- Email: [email protected]
Aegilops triuncialis introgression line
J. Plant Physiol. 161. 493 – 495 (2004) http://www.elsevier-deutschland.de/jplhp
Short Communication Root enzyme activities associated with resistance to Heterodera avenae conferred by gene Cre 7 in a wheat/Aegilops triuncialis introgression line María Jesús Montes, Isidoro López-Braña, Angeles Delibes* Departamento de Biotecnología, ETS Ing Agrónomos, UPM, Ciudad Universitaria s/n, Madrid, E-28040, Spain Received June 5, 2003 · Accepted October 1, 2003
Summary The effect of Cereal cyst nematode (Heterodera avenae) infection on the expression of putative root defence-related enzymes, peroxidase (PER), esterase (EST) and superoxide dismutase (SOD), was studied in roots of a wheat/Aegilops triuncialis introgression line TR-3531 carrying the Cre7 resistance gene. We analysed detoxificant isozyme changes within roots of the resistant line and their susceptible parent (H-10-15) as a control, during the early interaction with the pathotype Ha71 of H. avenae. Isoelectrofocusing (IEF) isozyme analysis, four and seven days after infection, revealed that PER, EST and SOD activities increased in the resistant line TR-3531 in comparison with the susceptible control. Moreover, four and seven days after infection, the TR-3531 line showed the expression of new PER isozymes, with pIs of 9.7, 9.0, 8.5, 6.5 and 5.0, and an increased activity of some constitutive isoforms. The intensity of some EST and SOD constitutive bands increased in the resistant line after infection. However, no new isoforms were detected for EST and SOD systems. Nematode-induced enzyme activity was minor (PER) or did not occur (EST and SOD) in the compatible interaction with H-10-15. The enhanced peroxidase and esterase activities may play a role in the lignification of cell walls, which assists in the resistance to penetration by the nematode. Key words: antioxidant enzymes – Cereal cyst nematode – resistance genes – wheat Abbreviations: AOS = active oxygen species. – CCN = Cereal cyst nematode. – Cre = cereal root eelworm. – EST = esterase. – HR = hypersensitive response. – pI = isoelectric point. – IEF = isoelectrofocusing. – PER = peroxidase. – SOD = superoxide dismutase
* E-mail corresponding author: [email protected] 0176-1617/04/161/04-493 $ 30.00/0
494
María Jesús Montes, Isidoro López-Braña, Angeles Delibes
Introduction Incompatible resistant interactions between plant and pathogen are often determined by the hypersensitive response (HR), which is characterised by a quick development of necrosis around the pathogen. The HR is preceded by the formation of active oxygen species (AOS) by the pathogen. Plants possess both enzymatic and non-enzymatic antioxidant defence systems to counteract AOS under stress conditions. The antioxidant enzymes include peroxidase (PER, EC 1.11.1.7), esterase (EST, EC 3.1.1.2) and superoxide dismutase (SOD, EC 1.15.1.1). Anionic PER are thought to be involved in host defence and stress-induced cell wall lignification and EST in an initial stage of the same program, which is considered an early event in the resistance response during nematode infection (Melillo et al. 1989, 1992). A protective role for SOD has also been hypothesised in plants infected by nematodes (Zacheo and Bleve-Zacheo 1988). In wheat root tissue, only the PER and EST isozymes encoded by genes of groups 2 and 3 chromosomes, respectively, are active (Liu et al. 1990, Liu and Gale 1994). To date the chromosome location of the SOD genes expressed in wheat roots has not been reported. Four and seven days after nematode infection, the roots of lines carrying Cre2 or Cre5 resistance genes from Ae. ventricosa for Heterodera avenae, showed a strong correlation between HR and PER and EST activities (Andrés et al.
2001, Montes et al. 2003). In this paper, we study changes in root enzyme activities after nematode infection of a Wheat/ Aegilops triuncialis introgression line (TR-3531) carrying Cre7 resistance gene to H. avenae (Romero et al. 1998). We focused on PER, EST, and SOD activities four and seven days after inoculation by the Ha71 pathotype, comparing resistant (with Cre7 gene) and susceptible infected and uninfected wheat roots.
Material and Methods The introgression line TR-3531, with 42 chromosomes, derived from the cross [Triticum turgidum H-1-1/Aegilops triuncialis A-1/Triticum aestivum cv. «Almatense, H-10-15»], has been previously described (Romero et al. 1998, Martín-Sanchez et al. 2003). The parent H-10-15 was used as a susceptible control (compatible interaction). Plants were grown and inoculated with Ha71 pathotype of H. avenae following procedures described elsewhere (Andrés et al. 2001, Montes et al. 2003). Root sections taken four and seven days after infection, and an equal quantity of uninfected material, were cut out and used for isozyme analysis. PER, EST, and SOD enzymes were extracted and fractionated by isoelectrofocusing (IEF) as described in Montes et al. (2003). A 3–7 pH range of ampholytes was used for IEF of all enzyme systems. In addition, a basic pH range (7–11) was also used for PER. The pIs of isozyme bands were determined before staining by taking pH readings across the gel at 5 mm intervals using a surface electrode. PER, EST, and SOD activities were visualised as described by
Figure 1. Peroxidase (A, B), Esterase (C) and Superoxide dismutase (D) isozyme IEF-patterns, of TR-3531 resistant line (3531) and their susceptible parent T. aestivum H-10-15 (H-10-15), of roots taken 7days after infection by H. avenae juveniles ( + ) and of uninfected roots as control ( – ). A was fractionated using a basic pH range of ampholytes (7–11) while B, C and D in an acidic range (3–7). New bands and those of increased intensity from infected roots of resistant line (TR-3531) are indicated by empty and full arrowheads, respectively.
Root enzyme activities associated with resistance to Heterodera avenae Liu et al. (1990), Liu and Gale (1994) and Neuman and Hart (1986), respectively. A minimum of six electrophoresis runs, using different extracts, were performed in order to confirm the reproducibility of phenotype patterns.
Results and Discussion In order to analyse quantitative and qualitative changes in antioxidant enzymes after wheat infection with pathotype Ha71 of H. avenae, root extracts were subjected to native IEF. Changes in isozyme patterns of infected roots of TR-3531 (incompatible interaction) were detected four-days after nematode infection (data not shown), but were yet more evident at seven days. The IEF-patterns for PER, EST, and SOD systems, in resistant and susceptible wheat roots infected (seven days post inoculation) and uninfected, are shown in Figure 1. Nematode infection preferentially enhanced the activity of PER system, with a notable increase in cationic (Fig. 1 A) and anionic (Fig. 1 B) isozymes, as previously described in other incompatible interactions (Melillo et al. 1992, Zacheo et al. 1993, Andrés et al. 2001, Montes et al. 2003). Both expression of new PER isozymes (pIs = 9.7, 9.0, 6.5, 5.0 and five bands around 8.5), indicated by empty arrowheads in Figure 1, and increased activity of constitutive isoforms (full arrowheads) were detected in response to the infection in the TR-3531 line. Several of these enhanced bands in TR-3531 (pIs close to 5.0, 7.0 and 9.0) were also increased in H-10-15, though with lesser intensity. Specific PER-isozymes are thought to catalyse the polymerisation of hydroxycinnamyl alcohols in the terminal step of lignin biosynthesis (Gross 1980) and their appearance could lead to a greater accumulation of lignin in the feeding site. Although the presence of novel PER bands and resistance were correlated, further investigations are necessary to elucidate whether they are directly involved with pathogenesis-related lignin deposition or if they have other functions. In contrast, infection had a minor effect on the activity of EST and SOD systems in roots of the resistant line, and no changes were detected in H-10-15 (Figs. 1 C, D). Differences between infected and uninfected roots of TR-3531 with respect to band intensity for both EST (pIs around 6.0–7.0 and 4.0 – 4.5) and SOD (pIs: 5.2, 6.3 and 7.0) were observed. However, new bands or pI differences were not detected. Changes in enzyme patterns following nematode infection indicated that root gene expression was altered in both sus-
495
ceptible and resistant wheat hosts, but they were greater and generally more rapidly expressed in the former. Results reported here for Cre7 are consistent with our previous findings when studying incompatible interactions between the pathotype Ha71 and wheat genotypes carrying Cre2 or Cre5 resistance genes introgressed from Ae. ventricosa (Andrés et al. 2001, Montes et al. 2003). However, specific isoforms, not visualised in the above-mentioned incompatible interactions, were induced in the TR-3531 line in response to the nematode infection. Purification and molecular characterisation of infection-related isozymes may provide further insight into the significance of individual isozymes to the defence response in the wheat-H. avenae system. In conclusion, the results suggest that PER, EST, and SOD present in wheat roots may play a role in Cre7-mediated resistance to H. avenae, either directly or indirectly, through the production of lignin. Acknowledgements. This research was supported by grant AGL 2001-3824-CO4-01 from the Comisión Interministerial de Ciencia y Tecnologia of Spain. We thank C. Gonzalez-Belinchón, J. Garcia and M. Gómez-Colmenarejo for their technical assistance and S. Moreno for critical reading of the manuscript.
References Andres MF, Melillo MT, Delibes A, Romero MD, Bleve-Zacheo T (2001) New Phytol 152: 343 – 354 Gross GG (1980) Adv Bot Res 8: 25 – 63 Liu CJ, Chao S, Gale MD (1990) Theor Appl Genet 79: 305 – 313 Liu CJ, Gale MD (1994) Theor Appl Genet 88: 796 – 802 Martín-Sánchez JA, Gómez-Colmenarejo M, Del Moral J, Sin E, Montes MJ, González-Belinchón C, López-Braña I, Delibes A (2003) Theor Appl Genet 106: 1248–1255 Melillo MT, Bleve-Zacheo T, Zacheo G, Gahan P (1989) Ann Appl Biol 114: 325 – 330 Melillo MT, Bleve-Zacheo T, Zacheo G (1992) Nematol Medit 20: 171– 179 Montes MJ, López-Braña I, Romero MD, Sin E, Andrés MF, MartínSánchez JA, Delibes A (2003) Theor Appl Genet 107: 611– 618 Neuman PR, Hart GE (1986) Biochem Gene 24: 435 – 446 Romero MD, Montes MJ, Sin E, Lopez-Braña I, Duce A, MartínSánchez JA, Andrés MF, Delibes A (1998) Theor Appl Genet 96: 1135–1140 Zacheo G, Bleve-Zacheo T (1988) Physiol Mol Plant Pathol 32: 313 – 322 Zacheo G, Orlando C, Bleve-Zacheo T (1993) J Nematol 25: 249 – 256
Short Communication Root enzyme activities associated with resistance to Heterodera avenae conferred by gene Cre 7 in a wheat/Aegilops triuncialis introgression line María Jesús Montes, Isidoro López-Braña, Angeles Delibes* Departamento de Biotecnología, ETS Ing Agrónomos, UPM, Ciudad Universitaria s/n, Madrid, E-28040, Spain Received June 5, 2003 · Accepted October 1, 2003
Summary The effect of Cereal cyst nematode (Heterodera avenae) infection on the expression of putative root defence-related enzymes, peroxidase (PER), esterase (EST) and superoxide dismutase (SOD), was studied in roots of a wheat/Aegilops triuncialis introgression line TR-3531 carrying the Cre7 resistance gene. We analysed detoxificant isozyme changes within roots of the resistant line and their susceptible parent (H-10-15) as a control, during the early interaction with the pathotype Ha71 of H. avenae. Isoelectrofocusing (IEF) isozyme analysis, four and seven days after infection, revealed that PER, EST and SOD activities increased in the resistant line TR-3531 in comparison with the susceptible control. Moreover, four and seven days after infection, the TR-3531 line showed the expression of new PER isozymes, with pIs of 9.7, 9.0, 8.5, 6.5 and 5.0, and an increased activity of some constitutive isoforms. The intensity of some EST and SOD constitutive bands increased in the resistant line after infection. However, no new isoforms were detected for EST and SOD systems. Nematode-induced enzyme activity was minor (PER) or did not occur (EST and SOD) in the compatible interaction with H-10-15. The enhanced peroxidase and esterase activities may play a role in the lignification of cell walls, which assists in the resistance to penetration by the nematode. Key words: antioxidant enzymes – Cereal cyst nematode – resistance genes – wheat Abbreviations: AOS = active oxygen species. – CCN = Cereal cyst nematode. – Cre = cereal root eelworm. – EST = esterase. – HR = hypersensitive response. – pI = isoelectric point. – IEF = isoelectrofocusing. – PER = peroxidase. – SOD = superoxide dismutase
* E-mail corresponding author: [email protected] 0176-1617/04/161/04-493 $ 30.00/0
494
María Jesús Montes, Isidoro López-Braña, Angeles Delibes
Introduction Incompatible resistant interactions between plant and pathogen are often determined by the hypersensitive response (HR), which is characterised by a quick development of necrosis around the pathogen. The HR is preceded by the formation of active oxygen species (AOS) by the pathogen. Plants possess both enzymatic and non-enzymatic antioxidant defence systems to counteract AOS under stress conditions. The antioxidant enzymes include peroxidase (PER, EC 1.11.1.7), esterase (EST, EC 3.1.1.2) and superoxide dismutase (SOD, EC 1.15.1.1). Anionic PER are thought to be involved in host defence and stress-induced cell wall lignification and EST in an initial stage of the same program, which is considered an early event in the resistance response during nematode infection (Melillo et al. 1989, 1992). A protective role for SOD has also been hypothesised in plants infected by nematodes (Zacheo and Bleve-Zacheo 1988). In wheat root tissue, only the PER and EST isozymes encoded by genes of groups 2 and 3 chromosomes, respectively, are active (Liu et al. 1990, Liu and Gale 1994). To date the chromosome location of the SOD genes expressed in wheat roots has not been reported. Four and seven days after nematode infection, the roots of lines carrying Cre2 or Cre5 resistance genes from Ae. ventricosa for Heterodera avenae, showed a strong correlation between HR and PER and EST activities (Andrés et al.
2001, Montes et al. 2003). In this paper, we study changes in root enzyme activities after nematode infection of a Wheat/ Aegilops triuncialis introgression line (TR-3531) carrying Cre7 resistance gene to H. avenae (Romero et al. 1998). We focused on PER, EST, and SOD activities four and seven days after inoculation by the Ha71 pathotype, comparing resistant (with Cre7 gene) and susceptible infected and uninfected wheat roots.
Material and Methods The introgression line TR-3531, with 42 chromosomes, derived from the cross [Triticum turgidum H-1-1/Aegilops triuncialis A-1/Triticum aestivum cv. «Almatense, H-10-15»], has been previously described (Romero et al. 1998, Martín-Sanchez et al. 2003). The parent H-10-15 was used as a susceptible control (compatible interaction). Plants were grown and inoculated with Ha71 pathotype of H. avenae following procedures described elsewhere (Andrés et al. 2001, Montes et al. 2003). Root sections taken four and seven days after infection, and an equal quantity of uninfected material, were cut out and used for isozyme analysis. PER, EST, and SOD enzymes were extracted and fractionated by isoelectrofocusing (IEF) as described in Montes et al. (2003). A 3–7 pH range of ampholytes was used for IEF of all enzyme systems. In addition, a basic pH range (7–11) was also used for PER. The pIs of isozyme bands were determined before staining by taking pH readings across the gel at 5 mm intervals using a surface electrode. PER, EST, and SOD activities were visualised as described by
Figure 1. Peroxidase (A, B), Esterase (C) and Superoxide dismutase (D) isozyme IEF-patterns, of TR-3531 resistant line (3531) and their susceptible parent T. aestivum H-10-15 (H-10-15), of roots taken 7days after infection by H. avenae juveniles ( + ) and of uninfected roots as control ( – ). A was fractionated using a basic pH range of ampholytes (7–11) while B, C and D in an acidic range (3–7). New bands and those of increased intensity from infected roots of resistant line (TR-3531) are indicated by empty and full arrowheads, respectively.
Root enzyme activities associated with resistance to Heterodera avenae Liu et al. (1990), Liu and Gale (1994) and Neuman and Hart (1986), respectively. A minimum of six electrophoresis runs, using different extracts, were performed in order to confirm the reproducibility of phenotype patterns.
Results and Discussion In order to analyse quantitative and qualitative changes in antioxidant enzymes after wheat infection with pathotype Ha71 of H. avenae, root extracts were subjected to native IEF. Changes in isozyme patterns of infected roots of TR-3531 (incompatible interaction) were detected four-days after nematode infection (data not shown), but were yet more evident at seven days. The IEF-patterns for PER, EST, and SOD systems, in resistant and susceptible wheat roots infected (seven days post inoculation) and uninfected, are shown in Figure 1. Nematode infection preferentially enhanced the activity of PER system, with a notable increase in cationic (Fig. 1 A) and anionic (Fig. 1 B) isozymes, as previously described in other incompatible interactions (Melillo et al. 1992, Zacheo et al. 1993, Andrés et al. 2001, Montes et al. 2003). Both expression of new PER isozymes (pIs = 9.7, 9.0, 6.5, 5.0 and five bands around 8.5), indicated by empty arrowheads in Figure 1, and increased activity of constitutive isoforms (full arrowheads) were detected in response to the infection in the TR-3531 line. Several of these enhanced bands in TR-3531 (pIs close to 5.0, 7.0 and 9.0) were also increased in H-10-15, though with lesser intensity. Specific PER-isozymes are thought to catalyse the polymerisation of hydroxycinnamyl alcohols in the terminal step of lignin biosynthesis (Gross 1980) and their appearance could lead to a greater accumulation of lignin in the feeding site. Although the presence of novel PER bands and resistance were correlated, further investigations are necessary to elucidate whether they are directly involved with pathogenesis-related lignin deposition or if they have other functions. In contrast, infection had a minor effect on the activity of EST and SOD systems in roots of the resistant line, and no changes were detected in H-10-15 (Figs. 1 C, D). Differences between infected and uninfected roots of TR-3531 with respect to band intensity for both EST (pIs around 6.0–7.0 and 4.0 – 4.5) and SOD (pIs: 5.2, 6.3 and 7.0) were observed. However, new bands or pI differences were not detected. Changes in enzyme patterns following nematode infection indicated that root gene expression was altered in both sus-
495
ceptible and resistant wheat hosts, but they were greater and generally more rapidly expressed in the former. Results reported here for Cre7 are consistent with our previous findings when studying incompatible interactions between the pathotype Ha71 and wheat genotypes carrying Cre2 or Cre5 resistance genes introgressed from Ae. ventricosa (Andrés et al. 2001, Montes et al. 2003). However, specific isoforms, not visualised in the above-mentioned incompatible interactions, were induced in the TR-3531 line in response to the nematode infection. Purification and molecular characterisation of infection-related isozymes may provide further insight into the significance of individual isozymes to the defence response in the wheat-H. avenae system. In conclusion, the results suggest that PER, EST, and SOD present in wheat roots may play a role in Cre7-mediated resistance to H. avenae, either directly or indirectly, through the production of lignin. Acknowledgements. This research was supported by grant AGL 2001-3824-CO4-01 from the Comisión Interministerial de Ciencia y Tecnologia of Spain. We thank C. Gonzalez-Belinchón, J. Garcia and M. Gómez-Colmenarejo for their technical assistance and S. Moreno for critical reading of the manuscript.
References Andres MF, Melillo MT, Delibes A, Romero MD, Bleve-Zacheo T (2001) New Phytol 152: 343 – 354 Gross GG (1980) Adv Bot Res 8: 25 – 63 Liu CJ, Chao S, Gale MD (1990) Theor Appl Genet 79: 305 – 313 Liu CJ, Gale MD (1994) Theor Appl Genet 88: 796 – 802 Martín-Sánchez JA, Gómez-Colmenarejo M, Del Moral J, Sin E, Montes MJ, González-Belinchón C, López-Braña I, Delibes A (2003) Theor Appl Genet 106: 1248–1255 Melillo MT, Bleve-Zacheo T, Zacheo G, Gahan P (1989) Ann Appl Biol 114: 325 – 330 Melillo MT, Bleve-Zacheo T, Zacheo G (1992) Nematol Medit 20: 171– 179 Montes MJ, López-Braña I, Romero MD, Sin E, Andrés MF, MartínSánchez JA, Delibes A (2003) Theor Appl Genet 107: 611– 618 Neuman PR, Hart GE (1986) Biochem Gene 24: 435 – 446 Romero MD, Montes MJ, Sin E, Lopez-Braña I, Duce A, MartínSánchez JA, Andrés MF, Delibes A (1998) Theor Appl Genet 96: 1135–1140 Zacheo G, Bleve-Zacheo T (1988) Physiol Mol Plant Pathol 32: 313 – 322 Zacheo G, Orlando C, Bleve-Zacheo T (1993) J Nematol 25: 249 – 256