INDUCTION OF PHOSPHOLIPASE ACTIVITY IN THE HYPERSENSITIVE RESPONSE OF WHEAT

Transcription

1 New Phytol. (1987), 107, 709-7H : 709 INDUCTION OF PHOSPHOLIPASE ACTIVITY IN THE HYPERSENSITIVE RESPONSE OF WHEAT BY C. A. O C A M P O * AND H. J. GRAMBOW Institut fiir Biologie III {Pflanzenphysiologie), Aachen University of Technology, Worringer Weg, D-5100 Aachen, Federal Republic of Germany {Accepted 24 July 1987) SUMMARY Phospholipase activity, probably C-type, increased when cultivars of wheat were infected in incompatible, but not in compatible, combinations with wheat stem rust (Puccinia graminis Pers. f. sp. tritici Eriks. & Henn) or with the non-pathogenic crown rust of barley (P. coronata Cda. f. sp. avenae Fraser & Led.). The effect was mimicked when an elicitor fraction isolated from germ tubes of the wheat stem rust was injected into leaves. The increase in phospholipase activity correlated with the hypersensitive response of leaves when infected with incompatible rust fungi or treated with fungal elicitor. This effect was not observed in healthy or in waterinjected control plants. The results suggest that membrane degradation is involved in the hypersensitive response. Key words: Elicitor, hypersensitive response, phospholipase, rust fungi. INTRODUCTION The hypersensitive response (HR), which results in lignification and cell death, is the most effective defence reaction of resistant wheat plants to avirulent races of the wheat stem rust fungus Puccinia graminis Pers. f. sp. tritici Eriks. & Henn. This reaction is observed within 48 h after fungal attack (Tiburzy, 1984). Biochemically, it is characterized by stimulation of enzymes involved in phenylpropanoid metabolism, phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamoyl alcohol dehydrogenase, and of peroxidases after elicitor application (Moerschbacher et al., 1986b). Cytologically, cells undergoing HR are characterized by a typical yellow autofluorescence thought to be due to lignin formation (Reisener et al., 1986). Alterations in membrane structure and function have been observed in various plant systems undergoing resistance reactions to pathogens (Novacky, 1983 ; Keppler & Novacky, 1987). Disorganization of internal structures within affected cells has been observed (Harder et al., 1979). The cell decompartmentalizes, i.e. a general membrane breakdown takes place during HR, suggesting that membrane-degrading enzymes are involved. We recently found that a rapid stimulation of lipoxygenase activity in wheat cells infected with avirulent rust fungi correlates with the appearance of HR (Ocampo, Moerschbacher & Grambow, 1986). This effect was mimicked by application to wheat leaves of an elicitor fraction isolated from germ tubes of the wheat stem rust fungus. It thus appears that, together with the stimulation of phenylpropanoid metabolism, a stimulation of membrane lipid breakdown also biochemically characterizes HR in wheat. To substantiate this assumption, we have measured the activities of enzymes involved in membrane phospholipid * Present address: Federacion de Cafeteros, Laboratorio de Investigaciones, Apartado Aereo 3938, Bogota, Colombia X/87/ $03.00/ The New Phytologist

2 7IO C. A. OcAMPO AND H. J. GRAMBOW metabolism. We describe the stimulation of a phospholipase activity in wheat plants infected with avirulent rust fungi and treated with fungal elicitor. MATERIALS AND METHODS Cultivars of wheat and species of rust The following wheat cultivars were used: the isogenic lines of wheat Triticum aestivum L. cv. Marquis carrying the allele Sr5 for resistance or sr5 for susceptibility to race 32 of the wheat stem rust fungus P. graminis f. sp. tritici (Pgt) and cv. 417/65, resistant to the same rust race. Wheat leaves were inoculated with urediniospores of Pgt or, alternatively, crown rust of barley Puccinia coronata Cda. f. sp. avenae Fraser & Led. (Pea) as described by Ocampo et al. (1986). Enzyme extraction Extracts were prepared in triplicate and measured independently. Each extract was prepared from two primary leaves. The leaves were weighed and ground in liquid nitrogen using a pestle and mortar. The material was further homogenized after addition of prechilled buffer (0-1 M Hepes/NaOH, ph 7, 1 mm EDTA, 20 mm dithiothreitol, 4 w/v). The homogenates were centrifuged for 15 min at x^ and 2 C, and the enzyme activity was measured in the supernatant fraction. Enzyme activity Phospholipase C activity was measured spectrophotometrically as described by Kurioka & Matsuda (1976). One hundred and sixty milligrams of substrate was dissolved in 50 ml 60 % sorbitol buffered with 25 mm Hepes/NaOH, ph 7-2. The reaction was started by adding 50 /ill of enzyme extract to 500 //I substrate solution in a glass microcuvette; the formation of p-nitrophenol (absorption coefficient of M~^ cm"^) was followed continuously at 400 nm. Great care was taken to ensure homogeneous mixing of enzyme extract with substrate (60% sorbitol), since the viscosities of the two solutions were very different. The reference cuvette contained all reagents without enzyme extract. Protein content was estimated by using the Biuret method. Elicitor application Isolation, partial characterization and biological assays of the elicitor fraction from Pgt have been described elsewhere (Moerschbacher et al., 1986a). A hypodermic syringe was used to inject an aqueous solution of elicitor into the intercellular spaces of leaves of wheat, cv. Marquis (Kogel et al., 1985). Controls were injected with distilled water. RESULTS In uninfected control plants, a slight but steady decrease in phospholipase activity was observed throughout the course of the experiments (Figs 1 & 2). This decrease can possibly be attributed to senescence of the leaves. In compatible infected plants, enzyme activity was fairly constant but slightly higher than in uninfected controls (Fig. 1).

3 Phospholipase and hyper sensitivity of wheat to rusts 711 In all incompatible systems tested, i.e. Marquis and Pea, 417/65 and Pgt, and 417/65 and Pea (Figs 1 and 2), phospholipase aetivity was enhanced about 200% at 54 h after penetration and this level was maintained. In these interactions, the fungi did not expand beyond the stage of two to four haustorium mother cells. At the time at which there was an increase in phospholipase activity, the fungi had completely stopped growing. This event correlates with the appearance of HR (see Figs 1 and 2 and Ocampo et al., 1986). In contrast, in the compatible interaction the rust spread throughout the leaf tissue until the onset of sporulation about 100 h after penetration. The protein content of the infected leaves in the compatible but not in the incompatible combination increased within this period (Table 1). Thus, the observed increase in phospholipase activity in the incompatible combination is most probably not of fungal origin. To test further for a correlation between phospholipase activity and HR, the hypersensitive reaction was induced in wheat leaves by injecting an elicitor fraction isolated from germ tubes of the wheat stem rust. The isogenic lines of cv Time after penetration (h) Fig. 1. Phosphohpase activity in interactions between wheat, cv. Marquis and rust. A. Inoculated controls;, infected with Puccinia graminis tritici (compatible interaction); #, infected with Puccinia coronata avenae (incompatible interaction). Arrows indicate the onset of a hypersensitive reaction in the incompatible interaction and the onset of sporulation in the compatible interaction. Each value represents an average of three independent determinations (sd ^ 5%). 30 5U Time after penetration Fig. 2. Phospholipase activity in interactions between wheat, cv. 417/65 and rust. A, Noninoculated controls;, infected with Puccinia graminis tritici (incompatible interaction); #, infected with Puccinia coronata avenae (incompatible interaction). The arrow indicates the onset of a hypersensitive reaction. Each value represents an average of three independent determinations (SD ^ 5 %). (h)

4 712 C. A. OCAMPO AND H. J. GRAMBOW Table 1. Protein content {\!ig mg ^ f. wt) in healthy and in rust-infected wheat leaves in a compatible {cv. Marquis and Puccinia graminis tritici) and an incompatible {cv. 417/65 and Puccinia graminis tritici) interaction {the data represent measurements obtained from one typical experiment) Time after cv. Marquis cv. 417/65 (h) Control Inoculated Control Inoculated i ^ 3 a.it D Treated with water M " " elicitor sr5 Sr5 T t3 o i-w LU Time after treatment (h) Fig. 3. Phospholipase activity after injection of an aqueous solution of elicitor (400/ig glucose equivalents ml"^) into 7 d old primary leaves of wheat cv. Marquis carrying either the allele for resistance (Sr5) or for susceptibility (sr5). Values represent an average of three independent determinations isd. Marquis were used in order to verify whether the elicitor specifically induces HR only in resistant plants. The elicitor was found to mimic the effect observed in plants infected with incompatible rust fungi, the effect being unspecific as regards the presence of the genes for resistance (Sr5) or susceptibility (sr5) (Fig. 3). Under the experimental conditions used (elicitor concentration was 400 fig glucose equivalents ml"^), the increase in phospholipase activity was about 50 % higher than that induced by the water control. DISCUSSION p-nitrophenylphosphorylcholine has been proposed as a suitable substrate for the specific determination of phospholipase C activity (Kurioka & Matsuda, 1976). Thus, the enzyme activity investigated here possibly represents C-type phospholipase, which hydrolyzes diacylglycerolphosphoryl lipids to diacylglycerol and the phosphoryl moiety. Nonetheless, some uncertainty remains as regards the identity of the enzyme measured. It has been shown that the combined action of acid phosphatase and of phospholipase D, which hydrolyzes diacylphosphoryl lipids to phosphatidic acid and the complex alcohol moiety, may lead to the formation of the same products as with phospholipase C if p-nitrophenyl-

5 Phospholipase and hypersensitivity of wheat to rusts 713 phosphorylcholine is used as substrate (Gupta & Wold, 1980). The results presented in this paper nonetheless clearly demonstrate that HR in wheat leaves correlates with an increase in a phospholipase activity. This, together with our previous report on the stimulation of lipoxygenase under similar conditions (Ocampo et al., 1986), leads us to propose that memhrane-degrading activities are involved in HR, a resistance reaction elicited by infection with avirulent rust fungi or by treatment with fungal elicitor in wheat. Several studies have focused on the participation of pathogen-derived phospholipases in host tissue damage (Faull & Gay, 1983 ; Moreau & Rawa, 1984), including that caused by necrotrophs, such as Botrytis spp. (Shepard & Pitt, 1976), which kill host tissue to obtain nutrients. It appears that phospholipases, together with other hydrolases, play a role in the pathogenesis of necrotrophic interactions (Wilson, 1973). However, conclusive evidence that these enzymes play a role in resistant reactions of plants infected with biotrophic organisms is still lacking (see also Bateman & Basham, 1976). The enzyme activity reported here is not likely to originate from the pathogen, since it is also stimulated in host tissue treated with the elicitor fraction in the absence of the rust fungus (Fig. 3). This conclusion is further supported by the lack of increase in the compatible interaction studied (Fig. 1), in which the fungus fully invades host tissues. Combined action of phospholipases and other enzymes responsible for the breakdown of membrane phospholipids may well be involved in the destruction of internal cell structures observed, at the electron microscope level, in many plant-parasite interactions in which hypersensitive cell death represents the prominent resistance reaction (Goodman & Plurad, 1971; Maclean et al., 1974; Harder et al., 1979). Such a combined action of phospholipases and lipoxygenase has been reported for potato extracts in an in vitro system (Galliard, 1970). In conjunction with the enzymes of phenylpropanoid metabolism, lipoxygenase and phospholipase activity may well be involved in the sequence of reactions leading to HR in wheat leaves infected with avirulent rust fungi. However, further studies are required to elucidate the chronology of biochemical events and the respective roles of the enzymes implicated in pathogen- or elicitor-induced HR. ACKNOWLEDGEMENTS This work was carried out while C. A. Ocampo was recipient of a graduate fellowship from the Deutscher Akademischer Austauschdienst. The Deutsche Forschungsgemeinschaft kindly provided financial support. We are very grateful to B. Moerschbacher for providing us with the elicitor. REFERENCES & BASHAM, H. G. (1976). Degradation of plant cell walls and membranes by microbial enzymes. In: Encyclopedia of Plant Physiology, vol. 4, Physiological Plant Pathology (Ed. by R. Heitefuss & P. H. Williams), pp Springer-Verlag, Berlin. FAULL, J. L. & GAY, J. L. (1983). Phospholipase activity in Erysiphe pisi. Physiological Plant Pathology, 22, GALLIARD, T. (1970). The enzymic breakdown of lipids in potato tuber by phospholipid- and galactolipidacyl hydrolase activities and by lipoxygenase. Phytochemistry, 9, GOODMAN, R. N. & PLURAD, S. B. (1971). Ultrastructural changes in tobacco undergoing the hypersensitive reaction caused by plant pathogenic bacteria. Physiological Plant Pathology, \, BATEMAN, D. F. 25 ANP 107

6 714 C. A. OcAMPO AND H. J. GRAMBOW GUPTA, M. N. & WOLD, F. (1980). A convenient spectrophotometric assay for phospholipase D using p-nitrophenyl-phosphocholine as substrate. Lipids, 15, HARDER, D. E., SAMBORSKI, D. J., ROHRINGER, R., RIMMER, S. R., KIM, W. K. & CHONG, J. (1979). Electron microscopy of susceptible and resistant near-isogenic (sr6/sr6) lines of wheat infected by Puccinia graminis f. sp. tritici. III. Ultrastructure in incompatible interactions. Canadian Journal of Botany, 57, KEPPLER, L. D. & NOVACKY, A. (1987). The initiation of membrane lipid peroxidation during bacteriainduced hypersensitive reaction. Physiological and Molecular Plant Pathology, 30, KOGEL, K. H., ScHRENK, F., SHARON, N. & REISENER, H. J. (1985). Suppression of the hypersensitive response in wheat stem rust interation by reagents with affinity for wheat plasma membranes glycoconjugates. Journal of Plant Physiology, 118, KuRiOKA, S. & MATSUDA, M. (1976). Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to study the metal and detergent requirement of the enzyme. Analytical Biochemistry, 75, MACLEAN, D. J., SARGENT, J. A., TOMMERUP, I. C. & INGRAM, D. S. (1974). Hypersensitivity as the primary event in resistance to fungal parasites. Nature, 249, MoERSCHBACHER, B., KoGEL, K. H., NoLL, U. & REISENER, H. J. (1986a). An elicitor of the hypersensitive lignification response in wheat leaves isolated from the rust fungus Puccinia graminis f. sp. tritici. I. Partial purification and characterization. Zeitschrift fiir Naturforschung, 41c, MOERSCHBACHER, B., HECK, B., KOGEL, K. H., OBST, O. & REISENER, H. J. (1986b). An elicitor of the hypersensitive lignification response in wheat leaves isolated from the rust fungus Puccinia graminis f. sp. tritici. II. Induction of enzymes correlated with the biosynthesis of lignin. Zeitschrift fiir Naturforschung, 41c, MoREAU, R. A. & RAWA, D. (1984). Phospholipase activity in cultures of Phytophthora infestans and in infected potato leaves. Physiological Plant Pathology, 24, NovACKY, A. (1983). The effects of disease on the structure and activity of membranes. In: Biochemical Plant Pathology (Ed. by J. A. Callow), pp John Wiley, New York. OcAMPO, C. A., MOERSCHBACHER, B. & GRAMBOW, H. J. (1986). Increased lipoxygenase activity is involved in the hypersensitive response of wheat cells infected with avirulent rust fungi or treated with fungal elicitor. Zeitschrift fiir Naturforschung, 41c, REISENER, H. J., TIBURZY, R., KOGEL, K. H., MOERSCHBACHER, B. & HECK, B. (1986). Mechanism of resistance of wheat against wheat stem rust in the Sr5/P5 interaction. In: NATO ASI series, vol. HI, Biology and Molecular Biology of Plant Pathogen Interactions (Ed. by J. Bailey), pp Springer- Verlag, Berlin. SHEPARD, D. V. & PITT, D. (1976). Purification of a phospholipase from Botrytis and its effects on plant tissues. Phytochemistry, 15, TIBURZY, R. (1984). Untersuchungen zur Bedeutung der hypersensitiven Reaktion befallener Weizenzellen als Resistenzfaktor gegen Puccinia graminis/. sp. tritici. Ph.D. thesis, Aachen University of Technology. WILSON, C. L. (1973). A lysosomal concept for plant pathology. Annual Review of Phytopathology, 11,

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