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A cytological assay reveals pathotype and resistance gene specific elicitors in leaf rust infections of wheat
Physiological and Molecular Plant Pathology (1998) 52, 25–34
A cytological assay reveals pathotype and resistance gene specific elicitors in leaf rust infections of wheat N. S and B. J. D Department of Crop Sciences, University of Sydney, N.S.W. 2006, Australia (Accepted for publication October 1997 )
Several different types of preparation from leaf rust-infected leaves were applied to uninfected wheat leaves in two bioassays. One bioassay used intact leaves and symptom formation visible to the unaided eye. The other used exposed mesophylls on leaf segments which were stained and examined by optical microscopy. The first bioassay revealed previously observed elicitation that was not specific for resistance genes, and the second suggested the presence of resistance-gene specific elicitors. The second bioassay was used with extracts from leaves infected with an avirulent pathotype with respect to Lr9 and avirulent or virulent pathotypes with respect to Lr28 and using wheats differing in the presence or absence of the Lr9 or Lr28 alleles for resistance. The results indicated the presence of elicitors specific for putative Avr9}Lr9 and Avr28}Lr28 interactions among other elicitors in the extracts. Preliminary tests showed that the specific elicitors were precipitated by ammonium sulphate. # 1998 Academic Press Limited
INTRODUCTION
The expression of incompatibility in many interactions between fungal pathotypes and plant cultivars is controlled by avirulence genes and their matching resistance genes. The molecular interpretation of such matching is that each avirulence gene encodes a specific elicitor which recognizes and}or interacts with the product of the corresponding resistance gene, thus triggering defence responses. Evidence for such elicitors in cell-free extracts has been obtained in very few interactions between fungal pathogens and plants. These include Cladosporium fulum (syn. Fulia fula [Cooke] Cif.) and tomato cultivars where the elicitors were first detected in intercellular fluids [7 ], Rhynchosporium secalis (Oudem.) J. J. Davis, and barley cultivars where an elicitor was obtained from culture filtrates of the fungus [12 ], and the rust fungus Uromyces ignae Barclay and cowpea cultivars, where the elicitors were found in extracts of germ-tubes from basidiospores [2, 3 ]. Elicitors specific for the Lr20 allele in wheat were detected in diffusates from wheat leaves infected with pathotypes of the leaf rust fungus avirulent with respect to this allele [14 ]. They were detected using a leaf sandwich technique whereby exposed mesophyll of the infected leaf was incubated on exposed mesophyll of an uninfected bioassay leaf. The Lr20-specific elicitors could not be obtained in intercellular washing fluids from infected leaves [4 ]. Other elicitors selective for a gene on chromosome 5A in wheat but not specific for any known resistance gene [5 ] were found in the 0885–5765}98}01002510 $25.00}0}pp970133
# 1998 Academic Press Limited
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N. Saverimuttu and B. J. Deverall
intercellular fluids [21 ] and they caused particularly strong chlorosis and necrosis in cv. Cappelle-Desprez [4 ]. These other elicitors, but not the Lr20-specific ones, were also obtained from germ-tubes of uredospores of the leaf rust fungus [21 ]. This paper reports the results of a new search for avirulence and resistance genespecific elicitors in cell-free extracts from interactions between the leaf rust fungus and wheat. The first aim was to find a source of the elicitors and to develop a bioassay for them. A subsequent aim was the isolation and characterization of the elicitors. The first step was isolation of haustoria from infected leaves [1, 11, 23 ], because haustoria of a biotrophic rust fungus are likely sites of elicitor production and action. The pathotypes and cultivars used were avirulent and resistant based on the expression of the Lr9 or Lr28 alleles in wheat. These alleles were chosen because their expression gives a series of rapid responses and hypersensitivity in the first cells entered by haustoria, in contrast to the Lr20 allele, which gives slower responses and hypersensitivity around the cells containing haustoria [17–20 ] and which had been used in earlier work [4, 14 ]. An expectation was that elicitors from isolated haustoria might cause rapid and clear symptoms in bioassays using leaves containing the Lr9 or Lr28 alleles and not in control cultivars lacking these alleles, and not in the cv. Cappelle-Desprez, which responded so strongly to the earlier detected non-specific elicitors [4 ]. In the light of the results reported herein that the isolated haustoria elicited strong responses in cv. Cappelle-Desprez and none visible to the unaided eye in Lr9 or Lr28containing cultivars, new steps were taken in the attempt to detect gene-specific elicitors. The new steps involved the use of homogenates of infected leaves as simply prepared sources of elicitors, and a more elaborate bioassay modified from that used in the leaf sandwich technique [14 ]. Exposed mesophylls were incubated under homogenates and then examined for capacity to take up a stain that has often been used in revealing wheat cells that have undergone hypersensitivity [15–17 ]. This new approach resulted in the detection of Lr9 and Lr28-specific elicitors as reported here. MATERIALS AND METHODS
Plants and fungi The main cultivars or lines of wheat (Triticum aestium L.) used were Morocco (University of Sydney Wheat accession number W1103), Little Club (W1), CappelleDesprez (W3055), Transfer (W2382) containing the Lr9 allele and CS 2D}2M (University of Sydney Wheat Cytogenetics Stock Accession Register C77.1) containing the Lr28 allele. Some additional tests were done on the Lr28-tester lines C77-1}4*Cook (National Cereal Rust Control line number 103217), C77-2}4*Oxley (50063) and C77-1}7*Teal (48874) and the parental cvs Cook, Oxley and Teal, which lack the Lr28. Puccinia recondita Rob. & Desm. f. sp. tritici Eriks. & Henn. pathotype 104-2,3,6,(7) (University of Sydney accession number 76694), avirulent with respect to Lr9 and Lr28, and pathotype 104-2,3,6,7,8 (80-L-3) avirulent with respect to Lr9 and virulent with respect to Lr28, were maintained on cv. Morocco. Conditions for growth, inoculation and incubation were as in Southerton and Deverall [17 ].
Cytological assay of leaf rust infections of wheat
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T 1 Assessment key for measuring stain uptake in mesophyll-exposed leaf segments Score
Description of pattern of stain uptake
0
Interveinal cells uniformly light blue ; veins and adjacent cells not stained Scattered darkly stained cells in the interveinal areas ; rare groups of darkly stained cells Small patches or rows of darkly stained cells (1–2 layers deep) often with collapsed protoplasts Large patches of darkly stained cells (" two layers deep) often with collapsed protoplasts and often as bands elongate for the full length of the segment Extensive bands of darkly stained cells often with collapsed protoplasts and nearly or completely covering the segment
1 2 3 4
Extractions Extracts of leaves were made from 11–15-day-old seedlings of Morroco inoculated 4–5 days earlier with pathotype 104-2,3,6,(7), unless stated that pathotype 104-2,3,6,(7),8 was used. Control extracts were made from uninoculated leaves of the same age. Extracts of haustoria from infected leaves were obtained by the procedure of Hahn and Mendgen [11 ]. Leaf tissue was homogenized (10 g fresh weight 50 ml−" homogenization buffer) and filtered through a double layer of muslin. The filtrate was centrifuged for 5 min at 5000 g and the pellet washed twice in suspension buffer and resuspended in the same buffer (25 ml). The suspension was washed into a ConASepharose affinity chromatography column (0±5 ml ml−" bed volume) and incubated for 15 min. Chloroplasts were eluted with 5 void volumes of suspension buffer and then haustoria were retrieved by agitating the column contents vigorously in suspension buffer (15 ml). Some parallel extracts of haustoria were also prepared by homogenizing leaves in de-ionized water. Samples were taken at each stage of separation and from each type of extract for bioassay in intact leaves. Routine homogenates were prepared subsequently by macerating 20 g fresh leaves in 50 ml chilled de-ionized water. The homogenates were filtered through two layers of muslin and allowed to stand for at least one hour at 4 °C. The sedimented uredospores and plant debris were discarded, and the supernatants used for bioassay on exposed mesophylls of leaf segments. Intercellular washing fluids were obtained as described [4 ]. Bioassays using an Hagborg deice Homogenates were introduced into the intercellular spaces of primary leaves of 7–8day-old seedlings of uninoculated wheat using an Hagborg device [10 ], which permits the flooding of areas in thin plant leaves by hydraulic pressure from a small rubber chamber without direct insertion of a hypodermic needle into the leaves and consequent damage. The infiltrated plants were then incubated at 20 °C under continuous illumination in a growth cabinet for 4 days. Infiltrated leaves were then examined under direct light for chlorosis and}or necrosis.
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Bioassays using mesophyll-exposed leaf segments Leaves harvested from 7–10-day-old uninoculated seedlings were brought to the laboratory in an insulated box containing crushed ice and lined with paper tissues. The lower epidermis of each leaf was removed with the help of a clean sharp razor blade and a pair of forceps. The mesophyll-exposed strips were then cut into segments of 8–10 mm length, covered with test solutions and placed between glass slides in Petri dishes lined with moist filter papers (10 segments 0±5 ml−" per dish). The segments were incubated at 20 °C under continuous illumination in a growth cabinet for 2–3 days (unless otherwise stated) and examined after clearing and staining in a solution of trypan blue in lactophenol at 70 °C for 1 min. The segments were then mounted in glycerine and observed under low power (¬4) microscopy. Mesophyll-exposed segments that had been treated with water were uniformly pale blue after staining, except for ridges of relatively unstained veinal regions. Dark blue staining in cells in each of the five interveinal areas in the centres of segments that had received other treatments was recorded using an assessment key (Table 1). The mean score for staining in each segment and the overall mean score and the standard error (SE) for the 10 segments in each treatment}cultivar combination were calculated. Analyses of variance were carried out to detect significant differences between treatments and overall mean scores for different treatments were compared by the Fisher’s least significant difference test. Precipitation of different protein fractions from homogenates of infected leaes Homogenates were brought successively to 0–20 %, 20–40 %, 40–60 % and 60–80 % with respect to ammonium sulphate. Each precipitate was collected separately by centrifugation and suspended in 5 ml water. The resulting solutions were dialysed overnight against water with continuous stirring.
RESULTS
Effects of injecting fractions from leaf homogenates into intact leaes in causing symptoms isible to the unaided eye Samples of fractions containing isolated haustoria, injected into healthy leaves using the Hagborg device, caused marked chlorosis and necrosis within 4 days in cv. CappelleDesprez, and paleness and sometimes more marked chlorosis in cv. Morocco, but no symptoms visible to the unaided eye in cvs Little Club, Transfer (Lr9) and the line CS 2D}2M (Lr28). Samples of original homogenates of infected leaves made in buffer or water and of supernatants and pellets taken at each stage of the haustorial isolation procedure also caused strong symptoms in cv. Cappelle-Desprez but not in the other cultivars. Comparable extracts from uninfected leaves caused no symptoms. Examination by staining and microscopy of intact leaes injected with fractions from leaf homogenates Infiltration with extracts of infected leaves using the Hagborg device was followed by examination with the unaided eye, and by subsequent staining and microscopy. The
Cytological assay of leaf rust infections of wheat
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strong symptom of necrosis in cv. Cappelle-Desprez correlated with groups of cells which had thickened walls and an apparent inability to take up trypan blue. In contrast, no visible symptoms appeared in line CS 2D}2M (Lr28) but scattered groups of cells stained intensely with trypan blue and appeared not to have thickened walls. No symptoms, no dark staining and no cell wall thickening occurred in cv. Little Club. The above results were obtained in repeated experiments and caused the abandonment of this approach to the detection of Lr9 or Lr28-specific elicitors.
Effects of applying leaf homogenates to exposed mesophylls on their subsequent capacity to take up stain as seen by microscopy Intense uptake of stain (mean score of 1±48³SE 0±086) by mesophyll cells in cv. Transfer (Lr9) followed the application of homogenates of infected leaves in water. The extent of stain uptake in this cultivar was much lower (mean score of 0±32³0±076) when comparable homogenates from healthy leaves were applied. Homogenates of infected leaves resulted in mean scores of 0±44³0±074 and 0±20³0±063 in cvs Morocco and Cappelle-Desprez, respectively. When the experiment was repeated several times, stain uptake was always significantly higher in the Lr9-containing cultivar treated with extracts of infected leaves than in the other cultivar}extract combinations. When similar extracts were applied to line CS 2D}2M (Lr28), more intense uptake of stain was caused by homogenates from infected leaves (mean score 1±24³0±062) than by those from healthy leaves (0±18³0±080). Mean scores for stain uptake in cvs Little Club and Cappelle-Desprez after treatment with extracts from infected leaves were 0±40³0±080 and 0±68³0±070, respectively. In repeated tests, stain uptake was always significantly higher in the Lr28-containing line treated with extracts of infected leaves than in the other cultivar}extract combinations. All further experiments used the procedures of applying homogenates to exposed mesophylls and their subsequent capacity to take up stain in order to test the pathotype and resistance gene specificity of the eliciting activity and to obtain preliminary information on the nature of the activity. Experiments with pathotypes airulent and irulent with respect to Lr28 Exposed mesophylls of line CS 2D}2M (Lr28) took up more stain after treatment with crude extracts of leaves inoculated by the avirulent pathotype than with those inoculated by the virulent pathotype (Table 2). Stain uptake increased with increasing time of exposure to the extracts. The cvs Little Club and Cappelle Desprez took up less stain after treatment with either extract.
Experiment using cultiars with and without Lr28 Exposed mesophylls from three Lr28-tester lines and from line CS 2D}2M (Lr28) took up more stain than five control cultivars after treatment with crude extracts of leaves inoculated with the avirulent pathotype (Table 3). Extracts from leaves inoculated with the virulent pathotype and from uninoculated leaves caused less potential to stain in all lines and cultivars.
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N. Saverimuttu and B. J. Deverall T 2 Effects of extracts from leaes inoculated with airulent or irulent pathotypes on capacity of exposed mesophylls to take up stain
Source of extract
Time of Mean score (³SE) for stain uptake exposure to In cultivar}line extracts (days) Cappelle-Desprez CS 2D}2M Little Club
Uninoculated leaves Avirulent interaction* Virulent interaction†
3
0±18³0±044 a
0±24³0±079 b
0±18³0±077 a
1 2 3 1 2 3
0±16³0±047 a 0±42³0±099 b 0±64³0±213 d 0±28³0±110 b 0±46³0±110 b 0±68³0±188 d
0±36³0±062 c 0±70³0±127 c 1±14³0±046 e 0±18³0±053 a 0±36³0±076 a 0±30³0±095 b
0±22³0±060 b 0±30³0±086 a 0±44³0±101 c 0±36³0±093 c 0±34³0±085 a 0±40³0±080 c
Values for a particular time of exposure denoted by different letters are statistically different at P ¯ 0±05. *cv. Morocco inoculated with pathotype 104–2,3,6,(7) (arivulent to Lr28) ; †cv. Morocco inoculated with pathotype 104–2,3,6,(7),8 (virulent to Lr28 ). T 3 Effects of extracts from leaes inoculated with pathotypes airulent or irulent towards Lr28 on the staining capacity of exposed mesophylls of cultiars with or without Lr28
Test cultivar}line Without Lr28 Cappelle-Desprez Little Club Oxley Teal Cook With Lr28 CS 2D}2M C77-2}4*Oxley C77-1}7*Teal C77-1}4*Cook
Mean score (³SE) for stain uptake Source of extracts Avirulent* Uninfected leaves infections Virulent† infections
0±14³0±049 a 0±12³0±051 a 0±20³0±075 a 0±22³0±066 a 0±36³0±068 b
0±34³0±094 b 0±32³0±110 b 0±26³0±097 a 0±24³0±074 a 0±28³0±012 a
0±42³0±103 b 0±34³0±090 b 0±28³0±095 a 0±26³0±094 a 0±36³0±105 b
0±18³0±052 a 0±16³0±047 a 0±15³0±015 a 0±30³0±065 b
1±06³0±106 c 0±96³0±074 c 1±24³0±116 d 0±90³0±099 c
0±26³0±070 a 0±28³0±086 a 0±18³0±060 a 0±30³0±071 b
Values followed by different letters are statistically different at P ¯ 0±05. *Extract of cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †extract of cv. Morocco inoculated with pathotype 104–2,3,6,(7),8.
Experiments on remoing intercellular washing fluids on actiities of extracts from leaes inoculated with airulent and irulent pathotypes Results in Table 4 show that there was high activity, before and after removal of intercellular washing fluids, in crude extracts of leaves inoculated with an avirulent pathotype. The activity was greatest on the Lr28-containing cultivar. The intercellular washing fluids had lower activity regardless of pathotype and test cultivar.
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T 4 Effects of remoing intercellular washing fluids on actiities of extracts from leaes inoculated with airulent or irulent pathotypes
Source of leaf extract Uninfected Avirulent* original IWF original minus IWF Virulent† original IWF original minus IWF
Mean score (³SE) for stain uptake In cultivar}line Cappelle-Desprez CS 2D}2M Little Club 0±18³0±044 a
0±24³0±055 b
0±18³0±077 a
0±64³0±067 d 0±26³0±094 b 0±46³0±113 c
1±14³0±186 e 0±44³0±093 c 1±20³0±175 e
0±44³0±101 c 0±42³0±076 c 0±38³0±060 c
0±68³0±235 d 0±50³0±194 c 0±52³0±130 c
0±18³0±066 a 0±14³0±064 a 0±18³0±096 a
0±28³0±076 b 0±28³0±087 b 0±40³0±098 c
Values followed by different letters are statistically different at P ¯ 0±05. *Extracts of cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †extracts of cv. Morocco inoculated with pathotype 104–2,3,6,(7),8. T 5 Effects of different protein fractions from extracts of inoculated leaes on exposed mesophylls
Extract and treatment Uninfected untreated Avirulent* untreated PF 2 (20–40 %)‡ PF 3 (40–60 %) PF 4 (60–80 %) Virulent† untreated PF 2 (20–40 %)‡ PF 3 (40–60 %) PF 4 (60–80 %)
Mean score (³SE) for stain uptake Cultivar}line Cappelle-Desprez CS 2D}2M Little Club
0±32³0±114 b
0±30³0±090 b
0±36³0±123 b
0±54³0±170 d 0±42³0±258 c 0±38³0±242 b 0±68³0±293 e
1±14³0±186 g 0±14³0±049 a 0±88³0±124 f 0±86³0±288 f
0±58³0±191 d 0±30³0±186 b 0±30³0±136 b 0±34³0±177 b
0±68³0±188 e 0±34³0±202 b 0±44³0±138 c 0±50³0±148 c
0±18³0±066 a 0±16³0±068 a 0±20³0±063 a 0±20³0±075 a
0±28³0±076 b 0±20³0±098 a 0±16³0±074 a 0±48³0±202 c
Values followed by different letters are significantly different at P ¯ 0±05. *From cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †from cv. Morocco inoculated with pathotype 104–2,3,6,(7),8 ; ‡with respect to ammonium sulphate.
Effects of different protein fractions from homogenates on exposed mesophylls Results in Table 5 showed that protein fractions 3 and 4 (precipitated at 40–60 % and 60–80 % concentrations of ammonium sulphate, respectively) from homogenates of leaves inoculated by the avirulent pathotype retained much of the original higher activity on line CS 2D}2M (Lr28). Generally, protein fraction 4 from infections by both
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pathotypes had slightly greater activities on the cv. Cappelle-Desprez and Little Club than did the other fractions. DISCUSSION
A consistently greater cellular response in Lr9- or Lr28-leaves to homogenates of wheat leaves inoculated with an avirulent pathotype than occurred in response to any other combination of leaves and extracts indicated the presence of elicitors specific for complementary resistance and avirulence alleles. Putative Avr9}Lr9- and Avr28}Lr28specific elicitors were thus distinguished from other elicitors of cellular change present in the extracts. The response was seen in a bioassay based on incubating exposed mesophylls with homogenates for up to 3 days and then observing the capacity of cells to take up trypan blue from a lactophenol solution at 70 °C. Remarkably, and as observed before [14 ], removal of the epidermis, incubation of uninfected mesophyll under water as a control and then immersion in heated trypan blue}lactophenol resulted in only a light and even uptake of stain by wheat cells, suggesting little or no damage to cell membranes by the apparently harsh treatments. A similar light and even staining has also been reported for wheat cells in parts of leaves remote from inoculation sites expressing hypersensitivity to rust pathogens [15, 17 ] in contrast to the intense staining at the sites. The intense trypan blue stain in Lr9 and Lr28-containing wheat cells entered by haustoria of avirulent pathotypes of the leaf rust fungus correlated precisely with autofluorescence of similar placed cells in leaves treated with a fluorochrome and examined by fluorescence microscopy, and was interpreted as a sign of necrosis [17 ]. Cellular autofluorescence has been used as an indicator of cowpea responses to specific elicitors from the cowpea rust fungus [2, 3, 8 ]. In the present work, incubation with extracts of uninfected leaves caused occasional cells to take up stain more strongly. Extracts of infected leaves often increased the frequency of cells that stained strongly. The stronger staining was interpreted as resulting from changes to membranes allowing stain intake and, in some cells, from deep staining of collapsed protoplasts associated with cell death, as has often been discussed for hypersensitivity to rust fungi [15–17, 22]. The strongest and most widespread staining in the present work correlated with extracts from leaves inoculated with avirulent pathotypes being incubated on Lr9 or Lr28bearing mesophylls, hence the claim for detecting gene-specific elicitors in those extracts. This is the first record of the detection, in cell-free extracts, of specific elicitors of rustresistance in wheat. In previous studies, elicitors specific for the Lr20 allele in wheat and for the complementary fungal avirulence allele were detected using a leaf-sandwich technique and subsequent staining with trypan blue for cellular necrosis [14 ]. Resistance specific elicitors were detected, however, in germ-tube extracts of germinating basidiospores of the cowpea rust fungus (Uromyces ignae Barclay) by the capacity to cause necrosis in resistant cowpea cultivars [2, 3 ], and this work has resulted in two of the elicitors being purified and characterized as peptides [8 ]. Other advances in characterization of specific elicitors from fungi have come with those found in intercellular spaces of tomato leaves infected by the leaf mould pathogen, C. fulum, and by their capacity to elicit hypersensitivity in resistant tomato cultivars [6, 7, 9, 13 ].
Cytological assay of leaf rust infections of wheat
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A problem in researching elicitation in wheat is the readily seen chlorotic and necrotic reaction of intact leaves in some cultivars to a widespread component of many types of extract from wheat rust fungi. Extracts from germ-tubes of stem and leaf rust fungi and from intercellular spaces of leaves infected by these fungi were active [21 ]. Similarly active were leaf homogenates and isolated haustoria in the present work. Reactions in intact leaves were particularly marked in cv. Cappelle-Desprez, and were encoded for by a gene that has no known relationship to rust resistance [5 ]. Necrosis in this cultivar was seen to be associated with an increased thickness of cell walls. In contrast, no visible responses to rust extracts occurred in intact leaves of cultivars chosen for their specific resistance to the fungal pathotypes tested. The use of exposed mesophylls and the uptake of trypan blue by this tissue in the more elaborate bioassay described here enabled progress to be made in revealing specific elicitors. Progress can now be made on the isolation and characterization of the specific elicitors. The precipitation of specific eliciting activity from fractions using high concentrations of ammonium sulphate suggests that protein may contribute to the activity. Isolation of specific elicitors should enable probes to be designed to test for their detection in or on haustoria of potentially avirulent pathotypes. Financial support from the Institutional Grants Scheme of the University of Sydney and the Australian Research Council is acknowledged. We thank colleagues, especially Dr R Park, at the University of Sydney Rust Research Laboratory, Plant Breeding Institute, Cobbitty, for providing wheat seed and rust pathogen cultures. REFERENCES 1. Cantrill LC, Deverall BJ. 1993. Isolation of haustoria from wheat leaves infected by the leaf rust fungus. Physiological and Molecular Plant Pathology 42 : 337–343. 2. Chen CY, Heath MC. 1990. Cultivar-specific induction of necrosis by exudates from basidiospore germlings of the cowpea rust fungus. Physiological and Molecular Plant Pathology 37 : 169–177. 3. Chen CY, Heath MC. 1993. Inheritance of resistance to Uromyces ignae in cowpea and the correlation between resistance and sensitivity to a cultivar-specific elicitor of necrosis. Phytopathology 83 : 224–230. 4. Deverall BJ, Deakin A-L. 1987. Genetic tests of the basis of wheat cultivar selectivity in symptom elicitation by preparations from rust pathogens. Physiological and Molecular Plant Pathology 30 : 225–232. 5. Deverall BJ, Saverimuttu N, Cantrill LC, McIntosh RA. 1994. Genetic control of responsiveness of wheat to elicitors in intercellular washing fluids from leaf rust infected leaves. Physiological and Molecular Plant Pathology 45 : 189–194. 6. de Wit PJGM 1992. Molecular characterization of gene-for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant pathogens. Annual Reiew of Phytopathology 30 : 391–418. 7. de Wit PJGM, Spikman G. 1982. Evidence for the occurrence of race and cultivar-specific elicitors of necrosis in intercellular fluids of compatible interactions of Cladosporium fulum and tomato. Physiological Plant Pathology 21 : 1–11. 8. D’Silva I, Heath MC. 1997. Purification and characterization of two novel hypersensitive responseinducing specific elicitors produced by the cowpea rust fungus. Journal of Biological Chemistry 272 : 3924–3927. 9. de Wit PJGM, Joosten MHA, Honee G, Vossen PJM, Cozijnsen, TJ, Koomangersmann M, Vogelsang R. 1995. Molecular aspects of avirulence genes of the tomato pathogen Cladosporium fulum. Canadian Journal of Botany 73 : S490–S494. 10. Hagborg, WAF. 1970. A device for injecting solutions and suspensions into thin leaves of plants. Canadian Journal of Botany 48 : 1135–1136. 11. Hahn M, Mendgen K. 1992. Isolation by conA binding of haustoria from different rust fungi and comparison of their surface qualities. Protoplasma 170 : 95–103. 12. Hahn M, Jungling S, Knogge W. 1993. Cultivar-specific elicitation of barley defense reactions by the phytotoxic peptide NIP1 from Rhynchosporium secalis. Molecular Plant Microbe Interactions 6 : 745–754.
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13. Honee G, Vandenackerveken GFJ, Vandenbroek HWJ, Cozijnsen TJ, Joosten MHA, Koomangersmann M, Vervoort J, Vogelsang R, Vossen P, Wubben JP, de Wit PJGM. 1994. Molecular characterization of the interaction between the fungal pathogen Cladosporium fulum and tomato. Euphytica 79 : 219–225. 14. Jones DR, Deverall BJ. 1978. The use of leaf transplants to study the cause of hypersensitivity to leaf rust, Puccinia recondita, in wheat carrying the Lr20 gene. Physiological Plant Pathology 12 : 311–320. 15. Rohringer R, Kim WK, Samborski DJ. 1979. A histological study of interactions between stem rust and wheat containing resistance genes Sr5, Sr6, Sr8 or Sr22. Canadian Journal of Botany 57 : 324–331. 16. Shipton WA, Brown JF. 1962. A whole-leaf clearing and staining technique to demonstrate hostpathogen relationships of wheat stem rust. Phytopathology 52 : 1313. 17. Southerton SG, Deverall BJ. 1989. Histological studies of the expression of the Lr9, Lr20 and Lr28 alleles for resistance to leaf rust in wheat. Plant Pathology 38 : 190–199. 18. Southerton SG, Deverall BJ. 1990. Changes in phenylalanine ammonia-lyase and peroxidase activities in wheat cultivars expressing resistance to the leaf-rust fungus. Plant Pathology 39 : 223–230. 19. Southerton SG, Deverall BJ. 1990. Changes in phenolic acid levels in wheat leaves expressing resistance to Puccinia recondita f sp tritici. Physiological and Molecular Plant Pathology 37 : 437–450. 20. Southerton SG, Deverall BJ. 1990. Histochemical and chemical evidence for lignin accumulation during the expression of resistance to leaf rust fungi in wheat. Physiological and Molecular Plant Pathology 36 : 483–494. 21. Sutherland MW, Deverall BJ, Moerschbacher B, Reisener HJ. 1989. Wheat cultivar and chromosomal selectivity of two types of eliciting preparation from rust pathogens. Physiological and Molecular Plant Pathology 35 : 535–541. 22. Tani T, Yamamoto H, Onoe T, Naito N. 1975. Initiation of resistance and host cell collapse in the hypersensitive reaction of oat leaves against Puccinia coronata aenae. Physiological Plant Pathology 7 : 231–242. 23. Tiburzy R, Martins EMF, Reisener HJ. 1992. Isolation of haustoria of Puccinia graminis f. sp. tritici from wheat leaves. Experimental Mycology 16 : 324–328.
A cytological assay reveals pathotype and resistance gene specific elicitors in leaf rust infections of wheat N. S and B. J. D Department of Crop Sciences, University of Sydney, N.S.W. 2006, Australia (Accepted for publication October 1997 )
Several different types of preparation from leaf rust-infected leaves were applied to uninfected wheat leaves in two bioassays. One bioassay used intact leaves and symptom formation visible to the unaided eye. The other used exposed mesophylls on leaf segments which were stained and examined by optical microscopy. The first bioassay revealed previously observed elicitation that was not specific for resistance genes, and the second suggested the presence of resistance-gene specific elicitors. The second bioassay was used with extracts from leaves infected with an avirulent pathotype with respect to Lr9 and avirulent or virulent pathotypes with respect to Lr28 and using wheats differing in the presence or absence of the Lr9 or Lr28 alleles for resistance. The results indicated the presence of elicitors specific for putative Avr9}Lr9 and Avr28}Lr28 interactions among other elicitors in the extracts. Preliminary tests showed that the specific elicitors were precipitated by ammonium sulphate. # 1998 Academic Press Limited
INTRODUCTION
The expression of incompatibility in many interactions between fungal pathotypes and plant cultivars is controlled by avirulence genes and their matching resistance genes. The molecular interpretation of such matching is that each avirulence gene encodes a specific elicitor which recognizes and}or interacts with the product of the corresponding resistance gene, thus triggering defence responses. Evidence for such elicitors in cell-free extracts has been obtained in very few interactions between fungal pathogens and plants. These include Cladosporium fulum (syn. Fulia fula [Cooke] Cif.) and tomato cultivars where the elicitors were first detected in intercellular fluids [7 ], Rhynchosporium secalis (Oudem.) J. J. Davis, and barley cultivars where an elicitor was obtained from culture filtrates of the fungus [12 ], and the rust fungus Uromyces ignae Barclay and cowpea cultivars, where the elicitors were found in extracts of germ-tubes from basidiospores [2, 3 ]. Elicitors specific for the Lr20 allele in wheat were detected in diffusates from wheat leaves infected with pathotypes of the leaf rust fungus avirulent with respect to this allele [14 ]. They were detected using a leaf sandwich technique whereby exposed mesophyll of the infected leaf was incubated on exposed mesophyll of an uninfected bioassay leaf. The Lr20-specific elicitors could not be obtained in intercellular washing fluids from infected leaves [4 ]. Other elicitors selective for a gene on chromosome 5A in wheat but not specific for any known resistance gene [5 ] were found in the 0885–5765}98}01002510 $25.00}0}pp970133
# 1998 Academic Press Limited
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N. Saverimuttu and B. J. Deverall
intercellular fluids [21 ] and they caused particularly strong chlorosis and necrosis in cv. Cappelle-Desprez [4 ]. These other elicitors, but not the Lr20-specific ones, were also obtained from germ-tubes of uredospores of the leaf rust fungus [21 ]. This paper reports the results of a new search for avirulence and resistance genespecific elicitors in cell-free extracts from interactions between the leaf rust fungus and wheat. The first aim was to find a source of the elicitors and to develop a bioassay for them. A subsequent aim was the isolation and characterization of the elicitors. The first step was isolation of haustoria from infected leaves [1, 11, 23 ], because haustoria of a biotrophic rust fungus are likely sites of elicitor production and action. The pathotypes and cultivars used were avirulent and resistant based on the expression of the Lr9 or Lr28 alleles in wheat. These alleles were chosen because their expression gives a series of rapid responses and hypersensitivity in the first cells entered by haustoria, in contrast to the Lr20 allele, which gives slower responses and hypersensitivity around the cells containing haustoria [17–20 ] and which had been used in earlier work [4, 14 ]. An expectation was that elicitors from isolated haustoria might cause rapid and clear symptoms in bioassays using leaves containing the Lr9 or Lr28 alleles and not in control cultivars lacking these alleles, and not in the cv. Cappelle-Desprez, which responded so strongly to the earlier detected non-specific elicitors [4 ]. In the light of the results reported herein that the isolated haustoria elicited strong responses in cv. Cappelle-Desprez and none visible to the unaided eye in Lr9 or Lr28containing cultivars, new steps were taken in the attempt to detect gene-specific elicitors. The new steps involved the use of homogenates of infected leaves as simply prepared sources of elicitors, and a more elaborate bioassay modified from that used in the leaf sandwich technique [14 ]. Exposed mesophylls were incubated under homogenates and then examined for capacity to take up a stain that has often been used in revealing wheat cells that have undergone hypersensitivity [15–17 ]. This new approach resulted in the detection of Lr9 and Lr28-specific elicitors as reported here. MATERIALS AND METHODS
Plants and fungi The main cultivars or lines of wheat (Triticum aestium L.) used were Morocco (University of Sydney Wheat accession number W1103), Little Club (W1), CappelleDesprez (W3055), Transfer (W2382) containing the Lr9 allele and CS 2D}2M (University of Sydney Wheat Cytogenetics Stock Accession Register C77.1) containing the Lr28 allele. Some additional tests were done on the Lr28-tester lines C77-1}4*Cook (National Cereal Rust Control line number 103217), C77-2}4*Oxley (50063) and C77-1}7*Teal (48874) and the parental cvs Cook, Oxley and Teal, which lack the Lr28. Puccinia recondita Rob. & Desm. f. sp. tritici Eriks. & Henn. pathotype 104-2,3,6,(7) (University of Sydney accession number 76694), avirulent with respect to Lr9 and Lr28, and pathotype 104-2,3,6,7,8 (80-L-3) avirulent with respect to Lr9 and virulent with respect to Lr28, were maintained on cv. Morocco. Conditions for growth, inoculation and incubation were as in Southerton and Deverall [17 ].
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T 1 Assessment key for measuring stain uptake in mesophyll-exposed leaf segments Score
Description of pattern of stain uptake
0
Interveinal cells uniformly light blue ; veins and adjacent cells not stained Scattered darkly stained cells in the interveinal areas ; rare groups of darkly stained cells Small patches or rows of darkly stained cells (1–2 layers deep) often with collapsed protoplasts Large patches of darkly stained cells (" two layers deep) often with collapsed protoplasts and often as bands elongate for the full length of the segment Extensive bands of darkly stained cells often with collapsed protoplasts and nearly or completely covering the segment
1 2 3 4
Extractions Extracts of leaves were made from 11–15-day-old seedlings of Morroco inoculated 4–5 days earlier with pathotype 104-2,3,6,(7), unless stated that pathotype 104-2,3,6,(7),8 was used. Control extracts were made from uninoculated leaves of the same age. Extracts of haustoria from infected leaves were obtained by the procedure of Hahn and Mendgen [11 ]. Leaf tissue was homogenized (10 g fresh weight 50 ml−" homogenization buffer) and filtered through a double layer of muslin. The filtrate was centrifuged for 5 min at 5000 g and the pellet washed twice in suspension buffer and resuspended in the same buffer (25 ml). The suspension was washed into a ConASepharose affinity chromatography column (0±5 ml ml−" bed volume) and incubated for 15 min. Chloroplasts were eluted with 5 void volumes of suspension buffer and then haustoria were retrieved by agitating the column contents vigorously in suspension buffer (15 ml). Some parallel extracts of haustoria were also prepared by homogenizing leaves in de-ionized water. Samples were taken at each stage of separation and from each type of extract for bioassay in intact leaves. Routine homogenates were prepared subsequently by macerating 20 g fresh leaves in 50 ml chilled de-ionized water. The homogenates were filtered through two layers of muslin and allowed to stand for at least one hour at 4 °C. The sedimented uredospores and plant debris were discarded, and the supernatants used for bioassay on exposed mesophylls of leaf segments. Intercellular washing fluids were obtained as described [4 ]. Bioassays using an Hagborg deice Homogenates were introduced into the intercellular spaces of primary leaves of 7–8day-old seedlings of uninoculated wheat using an Hagborg device [10 ], which permits the flooding of areas in thin plant leaves by hydraulic pressure from a small rubber chamber without direct insertion of a hypodermic needle into the leaves and consequent damage. The infiltrated plants were then incubated at 20 °C under continuous illumination in a growth cabinet for 4 days. Infiltrated leaves were then examined under direct light for chlorosis and}or necrosis.
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Bioassays using mesophyll-exposed leaf segments Leaves harvested from 7–10-day-old uninoculated seedlings were brought to the laboratory in an insulated box containing crushed ice and lined with paper tissues. The lower epidermis of each leaf was removed with the help of a clean sharp razor blade and a pair of forceps. The mesophyll-exposed strips were then cut into segments of 8–10 mm length, covered with test solutions and placed between glass slides in Petri dishes lined with moist filter papers (10 segments 0±5 ml−" per dish). The segments were incubated at 20 °C under continuous illumination in a growth cabinet for 2–3 days (unless otherwise stated) and examined after clearing and staining in a solution of trypan blue in lactophenol at 70 °C for 1 min. The segments were then mounted in glycerine and observed under low power (¬4) microscopy. Mesophyll-exposed segments that had been treated with water were uniformly pale blue after staining, except for ridges of relatively unstained veinal regions. Dark blue staining in cells in each of the five interveinal areas in the centres of segments that had received other treatments was recorded using an assessment key (Table 1). The mean score for staining in each segment and the overall mean score and the standard error (SE) for the 10 segments in each treatment}cultivar combination were calculated. Analyses of variance were carried out to detect significant differences between treatments and overall mean scores for different treatments were compared by the Fisher’s least significant difference test. Precipitation of different protein fractions from homogenates of infected leaes Homogenates were brought successively to 0–20 %, 20–40 %, 40–60 % and 60–80 % with respect to ammonium sulphate. Each precipitate was collected separately by centrifugation and suspended in 5 ml water. The resulting solutions were dialysed overnight against water with continuous stirring.
RESULTS
Effects of injecting fractions from leaf homogenates into intact leaes in causing symptoms isible to the unaided eye Samples of fractions containing isolated haustoria, injected into healthy leaves using the Hagborg device, caused marked chlorosis and necrosis within 4 days in cv. CappelleDesprez, and paleness and sometimes more marked chlorosis in cv. Morocco, but no symptoms visible to the unaided eye in cvs Little Club, Transfer (Lr9) and the line CS 2D}2M (Lr28). Samples of original homogenates of infected leaves made in buffer or water and of supernatants and pellets taken at each stage of the haustorial isolation procedure also caused strong symptoms in cv. Cappelle-Desprez but not in the other cultivars. Comparable extracts from uninfected leaves caused no symptoms. Examination by staining and microscopy of intact leaes injected with fractions from leaf homogenates Infiltration with extracts of infected leaves using the Hagborg device was followed by examination with the unaided eye, and by subsequent staining and microscopy. The
Cytological assay of leaf rust infections of wheat
29
strong symptom of necrosis in cv. Cappelle-Desprez correlated with groups of cells which had thickened walls and an apparent inability to take up trypan blue. In contrast, no visible symptoms appeared in line CS 2D}2M (Lr28) but scattered groups of cells stained intensely with trypan blue and appeared not to have thickened walls. No symptoms, no dark staining and no cell wall thickening occurred in cv. Little Club. The above results were obtained in repeated experiments and caused the abandonment of this approach to the detection of Lr9 or Lr28-specific elicitors.
Effects of applying leaf homogenates to exposed mesophylls on their subsequent capacity to take up stain as seen by microscopy Intense uptake of stain (mean score of 1±48³SE 0±086) by mesophyll cells in cv. Transfer (Lr9) followed the application of homogenates of infected leaves in water. The extent of stain uptake in this cultivar was much lower (mean score of 0±32³0±076) when comparable homogenates from healthy leaves were applied. Homogenates of infected leaves resulted in mean scores of 0±44³0±074 and 0±20³0±063 in cvs Morocco and Cappelle-Desprez, respectively. When the experiment was repeated several times, stain uptake was always significantly higher in the Lr9-containing cultivar treated with extracts of infected leaves than in the other cultivar}extract combinations. When similar extracts were applied to line CS 2D}2M (Lr28), more intense uptake of stain was caused by homogenates from infected leaves (mean score 1±24³0±062) than by those from healthy leaves (0±18³0±080). Mean scores for stain uptake in cvs Little Club and Cappelle-Desprez after treatment with extracts from infected leaves were 0±40³0±080 and 0±68³0±070, respectively. In repeated tests, stain uptake was always significantly higher in the Lr28-containing line treated with extracts of infected leaves than in the other cultivar}extract combinations. All further experiments used the procedures of applying homogenates to exposed mesophylls and their subsequent capacity to take up stain in order to test the pathotype and resistance gene specificity of the eliciting activity and to obtain preliminary information on the nature of the activity. Experiments with pathotypes airulent and irulent with respect to Lr28 Exposed mesophylls of line CS 2D}2M (Lr28) took up more stain after treatment with crude extracts of leaves inoculated by the avirulent pathotype than with those inoculated by the virulent pathotype (Table 2). Stain uptake increased with increasing time of exposure to the extracts. The cvs Little Club and Cappelle Desprez took up less stain after treatment with either extract.
Experiment using cultiars with and without Lr28 Exposed mesophylls from three Lr28-tester lines and from line CS 2D}2M (Lr28) took up more stain than five control cultivars after treatment with crude extracts of leaves inoculated with the avirulent pathotype (Table 3). Extracts from leaves inoculated with the virulent pathotype and from uninoculated leaves caused less potential to stain in all lines and cultivars.
30
N. Saverimuttu and B. J. Deverall T 2 Effects of extracts from leaes inoculated with airulent or irulent pathotypes on capacity of exposed mesophylls to take up stain
Source of extract
Time of Mean score (³SE) for stain uptake exposure to In cultivar}line extracts (days) Cappelle-Desprez CS 2D}2M Little Club
Uninoculated leaves Avirulent interaction* Virulent interaction†
3
0±18³0±044 a
0±24³0±079 b
0±18³0±077 a
1 2 3 1 2 3
0±16³0±047 a 0±42³0±099 b 0±64³0±213 d 0±28³0±110 b 0±46³0±110 b 0±68³0±188 d
0±36³0±062 c 0±70³0±127 c 1±14³0±046 e 0±18³0±053 a 0±36³0±076 a 0±30³0±095 b
0±22³0±060 b 0±30³0±086 a 0±44³0±101 c 0±36³0±093 c 0±34³0±085 a 0±40³0±080 c
Values for a particular time of exposure denoted by different letters are statistically different at P ¯ 0±05. *cv. Morocco inoculated with pathotype 104–2,3,6,(7) (arivulent to Lr28) ; †cv. Morocco inoculated with pathotype 104–2,3,6,(7),8 (virulent to Lr28 ). T 3 Effects of extracts from leaes inoculated with pathotypes airulent or irulent towards Lr28 on the staining capacity of exposed mesophylls of cultiars with or without Lr28
Test cultivar}line Without Lr28 Cappelle-Desprez Little Club Oxley Teal Cook With Lr28 CS 2D}2M C77-2}4*Oxley C77-1}7*Teal C77-1}4*Cook
Mean score (³SE) for stain uptake Source of extracts Avirulent* Uninfected leaves infections Virulent† infections
0±14³0±049 a 0±12³0±051 a 0±20³0±075 a 0±22³0±066 a 0±36³0±068 b
0±34³0±094 b 0±32³0±110 b 0±26³0±097 a 0±24³0±074 a 0±28³0±012 a
0±42³0±103 b 0±34³0±090 b 0±28³0±095 a 0±26³0±094 a 0±36³0±105 b
0±18³0±052 a 0±16³0±047 a 0±15³0±015 a 0±30³0±065 b
1±06³0±106 c 0±96³0±074 c 1±24³0±116 d 0±90³0±099 c
0±26³0±070 a 0±28³0±086 a 0±18³0±060 a 0±30³0±071 b
Values followed by different letters are statistically different at P ¯ 0±05. *Extract of cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †extract of cv. Morocco inoculated with pathotype 104–2,3,6,(7),8.
Experiments on remoing intercellular washing fluids on actiities of extracts from leaes inoculated with airulent and irulent pathotypes Results in Table 4 show that there was high activity, before and after removal of intercellular washing fluids, in crude extracts of leaves inoculated with an avirulent pathotype. The activity was greatest on the Lr28-containing cultivar. The intercellular washing fluids had lower activity regardless of pathotype and test cultivar.
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T 4 Effects of remoing intercellular washing fluids on actiities of extracts from leaes inoculated with airulent or irulent pathotypes
Source of leaf extract Uninfected Avirulent* original IWF original minus IWF Virulent† original IWF original minus IWF
Mean score (³SE) for stain uptake In cultivar}line Cappelle-Desprez CS 2D}2M Little Club 0±18³0±044 a
0±24³0±055 b
0±18³0±077 a
0±64³0±067 d 0±26³0±094 b 0±46³0±113 c
1±14³0±186 e 0±44³0±093 c 1±20³0±175 e
0±44³0±101 c 0±42³0±076 c 0±38³0±060 c
0±68³0±235 d 0±50³0±194 c 0±52³0±130 c
0±18³0±066 a 0±14³0±064 a 0±18³0±096 a
0±28³0±076 b 0±28³0±087 b 0±40³0±098 c
Values followed by different letters are statistically different at P ¯ 0±05. *Extracts of cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †extracts of cv. Morocco inoculated with pathotype 104–2,3,6,(7),8. T 5 Effects of different protein fractions from extracts of inoculated leaes on exposed mesophylls
Extract and treatment Uninfected untreated Avirulent* untreated PF 2 (20–40 %)‡ PF 3 (40–60 %) PF 4 (60–80 %) Virulent† untreated PF 2 (20–40 %)‡ PF 3 (40–60 %) PF 4 (60–80 %)
Mean score (³SE) for stain uptake Cultivar}line Cappelle-Desprez CS 2D}2M Little Club
0±32³0±114 b
0±30³0±090 b
0±36³0±123 b
0±54³0±170 d 0±42³0±258 c 0±38³0±242 b 0±68³0±293 e
1±14³0±186 g 0±14³0±049 a 0±88³0±124 f 0±86³0±288 f
0±58³0±191 d 0±30³0±186 b 0±30³0±136 b 0±34³0±177 b
0±68³0±188 e 0±34³0±202 b 0±44³0±138 c 0±50³0±148 c
0±18³0±066 a 0±16³0±068 a 0±20³0±063 a 0±20³0±075 a
0±28³0±076 b 0±20³0±098 a 0±16³0±074 a 0±48³0±202 c
Values followed by different letters are significantly different at P ¯ 0±05. *From cv. Morocco inoculated with pathotype 104–2,3,6,(7) ; †from cv. Morocco inoculated with pathotype 104–2,3,6,(7),8 ; ‡with respect to ammonium sulphate.
Effects of different protein fractions from homogenates on exposed mesophylls Results in Table 5 showed that protein fractions 3 and 4 (precipitated at 40–60 % and 60–80 % concentrations of ammonium sulphate, respectively) from homogenates of leaves inoculated by the avirulent pathotype retained much of the original higher activity on line CS 2D}2M (Lr28). Generally, protein fraction 4 from infections by both
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N. Saverimuttu and B. J. Deverall
pathotypes had slightly greater activities on the cv. Cappelle-Desprez and Little Club than did the other fractions. DISCUSSION
A consistently greater cellular response in Lr9- or Lr28-leaves to homogenates of wheat leaves inoculated with an avirulent pathotype than occurred in response to any other combination of leaves and extracts indicated the presence of elicitors specific for complementary resistance and avirulence alleles. Putative Avr9}Lr9- and Avr28}Lr28specific elicitors were thus distinguished from other elicitors of cellular change present in the extracts. The response was seen in a bioassay based on incubating exposed mesophylls with homogenates for up to 3 days and then observing the capacity of cells to take up trypan blue from a lactophenol solution at 70 °C. Remarkably, and as observed before [14 ], removal of the epidermis, incubation of uninfected mesophyll under water as a control and then immersion in heated trypan blue}lactophenol resulted in only a light and even uptake of stain by wheat cells, suggesting little or no damage to cell membranes by the apparently harsh treatments. A similar light and even staining has also been reported for wheat cells in parts of leaves remote from inoculation sites expressing hypersensitivity to rust pathogens [15, 17 ] in contrast to the intense staining at the sites. The intense trypan blue stain in Lr9 and Lr28-containing wheat cells entered by haustoria of avirulent pathotypes of the leaf rust fungus correlated precisely with autofluorescence of similar placed cells in leaves treated with a fluorochrome and examined by fluorescence microscopy, and was interpreted as a sign of necrosis [17 ]. Cellular autofluorescence has been used as an indicator of cowpea responses to specific elicitors from the cowpea rust fungus [2, 3, 8 ]. In the present work, incubation with extracts of uninfected leaves caused occasional cells to take up stain more strongly. Extracts of infected leaves often increased the frequency of cells that stained strongly. The stronger staining was interpreted as resulting from changes to membranes allowing stain intake and, in some cells, from deep staining of collapsed protoplasts associated with cell death, as has often been discussed for hypersensitivity to rust fungi [15–17, 22]. The strongest and most widespread staining in the present work correlated with extracts from leaves inoculated with avirulent pathotypes being incubated on Lr9 or Lr28bearing mesophylls, hence the claim for detecting gene-specific elicitors in those extracts. This is the first record of the detection, in cell-free extracts, of specific elicitors of rustresistance in wheat. In previous studies, elicitors specific for the Lr20 allele in wheat and for the complementary fungal avirulence allele were detected using a leaf-sandwich technique and subsequent staining with trypan blue for cellular necrosis [14 ]. Resistance specific elicitors were detected, however, in germ-tube extracts of germinating basidiospores of the cowpea rust fungus (Uromyces ignae Barclay) by the capacity to cause necrosis in resistant cowpea cultivars [2, 3 ], and this work has resulted in two of the elicitors being purified and characterized as peptides [8 ]. Other advances in characterization of specific elicitors from fungi have come with those found in intercellular spaces of tomato leaves infected by the leaf mould pathogen, C. fulum, and by their capacity to elicit hypersensitivity in resistant tomato cultivars [6, 7, 9, 13 ].
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A problem in researching elicitation in wheat is the readily seen chlorotic and necrotic reaction of intact leaves in some cultivars to a widespread component of many types of extract from wheat rust fungi. Extracts from germ-tubes of stem and leaf rust fungi and from intercellular spaces of leaves infected by these fungi were active [21 ]. Similarly active were leaf homogenates and isolated haustoria in the present work. Reactions in intact leaves were particularly marked in cv. Cappelle-Desprez, and were encoded for by a gene that has no known relationship to rust resistance [5 ]. Necrosis in this cultivar was seen to be associated with an increased thickness of cell walls. In contrast, no visible responses to rust extracts occurred in intact leaves of cultivars chosen for their specific resistance to the fungal pathotypes tested. The use of exposed mesophylls and the uptake of trypan blue by this tissue in the more elaborate bioassay described here enabled progress to be made in revealing specific elicitors. Progress can now be made on the isolation and characterization of the specific elicitors. The precipitation of specific eliciting activity from fractions using high concentrations of ammonium sulphate suggests that protein may contribute to the activity. Isolation of specific elicitors should enable probes to be designed to test for their detection in or on haustoria of potentially avirulent pathotypes. Financial support from the Institutional Grants Scheme of the University of Sydney and the Australian Research Council is acknowledged. We thank colleagues, especially Dr R Park, at the University of Sydney Rust Research Laboratory, Plant Breeding Institute, Cobbitty, for providing wheat seed and rust pathogen cultures. REFERENCES 1. Cantrill LC, Deverall BJ. 1993. Isolation of haustoria from wheat leaves infected by the leaf rust fungus. Physiological and Molecular Plant Pathology 42 : 337–343. 2. Chen CY, Heath MC. 1990. Cultivar-specific induction of necrosis by exudates from basidiospore germlings of the cowpea rust fungus. Physiological and Molecular Plant Pathology 37 : 169–177. 3. Chen CY, Heath MC. 1993. Inheritance of resistance to Uromyces ignae in cowpea and the correlation between resistance and sensitivity to a cultivar-specific elicitor of necrosis. Phytopathology 83 : 224–230. 4. Deverall BJ, Deakin A-L. 1987. Genetic tests of the basis of wheat cultivar selectivity in symptom elicitation by preparations from rust pathogens. Physiological and Molecular Plant Pathology 30 : 225–232. 5. Deverall BJ, Saverimuttu N, Cantrill LC, McIntosh RA. 1994. Genetic control of responsiveness of wheat to elicitors in intercellular washing fluids from leaf rust infected leaves. Physiological and Molecular Plant Pathology 45 : 189–194. 6. de Wit PJGM 1992. Molecular characterization of gene-for-gene systems in plant-fungus interactions and the application of avirulence genes in control of plant pathogens. Annual Reiew of Phytopathology 30 : 391–418. 7. de Wit PJGM, Spikman G. 1982. Evidence for the occurrence of race and cultivar-specific elicitors of necrosis in intercellular fluids of compatible interactions of Cladosporium fulum and tomato. Physiological Plant Pathology 21 : 1–11. 8. D’Silva I, Heath MC. 1997. Purification and characterization of two novel hypersensitive responseinducing specific elicitors produced by the cowpea rust fungus. Journal of Biological Chemistry 272 : 3924–3927. 9. de Wit PJGM, Joosten MHA, Honee G, Vossen PJM, Cozijnsen, TJ, Koomangersmann M, Vogelsang R. 1995. Molecular aspects of avirulence genes of the tomato pathogen Cladosporium fulum. Canadian Journal of Botany 73 : S490–S494. 10. Hagborg, WAF. 1970. A device for injecting solutions and suspensions into thin leaves of plants. Canadian Journal of Botany 48 : 1135–1136. 11. Hahn M, Mendgen K. 1992. Isolation by conA binding of haustoria from different rust fungi and comparison of their surface qualities. Protoplasma 170 : 95–103. 12. Hahn M, Jungling S, Knogge W. 1993. Cultivar-specific elicitation of barley defense reactions by the phytotoxic peptide NIP1 from Rhynchosporium secalis. Molecular Plant Microbe Interactions 6 : 745–754.
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