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Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat
J. Plant Physiol. 159. 1259-1261 (2002) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Short Communication Intercellular chitinase and peroxidase activities associated with resistance conferred by gene Lr35 to leaf rust of wheat Vesselina S. Anguelova-Merhar1 *, Amie J. van der Westhuizen, Zacharias A. Pretorius Department of Plant Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa Received April 21, 2002 · Accepted May 21, 2002
Summary The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust. Key words: intercellular chitinase – intercellular peroxidase – leaf rust resistance – Puccinia triticina – wheat
Introduction The Lr35 gene in wheat conditions resistance to diverse pathotypes of leaf rust (Puccinia triticina) (Kloppers et al. 1995). We recently demonstrated the involvement of intercellular β-1,3-glucanase in the resistance of Lr35-bearing wheat cultivar, Thatcher, to leaf rust infection (Anguelova et al. 1999). Intercellular chitinases alone or in combination with β-1,3-glucanases are likely to partially degrade fungal cell wall polysaccharides, thus restricting fungal growth (Boller * E-mail corresponding author: [email protected] 1 Present address: Plant Cell Biology Research Unit, School of Life and Environmental Sciences, George Campbell Building, University of Natal, Durban 4041, South Africa.
1993). Furthermore, the chitin and chitosan fragments released may act as elicitors of the consequent defence response (Vander et al. 1998). Peroxidases have been implicated in several defence-related events that occur in the intercellular spaces, including the generation of active oxygen species (AOS) (Bolwell and Wojtaszek 1997) and synthesis of lignin and suberin (Quiroga et al. 2000). The involvement of chitinase (Münch-Garthoff et al. 1997) and peroxidase (Moerschbacher et al. 1988) in the defence response of wheat to stem rust has been demonstrated. Induction of these enzymes in wheat leaves after treatment with elicitors (Vander et al. 1998), exogenous ethylene and mechanical wounding (Botha et al. 1998), has been reported also. Enhancement of the total peroxidase activity has been reported in wheat leaves after leaf rust infection (Southerton and Deverall 0176-1617/02/159/11-1259 $ 15.00/0
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Vesselina S. Anguelova-Merhar, Amie J. van der Westhuizen, Zacharias A. Pretorius
1990). However, the involvement of intercellular peroxidases and chitinases in the wheat-leaf rust interaction still has to be elucidated. Furthermore, the role of these enzymes in Lr35based expression of resistance has not yet been studied.
Materials and Methods Near-isogenic wheat (Triticum aestivum L.) lines, resistant (RL 6082 = Thatcher/Lr35) and susceptible (Thatcher) to leaf rust, were grown under controlled conditions in a glasshouse. Inoculation with uredospores of P. triticina (pathotype UVPrt9) occurred at seedling (14 days old), stem elongation (35 days old) and flag leaf (54 days old) stages of growth. Procedures for the growing and inoculation of plants have been described previously (Anguelova et al. 1999). Experiments were carried out with two sets of plants grown independently. Intercellular wash fluid (IWF) was collected from infected and uninfected leaves at 0, 24, 48, 72, 120 and 168 h post inoculation (HPI). Leaves were vacuum infiltrated with 50 mmol/L Tris-HCl buffer (pH 7.8), containing 5 mmol/L β-Mercaptoethanol and 0.5 mmol/L PMSF, and the IWF was collected by centrifugation at 2000 rpm for 10 min. Chitinase activity in the IWF was assayed spectrophotometrically (550 nm) as described by Wirth and Wolf (1990), using dye-labeled CM-Chitin-RBV (Loewe Biochemica GmbH, München, Germany) as substrate. Enzyme activity was expressed as A550 nm mg –1 protein min –1. Peroxidase activity was determined spectrophotometrically (470 nm) according to Zieslin and Ben-Zaken (1991) and expressed as µmol tetraguaiacol mg –1 protein min –1. Protein concentration in the IWF was determined according to Bradford (1976), using Bio-Rad Protein Assay reagent (Richmond, CA, USA) and gamma globulin (Sigma, St. Louis, MO, USA) as a standard.
Results and Discussion Results of one experiment only are presented. Similar trends were obtained in the second experiment. The changes of chitinase and peroxidase activities in resistant and susceptible, infected and uninfected with leaf rust wheat leaves are shown in Figure 1. The activities of these two enzymes were low in both infected and uninfected resistant and susceptible seedlings and remained almost constant during this investigation period. At stem elongation stage initial activities were higher in resistant than in susceptible plants, although to a lesser extent for peroxidases. Only after 24 HPI the chitinase activity in the susceptible genotype increased and reached levels similar to that of the uninfected resistant plants. Resistant plants at flag leaf stage had higher constitutively expressed chitinase and peroxidase activities than susceptible plants. These activities were much higher than during the stem elongation stage. In susceptible plants there was a significant decrease in chitinase activity in both infected and uninfected plants during the experiment. Intercellular chitinases and peroxidases are involved in the defence-related events that occur in the extracellular matrix during different host-pathogen interactions (Bolwell and Wojtaszek 1997, Van der Westhuizen et al. 1998). Since Lr35 conditions adult-plant resistance, Thatcher/Lr35 plants are susceptible to leaf rust at the seedling stage, intermediately resistant at the stem elongation stage and highly resistant at flag leaf stage. Thatcher plants are susceptible at all three
Figure 1. Effect of leaf rust infection on the intercellular chitinase and peroxidase activities in leaves from resistant (Thatcher/Lr35) and susceptible (Thatcher) wheat cultivars at seedling, stem elongation and flag leaf stages of plant growth.
Intercellular chitinase growth stages (Anguelova et al. 1999). The relatively low chitinase and peroxidase activities observed in seedlings may partly explain their susceptibility. The high level of resistance in flag leaves corresponded with high constitutive levels of intercellular chitinase and peroxidase activities. In addition, these plants have high constitutive levels of intercellular β-1,3glucanase activity (Anguelova et al. 1999). Presumably, chitinase and β-1,3-glucanase act synergistically to inhibit leaf rust growth in wheat, similar to results reported for other plant-pathogen interactions (Mauch et al. 1988). In addition to the degradation of fungal cell walls, cell wall fragments, which may act as signals in the elicitation of the host defence response, are released (Keen and Dawson 1992). Chitin and chitosan have been shown to be effective elicitors in the hypersensitive lignification response in wheat leaves (Vander et al. 1998). Constitutively expressed intercellular peroxidases might be involved in many events during the early stages of infection, including the hypersensitive response (HR) (Bolwell and Wojtaszek 1997). Previously, we have shown that the HR occurred 48 h after leaf rust infection in the Thatcher/Lr35 line (Anguelova et al. 1999). The presence of high constitutively expressed peroxidase activity in the IWF might have been involved in the early production of AOS, which is regarded as part of the HR (Bolwell and Wojtaszek 1997). Enhanced peroxidase activity has been shown to precede the collapse of wheat cells during leaf rust infection (Southerton and Deverall 1990). High intercellular peroxidase levels may also play an integral role in the lignification of cell walls, which assists in the resistance to penetration by fungal pathogens (Moerschbacher et al. 1988). Results support those of our previous studies (Anguelova et al. 1999, Anguelova-Merhar et al. 2001) showing that effective resistance of wheat to P. triticina coincides with increased constitutive expression of some intercellular defence-related enzymes. To our knowledge, this is the first evidence of constitutively expressed intercellular peroxidases in the defence response of wheat to P. triticina. The presence of constitutively expressed defence-related components in the cell walls may provide an early and fast response to pathogen attack. Progress in understanding the
1261
role of chitinase, peroxidase, β-1,3-glucanase and other constitutive compounds of resistance will be useful in developing new strategies not only for rust resistance in wheat, but for crop protection in general. This research was supported by the National Research Foundation (NRF) of South Africa and the University of the Free State.
References Anguelova VS, Van der Westhuizen AJ, Pretorius ZA (1999) Physiol Plant 106: 393 – 401 Anguelova-Merhar VS, Van der Westhuizen AJ, Pretorius ZA (2001) J Phytopathol 149: 381– 384 Boller T (1993) In: Fritig B, Legrand M (eds) Mechanisms of plant defense responses. Kluwer Academic Publishers, Dordrecht pp 391– 400 Bolwell GP, Wojtaszek P (1997) Physiol Mol Plant Pathol 51: 347– 366 Botha A-M, Nagel MAC, Van der Westhuizen AJ, Botha FC (1998) Bot Bull Acad Sin 39: 99–106 Bradford MM (1976) Anal Biochem 72: 248 – 254 Keen NT, Dawson WO (1992) In: Boller T, Meins F (eds) Genes involved in plant defense. Springer-Verlag, Wien, New York pp 85 – 114 Kloppers FJ, Pretorius ZA, Van Lill D (1995) S Afr J Plant & Soil 12: 55 – 58 Mauch F, Mauch-Mani B, Boller T (1988) Plant Physiol 88: 936 – 942 Moerschbacher BM, Noll UM, Flott BE, Reisener H-J (1988) Physiol Mol Plant Pathol 33: 33 – 46 Münch-Garthoff S, Neuhaus J-M, Boller T, Kemmerling B, Kogel K-H (1997) Planta 201: 235 – 244 Quiroga M, Guerrero C, Botella MA, Barceló A, Amaya I, Medina MI, Alonso FJ, De Forchetti SM, Tigier H, Valpuesta V (2000) Plant Physiol 122: 1119–1128 Southerton SG, Deverall BJ (1990) Plant Pathol 39: 223 – 230 Vander P, Vårum KM, Domard A, Gueddari NEE, Moerschbacher BM (1998) Plant Physiol 118: 1353–1359 Van der Westhuizen AJ, Qian X-M, Botha A-M (1998) Plant Cell Rep 18: 132–137 Wirth SJ, Wolf GA (1990) J Microbiol Methods 12: 197– 205 Zieslin N, Ben-Zaken R (1991) Plant Physiol Biochem 29: 147–151
Short Communication Intercellular chitinase and peroxidase activities associated with resistance conferred by gene Lr35 to leaf rust of wheat Vesselina S. Anguelova-Merhar1 *, Amie J. van der Westhuizen, Zacharias A. Pretorius Department of Plant Sciences, University of the Free State, P.O. Box 339, Bloemfontein 9300, South Africa Received April 21, 2002 · Accepted May 21, 2002
Summary The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust. Key words: intercellular chitinase – intercellular peroxidase – leaf rust resistance – Puccinia triticina – wheat
Introduction The Lr35 gene in wheat conditions resistance to diverse pathotypes of leaf rust (Puccinia triticina) (Kloppers et al. 1995). We recently demonstrated the involvement of intercellular β-1,3-glucanase in the resistance of Lr35-bearing wheat cultivar, Thatcher, to leaf rust infection (Anguelova et al. 1999). Intercellular chitinases alone or in combination with β-1,3-glucanases are likely to partially degrade fungal cell wall polysaccharides, thus restricting fungal growth (Boller * E-mail corresponding author: [email protected] 1 Present address: Plant Cell Biology Research Unit, School of Life and Environmental Sciences, George Campbell Building, University of Natal, Durban 4041, South Africa.
1993). Furthermore, the chitin and chitosan fragments released may act as elicitors of the consequent defence response (Vander et al. 1998). Peroxidases have been implicated in several defence-related events that occur in the intercellular spaces, including the generation of active oxygen species (AOS) (Bolwell and Wojtaszek 1997) and synthesis of lignin and suberin (Quiroga et al. 2000). The involvement of chitinase (Münch-Garthoff et al. 1997) and peroxidase (Moerschbacher et al. 1988) in the defence response of wheat to stem rust has been demonstrated. Induction of these enzymes in wheat leaves after treatment with elicitors (Vander et al. 1998), exogenous ethylene and mechanical wounding (Botha et al. 1998), has been reported also. Enhancement of the total peroxidase activity has been reported in wheat leaves after leaf rust infection (Southerton and Deverall 0176-1617/02/159/11-1259 $ 15.00/0
1260
Vesselina S. Anguelova-Merhar, Amie J. van der Westhuizen, Zacharias A. Pretorius
1990). However, the involvement of intercellular peroxidases and chitinases in the wheat-leaf rust interaction still has to be elucidated. Furthermore, the role of these enzymes in Lr35based expression of resistance has not yet been studied.
Materials and Methods Near-isogenic wheat (Triticum aestivum L.) lines, resistant (RL 6082 = Thatcher/Lr35) and susceptible (Thatcher) to leaf rust, were grown under controlled conditions in a glasshouse. Inoculation with uredospores of P. triticina (pathotype UVPrt9) occurred at seedling (14 days old), stem elongation (35 days old) and flag leaf (54 days old) stages of growth. Procedures for the growing and inoculation of plants have been described previously (Anguelova et al. 1999). Experiments were carried out with two sets of plants grown independently. Intercellular wash fluid (IWF) was collected from infected and uninfected leaves at 0, 24, 48, 72, 120 and 168 h post inoculation (HPI). Leaves were vacuum infiltrated with 50 mmol/L Tris-HCl buffer (pH 7.8), containing 5 mmol/L β-Mercaptoethanol and 0.5 mmol/L PMSF, and the IWF was collected by centrifugation at 2000 rpm for 10 min. Chitinase activity in the IWF was assayed spectrophotometrically (550 nm) as described by Wirth and Wolf (1990), using dye-labeled CM-Chitin-RBV (Loewe Biochemica GmbH, München, Germany) as substrate. Enzyme activity was expressed as A550 nm mg –1 protein min –1. Peroxidase activity was determined spectrophotometrically (470 nm) according to Zieslin and Ben-Zaken (1991) and expressed as µmol tetraguaiacol mg –1 protein min –1. Protein concentration in the IWF was determined according to Bradford (1976), using Bio-Rad Protein Assay reagent (Richmond, CA, USA) and gamma globulin (Sigma, St. Louis, MO, USA) as a standard.
Results and Discussion Results of one experiment only are presented. Similar trends were obtained in the second experiment. The changes of chitinase and peroxidase activities in resistant and susceptible, infected and uninfected with leaf rust wheat leaves are shown in Figure 1. The activities of these two enzymes were low in both infected and uninfected resistant and susceptible seedlings and remained almost constant during this investigation period. At stem elongation stage initial activities were higher in resistant than in susceptible plants, although to a lesser extent for peroxidases. Only after 24 HPI the chitinase activity in the susceptible genotype increased and reached levels similar to that of the uninfected resistant plants. Resistant plants at flag leaf stage had higher constitutively expressed chitinase and peroxidase activities than susceptible plants. These activities were much higher than during the stem elongation stage. In susceptible plants there was a significant decrease in chitinase activity in both infected and uninfected plants during the experiment. Intercellular chitinases and peroxidases are involved in the defence-related events that occur in the extracellular matrix during different host-pathogen interactions (Bolwell and Wojtaszek 1997, Van der Westhuizen et al. 1998). Since Lr35 conditions adult-plant resistance, Thatcher/Lr35 plants are susceptible to leaf rust at the seedling stage, intermediately resistant at the stem elongation stage and highly resistant at flag leaf stage. Thatcher plants are susceptible at all three
Figure 1. Effect of leaf rust infection on the intercellular chitinase and peroxidase activities in leaves from resistant (Thatcher/Lr35) and susceptible (Thatcher) wheat cultivars at seedling, stem elongation and flag leaf stages of plant growth.
Intercellular chitinase growth stages (Anguelova et al. 1999). The relatively low chitinase and peroxidase activities observed in seedlings may partly explain their susceptibility. The high level of resistance in flag leaves corresponded with high constitutive levels of intercellular chitinase and peroxidase activities. In addition, these plants have high constitutive levels of intercellular β-1,3glucanase activity (Anguelova et al. 1999). Presumably, chitinase and β-1,3-glucanase act synergistically to inhibit leaf rust growth in wheat, similar to results reported for other plant-pathogen interactions (Mauch et al. 1988). In addition to the degradation of fungal cell walls, cell wall fragments, which may act as signals in the elicitation of the host defence response, are released (Keen and Dawson 1992). Chitin and chitosan have been shown to be effective elicitors in the hypersensitive lignification response in wheat leaves (Vander et al. 1998). Constitutively expressed intercellular peroxidases might be involved in many events during the early stages of infection, including the hypersensitive response (HR) (Bolwell and Wojtaszek 1997). Previously, we have shown that the HR occurred 48 h after leaf rust infection in the Thatcher/Lr35 line (Anguelova et al. 1999). The presence of high constitutively expressed peroxidase activity in the IWF might have been involved in the early production of AOS, which is regarded as part of the HR (Bolwell and Wojtaszek 1997). Enhanced peroxidase activity has been shown to precede the collapse of wheat cells during leaf rust infection (Southerton and Deverall 1990). High intercellular peroxidase levels may also play an integral role in the lignification of cell walls, which assists in the resistance to penetration by fungal pathogens (Moerschbacher et al. 1988). Results support those of our previous studies (Anguelova et al. 1999, Anguelova-Merhar et al. 2001) showing that effective resistance of wheat to P. triticina coincides with increased constitutive expression of some intercellular defence-related enzymes. To our knowledge, this is the first evidence of constitutively expressed intercellular peroxidases in the defence response of wheat to P. triticina. The presence of constitutively expressed defence-related components in the cell walls may provide an early and fast response to pathogen attack. Progress in understanding the
1261
role of chitinase, peroxidase, β-1,3-glucanase and other constitutive compounds of resistance will be useful in developing new strategies not only for rust resistance in wheat, but for crop protection in general. This research was supported by the National Research Foundation (NRF) of South Africa and the University of the Free State.
References Anguelova VS, Van der Westhuizen AJ, Pretorius ZA (1999) Physiol Plant 106: 393 – 401 Anguelova-Merhar VS, Van der Westhuizen AJ, Pretorius ZA (2001) J Phytopathol 149: 381– 384 Boller T (1993) In: Fritig B, Legrand M (eds) Mechanisms of plant defense responses. Kluwer Academic Publishers, Dordrecht pp 391– 400 Bolwell GP, Wojtaszek P (1997) Physiol Mol Plant Pathol 51: 347– 366 Botha A-M, Nagel MAC, Van der Westhuizen AJ, Botha FC (1998) Bot Bull Acad Sin 39: 99–106 Bradford MM (1976) Anal Biochem 72: 248 – 254 Keen NT, Dawson WO (1992) In: Boller T, Meins F (eds) Genes involved in plant defense. Springer-Verlag, Wien, New York pp 85 – 114 Kloppers FJ, Pretorius ZA, Van Lill D (1995) S Afr J Plant & Soil 12: 55 – 58 Mauch F, Mauch-Mani B, Boller T (1988) Plant Physiol 88: 936 – 942 Moerschbacher BM, Noll UM, Flott BE, Reisener H-J (1988) Physiol Mol Plant Pathol 33: 33 – 46 Münch-Garthoff S, Neuhaus J-M, Boller T, Kemmerling B, Kogel K-H (1997) Planta 201: 235 – 244 Quiroga M, Guerrero C, Botella MA, Barceló A, Amaya I, Medina MI, Alonso FJ, De Forchetti SM, Tigier H, Valpuesta V (2000) Plant Physiol 122: 1119–1128 Southerton SG, Deverall BJ (1990) Plant Pathol 39: 223 – 230 Vander P, Vårum KM, Domard A, Gueddari NEE, Moerschbacher BM (1998) Plant Physiol 118: 1353–1359 Van der Westhuizen AJ, Qian X-M, Botha A-M (1998) Plant Cell Rep 18: 132–137 Wirth SJ, Wolf GA (1990) J Microbiol Methods 12: 197– 205 Zieslin N, Ben-Zaken R (1991) Plant Physiol Biochem 29: 147–151