G, arrow). Bars ¼ 20 mm.

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Table 2 Percentage epidermal cells with DAB-staining, indicating H2O2-accumulation in cvs Stakado and Sevin after inoculation with Septoria tritici dai

Stakado

Sevin

Odds ratio

1 3 5 7 9 11 13 15

0.7 1.4 3.5 2.2 4.4 4.3 4.0 2.3

0.1 0.4 1.1 0.4 0.1 0.2 79.5 81.1

11.35** 3.85** 3.56*** 7.30*** 34.28*** 18.92*** 0.01*** 0.00***

Odds ratio for comparison of Stakado and Sevin (Sevin used as a reference, odds ratio ¼ 1.00). The number of asterisks indicates the degree of significance. NS: non-significant difference, ***: significant at P # 0:001; **: significant at P # 0:01; *: significant at P # 0:05:

3.3. Cell death There were no indications of cell death in inoculated whole fresh leaf infiltrated with Evans blue in either cultivar until 11 dai. However, 11 dai, the mesophyll cells around penetrated substomatal cavities took up stain in Sevin but still looked intact. At 13 and 15 dai, an increasing number of cells took up stain and started to lose their original shape, indicating that they died. In Stakado there were no indications of cell death even at 13 and 15 dai. As control for the staining procedure, leaves of both Stakado and Sevin infected with P. striiformis f.sp. triticiwere also infiltrated with Evans blue. Here, strong positive staining reactions were observed in necrotic tissues (data not shown).

in the controls at all time points except 7 dai. Interestingly, this lack of significant difference 7 dai coincided with the decrease in H2O2 accumulation at this time (Table 2). Otherwise, the activity in inoculated plants increased at the same time as the H2O2 accumulation increased, except at 15 dai where low levels of H2O2 accumulated whereas peroxidase activity was high. In Sevin, peroxidase activity was generally also higher in inoculated than in the control plants, but the difference was only significant at 5 and 15 dai. At 5 dai, the increase in Sevin coincided with the onset of pathogen penetration (Table 1). After 5 dai, activity generally decreased in inoculated plants. Peroxidase in Flame accumulated in the same manner as in Stakado, with significantly higher activity in inoculated than in control plants at all time points. The activity was very low 1 dai in control plants and subsequently increased, however, not reaching the same level as in Stakado (data not shown). Catalase activity in Stakado and Sevin (Fig. 4) was significantly higher in uninoculated controls than in inoculated plants 1, 5 and 9 dai in both cultivars and furthermore at 15 dai in Stakado. At 7 dai, there was no significant difference between treatments in either cultivar and at 15 dai in Sevin, activity was very low but significantly higher in inoculated than in control plants. Also in Flame, significantly higher activity was seen in uninoculated control plants then after inoculation, but the level was not as high as in Stakado. Furthermore, the activity in control plants of Flame did not decrease as drastically as in Stakado control plants (data not shown). 3.5. Gene expression studies

3.4. Peroxidase and catalase activity Total peroxidase activity was measured in Stakado, Flame and Sevin with and without inoculation at 1, 5, 7, 9 and 15 dai. Data for Stakado and Sevin are presented in Fig. 3. The activity in the uninoculated control plants was rather high in Stakado except 1 dai whereas it was low in Sevin at all time points. In Stakado, total peroxidase activity was significantly higher in inoculated plants than

Table 3 Percentage of penetrations of Septoria tritici showing DAB-staining, indicating H2O2-accumulation in cvs Stakado and Sevin dai

Stakado

Sevin

Odds ratio

1 3 5 7

0.0 0.0 78.6 100.0

0.0 0.0 0.0 8.3

1.00NS 1.00NS 1** 1***

Odds ratio for comparison of Stakado and Sevin (Sevin used as a reference, odds ratio ¼ 1.00). The number of asterisks indicates the degree of significance. NS: non-significant difference, ***: significant at P # 0:001; **: significant at P # 0:01; *: significant at P # 0:05:

Transcript accumulation of an apoplastic peroxidase in inoculated Stakado plants was quite high at 12 hai and 3 and 7 dai whereas it was low at the other time points (Fig. 5). In control plants, activity was generally high 3 – 11 dai, especially 3 dai. In Sevin, transcript accumulation was generally low at all time points, but lower in control than in inoculated plants. In inoculated Stakado plants, transcript of the catalase accumulated already at 1 dai (Fig. 6). Gradually, accumulation increased until 15 dai with slightly lower accumulation at 5 and 9 dai. In control leaves, transcript accumulation started 5 dai and increased. Accumulation was slightly stronger than in inoculated plants until 9 dai. After this time, there was a stronger induction in inoculated than in control plants. In Sevin inoculated with S. tritici, strong catalase transcript accumulation occurred 13 dai whereas very little accumulation took place at other time points and in control plants. For both peroxidase and catalase blots, the S18 ribosomal RNA loading control was visualised using Methylene Blue and found to be equally loaded in all lanes (data not shown).

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Fig. 3. Time course of total peroxidase activity in Stakado and Sevin with and without inoculation with Septoria tritici, isolate IPO 323. Bars represent LSD0.95 values comparing activity between inoculated and control plants at each time point. NS ¼ non-significant difference.

Fig. 4. Time course of total catalase activity in Stakado and Sevin with and without inoculation with Septoria tritici, isolate IPO 323. Bars represent LSD95 values comparing activity between inoculated and control plants at each time point. NS ¼ non-significant difference.

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Fig. 5. Time course of transcript accumulation of an apoplastic peroxidase in cv. Stakado (incompatible interaction) and cv. Sevin (compatible interaction) following inoculation with isolate IPO 323 of Septoria tritici or water (control). Lanes represent days after inoculation.

Fig. 6. Time course of transcript accumulation of a catalase in cv. Stakado (incompatible interaction) and cv. Sevin (compatible interaction) following inoculation with isolate IPO 323 of Septoria tritici or water (control). Lanes represent days after inoculation.

4. Discussion Even though S. tritici is a very serious pathogen throughout the world, host defence responses are not understood. This is partly due to a lack of understanding of inheritance of resistance in the host and virulence in the pathogen. Only recently, it has been shown that a gene-forgene relationship is found in this host – pathogen system [7, 10,26]. Here, we present quantitative comparisons of the infection biology of S. tritici in a resistant (cv. Stakado) and a susceptible (cv. Sevin) cultivar of wheat and provide for the first time evidence for the involvement of H2O2 in the defence against this pathogen. The resistant cv. Flame was also included in the study to verify the most important observations made for cv. Stakado. Successful penetration of the host occurred through stomata, confirming previous reports [13,15,20,27,44]. However, formation of papilla-like structures and DABpositive reactions in periclinal cell walls indicated that direct penetrations were also attempted even though they were not successful. Direct penetrations have likewise been suggested and demonstrated in rare cases earlier [13,20,44].

Weber [53], however, reported that only direct penetrations took place. Cohen and Eyal [13] suggested that direct penetration was a secondary mechanism of invasion of the host, but it is not known which factors might trigger direct penetrations. Appressorium-like swellings were produced over stomata as well as periclinally and anticlinally and all stomatal penetrations took place from germ tubes with swellings. Swellings have been reported previously [13,15, 20,27], but their exact role in unclear, i.e. whether they serve as appressoria. Thus, Kema et al. [27] reported that swellings were not needed for penetration to occur in the interactions they studied. Pre-penetration growth and penetration was not very different for isolate IPO323 in Sevin and Stakado (Table 1). We provide quantitative estimates for these processes and thus substantiate previous qualitative observations comparing compatible and incompatible interactions between wheat and S. tritici [13,20]. After penetration, our observations confirmed the intercellular growth habit of S. tritici and initial slow and sparse colonization of the mesophyll in compatible interactions and inhibition of hyphal growth in incompatible interactions [13,20,27]. The observations that there were no marked differences in prepenetration growth and penetration of S. tritici between the cultivars and that the pathogen was generally restricted to the substomatal cavities in Stakado indicate that resistance operates after invasion of the host, i.e. in the mesophyll. As a host response, a higher percentage of cells accumulating autofluorescing substances were seen in Stakado than in Sevin. Accumulation of autofluorescing substances was previously reported as a resistance response in wheat by Cohen and Eyal [13]. This could indicate the presence of polyphenolic substances but neither lignin or polyphenolic substances have been observed previously [13,27]. However, autofluorescence could indicate callose accumulation in this system [13]. The nature and role of this response in resistance needs further investigation. As a conspicuous host response, H2O2 accumulation occurred to a significantly higher degree in the resistant cvs. Stakado and Flame than in the susceptible cv. Sevin after inoculation with S. tritici and significantly more penetrations were associated with H2O2 accumulation in Stakado (up to 100%) than in Sevin. Timing and localization of this accumulation thus correlate well with resistance, suggesting an active role of H2O2 in defence. Furthermore, pathogen hyphae were seen to accumulate H2O2 (Fig. 2E), and further growth of such hyphae was not seen. The production of AOS caused by elicitors or inoculation with pathogens has been studied in plant cell suspension culture systems, and here, two distinct phases are often seen [2,34]. The first phase is observed in both incompatible and compatible interactions and gives rise to a moderate, but transient increase in AOS production. After this first increase, a second, much larger increase occurs, but only in incompatible interactions [34,35]. A similar pattern was seen in wheat inoculated with S. tritici. Thus, a small,

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non-specific H2O2 accumulation took place in both cultivars 3 –6 hai, seen as numerous, small dots of DAB staining. This H2O2 accumulation probably represents an early recognition phenomenon. In Stakado, this was followed by a very long second increase 1 – 5 dai, coinciding with penetration of stomata and initial pathogen growth in the mesophyll. A third increase (9 –13 dai) was, however, also observed. This accumulation may take place as a response to pathogen hyphae, not initially stopped, when these attempt to establish in the mesophyll. In Sevin, very little H2O2 was seen until 13 dai when a massive accumulation took place in the mesophyll. This coincided with substantial fungal growth and was mainly found around the substomatal cavities where pycnidia formed and plant cells died. This massive H2O2 accumulation is most likely a stress response of the host, resulting from tissue and cell disorganization following pathogen colonisation. Cell death occurring in the last stages of infection during pycnidium formation has been reported earlier [15,27,53]. Cell death was suggested to be caused by toxic substances produced by the pathogen [20, 27]. However, toxins have so far not been isolated from S. tritici. According to Parbery [42], S. tritici is a predominantly biotrophic, hemibiotrophic pathogen, with a long biotrophic phase like other species of Septoria/Mycosphaerella. The growth pattern of S. tritici with infection through stomata, intercellular growth and no visible symptoms until pycnidium formation supports this view [44]. Furthermore, Rohel et al. [44] examined the nutrition of S. tritici and found that it metabolises soluble sugars present in the apoplast. Toxins are therefore not likely produced during the biotrophic phase of S. tritici. Biotrophy for S. tritici lasts until reproduction starts [42]. Progress towards reproduction is made during the biotrophic phase, however, symptom appearance coinciding with collapse of tissue has led to the conclusion that the parasite is necrotrophic or at least reproduces during a necrotrophic phase. What exactly happens during the transition from biotrophic to necrotrophic growth needs to be further investigated. This fundamental change in life style must be accompanied by a change in physiology. Toxins could be involved at this stage, thus explaining cell death and production/leakage of large amounts of H2O2. In support of this suggestion is the fact that a number of homologues to genes involved in toxin synthesis have been identified [41]. Another possible explanation for cell death and symptom appearance is that the large amount of fungal hyphae uses all available nutrients, thus resulting in cell collapse [41]. H2O2 is reported to be an effective factor in stopping growth of biotrophic pathogens like Blumeria graminis f.sp. hordei [50,52]. The fact that S. tritici has a long biotrophic phase supports our observation that H2O2 accumulation is in fact an efficient host defence mechanism stopping this pathogen. On the other hand, H2O2 and other AOS have been reported to be either produced by necrotrophic pathogens or to be utilized by them for successful pathogenesis [18,19,31]. This is plausible since

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necrotrophic or perthotrophic pathogens live from killing the host tissue. When S. tritici enters the necrotrophic phase, it is able to tolerate growing under a massive accumulation of H2O2 as evidenced from the compatible interaction with cv. Sevin. Possible explanations for the low H2O2 accumulation in Sevin could be failure of activation of H2O2 generation as the presence of S. tritici is not detected in the susceptible host, inhibition of the H2O2 generating enzymes, or that the pathogen scavenges the H2O2 produced. The ability of Sevin to produce H2O2 was checked by inoculating plants with the non-wheat pathogen B. oryzae. Here, it was found that large amounts of H2O2 accumulated as a response to infection attempts (data not shown). Therefore, we assume that S. tritici is able to colonize susceptible wheat plants by avoiding recognition, suppressing the production of H2O2 or scavenging H2O2. The latter possibility is supported by the observation that S. tritici has been found to produce catalase in culture [36] and S. tritici might therefore have the potential to scavenge H2O2. However, since a gene-for-gene relationship has been demonstrated in the wheat-S. tritici system [7,10,26], specific recognition of isolate IPO323 may also take place in Stakado, but not in Sevin. Previously, many reports have shown that H2O 2 accumulation is necessary for elicitation of HR [34,35,38, 50]. Other reports have shown that elicitors and pathogens triggered the oxidative burst but did not cause HR [23,45]. No HR is seen in the wheat-S. tritici system despite the fact that substantial amounts of H2O2 are produced, probably because the pathogen lives in the apoplast and cells are not penetrated directly. Thus, localised HR would not be expected to stop S. tritici growth. Therefore, H2O2 accumulation is regulated so it does not result in HR or otherwise reach such high levels that it could harm the plant itself. The antioxidant catalase is probably involved in this. Thus, catalase transcript accumulation (Fig. 6) followed accumulation of H2O2 (Table 2) in an inverse manner. When there was a high accumulation of H2O2 in Stakado, transcript accumulation of catalase decreased (5 and 9 dai). At 7 dai, H2O2 accumulation was low whereas there was a high accumulation of transcript. From 11 dai, a high induction of transcript was seen, coinciding with a decreasing accumulation of H2O2. In Sevin, transcript accumulation was induced only at 13 dai, coinciding with the massive H2O2 accumulation at this time. At 15 dai, catalase transcript accumulation was reduced. This could be due to suppression of catalase activity in this cultivar or the fact that progressively larger parts of the host tissue are killed. Total catalase activity and transcript accumulation did not correspond well, as the activity was generally downregulated whereas transcript accumulation was generally up-regulated with time. A great deal of the catalase activity in healthy plants is probably in the chloroplasts [54], and since the chloroplasts are eventually damaged during pathogenesis, this activity could be down-regulated whereas the extracellular and cytoplasmic activity might increase

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due to the stress imposed on the cells imposed by the pathogen. In addition, the levels of antioxidative enzymes are often post-transcriptionally regulated [39] thus often resulting in an unclear relationship between accumulation of transcript and enzyme activity. Total peroxidase enzyme activity was higher in control plants of Stakado and Flame than in Sevin even before inoculation. In Stakado and Flame, there was a significant increase in activity after inoculation with S. tritici compared to the uninoculated control plants. Increased peroxidase activity in plants is reported to be associated with resistance against pathogens [21,50], e.g. by oxidative polymerisation of phenolic compounds [30] and in both inoculated Stakado and Flame plants, the higher level of peroxidase correlated with an increased accumulation of autofluorescing substances. Several isozymes of peroxidase may be expressed during infection of wheat by S. tritici. A specific apoplastic peroxidase is known to be involved in resistance of barley against the biotrophic pathogen Blumeria graminis f.sp. hordei [30]. Hence, we tested it in the wheat-S. tritici interaction to see its possible role and found a high accumulation of transcript 12 hai, 3 and 7 dai. The first peak probably represented an early recognition of the pathogen by the host, as also evidenced by the non-specific H2O2 accumulation observed 3 and 6 hai. The peak 3 dai could represent an early, strong response to the first penetrating germ tubes, whereas the peak at 7 dai is likely a response to hyphae attempting to spread in the mesophyll. The transcript accumulation did not follow H2O2 accumulation very closely. Peaks at 3 and 7 dai correlated with low accumulation of H2O2, but the correlation was not obvious at 1 dai. This implies that this peroxidase was not directly involved in H2O2 production. Induction of apoplastic peroxidase transcript accumulation in the resistant cv. Stakado coincided with onset of penetration of S. tritici. This peroxidase was previously shown to be present in the mesophyll and is probably involved in deposition of phenylpropanoid compounds [30]. Production of phenylpropanoid radicals by H2O2 and peroxidase could form both a chemical and a physical barrier to the pathogen. Also in control plants, there was a strong accumulation of transcript of the apoplastic peroxidase (3 –11 dai). A prominent feature of Stakado is that it possesses a very high responsiveness to different stresses, thus resulting in, e.g. high transcript accumulation and high activity in enzyme assays even in control plants. Thus, macroscopically Stakado often reacts by forming tiny yellow spots following handling (wounding) as well as application of water and pathogens whereas, e.g. Flame does not. The role and accumulation pattern of the apoplastic peroxidase in the wheat-S. tritici interaction needs to be further investigated as does the presence of other peroxidase isozymes. Thus, the role of phenylpropanoid products in resistance should be investigated, especially considering that neither lignin, nor polyphenolic compounds have

previously been reported in this interaction even though autofluorescence has been seen. Likewise, the origin of the H2O2 should be analysed to determine whether peroxidase is involved. These studies will be the topic of further investigations into this host – pathogen interaction.

Acknowledgements The work was financed by the Royal Danish Ministry of Foreign Affairs through the Danida-ENRECA-project: ‘Systemic Acquired Resistance—an Eco-friendly Strategy for Managing Diseases in Rice and Pearl Millet’. We thank Dr G.H.J. Kema, Plant Research International B.V., The Netherlands, for providing S. tritici isolate IPO323. We also wish to thank Drs Eigil de Neergaard and Lisbeth Mikkelsen, The Royal Veterinary and Agricultural University as well as Professor H. Shekar Shetty, University of Mysore, for critically reviewing this manuscript.

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