Characterization of the parasitic interface between Erysiphe pisi and Pisum sativum using fluorescent probes

Physiological

Plant Pathology

(1984)

25, 259-276

Characterization of the parasitic interface between Erysiphe pisi and Pisum sativum using fluorescent probes J. M. CHAmf Department London

SW7

and J. L. GAY:

of Pure and ZBB, U.K.

iAcceptedforpublication

Applied

July

Biology,

Imperial

College

of Science and

Technology,

Prince

Consort

Road,

1984)

The constituents of the interface between a strain of Erysiph pixi and two susceptible and live resistant lines ofPisum satiuum were investigated by fluorescence microscopy after treatment with specilic reagents. Fragments of epidermes were detached so that the haustoria and extrahaustorial membranes were exposed directly to the reagents. The specificities of the 23 reagents employed included a- and P-linkages in polysaccharides; constituent sugars; aldehyde, amino, sulphydryl and disulphide groups; proteins, lysine, arginine, lipid, calcium and anions; and anion transport sites. The main conclusions are that in the susceptible cultivars the extrahaustorial membrane includes polysaccharides (pl4 linked) with small amounts of a-glucose, a-mannose and galactose, P-linked N-acetylglucosamine (haustorial face only), protein, arginine, amino and sulphydryl groups and calcium. The extrahaustorial matrix includes varying quantities of p-linked polysaccharides. Haustorial walls contain p-linked polysaccharides including u-glucose, cr-mannose, galactose and N-acetyl glucosamine (P-linked). The haustorial cytoplasm contains protein and rcleasrs fluorescein from fluorescein diacetate and 3-o-methyl fluorescein phosphate. The main differences between the susceptible lines and the resistant lines is that the resistant lines show enhanced reactivity of terminal glucose, mannose and N-acetylglucosamine groups in the extrahaustorial membranes of some lines. The last is probably due to greater accessibility 01’ lectin effected by rupture of extrahaustorial membranes. Constituents of extrahaustorial membranes fluorescing with 4-acetamido-4’-iso-thiocyanato-stilbene-2,2’-disulphonic acid dweloped more slowly and included less cross-linked protein. Calcium was rare in the extrahaustorial membranes and host cytoplasm in resistant lines. The results are discussed in relation to the molecular and functional properties of the interface.

INTRODUCTION

The region of the host plasmalemma which is invaginated around each fungal haustorium (extrahaustorial membrane) has an important r61e in haustorial physiology. tPrcsent address: Department of Microbiology, School of Agriculture, University of Edinburgh, West Mains Road, Edinburgh EH9 3JG. $Correspondence should be addressed to Dr John Gay. Abbreviations used in text: ANS, 8-anilino-1-naphthalene sulphonic acid; Con A, concanavalin agglutinin; CTC, chlortetracycline; DACM, N-(7-dimethylamino-4-methylcoumarinyl)-maleimide~ EHM, extrahaustorial membrane; FDA, fluorescein diacetate; FITC, fluorescein isothiocyanate; FMA, fluorescein mercuric acetate; MFP, 3-o-methyl fluorescein phosphate; PQ, phenanthrenequinone; PWM, pokeweed mitogen; RCA, Ricinus communis agglutinin; SBA, soybean agglutinin; SITS, 4-acetamido4’-iso-thiocyanato-stilbene-2,2’-disulphonic acid; UEA, Ulex ewoputw agglutinin; WGA, wheat germ agglutinin. 0048-4059/84/060259+

18 $03.00/O

0

1984 Academic

Press Inc.

(London)

Limited

260

J. M. Chard and J. L. Gay

This is because the membrane is attached to the surface of the haustorial neck forming a seal so that solutes entering the haustorium must first pass through the invagination. This conclusion has been reached in studies of powdery mildew and rust fungi [14, 16, 201 and results suggest that it is probably true for oomycete infections also [8, 471. Furthermore, it has been established in many haustorial infections involving all the groups of fungi mentioned above, that the structural and physiological properties of the invagination are distinct from those of the rest of the plasmalemma of the infected cell [3, 6, 8, 13, 16, 21, 25, 43, 471. It is likely that the special properties influence the transport of solutes into haustoria and this has been argued most strongly in respect of its deficiency in ATPase [43, 471. It follows that the development of these characteristrcs is important in the establishment of infection and conversely, in some instances, that their absence may be expected to be associated with host resistance. Most of the special characteristics of EHMs have been observed by electron microscopy which is time consuming and does not lend itself to examinations of the large numbers of samples necessary in studies of the mechanisms of resistance. Therefore the present work was undertaken with reagents which bind to specific compounds and, because they fluoresce, can be detected in small quantities by light microscopy. A comprehensive investigation was made with one cultivar of pea susceptible to Erysiphe pisi and, additionally, selected studies on another susceptible and several resistant cultivars have been carried out. In addition to providing information on the invaginated region of the plasmalemma, results have been obtained for the haustorium and other interfacial components and these are also reported. MATERIALS

AND

METHODS

Plants and inoculum Pi.wm sativum L. infected with E. pisi DC ex Saint Amans was used for the staining

procedures described. The susceptible cultivars used were Onward and BSlO and the resistant cultivars were BS 12, JI 1047, JI 1048, JI 1049, and JI 1050. All except Onward were initially obtained from Mr B. Snoad of the John Innes Institute, U.K. Plants were grown in a Fisons 600 G3 THTL growth cabinet with a 16 h photoperiod (21 “C during the light period and 16 “C during the dark period). The strain of E. pisi, which was used in previous investigations in this laboratory [including 15, IS, 29-31, 42, 431, was initially obtained from I.C.I., Plant Protection Division as K748, and was maintained on cv. Onward. Experimental plants were inoculated 20-22 days after germination by shaking previously infected plants over them. Treatments with reagents

Pieces of epidermis obtained from mature leaves of P. sativum which had been inoculated 2-7 days previously, were used for all the procedures. Treatments were usually carried out on unfixed, fresh material; where fixation was employed, this is specifically indicated. The surface mycelium was first removed by gently rolling away the hyphal mat with a finger and thumb. Epidermal strips were obtained from the upper surface of the leaves using fine forceps in such a way that the epidermal cell walls and cytoplasm were ruptured. Rupture was verified by treatment with neutral

Erysiphe-Pisum

interface

characterization

261 red and examination for the lack of plasmolysis of epidermal cells using 0.6 M sucrose. Immediately after their removal from the leaves, the epidermal pieces were placed in a reagent solution for the requisite period of time (see Table 1). Reactions were carried out at room temperature, unless indicated. Excess reagent was usually removed by washing and the pieces were mounted on microscope slides under coverglasses. When material was fixed, ethanol : acetic acid, 3 : 1, was used for 20-30 min. It was subsequently washed in 95% ethanol and then stained. Fluorescent reagents and controls

The preparation and use of these reagents are listed in Tables 1 and 2. Controls, usually to block the sites of reaction of the reagents, were carried out as follows. (1) Dansyl hydrazine; two pretreatments were used [19] : (a) thiosemicarbazide (Fluka A.G.) (1 mg ml-l in 95:5 cellosolve:acetic acid for 30 min and (b) dimethylamine borane (Koch Light Labs Ltd) in 50% ethanol (1 mg ml-l) for 30 min followed by aqueous potassium borohydride (1 mg ml-r) for 1 h. (2) Fluorescent lectins; tissues were treated with solutions of specific haptens from the following list: a-methyl-1-u-mannoside; ct-methyl-1-u-glucoside; N-acetyln-glucosamine; N-acetyl-o-galactosamine; D (+) galactose (BDH Laboratories Ltd). Ulex europaeusagglutinin (UEA) was tested with fucose instead of galactose. The final sugar concentrations were 0.2 M. (3) N- (7-dimethylamino-4-methylcoumarinyl) -maleimide (DACM) : pretreatment of tissues to block sulphydryl bonds involved incubation with 0.15 M N-ethyl maleimide in TAS (see Table 1) at 37 “C for 4 min followed by a wash in TAS for 3 min. Treatments to break disulphide bonds involved the above procedure followed by 0.5 mM EDTA, 100 mM dithiothreitol in TAS for 4 min at 37 “C. Samples were washed in TAS for 3 min and then reacted with DACM [34]. (4) Phenanthrenequinone (PQ : blockage of arginine groups was achieved using 0.679; cyclohexanedione in 0.1 N NaOH 80% ethanol for 3 h 1281. All the chemicals used in these controls were obtained from Sigma London Chemical Co. Ltd unless otherwise indicated. Microscopy

Material was examined with Zetopan (Reichert) and Microstar (American Optical Co.) microscopes. The former incorporated transmitted illumination by both visible and UV light and the latter was equipped with an epi-illumination system for UV light. The epi-illumination system incorporated filter modules with dichroic mirrors. The transmission characteristics of the components are given in Table 3. Photographs were taken on Ilford FP4 35 mm film with either a Vickers automatic exposure system (Zetopan microscope) or a manual system (Microstar microscope). RESULTS

The typical morphology of haustorial complexes in a fragment of epidermis is shown in Fig. 1(a). There was never any sign of plasmolysis when sample pieces of infected

FITC-Wheat germ agglutinin (LKB) * FITC~Pokeweed mitogen FITC-R&us communis agglutinin (M) * FITC-Soybean agglutinin (LKB) * FITC-Ulex europaeus agglutinin (LKB) * Fluorescein isothiocyanate Rhodamine B Fluorescamine (Sj * Dansyl chloride (S)

(Sj

(S) *

PBS PBS PBS PBS O,l~PB,pH7or8 O.l~PB,pH7or8 Water 95”;, Ethanol

lOOpgml-’ 100 pg ml-’

200 pg ml-’

lOOpgml-’

1 mg ml-’ 1 mg ml-’ 50 pg ml-’ O.lO”C

100 pg ml-’

0.01 M Tris-HCl, 0.15 M NaCI, 1 mM CaCl,, 1 mM MnCl,, pH 7.2 0.1 M PB, pH 7.2 or PBS

30 30 VI 240

45

45

45 45

45

45

200 pg ml-’

FITCXZoncanavalin

*

15&30 3545 60 30

0.1 M PB, pH 8 Water Water Cellosolve’

1% 0~01-0~05’~” Q. 1 %I 20/b’

A (LKB)

VP

SolventC 0.1 M PB, pH 8

Stain concentration

0,05q/,

b

Staining time (min)

Aniline blue-water soluble (C) Calcofluor white-MPR (PI) Tinopal BOPT (CG) Tinopal, Uvitex, AN (* *) Dansyl hydrazine (S)

Reagent (Supplier)

1

and fmcedures

TABLE Reagents”

95”,, rthanol; 100” 0 ethanol

PB PB

PBS

PBS

PBS PBS

PB or PBS

PB Water Water Reduction 01 hydrazine [19] Solvent

Solution

Wash

5 5

5

5

5 5

5

5

5 5 ~~

Time (min)

PBjwater PB/water Stain

PBS

PBS

PBS PBS

Water

Solvent

Water Water Water Water

Stain

Mountant

2073

2072 & 3 2074 2072 & 3

2072 & 3

2072 & 3

2072 & 3 2072 & 3

2072 & 3

2072 & 3

2072 2072 E4, Spl 2072

2072

Filters

39

37

II

II

II II

II

II

45 36 35 19

45

Reference

(S)

PM

1oopM 50 I(M 0.1 mg ml-’

100-500

0.8504 Acetate

0.01 rnMg

0.04 M PB, pH

7

0.4 M glucose, 0.1 0.1 M PB, pH 7 Water

0.1 M PB, pH7 M

PB, pH

7

0.1 M PB, pH 7 1 :4 : 1, 0.5 N NaOH : ethanol : stain solution (1 o/0 stock in DMF)’

NaC1, 5 rnM Trisbuffer, pH 6.8 (TAS)

0.6 M Sucrose,

500 p’M

30 7 6

1 O-30

VI 10

3 14 “Cl

PB Water

PB

3 x 959/o ethanol, rehydration

TAS

5 1

l-2

1

Stain PB Water

Stain 9 :2, Glycerol : 1 M NaOH Water

TAS

Sol\wlt

2073 2072 & 3 2072 & 3

2073

2072 & 3 2073

2073

2073

asolutions were prepared immediately before use except those asterisked, which were kept as stock solutions. See Table 2. “Suppliers of reagents: **, gift from Dr A. Paton, Aberdeen University; BDH, BDH Chemicals Ltd; CG, Ciba Geigy; G, Gurr; LKB, LKB Ltd; M, Miles Laboratories Ltd; 3M Co, Minnesota Mining & Manufacturing Carp; PI, Polysciences Incorporated; S, Sigma London Chemical Co Ltd. “PB, Phosphate buffer; water, distilled water; PBS, phosphate buffered saline, pH 7.1 [34. dVI, viewed immediately. ‘Fixed tissue used. ‘Supplied by Gurr. 83 min prewash (TAS). hSupplied by 3M Co. ‘DMF, Dimethyl formamide.

8-Anilino-1-Naphthalene sulphonic acid (S) Chlortetracycline (S) Fluoroscein diacetate (S) * 3-o-Methyl fluorescein phosphate (S)

4-Acetamido-4’-iso-thiocyanatostilbene-2,2’disulphonic acid (BDH) N-(7-Dimethylamino-4methylcoumarinyl)maleimide (S) * Fluorescein mercuric acetate Phenanthrenequinone (S)

23

15

32,40

41

9 28

34

26

J. M. Chard and J. L. Gay

264 TABLE Reagents prepared

2 as stock solutions

Concentration

Reagent Fluorescent lectins Fluorescamine DACM FDA

Solvent

1 mg ml-’ 5 mg ml-’ 5mM 5rnM

TABLE Characteristics

of.filters

Buffer (see Table dimethylsulphoxide Acetone Acetone

Transmitted Epi-illumination

Filter/module

no.

E4 SPl 2072 2073 2074

1)

~- 20 “C 4°C 4°C 4°C

3

usedfor&mscence

microscopy

Transmitted

Illumination

storage

Exciter flter (max.)

wavelengths

(nm)

Dichroic mirror (50°;1)

Barrier filter (50%)

>5OO >45O 2560

>470 >525 >475 >590

360 438 436 546

leaf epidermis were examined in 0.6 M sucrose, indicating that the cell walls and protoplasts had been disrupted during the stripping process. Thus the method partially isolated haustorial complexes and provided direct access of the stains to the cytoplasmic surface of each EHM. In many instances the reagents diffused into the complexes and reached the haustoria also. Reactions with reagents specific for carbohydrates

Five fluorescent dyes and six fluorescein-labelled lectins were reacted with infected epidermes of cv. Onward and the results of these tests are given in Table 4. FIG. 1. (a) Epidermal strip of Pisum sativum infected with Erysiphe pisi showing a stoma(s) and several haustoria; these show the haustorial neck (n) and also the extrahaustorial membrane (m). The granular appearance is due to lobes arising from, and surrounding, the haustorial body (b). (b) Treatment with Tinopal BOPT causes extrahaustorial membranes to fluoresce clearly and the bright patches may correspond to lobe attachments to them. (c) Treatment with Calcofluor causes bright fluorescence of haustorial walls and extrahaustorial membranes. .4 gradation in fluorescence of the extrahaustorial matrices (ma) from left to right is apparent. (d) -(o Epidermal strips treated with FITC-labelled Con A: (d) Haustoria with distended rxtrahaustorial membranes showing dull fluorescence of the membranes and in some cases faint fluorescence of the haustorial bodies; (e) Three haustoria (arrows) in an epidermal strip viewed with phase contrast optics; (r) Fluorescence micrograph of the same field as Fig. 5 showing fluorescence of the walls of one haustorium and the absence of fluorescence in the other two which had intact extrahaustorial membranes. (g) and (h) epidermal strips treated with FITC-labelled WGA: (g) Fluorescence of haustorial walls and part of the extrahaustorial membrane of one haustorium; two others (crosses) which had extended (intact) extrahaustorial membranes are not visible; (h) Fluorescence of the haustorial walls and extrahaustorial membrane of an haustorium. The membrane is clearly disrupted (arrow). (a)-(h) x 670.

266

J. M. Chard TABLE

and J. L. Gay

4

Reactions with reagents specifiG for carbohydrates Fluorescence Haustorial Reagent

Specificity”

Aniline blue Calcofluor white-MPR Tinopal BOPT Uvitex AN Dansyl hydra&e Fluorescent lectins Concanavalin A Wheat germ agglutinin Pokeweed mitogen Ricinus communis agglutinin Soybean agglutinin tilex eur0paeu.r agglutinin

Authority

B- 1 ,3-Glucans B-Linked polysaccharides B-Linked polysaccharides Anions Aldehydes

EHMb

Matrix -

Walls

12 27 2

+ +

It -

17 19

+ -.

-

+ -.

It +

Man, blc GlcNac GlcNac-GlcNac? Gal

Supplier Supplier Supplier Supplier

+ps +ES +ES * PS

i --.

+ + + It

GalNac Fuc

Supplier Supplier

~ -

--

-.

~Fuc, fucose; Gal, galactose; GalNac, glucosamine ; Man, mannose. bES, haustorial surface of extrahaustorial membrane.

N-acetylgalactosamine; membrane;

Glc,

PS, cytoplasmic

glucose; surface

Cytoplasm

GlcNac,

N-acetyl

of extrahaustorial

Staining with aqueous aniline blue did not initially result in any fluorescence of haustorial complexes except for the haustorial collars. However, after incubation for approximately 30 min, faint outlines of haustorial walls were detected. Extrahaustorial membranes fluoresced brightly with Calcofluor, Tinopal AN and Tinopal BOPT [Fig. 1 (b) and (c)l. Calcofluor and Tinopal AN also stained the haustorial walls and the extrahaustorial matrix fluoresced in some tests with Calcofluor [Fig. 1 (c)l. Fluorescein isothiocyanate (FITC) conjugates of the lectins concanavalin agglutinin (Con A), wheat germ agglutinin (WGA), pokeweed mitogen (PWM) and Ricinus communis agglutinin (RCA) stained some of the EHMs and also the walls, or parts of them, in some haustoria [Fig. 1 (d)-(h)]. Where fluorescence of the membrane occurred, it was always weaker than that of the haustorial walls and reactions with FITC-RCA were generally weaker than with FITC-Con A, FITC-WGA and FITC-PWM. In these tests the epidermal pieces were mounted in O-1 M buffer or distilled water and this caused swelling of extrahaustorial matrices and distension of most of the extrahaustorial membranes. Matrix swelling indicated that the extrahaustorial membrane was intact [16]. It was observed that swelling did not occur around haustoria whose walls were stained and then a break in the membrane or its remains could often be detected by bright field microscopy. Thus staining of components was partially determined by their access to the test reagents.

Erysiphe-Pisum

interface

characterization

267

Haustorial walls fluoresced brightly with FITC-Con A and FITC-RCA, and this obscured the weak fluorescence of the EHM, which could only be detected where the matrices were swollen and the intact extrahaustorial membranes precluded access to the walls [Fig. l(d) and (r)]. Pretreatment of FITC-Con A with the individual haptens, mannopyranose and glucopyranose led to a reduction in fluorescence, but other sugars had no effect. With FITC-RCA, a reduction in fluorescence only occurred with galactose. With FITC-WGA, an average of 28% of the haustorial complexes showed fluorescence of all or part of the haustorial walls. Of these, a small proportion (Sq/, of the total in samples taken 4 and 7 days after inoculation) had fluorescent EHMs. In most cases only parts of the membrane fluoresced and they corresponded to the intact regions ofbroken membranes [Fig. 1(g) and (h)]. The majority of the remaining haustoria had swollen matrices and neither their walls nor EHMs fluoresced with FITC-WGA. Similar reactions were seen in epidermal strips treated with FITCPWM, except that the outlines of haustorial walls were less distinct than with FITCWGA. This may have been due to fluorescence in the extrahaustorial matrix. Pretreatment of FITC-WGA and FITC-PWM with monosaccharides did not affect the fluorescence. Fluorescence of haustorial complexes was not detected with dansyl hydrazine, FITCsoybean agglutinin (SBA) nor FITC-UEA. However, the epidermal cell walls and especially the thickened walls of guard cells did fluoresce in preparations treated with dansyl hydrazine. The intensity of fluorescence was reduced in controls pretreated with thiosemicarbazide and eliminated by prior reduction with dimethylamine borane and aqueous potassium borohydride. Staining with FITC-SBA gave some greenish fluorescence of the host cytoplasm. With FITC-UEA, dull outlines of haustoria, similar to the auto-fluorescence in controls incubated in buffer alone, were observed. Reactions with protein-spec$ic

reagents

The results obtained with the nine fluorescent compounds used to detect proteins are shown in Table 5. Bright fluorescence of EHMs and haustorial and host cytoplasms was observed after treatment with FITC [Fig. 2(a)] and rhodamine B. A similar reaction was observed in fixed material treated with dansyl chloride. However, tests using fluorescamine did not result in fluorescence of any of the structures. Bright fluorescence of the EHM only was seen after staining with 4-acetamido-4’-iso-thiocyanato-stilbene2,2’-disulphonic acid (SITS) [Fig. 2 (b)]. Similar reactions occurred after treatments with sulphydryl-specific reagents. When epidermal strips were placed in DACM, an intense turquoise fluorescence of EHMs and especially of the haustorial cytoplasm, occurred in only 3 min [Fig. 2(c)]. This contrasts with the fluorescence of the haustorial cytoplasm which took 2&30 min to develop after treatment with fluorescein mercuric acid (FMA) [Fig. 2(e)], only the extrahaustorial membrane and remnants of host cytoplasm fluorescing immediately. This suggests slow penetration by FMA. Pretreatment of the strips to block sulphydryl groups greatly reduced the fluorescence with DACM. Extrahaustorial membranes fluoresced only after pretreatments

268

J. M. Chard and J. L. Gay TABLE

Reactions with reagents specijc

5

for proteins and constituent groups Fluorescence Haustorial

Reagent

Specificity

Fluorescein isothiocyanate Rhodamine B Fluorescamine Dansyl chloride SITS Fluorescein acetate DACM

mercuric

Phcnantbrenequinone ANS

Authority

EHM

Protein Protein NH,, Lysine NH,, Lysine NH,, Anion transport sites S-H

33 33 18 39 26

+

+ --

I

9

-

+d

SSH s:s Arginine Protein,

34 34 28 41

lipid aFluorescence

delayed

20-30

Matrix

\Valls

Cytoplasm

-t

+

4

i

t i r min.

with N-ethylmaleimide followed by EDTA and dithiothreitol to break disulphide bonds [Fig. 2(d)]. Th e fl uorescence of haustoria was less bright in this treatment than with DACM alone. It is uncertain whether the fluorescence of the EHM differed with the two treatments, because it may have been masked by the intense fluorescence of the haustorial cytoplasm which occurred when DACM was applied alone. The fluorescent arginine-specific dye PQ bound to EHMs and the haustorial cytoplasm in fixed material. However, this reaction was not diminished by pretreatment with cyclohexanedione to block arginine groups; indeed enhanced fluorescence occurred. The membrane probe 8-aniline-l -naphthalene sulphonic acid (ANS), which binds to proteins and lipids, also bound to the extrahaustorial membrane and accumulated in the haustorial cytoplasm. Reactions with other reagents Haustoria of E. pisi in epidermal

specific compound

chlortetracycline

strips of cv. Onward stained with the calcium(CTC). The EHMs fluoresced brightly as did

FIG. 2. !a) Treatment with FITC (pH 7) causes extrahaustorial membranes and cytoplasm of haustoria to fluoresce clearly. (bj Fluorescence of an extrahaustorial membrane after treatment with SITS. (CJ Treatment with DACM causes haustorial cytoplasm and extrahaustorial membranes to fluoresce brightly. (d) H austoria were pretreated wth reagents which block free sulphydryl bonds and break disulphide bonds and then reacted with DACM. Only the extrahaustorial membranes fluoresce. (ej An haustorium reacted with FMA showing fluorescence of the extrahaustorial membrane. Note also fluorescence of the remnants of host cytoplasm and a stoma. (0 Treatment with CTC causes patchy fluorescence of extrahaustorial membranes [arrows; and in one case of haustorial cytoplasm. Fluorescence of some host cytoplasm is also shown. iaim it) x 670.

Erysiphe-Pisum

interface

characterization

269

270

J. M. Chard

and J. L. Gay

the remnants of host cytoplasm in ruptured infected, and adjacent uninfected, cells [Fig. 2(f)]. I n t na ’ 1s with infected epidermes stripped from stems most of the cells remained intact and these showed fluorescent cells in patches restricted to infected areas. In epidermal strips from plants inoculated 4 and 6 days previously, and stained with fluorescein diacetate (FDA), the haustoria from the older infections showed a range of responses. Most fluoresced an intense green colour throughout the haustorial body and lobes, but the fluorescence faded rapidly and became diffuse in the surrounding zone. Other haustoria fluoresced less brightly or were negative. In contrast, few haustoria from the 4 day infected plants fluoresced. Similar results were obtained with another susceptible cultivar BSlO. In young infections (2-3 days) of this variety some haustoria fluoresced brightly, but most were dull. In a sample of 66 haustoria from 2 day infected leaves the ratio of bright : medium : dull : negative haustoria was approximately 1 :4 :4 : 1. However, by 4-7 days the majority of haustoria fluoresced brightly. In young infections and uninfected plants of both BSlO and Onward, bright fluorescence of epidermal guard cells was observed, whereas few guard cells fluoresced 4 days after inoculation. Treating epidermal strips from cv. Onward with 3-o-methyl fluorescein phosphate (MFP) resulted in bright green fluorescence of haustorial bodies similar to that observed with FDA. Comparisons of reactions in resistant and susceptible cultivars

Some of the reagents used in this investigation were also tested on resistant cvs (BS12, and JI1047 to JI1050) in which few haustoria develop and little subsequent growth occurs [4.5]. The reactions were compared directly with those in the susceptible cv. BSlO and sometimes with those in cv. Onward also. Calcofluor and Tinopal BOPT were used 3 and 7 days after inoculation and on each occasion both produced similar fluorescence of EHMs in both resistant and susceptible cultivars. Calcofluor also stained haustorial walls as described above. Most of the FITC labelled lectins gave reactions which were in general similar to those described for cv. Onward. Bright fluorescence of haustorial walls was observed both with FITC-Con A and FITC-WGA. With FITC-Con A, the extrahaustorial membranes were clearly visible around swollen matrices, but greater proportions of them fluoresced in some resistant cultivars (JI1047, 9.1 y0 ; JI1048, 10.3% ; JI1050, 13.6%) than in susceptible ones (BSl 0, 5.7% ; Onward, 1.5%). Some EHMs in resistant cultivars fluoresced with FITC-WGA as described above but fluorescence never occurred on distended membranes and was associated only with haustoria whose walls were fluorescing. Also, a larger proportion of extrahaustorial membranes fluoresced in the three resistant cultivars (JI 1047, 8.9% ; JI 1049, 7.496 ; JI1050, 9.1%) than in the two susceptible cvs (BSlO, 3.8% ; Onward, 5.8%). With FITC-RCA, the fluorescence of EHMs in resistant material was duller than in the susceptible cultivars. Fluorescence with FITCSBA and FITC-UEA on resistant material was even duller. Reactions with DACM were obtained with three resistant cultivars (JI1047, JI1048, JI1049). The fluorescence was as bright as that recorded for cv. Onward except after pretreatments to break disulphide bonds, when it was duller.

Erysiphe-Pisum

interface

characterization

271 TABLE

Fluorescent

haustoria

(%) following

SITS

6

treatment

of samples

Days Cultivar Total fluorescent BSlO BS12 JI1047 JI1048 JI1049 JI1050 Haustoria BSlO BS12 JI 1047 JI 1048 JI 1049 JI 1050

with

“Significantly

taken 2-7 days after inoculation

after inoculation

2

3

4

7

86.4 70.0 24.4a 62.7 35.4 58.3

87.4 70.4 73.0 87.7 48.9 64.4

85.1 60.7 84.5 89.7 84.1 82.2

93.6 81.2 82.4 94.5 90.1 83.3

49.5 13.3 0 * 31.9 7.2 50

59.8 42.5 30.3 46.0 26.5 33.2

59.6 34.0 61.2 48.2 42.8 44.3

78.6 47.9 54.0 70.3 69 55.9

haustoria

bright

fluorescence

different

from

BSlO at 95”)” confidence

limits.

Infected epidermal strips from all the resistant plants were tested with SITS. Samples were taken 2-7 days after inoculation (Table 6). At initial stages of infection 86:/i ofthe haustoria in BSlO had fluorescent EHMs. In all the resistant hosts a smaller proportion of the haustoria fluoresced and in cv. JI1047 very few were fluorescent (significantly different from BSlO at 95yo confidence limits). At later stages (4 days), fluorescing haustoria had reached similar percentages in all cultivars except BS12. This trend was even more marked when the proportion of haustoria exhibiting bright fluorescence was scored (Table 6). Few haustoria in resistant hosts fluoresced brightly after 2 days except in cvs JI1048 and JI1050, and throughout the experiment the haustoria in resistant plants had weaker fluorescence than those in the susceptible cv. BSlO. Bright fluorescence of haustoria in BSlO or in the resistant cultivars was seldom detected with CTC. This is in contrast to the intense fluorescence shown by EHMs and cytoplasmic remnants in cv. Onward. When epidermal strips (3 day) were treated with FDA the response of haustoria was similar to that in young infections of Onward. Duller reactions were recorded with 4 day infections but by 7 days haustoria in cvs BS12 and JI1047 had similar responses to those in cv. BSlO, with many fluorescing at medium intensities, whereas in cvs JI1048, JI1049 and JI1050 they showed a range of fluorescence from bright to none. DISCUSSION

The use of fluorescent probes in this study has added to our knowledge of the composition of the haustorial interface of E. pis;. Each component of the interface will be discussed in turn.

272

J. M. Chard and J. L. Gay

Extrahaustorial membrane Structuralproperties. Polysaccharides have already been demonstrated in this membrane by electron microscopy after reaction with silver methenamine and by light microscopy using Calcofluor fluorescence [6, 151. The presence of P-linked polysaccharides has now been confirmed in this investigation using Calcofluor. It is improbable that /3-1,3-linkages are involved because of the absence of fluorescence with aniline blue. The fabric brightener Tinopal BOPT, a stilbene derivative which also stained the membrane, binds to cellulosic material (Dr I. Price, personal communication) and indicates the presence of polysaccharides with B-1,4-linkages [2]. However, fibrillar material has not been recorded in the membrane and therefore other linkages may be involved. Detailed biochemical analysis is needed to identify these components. Small quantities of a-linked sugars were also demonstrated in the membrane by the fluorescence with FITC-labelled Con A which is specific for a-glucose and ~1mannose. The dull fluorescence indicated a paucity of exposed glucose and mannose groups and this also was seen for galactose by reactions with FITC-RCA. Fluorescence of the membrane with FITC-WGA and FITC-PWM was detected only in haustoria which had been damaged by the preparation method. It was therefore concluded that terminal N-acetyl glucosamine groups (P-linked) occurred only on the haustorial surface of the EHM (ES) [4]. I n as much as this sugar is characteristic of chitin, which is a component of ascomyceteous walls, the result suggests that the adjacent haustorium contributes to the EHM. However, this suggestion should be viewed with caution because N-acetylglucosamine is not uncommon in plant glycoproteins. Controls with monosaccharides did not reduce the fluorescence of FITC-WGA or FITCPWM, and thus oligosaccharides may have been necessary to block the reactions. Further work using lectins labelled for electron microscopy may locate the sugars more exactly. A recent electron microscopic study of Erysiphe graminis f. sp. hordei [IO] using lectins, showed similarities and differences in the staining patterns recorded here for E. pisi. In both species, labelling of the EHM occurred with FITCCon A but not with FITC-SBA or any lectins specific for fucose. However, FITC-WGA bound to the EHM only in E. pisi infections. The thickness and osmotic stability of the extrahaustorial membranes differ in E. graminis and E. pisi infections [3, 5, 6, 16, 441 and these properties may be related to the differences in composition indicated here. Proteins were present in the EHM of E. pisi. Fluorescence of the membrane with dansyl chloride and SITS indicates the presence of amino groups but the absence of fluorescence with fluorescamine, which also binds amino groups, was unexpected because it has been used successfully on both fixed and unfixed plant and animal tissues [18, 371. Sulphydryl and disulphide bonds were also demonstrated by DACM and FMA, the proteins indicated by the latter probably being cross-linked in the membrane. Calcium was also present and it is likely to be involved in membrane stabilization. Thus, together with the polysaccharide component, proteins and divalent cations probably contribute to the strength of the membrane referred to above. Arginine is also indicated to be present in the membrane proteins, as demonstrated by fluorescence with PQ A freeze fracture study of the extrahaustorial membrane of E. ptii showed that it lacked the intramembrane particles typical of normal plasma membranes [6, 151.

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Thus, the proteins detected in the present work are unlikeIy to have a closely folded (globular) structure. Properties which may relate to ion and solute transport. The membrane has properties which cause the binding of cations some of which may pass through it. Membrane fluorescence with Tinopal (Uvitex) AN, a cationic bis-benzoxazoyl methene derivative, is consistent with results of experiments in which uranyl ions bound to the cytoplasmic surface of the membrane [14] and uptake of Tinopal AN, as shown by haustorial fluorescence, is paralleled by uptake of the cations 2-hydroxyethylammonium and 1-methyl-pyridinium which accumulated in haustorial complexes against large concentration gradients [30]. Comparisons with studies on erythrocytes suggest that the membrane may have a more specific role in anion transport. This is indicated by the presence of arginyl residues, which have been implicated in anion recognition [38]. Thus, despite the implications of the transport hypothesis based on ATPase deficiency [43, 471, the membrane structure may contribute positively in transport processes. The fluorescence with SITS, which has been used to inactivate anion transport proteins [24] may also support this conclusion. However, this binding may not be specific because the cytoplasmic surface of the membrane was exposed to the reagent [7]. Extrahaustorial

Matrix

Staining of extrahaustorial matrices was variable and sometimes difficult to detect, probably because it was masked by the bright fluorescence of adjacent structures. The intensity of fluorescence with Calcofluor varied widely and this probably reflects different amounts of polysaccharides associated with young and old haustoria. Manners & Gay [31] showed that 64% of the 14C incorporated into isolated haustorial complexes was in trifluoracetic acid-soluble carbohydrate, some of which was probably in the matrix, and this conclusion has been substantiated by electron microscope autoradiography [42]. Diffuse staining around the haustorial walls was seen in ruptured haustoria treated with pokeweed mitogen (PWM) thus indicating the possibility of N-acetyl glucosamine in matrix polymers, but this was not seen with FITC-WGA. Haustorial

Walls

Haustorial walls fluoresced with Calcofluor indicating the presence of P-linked polysaccharides and the lectin-binding indicated terminal a-linked mannose and glucose, P-linked N-acetylglucosamine and a- and/or P-galactose residues. In contrast to these results with E. pisi, a-linked mannose and glucose were not detected in haustorial walls of E. graminis [IO]. Haustorial walls may be expected to have a composition similar to that of hyphal walls, i.e. mainly a- and P-linked glucans, chitin and some protein. The results of the present study support the reported carbohydrate composition, but it is not possible to be certain about protein components, since like the matrix they may have been masked by the strong fluorescence of other components. Haustorial

Cytoplasm

The bright fluorescence of haustorial cytoplasm with the protein-specific

dyes FITC,

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and J. L. Gay

rhodamine, dansyl chloride, DACM, FMA and ANS, is consistent with an active cytoplasm concerned with growth and rapid transport of assimilates. Staining with FDA and MFP also demonstrated the active nature of the haustorial cytoplasm. Fluorescence occurs only after their uptake and enzymic hydrolysis to liberate fluorescent ions. Haustoria obtained from plants 4 and 7 days after inoculation fluoresced more brightly in FDA than at earlier stages. I. Donaldson (personal communication) also found fluorescence in haustorial complexes isolated after 7 days. These results for haustorial complexes in the absence of a host cytoplasm contradict earlier observations [S, 151 that intact host cells are necessary for haustorial fluorescence. These variations in response warrant further investigation. It is noteworthy that fluorescence in the guard cells diminished after 4 days and this coincides with the impairment of stomata1 operation [I]. Reactions in resistant cultivars

The main results with haustorial complexes obtained from susceptible cultivars were also found in resistant hosts. However, differences were seen in reactions with two of the lectins (FITC-Con A and FITC-WGA), with SITS and with CTC. Greater proportions of EHMs fluoresced with FITC-Con A in three resistant cultivars than in the susceptible ones, indicating more terminal glucose and mannose groups in these membranes. Similarly, with FITC-WGA, a greater proportion of EHMs fluoresced in three of the resistant cultivan (two the same as with Con ‘2). In all three, the increase in fluorescent membranes corresponded to an increase in the numbers with breaks in them. This suggests that the EHMs in resistant hosts are less stable than in susceptible hosts. The decreased DACM fluorescence after pretreatments to break disulphide bonds in resistant cultivars is also consistent with this conclusion, fewer cross-linked proteins being indicated. In treatments using SITS, the proportion of haustoria with fluorescent EHMs was lower in resistant than in susceptible hosts. This confirms the results of Szot [45], but the present results show that the difference is limited to 2 days after inoculation. Subsequently, no differences were found. This suggests that specific molecules characteristic of EHMs are incorporated more slowly in resistant than susceptible plants. Tests for calcium also showed differences between susceptible and resistant reactions. The EHM and host cytoplasm of resistant hosts seldom reacted with CTC. Calcium has been shown to enhance the incidence of haustoria of E. graminis in barley [22, 461 and it seems likely that the two observations are connected and that Ca2+ is important at the interface in susceptible reactions. However, it must also be noted that the susceptible cv. BSIO did not react in the same way as cv. Onward. In conclusion, this investigation has significantly extended knowledge of the composition of the components of a host-haustorium interface. Good spatial resolution has been attained and differences between susceptible and resistant hosts have been demonstrated. Ultimately improved chemical resolution should result from subcellular fractionation and analysis. However, this will be applicable only to interfaces in susceptible hosts and for the small amounts of material available in the interfaces of resistant combinations it seems likely that the approach used in the present study will also be valuable.

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