Assessment of Lr20 gene-specificity of symptom elicitation by intercellular fluids from leaf rust-infected wheat leaves

P~ysiologzcal

Plant Pathology

(1985)

27,99%107

Assessment of Lr20 gene-specificity of symptom elicitation by intercellular fluids from leaf rust-infected wheat leaves B. J. DEVERALL Department

and A.-L.

DEAKIN

of Plant Pathology and Agricultural

!Arcepted forpublication

Entomology,

University

of Sydney,

N.S. W. 2006, Australia

April 1985)

Paleness, chlorosis and necrosis were caused in uninfected primary leaves of three UWbearing wheat cultivars after infiltration with fluids from intercellular spaces of leaves of Triticumaestiuum cv. Chinese Spring infected by mycelia of leaf rust (Puccinia recondita f.sp. tritki) either avirulent or virulent with respect to the L720 allele. No symptoms were caused in cv. Chinese Spring or two other cultivars, all of which lack Lr genes, or in cv. Brevit which bears the Lr2c allele. Symptoms were caused, however, in the Lrlla-bearing cv. Spica. Symptoms were not caused in a Chinese Spring/Axminster line in which chromosome 7A carrying the Lr20 allele had been substituted from the cv. Axminster. No symptoms were caused in any cultivar by fluids from uninfected k~\TS. The eliciting agents were partially thermostable macromolecules which were active after many-fold dilution and likely to have been derived from either walls of intercellular hyphae or hoat cells at the infection court.

INTRODUCTION Hypersensitivity to avirulent strains of leaf rust in wheat bearing the Lr20 allele for resistance shows a distinctive pattern of necrosis [II]. Necrosis begins in the mesophyll one or two cells ahead of apparently unaffected cells bearing the first haustoria arising from intercellular infection hyphae. Necrosis then develops in a wide band of cells around the avirulent colony. A possible cause of the necrosis is an elicitor diffusing from either the intercellular hyphae or from the cells bearing the haustoria. Evidence for the action of a diffusible elicitor was obtained in experiments with transplants of uninfected leaf tissue held for a period on infected tissue [13]. These experiments used a number of combinations of wheat genotypes as sources of transplants and as hosts for infection, and also used a number of avirulent and virulent strains of leaf rust. From the occurrence of necrosis in transplants, it was concluded that a toxin was released by avirulent strains of rust and was active only on LrZO-bearing wheat tissue. The hypothetical gene-specific agent was termed a toxin because it caused death of host cells but it can also be termed an elicitor of necrosis. Attempts failed to reveal the hypothetical specific elicitor in different types of aqueous extract from avirulent strains of leaf rust [2]. Th e extracts comprised leachates and macerates of germinated spores and diffusates from exposed mesophylls of leaves bearing established mycelia. The bioassay involved overlaying droplets of the extracts 00484059/85/O

10099 + 09 $03.00/O

0 1985 Academic

Press Inc.

(London)

Limited

100

B. J. Deverall and A.-L. Deakin

with exposed mesophyll of segments of uninfected leaves, prepared in the same way as the transplants. The most common result from many experiments was the detection of a low level of non-specific eliciting action on the resistant cultivar, Thew, and the susceptible cultivar, Chinese Spring. The research reported in this paper was directed to examine fluids from intercellular spaces of infected leaves, where the hyphothetical diffusible elicitor is likely to be released. The methods for obtaining fluids were adopted and modified from those used by de Wit & Spikman [q which were based on a report by Klement [15]. The bioassay for assessing activity in fluids was based on the use of a device for infiltrating solutions into leaves of cereals [7j. This paper contrasts activity in fluids from uninfected leaves with that in fluids from leaves infected with different strains of rust. The host cultivar used as a source of fluids was Chinese Spring, which possessesno known genes for leaf rust resistance in its seedling stages. It was used to permit equal development of a pair of rust strains regardless of their avirulence or virulence with respect to resistance genes in other wheat cultivars. Similar growth and development of the avirulent and virulent strains of leaf rust used here have been shown to occur in the cultivar Chinese Spring by Jones & Deverall [II]. The strains were allowed to grow to the mycelial and pre-uredial stage which yielded the specific elicitor from the avirulent mycelium in Chinese Spring in the transplant experiments [Z3]. In order to assessthe extent of leakage of intracellular contents into Auids, the combined activity of two plant and fungal cytosolic enzymes, namely glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was measured in fluids and homogenates of leaves. MATERIALS

AND METHODS

Wheat (Triticum aestivum L.) lines used were cultivars Thew (University of Sydney accession No. W203), Kenya 744 (W744), Norka (W578), Spica (W2341), Brevit (W974), Chinese Spring (W 1806)) M orocco (W 1103) and a line (W 1656) derived from Triticum timopheevi. Gene symbols for resistance given in the Results section are taken from an internationally agreed catalogue [27]. Seeds were sown (20 per 1O-cm pot) in a fertilized peat/sand mixture and grown in a controlled environment room at 19-20 “C under warm white fluorescent lights (18 000 lx at soil level) providing a 12 h photoperiod. Plants were inoculated or bioassayed with fluids 8 days after sowing. Puccinia recondita fsp. tritici strains used were 104-ANZ-1,2,3GT (University of Sydney accession No. 74606) virulent with respect to the Lr20 allele and 104-ANZ-2,3GT (74408) avirulent with respect to the Lr20 allele. Uredospores were produced on susceptible cultivars and stored in liquid nitrogen until required. Seedlings of cv. Chinese Spring were used for the production of intercellular fluids. A suspension of uredospores in distilled water (500 mg in 100 ml) was used to inoculate 960 seedlings using a gas-powered spray can (Jet Pack Power Unit, dichlorodifluoromethane gas). The pots were incubated in a perspex humidity chamber at 23 “C in the environment room for 24 h, starting at the beginning of the dark period. Pots were then removed to a growth cabinet providing 12 h photoperiods from cool-white fluorescent tubes at 25 000 lx for 5 days at 20 “C. Six days after inoculation, primary leaves bearing 2&25 chlorotic flecks per centimetre (length) were harvested.

Gene-specificity

of symptom

elicitation

101

The apical 2 cm of the leaves were discarded and the two successive 4 cm lengths from the remainder were collected. Leaf segments were placed in chilled distilled water ( 10 g fresh weight per 150 ml) and infiltrated under vacuum for three 1 min periods interrupted by 30s intervals. The water-soaked segments were dried between linen towels for 1 min before being wrapped in muslin as 4 cm lengths and 4 g fresh weight lots and placed vertically in 50 ml capped centrifuge tubes at 0 “C. The tubes were centrifuged at 1200 g for 10 min at 4 “C. Fluids were collected as approx. 1 ml volumes per tube, pooled and stored at - 10 “C as 1 ml aliquots. Control fluids were obtained by the same procedure from uninoculated leaves. The pH values of intercellular fluids were between 5.0 and 6.0. Bioassays were carried out on primary leaves of 8-day-old uninoculated plants. Six replicate leaves per treatment were infiltrated with intercellular fluids using a device made as described by Hagborg [il. The device was held across a leaf 3.5 cm from the tip and approx. 0.05 ml of fluid was injected from the syringe into the cavity. The length of the water-soaking was measured and noted on a tag attached to the plant. .After infiltration, the pots were placed in the growth cabinet at 20 “C under continuous illumination from cool-white fluorescent tubes (25 000 lx). Pots were watered daily from the base. Observations were made daily by examining infiltrated leaves under a direct light. Symptoms were recorded as no symptoms (0), slight paleness on one side of the leaf ( + ) and paleness on both sides of the leaf ( + + ) and, within these areas, occurrence of accentuated pale lesions ( x ), occurrence of yellow lesions ( x x ) and occurrence of necrosis ( -). A symbol (C) was added to each record when the general paleness extended throughout the area that had been infiltrated. The infiltrating device occasionally caused slight injury marks which were distinct from, and did not interfere with, symptoms caused by the fluids. For measurement of enzyme activity, fluids were prepared as above except that 1.5 ml of 50 mM Tris/HCl buffer, pH 7.8, containing 7 mM MgCl,, 5 mM EDTA, 7:~ w/v PVP and 10 mM B-mercaptoethanol was placed in the tubes before centrifugation of leaf segments. Extracts of whole leaves (0.2 g ml- ‘) were prepared by homogenization in the same buffer at 0 “C with a mortar and pestle; homogenates were centrifuged at 20 OOOg at 4 “C and the pellets were discarded. Enzyme activity was estimated from the rate of NADPH formation when glucose-6-phosphate was oxidized to 6-phosphogluconate, and 6-phosphogluconate to ribulose-5-phosphate. Fluid or supernatant ( 100 pl j was added to a cuvette with 2.9 ml of 50 mM Tris/HCl buffer, pH 7.8, containing 7 mM MgCl,, 5 tTIM EDTA, 2 mM glucose-6-phosphate, 2 mM 6-phosphogluconate and 1 mM NADP+ and the change in absorbance at 340 nm was measured. Activity units are defined as pmoles of NADPH produced min- ’ g- ’ fresh weight of leaf. RESULTS Response oJ‘T. aestivum cu. Thew tojuids centrzfigedfrom leaves The cultivar Thew was used as a standard in most experiments because it was the subject of most of the earlier research in the laboratory on the expression of the Lr20 allele [I, 3,11-131. Infiltrated fluids were observed from the appearance of water-soaking to move equidistantly on either side of the point of injection. The mean length of water-soaking

6. J. Deverall and A.-L. Deakin

102 TABLE

Response’ ofTriticum

aestivum

1

cu. Thew to intercellular~uidsfrom leaves of cu. Chinese Spring infected with virulent or avirulent strains of leaf rust Days from

Leaf rust strains

104-ANZ-1,2,3GT Virulent

104-ANZ-2,3GT Avirulent

Plant No. 1 2 3 4 5 6 7 8 9 10 11 12

I

4 + + + + 0 + 0 + + + + ++x

5 ++x ++x ++ ++x ++ ++ 0 ++ c+ + c+ c

++x

infiltration

6

c

c

c c+ c c+ c

++xx ++xx ++xx ++xx ++x ++xx 0 ++xxc + ++xx

7 c c c c c c

C c c c

++xx ++xx-c ++xx-c ++xx ++xx-c ++xx-c 0 ++xx ++ + + ++xx

8 c

c

c C c c c

++xx ++xx-c ++xx-c ++xx-c ++xx-C ++x 0 ++xx-c ++ + + ++xx

c

x-c

C C C c

aO, no symptoms; + , slight paleness on one side of the leaf; + + , paleness on both sides of the leaf; x , occurrence of pale lesions within pale regions; x x , occurrence of yellow lesions; the area that had been infiltrated. -> necrosis; C, general paleness extended throughout

in 96 leaves was 2.2 cm with a standard deviation of0.78. Water-soaking disappeared within a few hours. Fluids from uninfected leaves caused no symptoms in test leaves, a result obtained on numerous occasions. Fluids from infected leaves caused slight paleness visible, at least in patches, within the infiltrated area when viewed from above on day 4. This paleness was visible throughout most infiltrated areas and from both sides of the leaves on day 5. As indicated in Table 1, accentuated patches of chlorosis began to appear within the pale areas on day 5. These patches became yellow on day 6. Necrosis appeared as white or brown sunken lesions at the sites of these accentuated patches on day 7. Fluids from leaves infected with virulent strains were usually more active than those from avirulent strains in the speed and extent of symptom elicitation, as in the experiment shown in Table 1. Some variation between plants within a treatment occurred in speed and extent ofsymptom formation, as shown in Table 1. Combined activities of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in supernatants of homogenates were 396.0 and 614.0 units from uninfected and infected leaves, respectively. Combined activities in fluids centrifuged from uninfected and infected leaves were 2.4 and 3.1 units, respectively, representing about 0.59; of that in homogenates of comparable leaves. Comparison of the responses of cultiuars with or without the Lr20 allele The six cultivars chosen for this comparison were those used in experiments with transplants in earlier research in this laboratory [13]. The responses of replicate plants on dav 7 are shown in Table 2. The results are similar to those obtained in two other

Gene-specificity

of symptom

elicitation

103 TABLEP

Responses” after 7 days of cultiuars with or without the Ix20 allele to intercellular~uidsfrom with virulent or avirulent strains of leaf rust Cultivars Rust strains as sources of fluids

Thew Lr20 ++xx ++xx-c

104-ANZ-1,2,3GT Virulent

104-ANZ-2,3GT .4virulent

++xx-C ++xx ++xx-c r ++xx-c ++ ++ ++x + + +

Norka Lrl,Lr20 c

c

c c c C C

and designated

resistance

Kenya 744 Lr20

++ ++

c c

++ 0

++ ++ ++x ++ ++ 0 ++ ++ ++ ++

c c c c c

+ + + + ++ + ++xc + + 0

c c c c

Chinese Spring M.P.R.” 0 0

c c c c c c

c

0 0 0 0 0 0 0 0 0 0

leaves infected

genes

Brevit Lr2c 0 0 0 0 0 0 0 0 0 0 0 0

Spica Lrlla +x ++xx-c ++xx-c + 0 ++xx ++ ++x ++ ++ ++ ++

c

C c C c c C C C

“Responses of six replicate plants per treatment. 0, no symptoms; +, slight paleness on one side of the leaf + f , paleness on both sides of the leaf; x , occurrence of pale lesions within pale regions; X x , occurrence of yellow lesions; -, necrosis; C, general paleness extended throughout the area that had been infiltrated. %Jndesignated gene for mature plant resistance.

experiments, one using aliquots of the same preparation of fluids and the second using aliquots of a different preparation. The L&V-bearing cultivars responded to the fluids as did the Lrlla-bearing cultivar, Spica, but cvs Brevit (LrZc) and Chinese Spring did not. Comparison of the responses of T. aestivum cu. Chinese Spring and a substitution line bearing the 7A chromosome The substitution line Chinese Spring (Axminster 7A) was originally produced with the aid of five backcrosses by Dr E. R. Sears and then multiplied by Dr R. A. McIntosh. It possesses chromosome 7A, carrying the Lr20 allele, from cv. Axminster, and substituted for its Chinese Spring homologue. Samples of the line were checked by us for the characteristic expression of Lr20-based resistance after inoculation with strain 104-ANZ2,3GT of leaf rust. The substitution line and cv. Chinese Spring failed to give symptoms when uninfected leaves were infiltrated with fluids obtained from infected leaves of cv. Chinese Spring. The same result was obtained in several experiments with six replicate plants of each line and cultivar. The responses of other cultiuars lacking Lr genes Cultivar Chinese Spring was compared with line W1656, which also bears an undesignated gene for mature plant resistance to leaf rust, and cv. Morocco, which

104 possesses no known genes for resistance three cultivars using six replicate plants

B. J. Deverall and A.-L. Deakin to leaf rust. No symptoms on each of three occasions.

were caused

in the

Some properties of eliciting agents Tests were carried out on intercellular fluids obtained from leaves infected by strain 104-ANZ- 1,2,3GT and assessed by the responses of cv. Thew. Normal eliciting activity was found in 1 ml aliquots of fluids retainedin dialysis sacs (Selbys, 10 mm flat width, molecular weight cut-off approximately 12 000-14 000) after dialysis for 24 h against three changes (each 100 ml) of distilled water at 8 “C; no activity was found in the water outside, which had been concentrated to the original volume of the fluids (1 ml) by rotary film evaporation at 40 “C. Slightly diminished activity was found in fluids that had been positively filtered (MF-Millipore type GS, 0.22 pm pore size). The results of exposing fluids to a series of temperatures between 0 and 100 “C for 2 h, to 100 “C for different periods, and to autoclaving are shown in Table 3. Two-fold serial dilutions (to x 128) of the intercellular fluids obtained from leaves infected by each of the 104-strains of leaf rust were tested in three cultivars. Marked activity of the 104-ANZ-2,3GT strain occurred to x 16 in cv. Thew and to x 32 in cv. Spica; similar activity of the 104-ANZ-1,2,3GT strain occurred to x 32 in cv. Thew and x 128 in cv. Spica. No activity occurred in cv. Chinese Spring. DISCUSSION Detection of eliciting activity only in fluids from infected leaves implies that the source is either the rust fungus or the rust-host interaction. The very low levels of activity of two plant and fungal cytosolic enzymes in fluids indicates that host cells and hyphae are leaking very little of their contents. The eliciting activity is very likely to have come, therefore, from the intercellular spaces of infected leaves and particularly from the walls of intercellular rust hyphae or from adjacent host cell walls that have been affected by these hyphae. It is interesting to note that the level of glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities was much higher in homogenates of infected leaves than in those of uninfected leaves. This observation is consistent with other reports of enhanced activities of enzymes of the pentose phosphate pathway in infected plants [16,20]. The eliciting molecules are considered to be highly active. It should be recalled that they were obtained through a diluting process involving flushing intercellular spaces with water. Activity was retained after at least a further 32 serial dilutions. This suggests that the molecules are present in the infected leaf in adequate concentrations for symptom elicitation. It is not known if they can contact and affect host cells in the absence of the artificial process of flooding the intercellular spaces. The elicitors cause symptoms resembling those seen during rust infections of wheat but differing in some details of sequence of occurrence and in not being strain-specific. Paleness and chlorosis, but not necrosis, occur about 4-5 days after virulent infections at sites where uredial initials develop. Paleness is also seen later where intercellular hyphae grow around the uredia. Necrosis, but not paleness and chlorosis, occur about 3-4 days after avirulent infections, appearing as white-brown flecks that may become

4 8

Day

4 8

Day

c

++

Oto++x

0 x c

Autoclave for 15 min (103.5 kPa)

++xx-c

20

35

Oto+C +cto++x

15 min

c

‘l‘reatment .

45

oto+c Oto++xC

30 min

100 “C

temperaturesfor

Oto++C Oto++xx-c

for 120 min

at di$rent

and duration

(“C)

to,fluid?’ pretreated

Temperature

Thm

oto++c ++to++xx-c

aestivum

3

70

Oto+C Oto++

60 min

Oto++C +xto++xx

dz&ntperiods

aResponses after days shown of six replicates per treatment. 0, no symptoms; +, slight paleness on one side of the leaf; + + , paleness on both, sides of the leaf; x , occurrence of pair lesions within pale regions; x x , occurrence of yellow lesions; -, necrosis; C, general paleness extended throughout the area that had been infiltrated. %Iuids from cv. Chinese Spring infected by strain 104.ANZ-1,2,3GT.

++c ++cto++xx-c

22 “C for 120 min

++ ++xx-c

0

Responsesa of Triticum

TABLE

-c

0

0

120 min

++

Oto++x

Oto

100

x -

106

B. J. Deverall

and A.-L. Deakin

slightly sunken [11]. Necrosis occurs more slowly, therefore, after elicitor application and, furthermore, it does not occur in a strain-specific way. The major differences between the present experiments and the transplant experiments [13] were, firstly, the eliciting activity of intercellular fluids from the virulent as well as the avirulent strain, as discussed above, and, secondly, the action of the elicitors in affecting the Lrlla-bearing cv. Spica. The response of cv. Spica opposed the idea of specificity of the fluids for the Lr20 allele alone. Apart from this response, the sensitivity of the other tested cultivars encouraged the idea that the fluids contained an LrZOselective component. The idea of LrZO-selectivity was not sustained by the lack of response of the Chinese Spring chromosome substitution line bearing the Lr20 allele. It must be concluded, therefore, that the intercellular fluids are neither rust strain nor host gene-specific in their eliciting activity, unlike the agents detected in the transplant experiments [13]. The role of the eliciting activity in the regulation of the rust-wheat interactions cannot be understood at present, as discussed above, in contrast to that of the strain and cultivar-specific eliciting fluids in the Cladosporiumfulvum-tomato system [5,6]. The detection of symptom eliciting activity in preparations from rust fungi is, however, a rare event. It has previously been achieved with stem rust of wheat by Silverman [19] in the extraction and partial fractionation of a cultivar-selective chlorosis-elicitor, and by Olien [18] in displacing necrosis towards the anode when electrodes were applied to infected leaves. Necrosis-eliciting activity on soybean was obtained from germinating uredospores of the rust fungus Phakopsora pachyrhizi [4, 241. An elicitor of induced resistance and of phytoalexin accumulation in beans was isolated from germinated spores of the rust fungus Uromycesphaseoli [IO]. Suppressors of a response, that normally prevents the cowpea rust fungus U. phaseoli var. vignae from forming the first haustorium in bean leaves, have been obtained from intercellular fluids of bean leaves infected by U. phaseoli var. typica [S]. A suppressing factor has also been obtained from infection structures of the latter rust and one of the effects of the suppressors is to prevent formation ofsilicon-containing deposits normally elicited by the cowpea rust fungus [9]. Awareness that rust fungi produce elicitors and suppressors of processes associated with resistance encourages further exploration of the intercellular environment at infection sites. The transplant experiments [13] implied the diffusion of a gene-specific elicitor from avirulent leaf rust hyphae to transplants several cell widths away. The present experiments indicate that flushing the intercellular spaces with water extracts non-specific elicitors. Possibly the sought-after specific elicitors remain weakly bound to cell walls and, in future work, might be released by flushing with salt or buffer solutions. Specific elicitors may be extracted by water but in insufficient concentration to be revealed by the bioassay. It is conceivable that low concentrations of suppressors may be present in the fluids but that their action is masked. Further understanding may come from fractionation of the fluids and analysis of their components for biological activities. We acknowledge Dr R. A. McIntosh for advice and provision of seed, Dr J. R. Woodward for the enzyme assays, Mr J. D. Oates and Mr D. Gow for supply of seed and rust strains, and the Australian Research Grants Scheme for financial assistance.

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REFERENCES I,

B. J, (1980). The effects of light and a photosynthetic inhibitor on the of the Lr20 gene for resistance to leaf rust in wheat. Physiological Plant Pathology 16,415-423. DEVERALL., B. J. (1982). Elicitation ofnecrosis by rust fungi. In Actiue De&e Mechanisms of Plants, Ed. by R. K. S. Wood, pp. 326327. Plenum Press, New York and London. DEVERALL. B. J. & MCLEOD, S. (1980). Responses ofwheat cells around heat-inhibited rust mycelia and associated with the expression of the LrZO, St+ and Sr15 alleles for resistance. Physiological Plant PatholoQ 17,213p219. DEVERALL, B. J., KEOGH, R. C. & MCLEOD, S. (1977). Responses of soybean to infection by, and to germination fluids from, urediniospores of Phakopsora phachyrhizi. Transactions of the British Mycological Society 69,41 l-415. DE WIT, P. J. G. M. & SPIKMAN, G. (1982). Evidence for the occurrence of race and cultivar-specific elicitors of necrosis in intercellular fluids of compatible interactions of Cladosporiumfuluun and tomato. Pbsiological Plant Patholou 21, 1-l 1. DE WIT, P. J. G. M., HOFMAN, J. E. & AARTS, J. M. M. J. G. (1984). Origin of specific elicitors of chlorosis and necrosis occurring in intercellular fluids of compatible interactions of Cladosporium fulwn (syn. Fuluiafulva) and tomato. Physiological Plant Pathology 24, 17-23. HAGBORG, W. A. F. (1970). A device for injecting solutions and suspensions into thin leaves of plants. Canadian Journal ofBotany 48, 1135-l 136. HEATH, M. C. (1980). Effects of infection by compatible species or injection of tissue extracts on the susceptibility of nonhost plants to rust fungi. Phytopathology 70,356360. HEATH, M. C. (1981). Resistance ofplants to rust infection. Phytopatholou 71,971-974. HOPPE, H. H., HCMME, B. & HEITEFUSS, R. (1980). Elicitor induced accumulation of phytoalexins in healthy and rust infected leaves of Phaseolus vulgaris. Phytopathologische
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G. K. & DEVERALL,