Callose formation in Zea mays as a response to infection with Phytophthora cinnamomi

PhyGological

Plant Pathology

(1982)

21, 113-124

Callose formation in Zea mays as a response to infection with Phytophthora cinnamomi J.

M.

HINCH

Plant Cell Biology Parkville, Victoria

(Awptedfw

and A. E. Gentle, School 3052, Australia

CLARKE

of Botany,

University

of Melbourne,

publication May 1982)

Invasion of root tissue by the fungal pathogen Phytofihthora cinnamomi results in the production of papillae in the non-susceptible .ZJea mays but not in the susceptible Lupinus angurtifous. The papillae stain with aniline blue and hence contain callose. Cytochemical tests show the papillae contain carbohydrate but not protein, and are not lignified. Treatment of papillae with linkage specific /Sghlcan hydrolases indicates that the aniline blue-staining component contains glucan(s) with both I,3 and 1,4+glucosidic linkages, at least some of which are in the same linear chain.

INTRODUCTION

Callose, that is material identified by its fluorescent staining with decolourized aniline blue, is produced by higher plants in response to many physical and physiological stimuli [S]. Neither its nature nor its role in infection is precisely defined, but func:tions of blocking, or “walling-off” and isolation of an infected, or wounded area. of a plant have been suggested [I]. In many fungal infections of plants, the outcome, that is whether the plant ultimately survives or succumbs, depends to some extent on the speed with which the :plant can respond to initial contact with the invading pathogen [7j. One of the observable differences between successful and unsuccessful infections is the rapid induction of a hypersensitive response, during the latter types of infection [12, 41. This: response includes localized cell death and browning and may involve phytoalerin production at the site of infection. Another response is deformation or modification of host cells in contact with, or in the vicinity of, the invading fungal hyphae. This response is usually, but not exclusively observed in plants which are resistant to the particular pathogen [I, I,?]. The wall deformations are variously referred to as papillae, pads, lignitubers and callolsities; as they often stain with aniline blue they are said to contain “callose”. In this paper we report a comparison of callose production in roots of a resistant and a susceptible plant, in response to invasion by the fungal pathogen Phytophthora cinnamomi. This pathogen has a broad host and geographic range, infecting many horticultural, agricultural and Australian native plants [38].
16 803,00/O

@

1982

Academic

Press Inc.

(London)

Limited

114

J. M. Hinch and A. E. Clarke

staining. We refer to the wall modifications generally as papillae [I.!?] ; where these wall modifications stain with aniline blue, they are referred to as callose deposits. MATERIALS AND METHODS Seeds of <. mays (maize) and L. angustifolius (New Zealand blue lupin) were surface sterilized and germinated as previously described [13]. Roots were excised from 4-day-old seedlings and placed in zoospore suspensions ( IO4 spores ml -l in water) at 20 “C. Zoospores were prepared axenically [17] from a culture of P. cinnamomi isolated from the Brisbane Ranges, Victoria [14]. Fixation jwoceduresand embedding Cut roots were fixed [20] for 24 h, dehydrated and embedded in JB4 resin (Polysciences), and sectioned (1 to 4 urn) using a rocking microtome (Cambridge Instruments) with glass knives. Alternatively, sections of unfixed material were cut with a razor blade by hand. Callose tests Callose deposits were detected in either fresh hand sections or sections of embedded material by staining with resorcinol blue for light microscopy or with decolourized aniline blue or Calcofluor, for fluorescent microscopy. Resorcinol blue (1.5%) was prepared by adding 3 ml of 15 N ammonia to a solution of 3 g resorcinol (Sigma, St. Louis, MO.) in water (200 ml) [9]. Sections were stained for 5 min, washed with water, then with 100 mM acetate buffer, pH 5.1, for 2 min, and mounted in water for examination. Decolourized aniline blue (Merck, Darmstadt, West Germany) was prepared as a 0.1% solution in O-1 M KsPO,. Sections were mounted in decolourized aniline blue and examined directly by fluorescence microscopy. Calcofluor M2R New (American Cynamid Co., Bound, Brooklyn, NJ., U.S.A.) was prepared as a 0.01% solution in 0.15 M NaCl, 0.05 M phosphate buffer, pH 7.2 (phosphate buffered saline, PBS). Sections were mounted in Calcofluor and examined by fluorescence microscopy. Fluorescence microscopy was carried out using a mercury arc source (HBO 100) and Carl Zeiss (Oberkochen, West Germany) incident illuminator equipped with type 1 (BG 12) exciter filters and barrier filters adjusted to exclude light below 500 run. Histochmical tests Sections were stained with crystal violet (1 y0 in water) ; periodic acid-Schiff’s (PAS) stain (counter-stained with fast green); toluidine blue 0 (0.2% in acetate buffer pH 5.5) ; phloroglucinol-HCl ; aniline chloride; basic fuchsin; iodine-potassium iodide-H&SO,; Sudan IV; Coomassie blue; Congo red; [26, 181 and viewed in a Zeiss SMP-05 Scanning microscope photometer. Enzymic digestion of callose Sections of infected roots (43 h after inoculation) were treated with a number of p-glucan hydrolases with defined linkage specificities; the extent of callose hydrolysis was followed by the presence or absence of fluorescent staining with aniline blue. During the incubation, sections were rinsed with distilled water to remove end

Callose formation in Zea mays

115

products and a fresh portion of enzyme solution was applied; in this way 4changes of enzyme solution were made during the 40 h digestion. Sections (2 pm) were allowed to dry on slides, then 10 ~.tlof enzyme mixture was added to the section. Enzyme mixtures consisted of 50 pl of enzyme of specific activity given in Table 1, and 50 ~1 of 0.05 M acetate buffer, pH 5.5; the total protein in the mixture varied from 0.2 to 2 mg ml-l. Exceptions were the Rhizo@s enzyme, which was used at 1 mg ml-l in 0.05 M acetate buffer, pH 5.5, and Driselase, which was used at 4% in the same buffer. Slides were incubated at 37 “C. Each test was prepared in triplicate. The specificity of the batches of glycosidases used, which hydrolyse 1,3 and 1,4+glucosidic linkages, were as shown in Table 1. The specificity of each enzyme was checked against the polymeric 1,3 and 1,4+glucan substrates as well as the 1,3 : 1,4-&linked barley glucan. TABLE Source and spec$ci~

Enzyme

of f&g&an

hydmlases

hydrolase

Bndo-1,3+glucan hydlrolase (EC: 3.2.1.6)

used in

analysis of callosepreparation

Linkage specificity

Source

Exo-1,3-$-glucan (EC 3.2.1.58)

1

specific

activity

I

Euglena gradis

G3G J-

Bhizopus awhitus

0.004

15

-G3G3G

Reference

3

27

4 or I

-G3G4G-

4

1,3; 1,4+glucan (EC: 3.2.1.73)

hydrolase

Bacillus subtilis

-CXG3G4G

2.8

2

0.1

25

0.4

23

J

End.o-1,4+glucan hydrolase (ECi 3.2.1.4)

Streptomyces sp.

Endo-1,3-&glucan hydrolase (EC 3.2.1.39)

Nicotiana gluti?wsa

Driselase

Kyowa Kogyo Tokyo The &-glucan B. A. Stone, urn01 glucose

I

-iG4G

I 4

-G3G3-

I 4

Hakko Co.,

Mixture of cell wall degrading enzymea

hydrolases with defined linkage La Trobe University, Bundoora, s-r mg-r protein.

specificities Victoria.

were Specific

a gift from profascw activity expressed as

116

J. M. Hinch and A. E. Clarke

Solubility hm&ristics of callose Sections of infected roots were incubated separately with 1 M KOH, 6 M HCl and dimethyl sulphoxide (DMSO) in a similar manner to the enzymic digestions. Sections were placed in a moist chamber and three portions of each reagent were added during the 40 h incubation. Preparation of fungal culturejltrate Hyphal cultures were prepared by point inoculation into V-8 broth [17] and shaken at 120 r min-1 on an orbital shaker at 20 “C in the dark. After 1 week, the cultures were filtered, the filtrate dialysed exhaustively against distilled water in Visking tubing (mol. wt cut-off 6000 to 8000) and concentrated on a rotary film evaporator. The concentrated filtrate contained 5 mg ml-1 carbohydrate (as glucose) and 0.3 mg ml -1 protein (as bovine serum albumin). RESULTS

Time courseof infection of Zea and Lupinus roots Observations were made on roots 1, 2,4, 8, 16,24 and 48 h after infection. For both zea and Lupinus roots, zoospores had encysted and germ tubes had penetrated the root surface 1 h after infection (Plate 1A). In
Callose

formation

in Zea mays

117

were observed in epidermal and outer cortical cell layers of
118

J. M. Hinch and A. E. Clarke Staining

&muteristics

stain Aniline blue Resorcinol blue Calcofluor Congo red Periodic acid-fkhiff Scbiff-base Basic fuchsin Coomassie blue Toluidme blue Phloroglucinol-HCl Aniline chloride I-KI-H,SOI Sudan IV Ruthenium red

of

TABLE 2 callose paa3 produced in Zea mays Phytophthora cinnamomi

roots

Specificity Callose Callose Cell wall stain, believed to stain 1,4-S-glucans including cellulose Callose and other polysaccharides Carbohydrate containing vi&al hydroxyl groups Free aldehyde groups Free aldehyde groups + lignin Protein Charged groups (blue/red) Phenolic groups (green) Lignin Lignin Cellulose Suberin, lipids Pectin

48 h aftn

infection

Staining papillae

by

of

+ +’ + + +

deposits also stained with ruthenium red but not with Coomassie blue or toluidine blue. They gave a brilliant yellow fluorescence with decolourized aniline blue, intense brilliant white fluorescence with Calcofluor, and a deep cobalt-blue stain with resorcinol blue. Cell wall material of both the root tissue and the hyphae gave a weak yellow fluorescence with aniline blue but no fluorescence in the cytoplasm of either plant or fungal cell was detected. In contrast, sections stained with Calcofluor showed fluorescence of the cytoplasmic contents of both host and fungal cells as well as staining of both types of cell walls and the callose deposits. Some callose deposits fluoresced more brilliantly than others, possibly reflecting their thickness. Other dyes commonly used to detect callose, resorcinol blue and Congo red, stained the deposits but also stained other cell wall components. Of the stains used, aniline blue and resorcinol blue showed the greatest specificity for the deposits. Calcofluor was not so specific but was useful for examining the relationship between hyphae, host cells and pads. Reagents commonly used to detect lignin, phloroglucinol and aniline chloride, did not stain callose deposits formed 4 h and 48 h after infection, nor did the non-specific lignin stain, basic fuchsin. Under the same conditions, spiral thickenings of the xylem gave positive stains for lignin. The I-KI-HsSO, stain for cellulose was not effective in staining material embedded in JR4 resin, however, fast green (which stains non-lignified cell walls) gave a positive reaction at the outer perimeter of the deposits. Further insight into the chemical nature of the deposits was obtained by examining their solubility characteristics. Sections were treated separately with 1 M KOH, 6 M HCl and dimethyl sulphoxide (DMSO), and the staining reactions of callose deposits in the treated sections were examined (Table 3). The staining properties were only slightly altered in a few cases: for example there was less intense staining of deposits with aniline blue after KOH treatment; after acid treatment, both PAS and Calcofluor staining were weaker.

119

Callose formation in Zea mays

Enzymic hydroSysi.s of callosepads Staining of the deposits with both aniline blue and Calcofluor was lost during enzymic hydrolysis. The cell wall-degrading enzyme mixture Driselase, and the endo-1,3+glucan hydrolase from Rhizopus were most effective in destroying the aniline blue staining capacity. Sections treated with these enzymes for 20 h lost their capacity to give a yellow fluorescence with aniline blue; control sections and sections treated with other enzymes for the same time retained this capacity. Sections treated with exo- 1,3+glucan hydrolase from Euglena showed diminished yellow fluorescence after 38 h and after 40 h the endo-1,3 : 1,4-P-glucan hydrolase from Bacillus and the endo-1,3-P-glucan hydrolase from Ncotiana abolished the aniline blue staining capacity of the deposits (Plate 4). After 40 h, sections treated with the endo-1,4-P-glucan hydrolase from Streptomycesshowed less intense staining with aniline blue, but it was not abolished. TABLE

Effect

of enrymic

and chemical

3

mo&j!cation on callose staining Phytophthora cinnamomi Staining

Treatment Enz ymic Driselase

Aniline blue

Specificity

Mixture of cell wall degrading enzymes

E~&ena gracilis 1,3-a-glucan exohydrolase

I G3Gh

Rhizopus awhizus 1,3+glucan ertdohydrolase

I -G3G3G

in Zea

mays

roots infected

by

characteristics of “callose” pads after treatment Periodat* Calcofluor SchifYs test

-

-

+

i

-

+

-

-

+

-

+

f

+

+

-

-

+

z +

: +

z +

4 -G3GiG

Bacillus s&h 1,3; 1,4-b-glucan endohydrolase

or

?i -G4G3G4G4

St;reptomyces sp. l;q3-glucan endohydrolase

I -4G4G 4

Nicotiana glutinasa 1,3-fl-glucan endohydrolase

-G3G3-

4

Chemical HlCl (6 M) KOH (1 M) DIMS0 +,

Positive;

I

+,

weak;

-,

negative.

120

J. M. Hinch and A. E. Clarke

On the whole, the staining reaction of the callose deposits with aniline blue was less easily altered by enzymic treatment than the staining with Calcofluor. The brilliant white fluorescence of the deposits, when stained with Calcofluor, was abolished by every enzymic treatment except the endo-1,4+glucan hydrolase. Callose deposits treated with specific enzymes to the stage at which staining with aniline blue or Calcofluor was lost, retain their capacity to give a positive reaction with PAS. These results are summarized in Table 3. Induction of calloseformation in tissueslicesbyfungal culturejltraks Hand cut transverse sections of
PLATE 1.
[facing page I.201

PLATE 2. Lupines angustiflius root infected with Phytophthora cimamomi. Transverse section 48 h after infection, stained with toluidine blue. Hyphae (intensely stained) have penetrated the vascular tissue (Vt). Both intracellular (I) and extracellular (E) hyphae are apparent; (C), cortex. Scale bar=10 pm.

PLATE 3. Electron micrograph of callose deposit produced in
PLATE 4. Enzymic digestion of callose deposits formed in ze:ea roots 48 h after infection with Phytophthora cinnamomi. Longitudinal sections of roots at the zone of elongation were incubated with enzymes for 40 h as described in the text, washed and stained with aniline blue. Scale bar=20 pm. A. Control section (incubated in buffer) showing brilliant fluoresB. Section treated with .Stre@vnyces enzyme showing diminished cence (+ ) of callose deposits. (*) fluorescence. C. Section treated with Driselase enzyme showing absence (-) of fluorescence. Sections treated with the other specific enzymes showed aniline blue fluorescence similar to those depicted, i.e. either brilliant fluorescence (+), diminished fluorescence (+) or no fluorescence (-).

micrographs of hand PLATE 5. Induction of callose in tissue slices of
Cdlose

formation

in Zea mays

121

some of which were in the same linear chains [35]. The second approach, of examining staining of defined polymers by aniline blue, indicates that while 1,3-pglucans are stained, other carbohydrate polymers, including cellulose, and a 1,3 : 1,4+glucan also give fluorescent stains if presented in an appropriate physical form [3.?]. It seems that a positive fluorescent stain with aniline blue, indicates the presence of 1,3+glucosidic linkages, but allows that other glycosyl linkages may be present in the same polymer. Staining does not exclude the presence of other material associated with the aniline blue-staining material. On this basis the
Fwution of pa)dlae It is not clear whether papillae in general are involved in arrest of fungal growth within the host. In this study, papillae are apparently deposited after fungal penetration, that is, they are not physical barriers to fungal growth. They may, however, be formed as a wound response and allow repair of damaged cells, or provide a barrier to toxic products diffusing from the fungal hypha [I]. There is a close connection between unsuccessful infection and papilla formation in Phytophthoru-host interactions in -which root tissue is invaded (Table 4). Papillae are also formed during incompatible fungal infections of other members of the Gramineae [29].

122

J. M. Hinch and A. E. Clarke TABLE

Wall

modifzcations

associated

4

with Phytophthora

infections

Re&ant(R)

Pathogen

Sllzptible (S)

Host

P. cactorum

P. cap&i P. cinnamomi

P. drechsleri

P. infestans

Malus sp. fruit cv. Belle de Boskoop ” “Golden delicious” Capsicum fnrtescens (pepper fruit) Acacia pulchella root tissue Eucalypttuc spp. Carthamus tinctorius (safflower hypocotyls) Biggs NlO Solarium tuberosum (potato) tuber cv. Eba Bintje

P. infestans

Solarium tuberosum (potato) leaves cv. Orion Majestic

P. megas#erma var. sojae (i) race 1 (ii) race 4, race 6

Glycine max (soybean) roots hypocotyls cv. Altona

P. palmivora

Solarium melongem (egg plant fruit) Nicotiana tabacum (tobacco)

P. parasitic0 var. ntiotianae race

1, race 0

roots L-8 Burley

21

Wall modifications in response to infection”

R

Papillae

S

No papillae

formed

S

Cell border

lesions

R

Callose

Method of detection Reference

cm.

(=papillae)

24

e-m. 19

S+R

No callose

R S

Wall deposits None

R

Wall appositions (common) Wall appositions (r=e)

S

R S

S S+R

S

R-O Sl S

Papillae Papillae. both

occurred

little

34

1.m.

22

(i.e. callose)

e.m.

(i.e. callose)

Afb.

15

e.m.

30

le::

31 33

e.m.

6

e.m.

11

frequently

Papillae formed Cell wall deposits around pathogen in compatible reaction No sheaths formed

Higher frequency lesions j.;e;;rvzhyJho;;d;z

e.m.+ A.b. e.m.

of wall t

cell damage.

’ Description of wall modifications is that used by the authors e.m., Electron microscopy; l.m., light microscopy; A.b.,

cited. aniline

blue staining.

Little is known of how papilla deposition is initiated and controlled. The presence of diffuse aniline blue-positive droplets in the cytoplasm and the accumulation of cytoplasm at the site of callose deposition suggests cytoplasmic involvement in callose deposition [I, 121. It may be that material secreted from the hypha interacts with either a membrane- or a wall-located receptor to initiate a number of responses,

Callose

formation

123

in Zee mays

among which is papilla formation. We have made two observations relevant to this idea: firstly, some but apparently not all cells in contact with a hypha respond by papilla formation; secondly, in
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NEWHOOK,

an&f&s