Dual role of nitric oxide in Solanum spp.–Oidium neolycopersici interactions

Environmental and Experimental Botany 74 (2011) 37–44

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Dual role of nitric oxide in Solanum spp.–Oidium neolycopersici interactions Jana Piterková a , Jakub Hofman a , Barbora Mieslerová b , Michaela Sedláˇrová b , Lenka Luhová a , Aleˇs Lebeda b , Marek Petˇrivalsky´ a,∗ a b

Department of Biochemistry, Faculty of Science, Palack´ y University in Olomouc, Sˇ lechtitel˚ u 11, 78371 Olomouc-Holice, Czech Republic Department of Botany, Faculty of Science, Palack´ y University in Olomouc, Sˇ lechtitel˚ u 11, 78371 Olomouc-Holice, Czech Republic

a r t i c l e

i n f o

Article history: Received 25 May 2010 Received in revised form 22 April 2011 Accepted 29 April 2011 Keywords: Nitric oxide Tomato Solanum spp. Oidium neolycopersici Powdery mildew Hypersensitive reaction

a b s t r a c t The role of nitric oxide in the pathogenesis of Oidium neolycopersici was studied on leaf discs of three Solanum spp. genotypes differing in their susceptibility to powdery mildew infection. The germination of pathogen conidia, development of infection structures and reaction of host tissues were compared for S. lycopersicum (susceptible), S. chmielewskii (moderately resistant) and S. habrochaites f. glabratum (highly resistant genotype) in presence of compounds modulating NO levels. The effect of NO donor sodium nitroprusside varied among genotypes and studied time intervals whereas NO scavenger 2-phenyl4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide accelerated fungal development in all three Solanum spp. genotypes. The exposure of leaf discs to NOS inhibitor NG -nitro-l-arginine methyl ester decreased powdery mildew growth namely in S. chmielewskii. Confocal laser scanning microscopy using the fluorescent probe 4-amino-5-(N-methylamino)-2 ,7 -difluorofluorescein diacetate localised NO accumulation both in pathogen germ tubes and appressoria and in penetrated cells of resistant genotypes of S. chmielewskii and S. habrochaites f. glabratum. Our results confirm an essential role for NO in powdery mildew pathogenesis including the penetration of biotrophic pathogen and the initiation of hypersensitive reaction, and suggest the contribution of NO to molecular mechanisms of diversity in interactions of Solanum spp. with O. neolycopersici. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Nitric oxide (NO), an ubiquitous intra- and intercellular messenger, perform a broad spectrum of regulatory functions in plant growth, ontogeny and responses to multiple stress stimuli (Wendehenne et al., 2001; Lamattina et al., 2003; Neill et al., 2003; del Río et al., 2004). The crucial role of NO in both signalling and defence mechanisms of infected plants has been documented in their interactions with viruses (Durner et al., 1998; Danci et al., 2009), bacteria (Delledonne et al., 1998; Modolo et al., 2005; Mur et al., 2005; Johnson et al., 2008), oomycetes (Sedláˇrová et al., 2011) and fungi (Tada et al., 2004; Prats et al., 2005; Piterková et al., 2009). NO is indispensable for initiation and progress of plant hypersensitive response (HR), modification of gene expression, and synthesis of pathogenesis-related (PR) proteins (Wendehenne et al., 2004;

Abbreviations: cAMP, cyclic adenosine monophosphate; DAF-FM DA, 4-amino5-(N-methylamino)-2 ,7 -difluorofluorescein diacetate; hpi, hours post inoculation; HR, hypersensitive response; l-NAME, NG -nitro-l-arginine methyl ester; NOS, nitric oxide synthase; PR proteins, pathogenesis-related proteins; PTIO, 2-phenyl-4,4,5,5,tetramethylimidazoline-1-oxyl 3-oxide; ROS, reactive oxygen species; SNP, sodium nitroprusside. ∗ Corresponding author. Tel.: +420 58 563 4925; fax: +420 58 563 4933. ´ E-mail address: [email protected] (M. Petˇrivalsky). 0098-8472/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2011.04.016

Zeier et al., 2004; Mur et al., 2006; Zaninotto et al., 2006). The main source of NO in animals is nitric oxide synthase (NOS) while several enzymes were described to produce NO in plants, i.e. NOS-like enzyme, nitrate reductase, and nitrite:NO reductase, in addition to non-enzymatic generation of NO from nitrite (Yamasaki et al., 1999; del Río et al., 2004; Zemojtel et al., 2006; Arasimowicz and Floryszak-Wieczorek, 2007). Despite a decade of intensive research, the origin and function of NO in plants under physiological and stress conditions still awaits to be elucidated (Planchet et al., 2006; Neill et al., 2008; Wilson et al., 2008). Previous reports revealed an intimate interplay between NO and reactive oxygen species (ROS) during plant–pathogen interactions. Intensive ROS production in the infected plants triggers HR and plant cell wall reinforcement and is involved in microbe destruction (Bolwell and Wojtaszek, 1997; Neill et al., 2002; Wendehenne et al., 2004; Mur et al., 2008; Yoshioka et al., 2009). HR, attributed mainly to race-specific interactions, is conditioned by a rapid accumulation of both ROS (Keller et al., 1998) and NO (Delledonne et al., 1998). Synergistic action of NO and H2 O2 is believed to orchestrate the localized cell death thus restricting pathogen invasion (Zaninotto et al., 2006), although the mechanism of interaction between the molecules is still a matter of debate (De Gara et al., 2003; Delledonne et al., 2003; Tada et al., 2004; Mur et al., 2006; Wilson et al., 2008). Moreover, both ROS and NO are produced also

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by microorganisms, especially those with mycelial growth (Johnson et al., 2008; Prats et al., 2008; Sedláˇrová et al., 2011). This facilitates active penetration of oomycetes and fungi into host cells. However, the specific role of NO and ROS can vary among pathosystems, influenced by metabolism of the host plant, life strategy of the pathogen, and environmental conditions (Shetty et al., 2008). Oidium neolycopersici (Kiss et al., 2001), an epiphytic biotrophic pathogen positioned among ascomycete fungi, has caused epidemic infections on glasshouse tomato crops since the 1980s (Jones et al., 2001; Mieslerová and Lebeda, 1999; Mieslerová et al., 2002). Most of tomato cultivars (Solanum lycopersicum) are considered highly susceptible to the tomato powdery mildew. However, extensive screening revealed many valuable sources of potential resistance among wild Solanum spp. (Lebeda and Mieslerová, 2002). Histological studies revealed that resistant Solanum species utilize HR to prevent O. neolycopersici spread to non-infected parts of the plant. Though HR does not abolish completely the growth of mycelium, it usually suppresses the pathogen reproduction (Huang et al., 1998; Mieslerová et al., 2004). Model interactions of S. lycopersicum (Amateur), S. chmielewskii (LA 2663) and S. habrochaites (LA 2128) with O. neolycopersici have been studied in our laboratory for several years from anatomical, physiological and molecular perspectives. Previous studies e.g. showed that moderately resistant S. chmielewskii expressed HR more intensively than highly resistant S. habrochaites (Mieslerová et al., 2004). Timing as well as intensity of antioxidant enzymes activity and ROS production in Solanum spp. genotypes during powdery mildew pathogenesis correlated with degree of their resistance (Mlíˇcková et al., 2004; Tománková et al., 2006). Additionally, production of secondary metabolites (alkaloids, saponins, phenol compounds, etc.) was predicted to influence interactions within this pathosystem (Mieslerová et al., 2004). Recently, we demonstrated NO production by a NOS-like arginine-dependent enzyme related to the activation of both local and systemic resistance mechanisms (Piterková et al., 2009). We hypothesized interaction of NO and H2 O2 in response to powdery mildew can form molecular basis of Solanum spp. resistance to powdery mildew. Preliminary results indicated also NO production by mycelium of O. neolycopersici (Piterková et al., 2009), similarly to that reported for barley powdery mildew, Blumeria graminis f. sp. hordei (Prats et al., 2008) or lettuce downy mildew, Bremia lactucae (Sedláˇrová et al., 2011). Herein we present the study of NO role over O. neolycopersici development on three Solanum spp. genotypes with various reaction patterns to powdery mildew, using compounds modulating endogenous NO level in a leaf disc experiments during 72 h post inoculation. Our objective was to determine the relation between increased or decreased NO levels and the development of pathogen structures on leaf discs. To this purpose we tested the hypothesis that: (1) NO is produced both by pathogen and plant cells during various stages of pathogenesis and (2) the effects of NO level modulation are variable among plant genotypes depending on their resistance mechanism to the biotrophic pathogen.

2. Materials and methods 2.1. Plant material Three genotypes of Solanum spp. expressing differential level of resistance to O. neolycopersici were used: highly susceptible Solanum lycopersicum L. cv. Amateur, moderately resistant S. chmielewskii (Rick, Kesickii, Forbes and Holle) Spooner, Anderson and Jansen (LA 2663) and highly resistant S. habrochaites S. Knapp & D.M. Spooner f. glabratum (LA 2128) (Mieslerová et al., 2004). Seeds were sown on moistened Perlite (Agroperlite, Novy´ Jiˇcín,

Czech Republic). Seedlings were transferred into a garden soil-peat mixture (2:1, v/v) in plastic pots (7 cm in diameter) and grown in a growth chamber with 12-h light/dark cycles with light intensity of 100 ␮mol photons m−2 s−1 at 20/18 ◦ C. Plants aged approximately 10 weeks were used for the following experiments. 2.2. Application of compounds modulating NO metabolism Leaf discs (12 mm in diameter) were cut out of 4th true leaves of tomato plants by a cork borer and laid adaxial side up in Petri dishes (16 discs per dish) with filter paper moistened by 10 ml of one of the following solutions: distilled water (control); 0.1 mM SNP (sodium nitroprusside); 1 mM l-NAME (NG -nitro-l-arginine methyl ester); 0.1 mM PTIO (2–phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3oxide). All solutions were prepared freshly prior to their application. 2.3. Pathogen isolate, inoculation and incubation O. neolycopersici Kiss (isolate C-2) from the collection of the Department of Botany, Palacky´ University in Olomouc, included in the Czech National Collection of Microorganisms (collection number UPOC-FUN-127) was used for the experiments (Mieslerová et al., 2004). The pathogen was maintained and multiplied on plants of susceptible S. lycopersicum cv. Amateur aged 5–8 weeks, grown under plastic covers in a growth chamber at 20/18 ◦ C, 12/12 h day/night photoperiod and light intensity of 100 ␮mol photons m−2 s−1 (Tománková et al., 2006). Adaxial side of each leaf disc was inoculated by a surface contact (dusting/tapping) with leaves bearing fresh sporulating mycelium of tomato powdery mildew. The average number of powdery mildew conidia delivered to leaf discs reached 65 ± 15 mm−2 . Following inoculation, Petri dishes with leaf discs were incubated in a growth chamber at 20/18 ◦ C and 12 h photoperiod (light/dark). 2.4. Pathogen germination and growth Leaf discs were collected 8, 24, 48 and 72 h post inoculation (hpi), immersed in 100% acetic acid for 48 h, mounted in glycerol and prior to light microscopy (Olympus BX50 equipped with CCD digital camera Olympus DP70) stained with 1% cotton blue (Lebeda and Reinink, 1994). For each treatment following parameters were studied on four leaf discs per time interval: number of germ tubes per conidia and the length of individual germ tubes. A minimum of 120 conidia, randomly selected on leaf discs, were evaluated per each treatment and time interval. Values are given as mean and standard error. 2.5. Histochemical localization of NO by confocal microscopy Leaf discs incubated with distilled water or solutions of NO modulators were collected 24, 48, and 72 hpi and incubated in 10 ␮M solution of DAF-FM DA (4-amino-5-(N-methylamino)-2 ,7 difluorofluorescein diacetate) for 30 min, mounted on microscopic slides and subjected to confocal laser scanning microscopy on Olympus Fluorview 1000 attached to inverted microscope IX81 (Olympus, Japan). Excitation was provided by the 488 nm line of an argon ion laser; a 505 nm dichroic filter and 519 nm longpass emission filter were used. To adjust the right intensity of lasers the samples from control uninoculated plants were examined in the beginning of experiment. 2.6. Statistical analysis Statistical significance of differences among variants were evaluated by one-way analysis of variance (ANOVA; P < 0.05), followed

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Early stages of O. neolycopersici development (72 hpi) are characterized by similar dynamics on leaf discs of all three genotypes, although slightly decreased growth on resistant Solanum spp. compared to susceptible tomato can be observed (Fig. 2). The longest germ tubes were detected on susceptible S. lycopersicum cv. Amateur. Length of O. neolycopersici germ tubes was limited on resistant genotypes, namely for the 2nd and the 3rd germ tubes at 48 and 72 hpi (Fig. 2). 3.2. Effect of NO modulators on O. neolycopersici development Application of compounds known to modulate endogenous NO level, i.e. NO donor sodium nitroprusside (SNP), NO scavenger 2phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO), and inhibitor of animal NO synthase NG -nitro-l-arginine methyl ester (l-NAME), exerted varied influence to the development of O. neolycopersici on inoculated leaf discs (Figs. 3–5). Incubation of leaf discs with NO donor SNP affected pathogen growth in different manner

Fig. 1. Schematic representation of Oidium neolycopersici development at 8, 24 and 72 hpi on leaf discs of susceptible genotype Solanum lycopersicum cv. Amateur.

by a comparison of means using the Bonferroni test, in NCSS 2000 software (Statistical Solutions Ltd., Cork, Ireland). 3. Results 3.1. Development of O. neolycopersici during 72 hpi O. neolycopersici development includes formation of typical powdery mildew infection structures, i.e. short primary germ tube with conspicuous lobate appressorium, and longer secondary and tertiary germ tubes with nipple-shaped appressoria (Fig. 1). Nongerminated conidia as well as conidia with the 1st germ tube, either with or without formed appressoria, were found on leaf discs at 8 hpi. Conidia with two germ tubes prevailed on leaf discs collected 24 hpi, whereas those with two and three germ tubes were mostly found at 48 and 72 hpi. Intensive mycelium branching and formation of haustoria was recorded at 72 hpi.

Germ tube length [ µm]

300 S. lycopersicum S. chmielewskii 200

S. habrochaites

100

0 8 hpi

24 hpi

48 hpi

1st germ tube

72 hpi

24 hpi

48 hpi

72 hpi

2nd germ tube

48 hpi

72 hpi

3rd germ tube

Fig. 2. Length of primary, secondary and tertiary germ tube of O. neolycopersici on leaf discs of susceptible S. lycopersicum cv. Amateur (black), moderately resistant S. chmielewskii (dark grey) and resistant genotype S. habrochaites (grey).

Fig. 3. Changes in length of O. neolycopersici germ tubes caused by the application of modulators of endogenous NO level: (A) NO donor SNP, (B) NO scavenger PTIO and (C) NOS inhibitor l-NAME on leaf discs of susceptible S. lycopersicum cv. Amateur (black), moderately resistant S. chmielewskii (dark grey), and resistant S. habrochaites (grey). Values are expressed as percentage of control value (length of O. neolycopersici germ tubes on leaf discs incubated with distilled water and evaluated for each genotype individually).

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Fig. 4. Representative images of O. neolycopersici germination 48 hpi. Leaf discs of susceptible S. lycopersicum cv. Amateur (A–D) and moderately resistant S. chmielewskii (E–H) were incubated with distilled water (A, E), NO donor SNP (B, F), NO scavenger PTIO (C, G) and NO synthase inhibitor l-NAME (D, H). Bar represents 200 ␮m.

Fig. 5. Relative frequency of O. neolycopersici conidia forming one, two or three germ tubes on leaf discs of Solanum spp. genotypes (A) 8 hpi, (B) 24 hpi and (C) 48 hpi. Leaf discs were incubated with water, NO donor SNP, NO scavenger PTIO or NO synthase inhibitor l-NAME.

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Fig. 6. Localization of NO production by confocal microscopy in Solanum spp.-Oidium neolycopersici interactions; S. lycopersicum (cv. Amateur) (A–E), S. chmielewskii (LA 2663) (F–J) and S. habrochaites f. glabratum (LA 2128) (K–O) at 24 hpi (A, D, E, F, I, J, K, N, O), 48 hpi (B, G, L) and 72 hpi (C, H, M). Some leaf discs were pre-treated with NO scavenger PTIO (D, I, N) or NO synthase inhibitor l-NAME (E, J, O) 30 min prior to the staining with DAF-FM probe. Pathogen infection structures: c, conidium; gt, germ tube; a, appressorium; hy, hypha. Bar represents 50 ␮m.

on susceptible and resistant genotypes (Fig. 3A). SNP suppressed the growth of germ tubes on highly resistant S. habrochaites during 8–72 hpi (Fig. 3A). On the contrary, SNP application resulted in longer germ tubes compared to control leaf discs on moderately resistant S. chmielewskii starting from 24 hpi, where NO donor accelerated especially growth of 1st and 2nd germ tubes at 48 hpi. Interestingly, variable effect of SNP on conidial germination was recorded for susceptible S. lycopersicum cv. Amateur, which showed induced germ tubes growth by SNP 8 and 24 hpi but suppressed for all three germ tubes at 48 hpi and also for primary germ tube at 72 hpi (Fig. 3A). NO scavenger PTIO accelerated in general the growth of powdery mildew germ tubes on all tomato genotypes compared to the control leaf discs incubated with distilled water (Fig. 3B). Longer germ tubes were found up to 72 hpi on susceptible S. lycopersicum cv. Amateur. Shortening of primary germ tube 8–24 hpi in the presence of NO scavenger was followed by a significant prolongation of all germ tubes on both moderately resistant and resistant genotypes at 48 and 72 hpi. l-NAME, a competitive inhibitor of animal NO synthase, did not cause any significant effect on pathogen germination 8 hpi (Fig. 3C). Primary germ tubes were prolonged while secondary and tertiary tubes shortened 24 hpi, whereas at 48–72 hpi all three germ tubes were shorter than in control samples. Striking retardation of the pathogen growth was observed namely for moderately resistant S. chmielewskii (Fig. 4), however, l-NAME treatment had limited

impact on pathogen development on resistant S. habrochaites f. glabratum. Additionally to observed effects to germ tube lengths, NO modulators also affected the timing of pathogen germination as evident from relative distribution of primary, secondary and tertiary germ tubes during 8, 24 and 48 hpi time intervals (Fig. 5). Reduction in the velocity of conidial germination due to NO donor treatment was noted for susceptible S. lycopersicum and resistant S. habrochaites f. glabratum at 24 and 48 hpi (Fig. 5B,C). Conversely, SNP accelerated O. neolycopersici development on moderately resistant S. chmielewskii. NO scavenger PTIO had positive effect on the timing of O. neolycopersici germination on leaf discs of all genotypes, especially the formation of secondary and tertiary germ tube at 24 and 48 hpi, respectively, was stimulated in both resistant genotypes. NO synthase inhibitor l-NAME retarded the pathogen development, most intensely on moderately resistant S. chmielewskii where most conidia were arrested in the stage of 1st germ tube at 24 or even 48 hpi (Fig. 5A–C). 3.3. Localization of NO accumulation by confocal microscopy In vivo NO production within O. neolycopersici infection structures and inoculated host tissues was localized using cellpermeable fluorescent probe DAF-FM DA and confocal laser scanning microscopy. In general, signal for NO was detected in O. neolycopersici conidia, germ tubes and appressoria on leaf discs

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Table 1 Summary of the effect of external application of compounds modulating NO level to the development of Oidium neolycopersici on three Solanum spp. Leaf discs were laid in Petri dishes with filter paper moistened by distilled water (control), 0.1 mM SNP, 1 mM l-NAME, 0.1 mM PTIO (for details see Section 2.2). Effects: +, slightly positive, ++, positive, +++, highly positive, −, slightly negative, − −, negative, − − −, highly negative.

S. lycopersicum S. chmielewskii S. habrochaites

SNP

PTIO

l-NAME

−− −− +++

+ ++ +++

−− − −−−

of all Solanum spp. genotypes at 24 and 48 hpi (Fig. 6A, B, F, G, K, L), whereas it was absent in the pathogen structures on susceptible S. lycopersicum cv. Amateur (Fig. 6C). On the other hand, presence of NO was evident in infected cells of moderately resistant S. chmielewskii and resistant S. habrochaites f. glabratum at 72 hpi (Fig. 6H, M). Specificity of the fluorescence signal was confirmed by pre-incubation of control samples with NO scavenger PTIO (Fig. 6D, I, N). NOS-like enzyme activity was confirmed to be the main NO source both in pathogen and plant cells, as the NOS inhibitor l-NAME abolished the fluorescence signal (Fig. 6E, J, O). Current knowledge of NO effect on pathogen development in Solanum spp. – O. neolycopersici interactions is summarized in Table 1. 4. Discussion We present a detailed study of O. neolycopersici development in relation to the modulation of endogenous NO level within leaf disc tissues of Solanum spp. These experiments extend previous research focused on NO involvement in Solanum spp.–O. neolycopersici interactions on whole plant system (Piterková et al., 2009). Both local and systemic increase in NO production by an NOS-like enzyme detected in moderately and highly resistant genotypes within 216 hpi indicated an important role of NO levels in the determination of the pathogen fate (Piterková et al., 2009). As demonstrated previously, the key stages of tomato powdery mildew infection include conidial germination (3–9 hpi), development of appressoria (6–12 hpi), formation of haustoria (12–24 hpi), and the initiation of intensive germ tubes branching (48–72 hpi) (Mieslerová et al., 2004; Mlíˇcková et al., 2004). However, major differences in powdery mildew development on the three genotypes appear later, in relation to mycelium growth and particularly asexual reproduction. Powdery mildew on highly susceptible S. lycopersicum cv. Amateur usually expresses intensive sporulation from 7 days following inoculation, whereas sporadic conidiophores’ formation occurs on moderately resistant S. chmielewskii (LA 2663) and none on resistant S. habrochaites f. glabratum (LA 2128) in both cases accompanied by HR. Similarly, in glasshouse conditions under natural infection pressure throughout growing season S. chmielewskii (LA 2663) and S. habrochaites f. glabratum (LA 2128) do not show symptoms of tomato powdery mildew opposite to serious infection of S. lycopersicum cv. Amateur (Mieslerová et al., 2004). Biotrophic pathogens, such as O. neolycopersici, cannot be grown as in vitro cultures (Panstruga, 2003) which would enable to target endogenous NO levels directly in pathogen structures independently of plant cells. Therefore translaminar application of substances modulating NO concentration via leaf discs was used (Sedláˇrová et al., 2011). On this model system, as expected, the pathogen growth on susceptible genotype was slightly limited compared to resistant genotypes (Fig. 2). Increased NO level by incubation with SNP was expected to inhibit pathogen development as a consequence of stimulated NO-dependent plant defence mechanisms (Wang and Higgins, 2005). This was clearly observed

only in highly resistant S. habrochaites (Fig. 4) which is characterized by less extensive HR than moderately resistant genotype S. chmielewskii (Mieslerová et al., 2004). NO donor stimulated pathogen growth on S. chmielewskii where stronger signal of NO fluorescent probe points to an increased NO level within pathogen infection structures. Expected acceleration of O. neolycopersici development following NO scavenger application was proven in particular for resistant genotypes, previously described as a consequence of the knock-down of NO-linked plant defence responses (Delledonne et al., 1998; Bolwell, 1999; Wendehenne et al., 2001). The strongest effect was found in leaf discs of S. chmielewskii, a genotype with the highest amount of NO produced locally in infected tissues during 72 hpi (Piterková et al., 2009). By contrast, highly resistant S. habrochaites f. glabratum displayed the first culmination of NO at 8 hpi and pronounced continual increase in NO production from 96 hpi. In the susceptible genotype, elevated NO production was observed only during the early interval following inoculation, at 4–8 hpi (Piterková et al., 2009). For exact explanation of exogenous NO influence on S. chmielewskii we will continue detailed studies. The potential decrease of NO production by NOS inhibitor lNAME was reported to correlate with intensive fungal growth (Wang and Higgins, 2005). In contrast, we observed l-NAME arrested O. neolycopersici germination and development, in significant extent mainly on leaf discs of S. chmielewskii. This differential effect of NOS inhibitor on Solanum spp. genotypes cannot be simply explained by a possible inhibition of the fungal NOS activity and decrease of NO level necessary for pathogen signalling and development. We propose NO likely interacts with ROS to control fungal germination as in other pathogenesis models (Wang and Higgins, 2005; Gessler et al., 2007). To verify our hypothesis we studied the effects of rutin, unspecific scavenger of reactive nitrogen and oxygen species, on the pathogen growth (data not shown). The application of rutin on inoculated leaf discs of Solanum spp. caused only minor acceleration of powdery mildew germination at 8 hpi and did not influence later development. NOS-like activity production was previously detected in extracts of O. neolycopersici conidia by oxyhaemoglobin method (Piterková et al., 2009). In present study, the localization of NO by fluorescent probe DAF-FM DA confirmed its presence mainly in apical parts of germ tubes and appressoria (Fig. 6). Germination of fungal spores has been extensively studied in many plant–pathogen interactions. Number of signalling pathways including cAMP, phospholipase C and mitogen-activated protein kinase coordinates the spore germination and development of appressoria (Xu, 2000; Tucker and Talbot, 2001; Barhoom and Sharon, 2004). Similarly, NO was proposed to play role in the differentiation of Blumeria graminis appressoria (Prats et al., 2008) and development of haustoria and secondary hyphae (Prats et al., 2005). NO production by several phytopathogenic fungi has been reported. Conrath et al. (2004) demonstrated nitrite-dependent NO production in cultures of Pythium, Botrytis, and Fusarium spp. NO burst was associated with conidial germination of Colletotrichum coccodes (Wang and Higgins, 2005). NO apparently regulates sporangiophore development in Phycomyces blakesleeanus (Maier et al., 2001). By contrast, exogenous NO delayed germination of fungal spores and shortterm exposure to a low concentration of NO gas was able to inhibit subsequent growth of Aspergillus niger, Monilinia fructicola and Penicillium italicum (Lazar et al., 2008). The germination of Aspergillus fumigatus was inhibited in the presence of various NO donors (Kunert, 1995). The only non-animal NOS gene characterized so far was found in slime mould Physarum polycephalum (Golderer et al., 2001). Its expression increases during starvation preceding sporangium differentiation in this protozoan. Based on presented results it should be now widely accepted that both partners in plant–pathogen interactions produce NO with

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significant spatio-temporal variations dependent on pathogen and plant genotype (Mur et al., 2006; Prats et al., 2008; Piterková et al., 2009). In Solanum spp. genotypes, that differ in the extent and timing of HR (Mieslerová et al., 2004; Tománková et al., 2006), NO production was proposed to be involved mainly in local defence to powdery mildew by initiation of HR, although an systemic increase of NO production was detected also in distal non-infected tissues (Piterková et al., 2009). Changes in fluorescent signal intensity of DAF-FM DA probe localized to infected cells of resistant genotypes preceding visible symptoms of necrotization (Fig. 6). Synergistic effect of ROS and NO on arrest of pathogen development has been already suggested (De Gara et al., 2003; Tada et al., 2004; Mur et al., 2006). Compared to the knowledge on biotrophic fungal pathogens, roles of ROS and NO in plant resistance to necrotrophic pathogens are neither fully understood. Nitric oxide was shown to play a key role in the basal resistance against Botrytis cinerea, in contrast to ROS which were reported to have a negative function in the resistance of Nicotiana benthamiana (Asai and Yoshioka, 2009; Asai et al., 2010). Recently, modulation of NO production was shown to promote the colonization of plant tissue by B. cinerea and NO diffusing outside the fungal cell was suggested to influence plant defence against this necrotrophic pathogen (Turrion-Gomez and Benito, 2011). The spatio-temporal aspects of HR in Solanum spp. – O. neolycopersici interactions correspond with changes of hydrogen peroxide concentration localized both histochemically within tissues and quantified in plant extracts (Mlíˇcková et al., 2004; Tománková et al., 2006), and NO production detected by the oxyhaemoglobin method in plant extracts (Piterková et al., 2009). We expect the cytotoxic effects of increased NO and ROS level in tomato HR to pathogen challenge are probably derived from the diffusion-limited reaction of NO with O2 − to form the peroxynitrite anion (ONOO− ), a molecule known to damage lipids, proteins and nucleic acids in animal models. 5. Conclusion Altogether, presented data indicate multivalent role of NO in fungal ontogeny with a dose-dependent effect. Our results demonstrated the necessity of nitric oxide for signalling in powdery mildew development, recognition by a host plant and expression of defence mechanisms. Nevertheless, further experiments are required to understand the delicate balance among NO, ROS and other factors that facilitate germination of the fungal pathogen and host plant responses, namely hypersensitive reaction. In particular S. chmielewskii, the genotype expressing moderate resistance in experimental conditions but possessing field resistance to tomato powdery mildew, exhibits pronounced changes in NO and ROS levels as a part of stress metabolism and thus represents an interesting germplasm for the crop breeding. Acknowledgments This study was supported by the Czech Ministry of Education, Youth and Sports grants (MSM 6198959215 and 2E08018). The cooperation with Olympus Czech Group (Prague, Czech Republic) is gratefully acknowledged. References Arasimowicz, M., Floryszak-Wieczorek, J., 2007. Nitric oxide as a bioactive signalling molecule in plant stress responses. Plant Sci. 172, 876–887. Asai, S., Yoshioka, H., 2009. Nitric oxide as a partner of reactive oxygen species participates in disease resistance to necrotrophic pathogen Botrytis cinerea in Nicotiana benthamiana. Mol. Plant: Microbe Interact. 22, 619–629. Asai, S., Mase, K., Yoshioka, H., 2010. Role of nitric oxide and reactive oxide species in disease resistance to necrotrophic pathogens. Plant Signal. Behav. 5, 872–874.

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