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Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea
Agricultural Sciences in China
March 2010
2010, 9(3): 378-382
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea LI Hong-li, WANG Hai-yan, HAO Xin-yuan, SONG Xiao-he and MA Qing College of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University/Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Yangling 712100, P.R.China
Abstract The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and microfilaments, serving a multiplicity of functions in plant cells. To reveal the relationship between the cytoskeleton in wheat (Triticum aestivum L.) cv. Suwon 11 attacked by the non-host pathogen Sphaerotheca fuliginea and the initiation of the hypersensitive response, the microtubule inhibitor oryzalin was injected into the wheat leaves immediately prior to inoculation. The incidence of hypersensitive cell death was significantly lower than that in water-treated control. In addition, the occurrence of hypersensitive cell death was also delayed and S. fuliginea was able to penetrate and form haustoria in epidermal tissues of wheat. All the results above indicated that hypersensitive cell death was associated with depolymerisation of microtubules, suggesting that microtubules might play an important role in the expression of non-host resistance of wheat. Key words: cytoskeleton, non-host resistance, hypersensitive cell death, oryzalin
INTRODUCTION Plants are liberally exposed to a vast spectrum of potential pathogens. As a result, they have evolved intricate defense mechanisms to recognize certain pathogen elicitors and to trigger a set of defense responses against pathogens (Mysore and Ryu 2004). Non-host resistance is the most common and a durable form of disease resistance exhibited by plants (Heath 2000). A universal feature of plant-pathogen interactions is manifested as rapid localized cell death at the site of infection, termed the hypersensitive response (HR), an important defense measure in incompatible and non-host interactions (Kombrink and Schmelzer 2001; Schmelzer 2002; Ma and Shang 2009). Plant cytoskeleton, including microfilaments and microtubules, is a highly conservative subcellular struc-
ture and serves a multiplicity of functions in plant biology. In many cases, it is instrumental in mediating the plant response. For example, cytoskeletal elements are responsible for cytoplasmic aggregation, organelle movements, and cell wall apposition development beneath the infection sites (Williamson 1993; Schmelzer 2002; Hardham et al. 2007). The research on the applications of cytoskeleton-disrupting drugs could provide broad insights into the participation of microtubule or microfilament arrays in specific cell functions, especially in the non-host resistance (Mysore and Ryu 2004). Cytochalasins, phalloidin and oryzalin are widely used to prevent cytoskeleton polymerization and depolymerization (Schmidt and Panstruga 2007). The dynamic reorganization of cytoskeleton was repeatedly found to be required for the execution of HR-like cell death (Kobayashi et al. 1994; Hou et al. 2002). The roles of microtubules on the hypersensitive response,
Received 1 July, 2009 Accepted 9 September, 2009 LI Hong-li, MSc, E-mail: [email protected]; Correspondence MA Qing, Professor, Tel: +86-29-87091342, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S1671-2927(09)60107-3
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host
however, have not been reported in the wheat-cucumber powdery mildew system yet. Sphaerotheca fuliginea, the causal agent of the powdery mildew of cucumber, is an obligate biotrophic fungus that exclusively infects epidermal leaf tissue. In this paper, the microtubule polymerization inhibitor oryzalin was used to investigate the effects of microtubules on the hypersensitive response of non-host resistance in the wheat-cucumber powdery mildew system.
MATERIALS AND METHODS Plant materials Wheat (Triticum aestivum L.) cv. Suwon 11 was used in the present study. Wheat seeds were grown in organic soil in 10 cm pots in a growth chamber at 16°C, RH 70%, and a photoperiod of 16 h:8 h (L:D). Sevend-old seedlings were used for inoculation.
Treatment with microtubule inhibitor Oryzalin (Sigma-Aldrich, USA) was dissolved in dimethylsulfoxide (DMSO, Amresco, USA) as 400 mg mL-1 stock solution, stored at -20°C until use and diluted to designated concentrations (400 μg mL-1) with ddH2O just before use. For inhibitor treatments, the oryzalin solution was injected into the primary leaves of 7-d-old wheat seedlings by vacuum infiltration (Hou et al. 2002).
Inoculation with S. fuliginea Sphaerotheca fuliginea was obtained from naturally infected field-grown cucumber plants, and maintained on a growth chamber-grown cucumber cultivar Changchun Thorn at temperature of 23°C, RH 70%, and a photoperiod of 16 h:8 h (L:D). In order to ensure the freshness and the same age, spores were shaken 24 h prior to inoculation. After being treated with oryzalin, the upper surface of the wheat seedlings was inoculated with freshly harvested conidia of S. fuliginea with a paintbrush. After inoculation, seedlings were kept at high humidity in the dark for 24 h at 23°C before being transferred to the growth chamber at the same conditions as cucumber.
379
Localization of hypersensitive cell death Hypersensitive cell death was observed after staining with trypan blue (Tang et al. 1999). At 24, 48 and 72 h after inoculation (hai), the inoculated leaves were cut into 1 cm length segments, discarding the end parts of the leaves before staining. The leaf pieces were vacuum-infiltrated for 5 min with trypan blue solution in a desiccator equipped with a vacuum pump. Leaf sections were fixed and decolorized in boiling trypan blue for 10 min, then were kept at room temperature for 8 h, and cleared overnight in saturated chloral hydrate before being stored in 50% glycerol. For microscopy observation, cleared leaf segments were mounted on microscope slides, examined under a light microscope. Images were obtained using a charge-coupled device (CCD) digital camera mounted on the microscope. The formation of penetration sites was calculated as the percentage of appressoria examined. At least 50 penetration sites were examined for each time point.
RESULTS Growth and development of S. fuliginea on cucumber Sphaerotheca fuliginea, an obligate fungal pathogen, is able to establish complex and intimate structures on cucumber. To visualize the pathogenic structure, cucumber leaves were excised at different times. Conidia germinated several hours after they contacted the leaf surface of cucumber. After appressoria formed and the penetrating peg developed, an oval haustorium soon formed in the epidermal host cell (Fig.1-A). More haustoria formed with the fungal development, but no hypersensitive cell death occurred during the infection process.
Hypersensitive response in the wheat-S. fuliginea interaction (water treatment) To reveal non-host resistance responses, conidia of the cucumber powdery mildew were dusted onto the leaves of wheat. With trypan blue staining, germ tubes and appressoria were observed on both water and oryzalin
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
380
LI Hong-li et al.
A
B
Co
App
HR App Co H
20 μm
20 μm
C
D HR App H Co Co 20 μm
20 μm
E
F Co H
App HR App Co 20 μm
20 μm
Fig. 1 Hypersensitive cell death in wheat cells with water or oryzalin treatment before inoculation with non-pathogen cucumber powdery mildew. A, infection structures of Sphaerotheca fuliginea on its host cucumber, 24 hai. B, the whole cell had undergone hypersensitive response after water treatment, 24 hai. C, two fully stained cells at the penetration site and nearby with water treatment, 48 hai. D, E, successful invasion of S. fuliginea and haustorial formation on wheat epidermal cells after oryzalin treatment, 48 hai. F, incomplete HRlike cell death in epidermal cells after oryzalin treatment, 48 hai. H, haustorium; Co, conidium; App, appresorium; HR, hypersensitive response.
treatment leaves, and attempted to penetrate into the underlying host cells in infection process similar to that on its host cucumber. Occasionally, one spore could produce two germ tubes. At 24 hai, some epidermal cells near the penetration sites showed HR after inoculation with S. fuliginea on wheat leaves (Fig.1-B). The non-host pathogen S. fuliginea generally failed to penetrate into the wheat cells, with papillae being observed at penetration sites beneath appressoria. At 48 hai, an increasing number of host cells took up stain, and with advancing incubation time, the percentage of cells that had undergone hypersensitive cell death increased dramatically (Fig.2). At 72 hai, the number of stained cells was significantly higher than that at 48 hai. However, the haustoria of S. fuliginea were rarely observed in the epidermal cells of wheat at all indicated times.
Effects of microtubule inhibitor oryzalin on hypersensitive response In the preliminary experiment, different concentrations of oryzalin (50, 100, 200, and 400 μg mL-1) were used to reveal their effects on the hypersensitive response. The results showed that the effects of oryzalin treatment on the wheat plants attacked by the non-pathogen were dependent on both drug concentrations and inoculation time (data not shown). The oryzalin concentration (400 μg mL-1) used in this study was based on the preliminary experiment. For microscopy analysis, inoculated leaves injected with oryzalin (400 μg mL-1) were excised at the time points and stained with trypan blue. Treatment with
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host
Fig. 2 Percentage of infection sites of wheat epidermal cells exhibiting trypan blue staining with water and oryzalin treatment.
oryzalin delayed the HR to such an extent that the number of stained cells was significantly lower and the cells showed incomplete HR, only some parts of the cells stained in comparison with water treated control (Fig. 1-D, E and F). For example, at 48 hai, only a low percentage of cells (14.2%) displayed trypan blue staining, in contrast with 48.3% in the water treatment. At 48 hai, S. fuliginea penetrated successfully in a few cases, and formed haustoria in oryzalin treated epidermal tissues of wheat (Fig.1-D, E), but haustoria were rarely observed in living cells in the water treated ones (Fig.1-B, C). Haustorial formation is an essential step for successful colonization of plant epidermal cells. Although the haustoria formed in some infection sites of oryzalin-treated wheat leaves, they could not develop further to form colonies.
DISCUSSION For fungi that attempt to penetrate directly into epidermal cells of plants, quite a number of evidence points to the cell wall as a primary site of non-host resistance expression (Heath 2000). Wall-associated defenses include the translocation of cytoplasm and of the cell nucleus to the fungal penetration site, extracellular H2O2 generation and callose-containing papillae, which combine to produce physical barriers to infection (Lamb and Dixon 1997). However, when a papilla is lacking or fails for whatever reason to be an effective local defense against pathogen invasion, hypersensitive cell
381
death occurs as the last and the most effective defense measure in incompatible and non-host interactions (Schmelzer 2002). In our research of the compatible host-pathogen combination, no hypersensitive response occurs, but in the wheat-S. fuliginea interaction, hypersensitive response occurred either in water or oryzalin-treated leaves. Recent studies revealed that rearrangements of the cytoskeleton play an important role in cellular defense (Takemoto and Hardham 2004). Experiments involving pharmacological inhibitors of cytoskeleton integrity provide an acceptable approach to investigate the cytoskeleton function in plant-microbe interaction. In the powdery mildew-cowpea interactions, the actin microfilament-disrupting agent, cytochalasin E, was efficient at reducing H 2O 2 generation and phenolic accumulation, and increases in penetration efficiency, suggesting that the induction of H2O2 generation appeared to require the presence of an intact actin cytoskeleton (Mellersh et al. 2002). Cytochalasin A treatment allowed other non-host pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells and formed infection hyphae (Kobayashi et al. 1997). Furthermore, a combination of loss of actin cytoskeletal function and EDS1 activity severely compromises non-host resistance in Arabidopsis against the wheat powdery mildew (Blumeria graminis f. sp. tritici) (Yun et al. 2003). Our investigation showed that depolymerisation of microtubules in wheat leaves inoculated with S. fuliginea caused a marked reduction of cells undergoing HR. Treated with oryzalin, the non-pathogen S. fuliginea could penetrate successfully, forming haustoria in epidermal tissues of the non-host wheat. Similar result was reported in wheat-leaf rust interaction (Hou et al. 2002). These data further confirm that the plant cytoskeleton plays a significant role in non-host resistance. Cloning of cytoskeleton regulatory protein genes and application of green fluorescent protein (GFP)-based in vivo visualization of the cytoskeleton might provide new insights in the role of the cytoskeleton in plantmicrobe interactions for the future researches.
Acknowledgements This research was financially supported by the National Natural Science Foundation of China (30771398) and
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
382
LI Hong-li et al.
the 111 Project from Ministry of Education of China (B07049).
References Hardham A R, Jones D A, Takemoto D. 2007. Cytoskeleton and cell wall function in penetration resistance. Current Opinion in Plant Biology, 10, 342-348. Heath M C. 2000. Nonhost resistance and nonspecific plant defenses. Current Opinion in Plant Biology, 3, 315-319. Hou C Y, Wang D M, Li X J, Han S F, Liu J, Wang Z X. 2002. The effects of depolymeric cytoskeleton on hypersensitive reaction induced by the wheat-leaf rust fungus interaction. Acta Phytopathologica Sinica, 32, 147-152. (in Chinese) Kobayashi I, Kobayashi Y, Hardham A R. 1994. Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection. Planta, 195, 237-247. Kobayashi Y, Kobayashi I, Funaki Y, Fujimoto S, Takemoto T, Kunoh H. 1997. Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells. The Plant Journal, 11, 525-537. Kombrink E, Schmelzer E. 2001. The hypersensitive response and its role in local and systemic disease resistance. European Journal of Plant Pathology, 107, 69-78. Lamb C, Dixon R A. 1997. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 251-275.
Ma Q, Shang H S. 2009. Ultrastructure of stripe rust (Puccinia striiformis f. sp. tritici) interacting with slow-rusting, highly resistant, and susceptible wheat cultivars. Journal of Plant Pathology, 91, 597-606. Mellersh D G, Foulds I V, Higgins V J, Heath M C. 2002. H2O2 plays different roles in determining penetration failure in three diverse plant-fungal interactions. The Plant Journal, 29, 257-268. Mysore K S, Ryu C M. 2004. Nonhost resistance: how much do we know? Trends in Plant Science, 9, 97-104. Schmelzer E. 2002. Cell polarization, a crucial process in fungal defence. Trends in Plant Science, 7, 411-415. Schmidt S M, Panstruga R. 2007. Cytoskeleton functions in plant-microbe interactions. Physiological and Molecular Plant Pathology, 71, 135-148. Takemoto D, Hardham A R. 2004. The cytoskeleton as a regulator and target of biotic interactions in plants. Plant Physiology, 136, 3864-3876. Tang X, Xie M, Kim Y J, Zhou J, Klessig D F, Martin G B. 1999. Overexpression of Pto activates defense responses and confers broad resistance. The Plant Cell, 11, 15-29. Williamson R E. 1993. Organelle movements. Annual Review of Plant Physiology and Plant Molecular Biology, 44, 181-202. Yun B W, Atkinson H A, Gaborit C, Greenland A, Read N D, Pallas J A, Loake G J. 2003. Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. The Plant Journal, 34, 768-777. (Managing editor ZHANG Juan)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
March 2010
2010, 9(3): 378-382
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host Pathogen Sphaerotheca fuliginea LI Hong-li, WANG Hai-yan, HAO Xin-yuan, SONG Xiao-he and MA Qing College of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture, Northwest A&F University/Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Yangling 712100, P.R.China
Abstract The plant cytoskeleton is a highly dynamic and versatile intracellular scaffold composed of microtubules and microfilaments, serving a multiplicity of functions in plant cells. To reveal the relationship between the cytoskeleton in wheat (Triticum aestivum L.) cv. Suwon 11 attacked by the non-host pathogen Sphaerotheca fuliginea and the initiation of the hypersensitive response, the microtubule inhibitor oryzalin was injected into the wheat leaves immediately prior to inoculation. The incidence of hypersensitive cell death was significantly lower than that in water-treated control. In addition, the occurrence of hypersensitive cell death was also delayed and S. fuliginea was able to penetrate and form haustoria in epidermal tissues of wheat. All the results above indicated that hypersensitive cell death was associated with depolymerisation of microtubules, suggesting that microtubules might play an important role in the expression of non-host resistance of wheat. Key words: cytoskeleton, non-host resistance, hypersensitive cell death, oryzalin
INTRODUCTION Plants are liberally exposed to a vast spectrum of potential pathogens. As a result, they have evolved intricate defense mechanisms to recognize certain pathogen elicitors and to trigger a set of defense responses against pathogens (Mysore and Ryu 2004). Non-host resistance is the most common and a durable form of disease resistance exhibited by plants (Heath 2000). A universal feature of plant-pathogen interactions is manifested as rapid localized cell death at the site of infection, termed the hypersensitive response (HR), an important defense measure in incompatible and non-host interactions (Kombrink and Schmelzer 2001; Schmelzer 2002; Ma and Shang 2009). Plant cytoskeleton, including microfilaments and microtubules, is a highly conservative subcellular struc-
ture and serves a multiplicity of functions in plant biology. In many cases, it is instrumental in mediating the plant response. For example, cytoskeletal elements are responsible for cytoplasmic aggregation, organelle movements, and cell wall apposition development beneath the infection sites (Williamson 1993; Schmelzer 2002; Hardham et al. 2007). The research on the applications of cytoskeleton-disrupting drugs could provide broad insights into the participation of microtubule or microfilament arrays in specific cell functions, especially in the non-host resistance (Mysore and Ryu 2004). Cytochalasins, phalloidin and oryzalin are widely used to prevent cytoskeleton polymerization and depolymerization (Schmidt and Panstruga 2007). The dynamic reorganization of cytoskeleton was repeatedly found to be required for the execution of HR-like cell death (Kobayashi et al. 1994; Hou et al. 2002). The roles of microtubules on the hypersensitive response,
Received 1 July, 2009 Accepted 9 September, 2009 LI Hong-li, MSc, E-mail: [email protected]; Correspondence MA Qing, Professor, Tel: +86-29-87091342, E-mail: [email protected]
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S1671-2927(09)60107-3
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host
however, have not been reported in the wheat-cucumber powdery mildew system yet. Sphaerotheca fuliginea, the causal agent of the powdery mildew of cucumber, is an obligate biotrophic fungus that exclusively infects epidermal leaf tissue. In this paper, the microtubule polymerization inhibitor oryzalin was used to investigate the effects of microtubules on the hypersensitive response of non-host resistance in the wheat-cucumber powdery mildew system.
MATERIALS AND METHODS Plant materials Wheat (Triticum aestivum L.) cv. Suwon 11 was used in the present study. Wheat seeds were grown in organic soil in 10 cm pots in a growth chamber at 16°C, RH 70%, and a photoperiod of 16 h:8 h (L:D). Sevend-old seedlings were used for inoculation.
Treatment with microtubule inhibitor Oryzalin (Sigma-Aldrich, USA) was dissolved in dimethylsulfoxide (DMSO, Amresco, USA) as 400 mg mL-1 stock solution, stored at -20°C until use and diluted to designated concentrations (400 μg mL-1) with ddH2O just before use. For inhibitor treatments, the oryzalin solution was injected into the primary leaves of 7-d-old wheat seedlings by vacuum infiltration (Hou et al. 2002).
Inoculation with S. fuliginea Sphaerotheca fuliginea was obtained from naturally infected field-grown cucumber plants, and maintained on a growth chamber-grown cucumber cultivar Changchun Thorn at temperature of 23°C, RH 70%, and a photoperiod of 16 h:8 h (L:D). In order to ensure the freshness and the same age, spores were shaken 24 h prior to inoculation. After being treated with oryzalin, the upper surface of the wheat seedlings was inoculated with freshly harvested conidia of S. fuliginea with a paintbrush. After inoculation, seedlings were kept at high humidity in the dark for 24 h at 23°C before being transferred to the growth chamber at the same conditions as cucumber.
379
Localization of hypersensitive cell death Hypersensitive cell death was observed after staining with trypan blue (Tang et al. 1999). At 24, 48 and 72 h after inoculation (hai), the inoculated leaves were cut into 1 cm length segments, discarding the end parts of the leaves before staining. The leaf pieces were vacuum-infiltrated for 5 min with trypan blue solution in a desiccator equipped with a vacuum pump. Leaf sections were fixed and decolorized in boiling trypan blue for 10 min, then were kept at room temperature for 8 h, and cleared overnight in saturated chloral hydrate before being stored in 50% glycerol. For microscopy observation, cleared leaf segments were mounted on microscope slides, examined under a light microscope. Images were obtained using a charge-coupled device (CCD) digital camera mounted on the microscope. The formation of penetration sites was calculated as the percentage of appressoria examined. At least 50 penetration sites were examined for each time point.
RESULTS Growth and development of S. fuliginea on cucumber Sphaerotheca fuliginea, an obligate fungal pathogen, is able to establish complex and intimate structures on cucumber. To visualize the pathogenic structure, cucumber leaves were excised at different times. Conidia germinated several hours after they contacted the leaf surface of cucumber. After appressoria formed and the penetrating peg developed, an oval haustorium soon formed in the epidermal host cell (Fig.1-A). More haustoria formed with the fungal development, but no hypersensitive cell death occurred during the infection process.
Hypersensitive response in the wheat-S. fuliginea interaction (water treatment) To reveal non-host resistance responses, conidia of the cucumber powdery mildew were dusted onto the leaves of wheat. With trypan blue staining, germ tubes and appressoria were observed on both water and oryzalin
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
380
LI Hong-li et al.
A
B
Co
App
HR App Co H
20 μm
20 μm
C
D HR App H Co Co 20 μm
20 μm
E
F Co H
App HR App Co 20 μm
20 μm
Fig. 1 Hypersensitive cell death in wheat cells with water or oryzalin treatment before inoculation with non-pathogen cucumber powdery mildew. A, infection structures of Sphaerotheca fuliginea on its host cucumber, 24 hai. B, the whole cell had undergone hypersensitive response after water treatment, 24 hai. C, two fully stained cells at the penetration site and nearby with water treatment, 48 hai. D, E, successful invasion of S. fuliginea and haustorial formation on wheat epidermal cells after oryzalin treatment, 48 hai. F, incomplete HRlike cell death in epidermal cells after oryzalin treatment, 48 hai. H, haustorium; Co, conidium; App, appresorium; HR, hypersensitive response.
treatment leaves, and attempted to penetrate into the underlying host cells in infection process similar to that on its host cucumber. Occasionally, one spore could produce two germ tubes. At 24 hai, some epidermal cells near the penetration sites showed HR after inoculation with S. fuliginea on wheat leaves (Fig.1-B). The non-host pathogen S. fuliginea generally failed to penetrate into the wheat cells, with papillae being observed at penetration sites beneath appressoria. At 48 hai, an increasing number of host cells took up stain, and with advancing incubation time, the percentage of cells that had undergone hypersensitive cell death increased dramatically (Fig.2). At 72 hai, the number of stained cells was significantly higher than that at 48 hai. However, the haustoria of S. fuliginea were rarely observed in the epidermal cells of wheat at all indicated times.
Effects of microtubule inhibitor oryzalin on hypersensitive response In the preliminary experiment, different concentrations of oryzalin (50, 100, 200, and 400 μg mL-1) were used to reveal their effects on the hypersensitive response. The results showed that the effects of oryzalin treatment on the wheat plants attacked by the non-pathogen were dependent on both drug concentrations and inoculation time (data not shown). The oryzalin concentration (400 μg mL-1) used in this study was based on the preliminary experiment. For microscopy analysis, inoculated leaves injected with oryzalin (400 μg mL-1) were excised at the time points and stained with trypan blue. Treatment with
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Effects of Microtubule Polymerization Inhibitor on the Hypersensitive Response of Wheat Induced by the Non-Host
Fig. 2 Percentage of infection sites of wheat epidermal cells exhibiting trypan blue staining with water and oryzalin treatment.
oryzalin delayed the HR to such an extent that the number of stained cells was significantly lower and the cells showed incomplete HR, only some parts of the cells stained in comparison with water treated control (Fig. 1-D, E and F). For example, at 48 hai, only a low percentage of cells (14.2%) displayed trypan blue staining, in contrast with 48.3% in the water treatment. At 48 hai, S. fuliginea penetrated successfully in a few cases, and formed haustoria in oryzalin treated epidermal tissues of wheat (Fig.1-D, E), but haustoria were rarely observed in living cells in the water treated ones (Fig.1-B, C). Haustorial formation is an essential step for successful colonization of plant epidermal cells. Although the haustoria formed in some infection sites of oryzalin-treated wheat leaves, they could not develop further to form colonies.
DISCUSSION For fungi that attempt to penetrate directly into epidermal cells of plants, quite a number of evidence points to the cell wall as a primary site of non-host resistance expression (Heath 2000). Wall-associated defenses include the translocation of cytoplasm and of the cell nucleus to the fungal penetration site, extracellular H2O2 generation and callose-containing papillae, which combine to produce physical barriers to infection (Lamb and Dixon 1997). However, when a papilla is lacking or fails for whatever reason to be an effective local defense against pathogen invasion, hypersensitive cell
381
death occurs as the last and the most effective defense measure in incompatible and non-host interactions (Schmelzer 2002). In our research of the compatible host-pathogen combination, no hypersensitive response occurs, but in the wheat-S. fuliginea interaction, hypersensitive response occurred either in water or oryzalin-treated leaves. Recent studies revealed that rearrangements of the cytoskeleton play an important role in cellular defense (Takemoto and Hardham 2004). Experiments involving pharmacological inhibitors of cytoskeleton integrity provide an acceptable approach to investigate the cytoskeleton function in plant-microbe interaction. In the powdery mildew-cowpea interactions, the actin microfilament-disrupting agent, cytochalasin E, was efficient at reducing H 2O 2 generation and phenolic accumulation, and increases in penetration efficiency, suggesting that the induction of H2O2 generation appeared to require the presence of an intact actin cytoskeleton (Mellersh et al. 2002). Cytochalasin A treatment allowed other non-host pathogens, Colletotrichum lagenarium and Alternaria alternata, to penetrate successfully into the non-host barley cells and formed infection hyphae (Kobayashi et al. 1997). Furthermore, a combination of loss of actin cytoskeletal function and EDS1 activity severely compromises non-host resistance in Arabidopsis against the wheat powdery mildew (Blumeria graminis f. sp. tritici) (Yun et al. 2003). Our investigation showed that depolymerisation of microtubules in wheat leaves inoculated with S. fuliginea caused a marked reduction of cells undergoing HR. Treated with oryzalin, the non-pathogen S. fuliginea could penetrate successfully, forming haustoria in epidermal tissues of the non-host wheat. Similar result was reported in wheat-leaf rust interaction (Hou et al. 2002). These data further confirm that the plant cytoskeleton plays a significant role in non-host resistance. Cloning of cytoskeleton regulatory protein genes and application of green fluorescent protein (GFP)-based in vivo visualization of the cytoskeleton might provide new insights in the role of the cytoskeleton in plantmicrobe interactions for the future researches.
Acknowledgements This research was financially supported by the National Natural Science Foundation of China (30771398) and
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
382
LI Hong-li et al.
the 111 Project from Ministry of Education of China (B07049).
References Hardham A R, Jones D A, Takemoto D. 2007. Cytoskeleton and cell wall function in penetration resistance. Current Opinion in Plant Biology, 10, 342-348. Heath M C. 2000. Nonhost resistance and nonspecific plant defenses. Current Opinion in Plant Biology, 3, 315-319. Hou C Y, Wang D M, Li X J, Han S F, Liu J, Wang Z X. 2002. The effects of depolymeric cytoskeleton on hypersensitive reaction induced by the wheat-leaf rust fungus interaction. Acta Phytopathologica Sinica, 32, 147-152. (in Chinese) Kobayashi I, Kobayashi Y, Hardham A R. 1994. Dynamic reorganization of microtubules and microfilaments in flax cells during the resistance response to flax rust infection. Planta, 195, 237-247. Kobayashi Y, Kobayashi I, Funaki Y, Fujimoto S, Takemoto T, Kunoh H. 1997. Dynamic reorganization of microfilaments and microtubules is necessary for the expression of non-host resistance in barley coleoptile cells. The Plant Journal, 11, 525-537. Kombrink E, Schmelzer E. 2001. The hypersensitive response and its role in local and systemic disease resistance. European Journal of Plant Pathology, 107, 69-78. Lamb C, Dixon R A. 1997. The oxidative burst in plant disease resistance. Annual Review of Plant Physiology and Plant Molecular Biology, 48, 251-275.
Ma Q, Shang H S. 2009. Ultrastructure of stripe rust (Puccinia striiformis f. sp. tritici) interacting with slow-rusting, highly resistant, and susceptible wheat cultivars. Journal of Plant Pathology, 91, 597-606. Mellersh D G, Foulds I V, Higgins V J, Heath M C. 2002. H2O2 plays different roles in determining penetration failure in three diverse plant-fungal interactions. The Plant Journal, 29, 257-268. Mysore K S, Ryu C M. 2004. Nonhost resistance: how much do we know? Trends in Plant Science, 9, 97-104. Schmelzer E. 2002. Cell polarization, a crucial process in fungal defence. Trends in Plant Science, 7, 411-415. Schmidt S M, Panstruga R. 2007. Cytoskeleton functions in plant-microbe interactions. Physiological and Molecular Plant Pathology, 71, 135-148. Takemoto D, Hardham A R. 2004. The cytoskeleton as a regulator and target of biotic interactions in plants. Plant Physiology, 136, 3864-3876. Tang X, Xie M, Kim Y J, Zhou J, Klessig D F, Martin G B. 1999. Overexpression of Pto activates defense responses and confers broad resistance. The Plant Cell, 11, 15-29. Williamson R E. 1993. Organelle movements. Annual Review of Plant Physiology and Plant Molecular Biology, 44, 181-202. Yun B W, Atkinson H A, Gaborit C, Greenland A, Read N D, Pallas J A, Loake G J. 2003. Loss of actin cytoskeletal function and EDS1 activity, in combination, severely compromises non-host resistance in Arabidopsis against wheat powdery mildew. The Plant Journal, 34, 768-777. (Managing editor ZHANG Juan)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.