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Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
Agricultural Sciences in China
January 2010
2010, 9(1): 64-70
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance JIANG Xuan-li1 and KANG Zhen-sheng2 1 2
Department of Plant Protection, Agricultural College, Guizhou University, Guiyang 550025, P.R.China Plant Protection College, Northwest A&F University, Yangling 712100, P.R.China
Abstract Ultrastructural changes in both pathogen and host cells in the interaction between Puccinia striiformis and wheat cultivar (Libellula) with slow-rusting resistance were observed by transmission electron microscopy. Observations revealed marked changes in ultrastructure of both pathogen and host cells. In the pathogen respect, there were many vesicles appeared in the intercellular hyphae and gradually fused into bigger vacuoles, a number of fat drops and electron-dense granules accumulated, mitochondria became swollen and some of them degraded into vesicles, and the plasmalemma of intercellular hyphae became dark. In the haustoria, the cytoplasm degraded gradually and developed a vacuole in the center, fat drops increased, the extrahaustorial matrix widened with a great amount of electron-dense fibrillar and granular materials, and most of the haustoria died with in conjunction with the disappearance of fat drops and other organelles. Structural defense of the host, including formation of cell wall apposition, collar and papilla, occurred in the host respect. Host resistance expression and cytological features occurring in the slow-rusting resistance were discussed. Key words: wheat, Puccinia striiformis, ultrastructure, slow-rusting resistance
INTRODUCTION Stripe rust of wheat (Triticum aestivum L.), caused by Puccinia striiformis f. sp. tritici Eriks., is one of the most destructive and widely distributed diseases of wheat (Stubbs 1988; Line 2002). The use of genetic resistance is the most economical and environmentally friendly way to control this disease. Although a number of race-specific genes that confer resistance to stripe rust have been utilised by breeders as they are easy to manipulate (McIntosh et al. 2003), these genes have been quickly overcome by new pathotypes of the pathogen. The result has been the development of significant epidemics especially when single resistance gene(s)
Received 6 March, 2009
have been deployed over large areas (Rosewarne et al. 2006). Consequently, breeders are now more inclined to use non-specific stripe rust resistance (Kolmer 1996). As described by Caldwell (1968), slow-rusting resistance can be characterized by slow disease development in the field despite a susceptible infection type, and by the presence of one or more resistance components, such as longer latent period, lower receptivity or infection frequency, smaller uredium size, and reduced spore production. Slow-rusting resistance genes are expressed quantitatively and are typically small in genetic effect thereby requiring multiple genes to provide adequate protection against pathogens. These effects are valuable and are generally considered to confer durable resistance (Rosewarne et al. 2008; Bjarko
Accepted 16 June, 2009
JIANG Xuan-li, Professor, Tel: +86-851-3855894, E-mail: [email protected]; Corresponndence KANG Zhen-sheng, Professor, Tel: +86-29-87091312, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60068-7
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
and Line 1988; Caldwell 1968). Many studies have reported slow-rusting resistance to stripe rust in wheat, but most of the work focus on genetic analysis of slow-rusting resistance gene(s) (Suenaga et al. 2003; Navabi et al. 2005; Rosewarne et al. 2006, 2008; Guo et al. 2008), identification of cultivars (lines) with slow-rusting resistance and analysis of components of slow-rusting resistance (Li et al. 2006; McCallum et al. 2007; Feng et al. 2007). Reports on the ultrastructure of slow-rusting resistance are scarce. The objective of this study was to determine the ultrastructural changes of both host and the pathogen in the interaction between a slow-rusting resistant wheat host and P. striiformis.
65
inoculating. Samples of infected tissue were cut into small pieces (1 mm × 2 mm) and fixed with 3% (v/v) glutaraldehyde in 50 mmol L-1 phosphate buffer (pH 6.8) overnight at 4°C, rinsed thoroughly with the same buffer, and post-fixed with 1% (v/v) osmium tetroxide for 2 h at room temperature. The samples were dehydrated in a graded acetone series, embedded in Eponaraldite and kept 1 d at 60°C for polymerization. Ultrathin sections of the samples were cut with a diamond knife and placed on 200-mesh copper grids. Sections were stained with 2% uranyl acetate for 15 min, washed in double-distilled water and post-stained in lead citrate for 5 min, and washed in double-distilled wate. The grids were subsequently viewed with a JEM-100CX II transmission electron microscope at 80 kV.
MATERIALS AND METHODS RESULTS Materials Chinese strain CY31 of P. striiformis was used in this study. Uredospores were produced on a susceptible wheat cultivar Mingxian 169 with no resistance genes against wheat stripe rust. Wheat (T. aestivum) cv. Libellula with typical slow-rusting resistance to stripe rust was used to investigate the ultrastructure of slowrusting resistance. Wheat seeds were sown in organic soil in 10 cm pots in a growth chamber at 15°C and with a 16 h photoperiod. 10-d-old seedlings were used for inoculation when the primary leaves were fully expanded.
Inoculation Freshly collected urediospores were applied with a fine paintbrush to the adaxial surface of the first leaf of wheat seedlings. Inoculated seedlings were kept in a humid chamber for 24 h at 15°C in the dark, and were then returned to the growth chamber. Noninoculated pots served as controls, and were maintained in the humid chamber and the growth chamber under the same conditions as the inoculated pots.
Transmission electron microscopy Inoculated leaves were sampled 3, 4, 5, and 6 d after
In the susceptible wheat cultivar Mingxian 169, the appearance of chlorotic lesions just first begins at 4 d after inoculating, and chlorotic lesions were more clearly at 5 d post-inoculation. In the wheat cultivar Libellula with slow-rusting resistance, the appearance of chlorotic lesions just first begins at 5 d post-inoculation and chlorotic lesions were more clearly at 6 d after inoculating. Although the infection process of P. striiformis in wheat with slow-rusting resistance is similar to infection of susceptible wheat, including the formation of substomatal vesicles, intercellular hyphae, haustorial mother cell, and haustoria, both the pathogen and the host cells displayed notable differences at the ultrastructural level. In particular, the host formed distinct resistance-related structures and materials.
Ultrastructural changes of the pathogen cell In the susceptible host (wheat cultivar Mingxian 169) at 3 d post-inoculation, the fungal hyphae contained numerous ribosomes, mitochondria, endoplasmic reticulum, and nuclei. The haustorium consisted typically of a neck and a rotund body. The extrahaustorial matrix was narrow, with none or just a little material deposited. An electron-opaque neckband was observed along the haustorial neck. But in the wheat cultivar Libellula with slow-rusting resistance to stripe rust at 3 d
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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post-inoculation, the ultrastructure of intercellular hyphae were basically normal in the early development, there are several organelles, such as ribosomes, mitochondria, endoplasmic reticulum, vesicles, fat drops, and nuclei in the cytoplasm (Fig.1-A). With the development of intercellular hyphae and expression of the resistance at 4, 5 and 6 d post-inoculation respectively, many vesicles appeared and gradually fused into a bigger vacuole (Fig.1-C, E, and F). At the same time, many fat drops accumulated (Fig.1-D). The num-
A
ber of electron dense granules began to increase in the spaces between the wall and the cytoplasm membrane and in the central cell (Fig.1-D, E). The mitochondria became swollen and some degraded into vesicles. The plasmalemma of the intercellular hyphae became dark. The wall of the intercellular hyphae became thicker and the constructures of the wall became loosen (Fig.1-E, F). Although the pathogen haustoria were able to develop and expand in size in the wheat cultivar Libellula with slow-rusting resistance to stripe rust, the shape of
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Fig. 1 The ultrastructural changes of the pathogen cell in the slow rusting cultivar Libellula. A, the ultrastructures of intercellular hyphae in the early development at 3 d post-inoculation, × 10 000. B, the shape and ultrastructures of the haustorium at 3 d post-inoculation, × 5 800. C, many vesicles appeared and the wall became slightly darker at 4 d post-inoculation, × 10 000. D, fit drops and electron dense granules increase at 5 d post-inoculation, ×10 000. E, a number of electron dense granules, were accumulaled on the wall and the wall became even darker at 6 d post-inoculation, × 10 000. F, many vesicles appeared and fused into big vacuole in the cytoplasm. Electron dense granules were accumulated in the cytoplasm and on the membrane at 6 d post-inoculation, × 7 200.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
the haustorial body was abnormally rotund (Fig.1-B; Fig.2-A-D). With the development of haustoria and expression of resistance at 4, 5 and 6 d post-inoculation, respectively, the cytoplasm of the haustoria gradually degraded and established a vacuole in the center (Fig.2-A-D). At the same time, fat drop density increased, and drops fused gradually into several bigger ones (Fig.2-A-D). The haustorial wall became thickened and darker (Fig.2-A-D). With the increase of duration of post-inoculation, a number of mitochondria increased, and became swollen and degraded into vesicles gradually (Fig.2-C, D, and F). Another marked change in haustoria was a widening of the extrahaustorial matrix with a great amount of electron dense fibrillar and granular materials (Fig.2-F). Eventually, most of the haustoria had died coincident with the disappearance with of fat drops and other organelles (Fig.2-E).
Ultrastructural changes of the host cell In the susceptible host (wheat cultivar Mingxian 169) at 3 d post-inoculation, the mesophyll cells directly in contact with intercellular hyphae had no any response, still kept natural shapes and constructures. In the haustorium-bearing mesophyll cells of Mingxian 169, the host nuclei, en-doplasmic reticulum, chloroplast, and Golgi bodies were associated with haustorium, deposA
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ited collar around neck of haustorium. But in the wheat cultivar Libellula with slow-rusting resistance to stripe rust, one of the early host responses observed was plasmolysis at 3 d post-inoculation. The mesophyll cells directly in contact with intercellular hyphae and existing haustorium had plasmolyzed (Fig.1-A, Fig.3-A). In the mesophyll cells directly in contact with intercellular hyphae, there few starch granules accumulated, and most of the organelles retained their normal shape (Fig.1-A). But in the haustorium-bearing mesophyll cells, there were more starch granules accumulated than other mesophyll cells (Fig.3-A). These starch granules showed various changes during the infected period. Mitochondrial numbers increased. More callose accumulated, but did not surround the whole haustorium (Fig.3-A, B). In addition, the apposition of electron-dense materials was formed on the inner surface of host cell walls, directly in contact with the haustorial mother cell (Fig.3-B). With the expression of resistance at 4, 5 and 6 d post-inoculation respectively, notable changes took place in haustorium-bearing mesophyll cells. The chloroplasts became rotund, the chloroplast membranes remained intact but the granum membrane became disordered and indistinct, the stromal membrane broke, then the granum degenerated and the matrix became densely stained. At the same time, the starch granules that had
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Fig. 2 The ultrastructural changes of the haustorium in the slow rusting cultivar Libellula. A, the huastorial wall became darker, extrahaustorial membrane was wrinkled, extrahaustorial matrix was widened and there was a lightly staining zone in the cytoplasm at 3 d post-inoculation, × 10 000. B, the cytoplasm became aggregating and great amount of fit drops were accumulated, and there was a even lighter staining zone in the cytoplasm at 4 d post-inoculation, × 5 800. C, a big vesicle appeared in the central zone of haustorium at 5 d post-inoculation, × 5 800. D, number of mitochondria increased at 5 d post-inoculation, × 10 000. E and F showed the ultrastructure of the haustoria at 6 d post-inoculation; E, a great amount of vesicles appeared in the cytoplasm, × 10 000; F, mitochondria and endoplasmic reticulum became swollen, and there were great amount of fibril deposits accumulated in the extrahaustorial matrix, × 48 000.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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Fig. 3 The ultrastructural changes of the host cell in the slow rusting cultivar Libellula. A and B were early host cell responses at 3 d postinoculation; A, plasmolysis of haustorium-bearing mesophyll cells and more starch granules accumulated, ×4 800; B, more callose accumulated, × 7 200; C-F, the ultrastructural changes of the host cell in the later. C, at 4 d post-inoculation, × 3 600. D, at 4 d post-inoculation, × 7 200. E, at 5 d post-inoculation, × 10 000. F, at 6 d post-inoculation, × 2 900.
accumulated decreased gradually until they disappeared. Mitochondria increased, swollen and degraded into vesicles until disappearance (Fig.2-B-D, Fig.3-C-E). Lastly, the envelope membrane of the chloroplast broke and the cell died (Fig.3-F).
DISCUSSION We observed whether intercellular hyphae or haustoria have marked change in their structures in Libellula with typical slow-rusting resistance to stripe rust, such as, in the intercellular hyphae, many vesicles appeared and finally fused into bigger vacuole gradually, a great amount of fat drops and electron dense granules accumulated, mitochondria began to became swollen
and some of them degrade into vesicles, the plasmalemma of intercellular hyphae became dark, the wall became thicker and loosen. In the haustoria, the cytoplasm of haustorium degraded gradually and occurred a vacuole in the central part, fat drops became more, widening of the extrahaustorial matrix with great amount of electron dense fibrillar and granular materials, most of the haustoria had died with disappearing of fat drops and other organelles. In addition, structural defense of the host, such as formation of cell wall apposition, collar or papilla, occurred. These structural changes of both host cell and the pathogen P. striiformis are similar to Jacobs’ research results in the interaction between P. recondita and wheat with partial resistance (Jacobs 1989). These structures led the pathogens to grow and develop slowly necessarily, longer latent
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
period, lower receptivity or infection frequency, smaller uredium size, and reduced spore production. Therefore, these structures may be one of the cytological mechanisms of slow-rusting resistance. The necrotic response of the host cells is considered as a typically resistant mechanism, and is a common phenomenon in incompatible combinations. But there were different conclusions about the sequence of death of haustoria and host cells in the interaction between the pathogen and its host. Harder et al. (1979) reported that haustorium and host cells had occurred almost the same times in incompatible combinations of P. graminis-wheat, Heath M C and Heath I B (1971) drew the same conclusion in incompatible combinations of U. appendiculatus-cowpea. Rohring et al. (1979) reported that infected host cells died earlier than the haustoria. Ma and Shong (2004) reported that in P. striifomis-infected wheat with HTAR, haustorium-bearing host cells remained alive in host tissues although the haustoria were necrotic. Skipp et al. (1974) and Prushy et al. (1980) drew the same conclusions in other rusts-cereal incompatible combinations. Our previous studies (Kang et al. 2003) reported that haustoria of the pathogen died earlier than infected host cells in incompatible combinations between P. striiformis and wheat with resistance of low reaction type. In the present study, we observed not only the pathogens, but also haustorium-bearing host cells had died in the interaction between P. striiformis and wheat with slow-rusting resistance, and the death of both the haustoria and haustorium-bearing host cells occurred almost the same times. Therefore, the order of death of haustoria and host cells in the interaction between pathogen and its host is involved in pathogen, host and resistant gene(s).
Acknowledgements The authors wish to thank the Academician Li Zhenqi for looking after the trial over the past four years. This study was supported by Provincial Key Scientific and Technological Project of Guizhou, China ([2007] 5003) and Guizhou Province Scientific and Technological Research, China ([2007] 2051).
References Bjarko M E, Line R F. 1988. Heritability and number of genes
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controlling leaf rust resistance in four cultivars of wheat. Phytopathology, 78, 457-461. Caldwell R M. 1968. Breeding for general and/or specific plant disease resistance. In: Finlay K W, Shepherd K W, eds, Proceeding of International Wheat Genetics Symposium. 3rd. Australian Academic Science Press, Canberra, Australia. pp. 263-272. Feng J, Zhang Z J, Li G, Zhou Y, Guo Q, Sun J. 2007. Components of quantitative, durable resistance to stripe rust in five wheat cultivars and the genetic distance among the cultivars. Acta Phytopathology Sinica, 37, 175-183. (in Chinese) Guo Q, Zhang Z J, Xu Y B, Li G H, Feng J, Zhou Y. 2008. Quantitative trait loci for high-temperature adult-plant and slow-rusting resistance to Puccinia striiformis f. sp. tritici in wheat cultivars. Phytopathology, 98, 803-809. Harder D, Samborski D J, Rohringer R, Kim W K, Chong J. 1979. Electron microscopy of susceptible and resistant nearisogenic (sr6/Sr6) lines of wheat infected by Puccinia graminis tritici. III. Ultrastructure of incompatible interactions. Canadian Journal of Botany, 57, 2626-2634. Heath M C, Heath I B. 1971. Ultrastructure of an immune and a susceptible reaction of cowpea leaves to rust infection. Physiological Plant Pathology, 1, 277-287. Jacobs T H. 1989. Haustorium formation and cell wall appositios in susceptible and partially resistant wheat and barley seedlings infected with wheat leaf rust. Journal of Phytopathology, 127, 250-261. Kang Z S, Wang Y, Huang L L, Wei G R, Zhao J. 2003. Histology and ultrastructure of incompatible combination between Puccinia striiformis and wheat cultivars with resistance of low reaction type. Scientia Agricultura Sinica, 36, 10261031. (in Chinese) Kolmer J A. 1996. Genetics of resistance to wheat leaf rust. Annual Review of Phytopathology, 34, 435-455. Li Z F, Xia X C, Zhou X C, Niu Y C, He Z H, Zhang Y, Li G Q, Wan A M, Wang D S, Chen X M, Lu Q L, Singh R P. 2006. Seedling and slow-rusting resistance to stripe rust in Chinese common wheats. Plant Disease, 90, 1302-1312. Line R F. 2002. Stripe rust of wheat and barley in North America: a retrospective historical review. Annual Review of Phytopathology, 40, 75-118. Ma Q, Shong H S. 2004. Ultrastructural analysis of the interaction between Puccinia striiformis f. sp. Tritici and wheat after thermal induction of resistance. Journal of Plant Pathology, 86, 19-26. McCallum B D, Chen X, Shorter S, Sadasivaiah R S, Tewari J P. 2007. Stripe rust reaction of 28 Canadian wheat cultivars. Canadian Journal of Plant Pathology, 29, 401-407. McIntosh R A, Yamazaki Y, Devos K M, Dubkowsky J, Rogers
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W J, Appels R. 2003. Catalogue of gene symbols for wheat. In: Pogna N E, Romano M, Pogna E A, Galterio G, eds, Proceedings of the 10th International Wheat Genetics Symposium. Instituto Sperimentale per la Cerealicoltura, Pasteum, Rome, Italy. 4, 1-34. Navabi A, Tewari J P, Singh R P, McCallum B, Laroche A, Briggs K G. 2005. Inheritance and QTL analysis of durable resistance to stripe and leaf rusts in an Australian cultivar, Triticum aestivum “Cook”. Genome, 48, 97-107. Prusky D, Dinoor A, Jacoby B. 1980. The sequence of death of haustoria and host cells during the hypersensitive reaction of oat to crown rust. Physiology and Plant Pathology, 17, 3340. Rohringer R, Kim W K, Samborski D J. 1979. A histological study of interaction between avirulent races of stem rust and wheat containing resistance genes Sr6, Sr8, or Sr22. Canadian Journal of Botany, 57, 324-331. Rosewarne G M, Singh R P, Huerta-Espino J, Rebetzke G J. 2008. Quantitative trait loci for slow-rusting resistance in wheat to leaf rust and stripe rust identified with multi-
environment analysis. Theoretical and Applied Genetics, 116, 1027-1034. Rosewarne G M, Singh R P, Huerta-Espino J, William H M, Bouchet S, Cloutier S, McFadden H, Lagudah E S. 2006. Leaf tip necrosis, molecular markers and b1-proteasome subunits associated with the slow-rusting resistance genes Lr46/Yr29. Theoretical and Applied Genetics, 112, 500-508. Skipp R A, Harder D E, Samboski D J. 1974. Electron microscopy studies on infection of resistant (Sr6 gene) and susceptible near-isogenic wheat lines by Puccinia graminis f. sp. tritici. Canadian Journal of Botany, 52, 2615-2620. Stubbs R W. 1988. Pathogenicity analysis of yellow (stripe) rust of wheat and its significance in a global context. In: Simmonds N W, Rajaram S, eds, Breeding Strategy for Resistance to the Rusts of Wheat. CIMMYT, Mexico DF. pp. 23-38. Suenaga K, Singh R P, Huerta-Espino J, William H M. 2003. Microsatellite markers for genes Lr34/Yr18 and other quantitative trait loci for leaf rust and stripe rust resistance in bread wheat. Phytopathology, 93, 881-890. (Managing editor ZHANG Juan)
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
January 2010
2010, 9(1): 64-70
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance JIANG Xuan-li1 and KANG Zhen-sheng2 1 2
Department of Plant Protection, Agricultural College, Guizhou University, Guiyang 550025, P.R.China Plant Protection College, Northwest A&F University, Yangling 712100, P.R.China
Abstract Ultrastructural changes in both pathogen and host cells in the interaction between Puccinia striiformis and wheat cultivar (Libellula) with slow-rusting resistance were observed by transmission electron microscopy. Observations revealed marked changes in ultrastructure of both pathogen and host cells. In the pathogen respect, there were many vesicles appeared in the intercellular hyphae and gradually fused into bigger vacuoles, a number of fat drops and electron-dense granules accumulated, mitochondria became swollen and some of them degraded into vesicles, and the plasmalemma of intercellular hyphae became dark. In the haustoria, the cytoplasm degraded gradually and developed a vacuole in the center, fat drops increased, the extrahaustorial matrix widened with a great amount of electron-dense fibrillar and granular materials, and most of the haustoria died with in conjunction with the disappearance of fat drops and other organelles. Structural defense of the host, including formation of cell wall apposition, collar and papilla, occurred in the host respect. Host resistance expression and cytological features occurring in the slow-rusting resistance were discussed. Key words: wheat, Puccinia striiformis, ultrastructure, slow-rusting resistance
INTRODUCTION Stripe rust of wheat (Triticum aestivum L.), caused by Puccinia striiformis f. sp. tritici Eriks., is one of the most destructive and widely distributed diseases of wheat (Stubbs 1988; Line 2002). The use of genetic resistance is the most economical and environmentally friendly way to control this disease. Although a number of race-specific genes that confer resistance to stripe rust have been utilised by breeders as they are easy to manipulate (McIntosh et al. 2003), these genes have been quickly overcome by new pathotypes of the pathogen. The result has been the development of significant epidemics especially when single resistance gene(s)
Received 6 March, 2009
have been deployed over large areas (Rosewarne et al. 2006). Consequently, breeders are now more inclined to use non-specific stripe rust resistance (Kolmer 1996). As described by Caldwell (1968), slow-rusting resistance can be characterized by slow disease development in the field despite a susceptible infection type, and by the presence of one or more resistance components, such as longer latent period, lower receptivity or infection frequency, smaller uredium size, and reduced spore production. Slow-rusting resistance genes are expressed quantitatively and are typically small in genetic effect thereby requiring multiple genes to provide adequate protection against pathogens. These effects are valuable and are generally considered to confer durable resistance (Rosewarne et al. 2008; Bjarko
Accepted 16 June, 2009
JIANG Xuan-li, Professor, Tel: +86-851-3855894, E-mail: [email protected]; Corresponndence KANG Zhen-sheng, Professor, Tel: +86-29-87091312, E-mail: [email protected] © 2010, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S1671-2927(09)60068-7
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
and Line 1988; Caldwell 1968). Many studies have reported slow-rusting resistance to stripe rust in wheat, but most of the work focus on genetic analysis of slow-rusting resistance gene(s) (Suenaga et al. 2003; Navabi et al. 2005; Rosewarne et al. 2006, 2008; Guo et al. 2008), identification of cultivars (lines) with slow-rusting resistance and analysis of components of slow-rusting resistance (Li et al. 2006; McCallum et al. 2007; Feng et al. 2007). Reports on the ultrastructure of slow-rusting resistance are scarce. The objective of this study was to determine the ultrastructural changes of both host and the pathogen in the interaction between a slow-rusting resistant wheat host and P. striiformis.
65
inoculating. Samples of infected tissue were cut into small pieces (1 mm × 2 mm) and fixed with 3% (v/v) glutaraldehyde in 50 mmol L-1 phosphate buffer (pH 6.8) overnight at 4°C, rinsed thoroughly with the same buffer, and post-fixed with 1% (v/v) osmium tetroxide for 2 h at room temperature. The samples were dehydrated in a graded acetone series, embedded in Eponaraldite and kept 1 d at 60°C for polymerization. Ultrathin sections of the samples were cut with a diamond knife and placed on 200-mesh copper grids. Sections were stained with 2% uranyl acetate for 15 min, washed in double-distilled water and post-stained in lead citrate for 5 min, and washed in double-distilled wate. The grids were subsequently viewed with a JEM-100CX II transmission electron microscope at 80 kV.
MATERIALS AND METHODS RESULTS Materials Chinese strain CY31 of P. striiformis was used in this study. Uredospores were produced on a susceptible wheat cultivar Mingxian 169 with no resistance genes against wheat stripe rust. Wheat (T. aestivum) cv. Libellula with typical slow-rusting resistance to stripe rust was used to investigate the ultrastructure of slowrusting resistance. Wheat seeds were sown in organic soil in 10 cm pots in a growth chamber at 15°C and with a 16 h photoperiod. 10-d-old seedlings were used for inoculation when the primary leaves were fully expanded.
Inoculation Freshly collected urediospores were applied with a fine paintbrush to the adaxial surface of the first leaf of wheat seedlings. Inoculated seedlings were kept in a humid chamber for 24 h at 15°C in the dark, and were then returned to the growth chamber. Noninoculated pots served as controls, and were maintained in the humid chamber and the growth chamber under the same conditions as the inoculated pots.
Transmission electron microscopy Inoculated leaves were sampled 3, 4, 5, and 6 d after
In the susceptible wheat cultivar Mingxian 169, the appearance of chlorotic lesions just first begins at 4 d after inoculating, and chlorotic lesions were more clearly at 5 d post-inoculation. In the wheat cultivar Libellula with slow-rusting resistance, the appearance of chlorotic lesions just first begins at 5 d post-inoculation and chlorotic lesions were more clearly at 6 d after inoculating. Although the infection process of P. striiformis in wheat with slow-rusting resistance is similar to infection of susceptible wheat, including the formation of substomatal vesicles, intercellular hyphae, haustorial mother cell, and haustoria, both the pathogen and the host cells displayed notable differences at the ultrastructural level. In particular, the host formed distinct resistance-related structures and materials.
Ultrastructural changes of the pathogen cell In the susceptible host (wheat cultivar Mingxian 169) at 3 d post-inoculation, the fungal hyphae contained numerous ribosomes, mitochondria, endoplasmic reticulum, and nuclei. The haustorium consisted typically of a neck and a rotund body. The extrahaustorial matrix was narrow, with none or just a little material deposited. An electron-opaque neckband was observed along the haustorial neck. But in the wheat cultivar Libellula with slow-rusting resistance to stripe rust at 3 d
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
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post-inoculation, the ultrastructure of intercellular hyphae were basically normal in the early development, there are several organelles, such as ribosomes, mitochondria, endoplasmic reticulum, vesicles, fat drops, and nuclei in the cytoplasm (Fig.1-A). With the development of intercellular hyphae and expression of the resistance at 4, 5 and 6 d post-inoculation respectively, many vesicles appeared and gradually fused into a bigger vacuole (Fig.1-C, E, and F). At the same time, many fat drops accumulated (Fig.1-D). The num-
A
ber of electron dense granules began to increase in the spaces between the wall and the cytoplasm membrane and in the central cell (Fig.1-D, E). The mitochondria became swollen and some degraded into vesicles. The plasmalemma of the intercellular hyphae became dark. The wall of the intercellular hyphae became thicker and the constructures of the wall became loosen (Fig.1-E, F). Although the pathogen haustoria were able to develop and expand in size in the wheat cultivar Libellula with slow-rusting resistance to stripe rust, the shape of
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Fig. 1 The ultrastructural changes of the pathogen cell in the slow rusting cultivar Libellula. A, the ultrastructures of intercellular hyphae in the early development at 3 d post-inoculation, × 10 000. B, the shape and ultrastructures of the haustorium at 3 d post-inoculation, × 5 800. C, many vesicles appeared and the wall became slightly darker at 4 d post-inoculation, × 10 000. D, fit drops and electron dense granules increase at 5 d post-inoculation, ×10 000. E, a number of electron dense granules, were accumulaled on the wall and the wall became even darker at 6 d post-inoculation, × 10 000. F, many vesicles appeared and fused into big vacuole in the cytoplasm. Electron dense granules were accumulated in the cytoplasm and on the membrane at 6 d post-inoculation, × 7 200.
© 2010, CAAS. All rights reserved. Published by Elsevier Ltd.
Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
the haustorial body was abnormally rotund (Fig.1-B; Fig.2-A-D). With the development of haustoria and expression of resistance at 4, 5 and 6 d post-inoculation, respectively, the cytoplasm of the haustoria gradually degraded and established a vacuole in the center (Fig.2-A-D). At the same time, fat drop density increased, and drops fused gradually into several bigger ones (Fig.2-A-D). The haustorial wall became thickened and darker (Fig.2-A-D). With the increase of duration of post-inoculation, a number of mitochondria increased, and became swollen and degraded into vesicles gradually (Fig.2-C, D, and F). Another marked change in haustoria was a widening of the extrahaustorial matrix with a great amount of electron dense fibrillar and granular materials (Fig.2-F). Eventually, most of the haustoria had died coincident with the disappearance with of fat drops and other organelles (Fig.2-E).
Ultrastructural changes of the host cell In the susceptible host (wheat cultivar Mingxian 169) at 3 d post-inoculation, the mesophyll cells directly in contact with intercellular hyphae had no any response, still kept natural shapes and constructures. In the haustorium-bearing mesophyll cells of Mingxian 169, the host nuclei, en-doplasmic reticulum, chloroplast, and Golgi bodies were associated with haustorium, deposA
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ited collar around neck of haustorium. But in the wheat cultivar Libellula with slow-rusting resistance to stripe rust, one of the early host responses observed was plasmolysis at 3 d post-inoculation. The mesophyll cells directly in contact with intercellular hyphae and existing haustorium had plasmolyzed (Fig.1-A, Fig.3-A). In the mesophyll cells directly in contact with intercellular hyphae, there few starch granules accumulated, and most of the organelles retained their normal shape (Fig.1-A). But in the haustorium-bearing mesophyll cells, there were more starch granules accumulated than other mesophyll cells (Fig.3-A). These starch granules showed various changes during the infected period. Mitochondrial numbers increased. More callose accumulated, but did not surround the whole haustorium (Fig.3-A, B). In addition, the apposition of electron-dense materials was formed on the inner surface of host cell walls, directly in contact with the haustorial mother cell (Fig.3-B). With the expression of resistance at 4, 5 and 6 d post-inoculation respectively, notable changes took place in haustorium-bearing mesophyll cells. The chloroplasts became rotund, the chloroplast membranes remained intact but the granum membrane became disordered and indistinct, the stromal membrane broke, then the granum degenerated and the matrix became densely stained. At the same time, the starch granules that had
B
C H
H
H
H
D
E
F
EX EM
H H
M
ER H
H
Fig. 2 The ultrastructural changes of the haustorium in the slow rusting cultivar Libellula. A, the huastorial wall became darker, extrahaustorial membrane was wrinkled, extrahaustorial matrix was widened and there was a lightly staining zone in the cytoplasm at 3 d post-inoculation, × 10 000. B, the cytoplasm became aggregating and great amount of fit drops were accumulated, and there was a even lighter staining zone in the cytoplasm at 4 d post-inoculation, × 5 800. C, a big vesicle appeared in the central zone of haustorium at 5 d post-inoculation, × 5 800. D, number of mitochondria increased at 5 d post-inoculation, × 10 000. E and F showed the ultrastructure of the haustoria at 6 d post-inoculation; E, a great amount of vesicles appeared in the cytoplasm, × 10 000; F, mitochondria and endoplasmic reticulum became swollen, and there were great amount of fibril deposits accumulated in the extrahaustorial matrix, × 48 000.
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JIANG Xuan-li et al.
A
B
IH CH C
H MC
H H
C
C
D H CH
CH
C
E
F
MC
MC H
H CH
Fig. 3 The ultrastructural changes of the host cell in the slow rusting cultivar Libellula. A and B were early host cell responses at 3 d postinoculation; A, plasmolysis of haustorium-bearing mesophyll cells and more starch granules accumulated, ×4 800; B, more callose accumulated, × 7 200; C-F, the ultrastructural changes of the host cell in the later. C, at 4 d post-inoculation, × 3 600. D, at 4 d post-inoculation, × 7 200. E, at 5 d post-inoculation, × 10 000. F, at 6 d post-inoculation, × 2 900.
accumulated decreased gradually until they disappeared. Mitochondria increased, swollen and degraded into vesicles until disappearance (Fig.2-B-D, Fig.3-C-E). Lastly, the envelope membrane of the chloroplast broke and the cell died (Fig.3-F).
DISCUSSION We observed whether intercellular hyphae or haustoria have marked change in their structures in Libellula with typical slow-rusting resistance to stripe rust, such as, in the intercellular hyphae, many vesicles appeared and finally fused into bigger vacuole gradually, a great amount of fat drops and electron dense granules accumulated, mitochondria began to became swollen
and some of them degrade into vesicles, the plasmalemma of intercellular hyphae became dark, the wall became thicker and loosen. In the haustoria, the cytoplasm of haustorium degraded gradually and occurred a vacuole in the central part, fat drops became more, widening of the extrahaustorial matrix with great amount of electron dense fibrillar and granular materials, most of the haustoria had died with disappearing of fat drops and other organelles. In addition, structural defense of the host, such as formation of cell wall apposition, collar or papilla, occurred. These structural changes of both host cell and the pathogen P. striiformis are similar to Jacobs’ research results in the interaction between P. recondita and wheat with partial resistance (Jacobs 1989). These structures led the pathogens to grow and develop slowly necessarily, longer latent
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Ultrastructural Changes in the Interaction Between Puccinia striiformis and Wheat Cultivar with Slow-Rusting Resistance
period, lower receptivity or infection frequency, smaller uredium size, and reduced spore production. Therefore, these structures may be one of the cytological mechanisms of slow-rusting resistance. The necrotic response of the host cells is considered as a typically resistant mechanism, and is a common phenomenon in incompatible combinations. But there were different conclusions about the sequence of death of haustoria and host cells in the interaction between the pathogen and its host. Harder et al. (1979) reported that haustorium and host cells had occurred almost the same times in incompatible combinations of P. graminis-wheat, Heath M C and Heath I B (1971) drew the same conclusion in incompatible combinations of U. appendiculatus-cowpea. Rohring et al. (1979) reported that infected host cells died earlier than the haustoria. Ma and Shong (2004) reported that in P. striifomis-infected wheat with HTAR, haustorium-bearing host cells remained alive in host tissues although the haustoria were necrotic. Skipp et al. (1974) and Prushy et al. (1980) drew the same conclusions in other rusts-cereal incompatible combinations. Our previous studies (Kang et al. 2003) reported that haustoria of the pathogen died earlier than infected host cells in incompatible combinations between P. striiformis and wheat with resistance of low reaction type. In the present study, we observed not only the pathogens, but also haustorium-bearing host cells had died in the interaction between P. striiformis and wheat with slow-rusting resistance, and the death of both the haustoria and haustorium-bearing host cells occurred almost the same times. Therefore, the order of death of haustoria and host cells in the interaction between pathogen and its host is involved in pathogen, host and resistant gene(s).
Acknowledgements The authors wish to thank the Academician Li Zhenqi for looking after the trial over the past four years. This study was supported by Provincial Key Scientific and Technological Project of Guizhou, China ([2007] 5003) and Guizhou Province Scientific and Technological Research, China ([2007] 2051).
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