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Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia A. M. BERRY Department of Environmental Horticulture, University of California, Davis, CA 95616, U.S.A. AND M. E. MCCULLY Biology Department, Carleton University, Ottawa, Ont., Canada KlS 5B6 Received July 14, 1989 BERRY, A. M., and MCCULLY, M. E. 1990. Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia. Can. J. Bot. 68: 798-802. Light microscopy, aniline-blue fluorescence histochemistry, and transmission electron microscopy were used to elucidate the nature of localized wall deposition in infected and uninfected root hairs on nodulated roots of Alnus rubra Bong. inoculated with the nitrogen-fixing symbiont, Frankia HFPArl3. Callose-containing papillae were found only in epidermal hair cells and not in cortical or vascular tissue. At the site of successful root-hair wall penetration, transfer cell-like wall ingrowths were elaborated, but callose was not detected. At sites of arrested root-hair infections, complex deposits consisting of callose, fibrillar components, and electron-dense material surrounded the incipient hyphal infection. The cytoplasm of root hairs containing arrested infections was deteriorated compared with successfully infected root hairs. BERRY, A. M., et MCCULLY, M. E. 1990. Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia. Can. J. Bot. 68 : 798-802. La microscopie photonique, l'histochirnie par fluorescence au bleu d'aniline et la microscopie Clectronique a transmission ont CtC utilisCes pour Clucider la nature du dCpBt pariCtal localisC chez les poils absorbants infest& et non-infest& des racines du Alnus rubra Bong., portant des nodositCs et inoculCes avec le symbiote fixateur d'azote, Frankia HFPArl3. Des papilles a callose ont etC trouvkes seulement dans les trichoblastes Cpidermiques et non dans le tissue cortical ni dans le tissu vasculaire. Au site d'une pCnCtration rCussie du poll absorbant, des replis parietaux ressemblant a ceux des cellules de transfert sont apparus, mais la prCsence de callose n'a pas CtC dCcelCe. Aux sites d'infections avortkes, les hyphes d'infection naissants Ctaient entouris de dCp8ts complexes constituCs de callose, de composants fibrillaires et de materiel dense aux electrons. Le cytoplasme des poils absorbants contenant des infections avortCes Ctait dCgCnCr6 par rapport a celui des poils absorbants a infections reussies. [Traduit par la revue] Introduction reported a callosic component at the site of infection-thread Among the structural features of plant cell response to infec- tion by pathogenic microorganisms, "papillae" frequently occur at the sites of invasion through the host cell wall. Such local deposits are associated with a range of microbial infec- tions in plant tissues (Aist and Williams 1971; Hinch and Clarke 1982; Lazarovits and Higgins 1976; Littlefield and Bracker 1972; Mourichon and Sall6 1981). Papillae are complex deposits of which the 1,3-P-glucan callose is a major, readily detected component. Callosic papillae may also contain cellulose, phenolic compounds, membane fragments, and other substances (Aist 1983; Hinch and Clarke 1982; Hachler and Hohl 1982). The extent to which specific components of papillae partic- ipate in resistance to pathogen infection is not yet understood. Experimentally induced papillae prevented cell-wall penetra- tion by fungal parasites in otherwise compatible hosts (Aist 1977; Aist et al. 1979), yet infection was still blocked pref- erentially at sites of similar papillae even after enzymatic treatment, which removed aniline-blue positivity and auto- fluorescence (Smart et al. 1986). In symbiotic systems such as nitrogen-fixing root nodules, the occurrence of callose-containing deposits at the site of bac- terial penetration of host cell walls has been investigated in two studies of the Rkizobiunz-clover infection process with aniline-blue fluorescence as an indicator, but the observations have been conflicting. Kumarasinghe and Nutman (1977) initiation in most hairs examined, although they did not deter- mine whether the root-hair infections led to nodule formation or represented arrested infections. Callaham (1979), using stringent procedural conditions, was unable to demonstrate callose at sites of successful infection in clover root hairs. The occurrence of callose-containing papillae in the early stages of nodulation of actinorhizal plants has not yet been characterized. This report presents histochemical and ultra- structural evidence on the nature of papillae in root hairs dur- ing early infection of Alnus rubra by Frankia. Materials and methods Seedlings of A. rubra were grown in washed sand as described previously (Beny and Torrey 1983) and transplanted into aeroponics culture (Zobel et al. 1976). The roots were inoculated with a homog- enized suspension of Frankia sp. HFPArl3 (Beny and Torrey 1979). As soon as swellings were noted on the roots, the prenodules were excised and fixed overnight in 2.5% glutaraldehyde in 0.025 M potas- sium phosphate buffer, pH 6.8-7.0, at 4°C. Specimens were rinsed in buffer and postfixed for 2 h in aqueous 2% OsQ,. Prenodules were then dehydrated in a graded acetone series, infiltrated, and embedded in Spurr's low-viscosity resin (Spurr 1969). Alternating thick and thin and serial sections of the prenodule zone were cut with a diamond knife. Thick plastic sections for light microscopic examination were stained with 0.1% toluidine blue O in 1% Na borate, pH 11. For electron microscopy, gold sections were mounted on Formvar-coated grids and stained with uranyl acetate and lead citrate. Ten nodules Printed in Canada i Imprime au Cannda Can. J. Bot. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 11/11/14 For personal use only.

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Page 1: Callose-containing deposits in relation to root-hair infections of               Alnus rubra               by               Frankia

Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia

A. M. BERRY Department of Environmental Horticulture, University of California, Davis, CA 95616, U.S.A.

AND

M. E. MCCULLY Biology Department, Carleton University, Ottawa, Ont., Canada KlS 5B6

Received July 14, 1989

BERRY, A. M., and MCCULLY, M. E. 1990. Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia. Can. J. Bot. 68: 798-802.

Light microscopy, aniline-blue fluorescence histochemistry, and transmission electron microscopy were used to elucidate the nature of localized wall deposition in infected and uninfected root hairs on nodulated roots of Alnus rubra Bong. inoculated with the nitrogen-fixing symbiont, Frankia HFPArl3. Callose-containing papillae were found only in epidermal hair cells and not in cortical or vascular tissue. At the site of successful root-hair wall penetration, transfer cell-like wall ingrowths were elaborated, but callose was not detected. At sites of arrested root-hair infections, complex deposits consisting of callose, fibrillar components, and electron-dense material surrounded the incipient hyphal infection. The cytoplasm of root hairs containing arrested infections was deteriorated compared with successfully infected root hairs.

BERRY, A. M., et MCCULLY, M. E. 1990. Callose-containing deposits in relation to root-hair infections of Alnus rubra by Frankia. Can. J. Bot. 68 : 798-802.

La microscopie photonique, l'histochirnie par fluorescence au bleu d'aniline et la microscopie Clectronique a transmission ont CtC utilisCes pour Clucider la nature du dCpBt pariCtal localisC chez les poils absorbants infest& et non-infest& des racines du Alnus rubra Bong., portant des nodositCs et inoculCes avec le symbiote fixateur d'azote, Frankia HFPArl3. Des papilles a callose ont etC trouvkes seulement dans les trichoblastes Cpidermiques et non dans le tissue cortical ni dans le tissu vasculaire. Au site d'une pCnCtration rCussie du poll absorbant, des replis parietaux ressemblant a ceux des cellules de transfert sont apparus, mais la prCsence de callose n'a pas CtC dCcelCe. Aux sites d'infections avortkes, les hyphes d'infection naissants Ctaient entouris de dCp8ts complexes constituCs de callose, de composants fibrillaires et de materiel dense aux electrons. Le cytoplasme des poils absorbants contenant des infections avortCes Ctait dCgCnCr6 par rapport a celui des poils absorbants a infections reussies.

[Traduit par la revue]

Introduction reported a callosic component at the site of infection-thread

Among the structural features of plant cell response to infec- tion by pathogenic microorganisms, "papillae" frequently occur at the sites of invasion through the host cell wall. Such local deposits are associated with a range of microbial infec- tions in plant tissues (Aist and Williams 1971; Hinch and Clarke 1982; Lazarovits and Higgins 1976; Littlefield and Bracker 1972; Mourichon and Sall6 1981).

Papillae are complex deposits of which the 1,3-P-glucan callose is a major, readily detected component. Callosic papillae may also contain cellulose, phenolic compounds, membane fragments, and other substances (Aist 1983; Hinch and Clarke 1982; Hachler and Hohl 1982).

The extent to which specific components of papillae partic- ipate in resistance to pathogen infection is not yet understood. Experimentally induced papillae prevented cell-wall penetra- tion by fungal parasites in otherwise compatible hosts (Aist 1977; Aist et al. 1979), yet infection was still blocked pref- erentially at sites of similar papillae even after enzymatic treatment, which removed aniline-blue positivity and auto- fluorescence (Smart et al. 1986).

In symbiotic systems such as nitrogen-fixing root nodules, the occurrence of callose-containing deposits at the site of bac- terial penetration of host cell walls has been investigated in two studies of the Rkizobiunz-clover infection process with aniline-blue fluorescence as an indicator, but the observations have been conflicting. Kumarasinghe and Nutman (1 977)

initiation in most hairs examined, although they did not deter- mine whether the root-hair infections led to nodule formation or represented arrested infections. Callaham (1 979), using stringent procedural conditions, was unable to demonstrate callose at sites of successful infection in clover root hairs.

The occurrence of callose-containing papillae in the early stages of nodulation of actinorhizal plants has not yet been characterized. This report presents histochemical and ultra- structural evidence on the nature of papillae in root hairs dur- ing early infection of Alnus rubra by Frankia.

Materials and methods

Seedlings of A. rubra were grown in washed sand as described previously (Beny and Torrey 1983) and transplanted into aeroponics culture (Zobel et al. 1976). The roots were inoculated with a homog- enized suspension of Frankia sp. HFPArl3 (Beny and Torrey 1979). As soon as swellings were noted on the roots, the prenodules were excised and fixed overnight in 2.5% glutaraldehyde in 0.025 M potas- sium phosphate buffer, pH 6.8-7.0, at 4°C. Specimens were rinsed in buffer and postfixed for 2 h in aqueous 2% OsQ,. Prenodules were then dehydrated in a graded acetone series, infiltrated, and embedded in Spurr's low-viscosity resin (Spurr 1969). Alternating thick and thin and serial sections of the prenodule zone were cut with a diamond knife. Thick plastic sections for light microscopic examination were stained with 0.1% toluidine blue O in 1% Na borate, pH 11. For electron microscopy, gold sections were mounted on Formvar-coated grids and stained with uranyl acetate and lead citrate. Ten nodules

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BERRY AND MCCULLY 799

were sectioned serially and examined with light microscopy. Several representative sections were prepared for transmission electron microscopy (TEM) and fluorescence microscopy.

For fluorescence microscopy, resin was removed from thick (0.44.5 pm) sections by soaking in sodium ethoxide (a saturated solution of NaBH in anhydrous ethanol) for 2-3 min. Sections were mounted in 0.067 M potassium phosphate buffer, pH 8.5, and exam- ined for background autofluorescence. Slicks containing sections were next flooded with aniline blue (0.05% in 0.067 M potassium phos- phate buffer, pH 8.5) and viewed with an Qlympus 40 x UVFl oil- immersion lens on a Zeiss Universal microscope equipped with epi- fluorescence optics. Excitation filter BG12, F1 beam splitter, and barrier filter 530 were used. Material was photographed onto Kodak Plus X film. Exposure times varied from 30 s for aniline-blue reactive papillae (e .g . , Figs. 4 4 ) to 5 min for autofluorescence (Fig. 7).

Results General obsewations

Observations were made of root-hair wall appositions in nodulated roots, in the region of prenodules (Figs. 1-3). Papillae were observed in epidermal hair cells and were never observed in cortical cells or in the vascular tissue. The origin of Frankia hyphae within the prenodule cortex was traced to a single root-hair infection in each prenodule. Papillae were observed predominantly in root hairs near the successfully infected root hair (as in Fig. 3) and occasionally were present in root hairs some distance from the infection site (as in Fig. 1, 200-300 pm proximal to the successfully infected root hair).

Root-hair papillae were localized deposits formed along the side walls of root-hair lobes or branches. Papillae were not observed with any frequency at the tips of root hairs. Occa- sionally, papillae were located between primary and secondary wall layers. When stained with toluidine blue 8, the papillae appeared bright blue to green. Cell walls of successfully infected root hairs also appeared blue-green, whereas cell walls in uninfected hairs or hairs lacking papillae were dark blue. Wall ingrowths at the sites of successful infection by Frankia (Fig. 2) stained dark blue to purple.

Fluorescence microscopy In Alnus root hairs, intense aniline-blue induced fluores-

cence was localized in wall papillae observed in adjacent sec- tions with bright-field optics (Figs. 1 and 4, 2 and 5, and 3 and 6). In successfully infected root hairs, the extensive wall proliferation in the zone of Frankia penetration was not ani- line-blue fluorescent (Fig. 5, iz). Root hairs containing arrested infections, that is, intracellular infections by Frankia that were confined to a small region of the root hair and did not lead to nodule formation, were observed microscopically, usually near the successfully infected root hair. Papillae were observed at the sites of such arrested infections (Fig. 3) and these were intensely fluorescent with aniline blue (Fig. 6).

Aniline-blue positive deposits were also noted in uninfected hairs, or even within the infected hair, which were not asso- ciated with Frankia wall penetration or subsequent intracel- lular proliferation (p, Figs. 4 and 5; also see Fig. 1 1). Colonies of rhizosphere bacteria were observed around the root hairs, but no definitive correlations could be made between the pres- ence of rhizosphere bacteria and callosic deposits in root hairs.

Diffuse aniline-blue positive fluorescence was present in the cell walls of hairs containing callosic wall appositions, espe- cially in the vicinity of the appositions (Figs. 4 and 5).

Very faint autofluorescence was noted in cell walls of nod-

ule and other root tissue, including root hairs, and in some of the cell contents, notably amyloplasts (Fig. 7).

Ultrastructural obsewations Structural and subcellular details of papillae were examined

in relation to successful and arrested root-hair infection by Frankia. In the infection zone, the wall ingrowths were com- posed of electron-dense, finely fibrillar material (Fig. 8), char- acteristic of transfer-cell walls (Gunning and Pate 1969). No aniline-blue positive fluorescence was noted.

In the case of hairs containing partial infections, callose- containing papillae as observed in Fig. 6 were located at sites of arrested Frankia infection. At the ultrastructural level (Fig. 9), the papillae were composed of electron-lucent mate- rial interspersed with electron-dense areas.

The cytoplasm of root hairs, which had sustained partial or arrested infection by Frankia, exhibited varying degrees of disintegration (Figs. 9 and 10) as compared with successfully infected root hairs similarly prepared (Fig. 8) and contained few or no recognizable organelles. The partially invaded cells contained higher levels of electron-dense material than nearby uninfected cells.

As discussed in the previous section, callosic papillae also occurred in areas where no Frankia wall penetration, either successful or arrested, was observed (Fig. 11). Such sites were not associated with cytoplasmic degradation.

Discussion

In root-hair infections of A. rubra leading to nodulation, we demonstrate that cell-wall elaboration in the zone of Frankia penetration is nonreactive with aniline blue. Thus, callose is not a major component of the cell wall at the site of successful Frankia infection. The results reported in the present work correspond to similar observations of Callaham (1979) for Rhizobium infection of clover root hairs.

Even though callose is not found at sites of successful root- hair infection by Frankia, callose-containing papillae surround Frankia hyphae where these penetrate the host wall but where subsequent stages of infection are arrested. The cytoplasm in these root hairs appears senescent or deteriorated. A similar structural pattern of response to symbiotic infection has been described in ectomycorrhizal infections on several host species (Ashford and Allaway 1985; Nylund et al. 1982). Papillae were observed in regions of cell-wall penetration or disturbance by the fungal hyphae. Wall penetration was associated with senescing tissues or with specific senescing cells. The invaded host cells exhibited deterioration of the cytoplasm.

Callose-containing wall appositions also occur in Alnus root hairs as local depositions not in obvious relation to any intra- cellular penetration. It is not possible to determine from struc- tural evidence whether such wall appositions in Alnus root hairs are induced to form by the presence of rhizosphere microor- ganisms. Callose can form in root hairs through mechanical disturbance (Aist 1977), under axenic conditions (Clarke and McCully 1985), or in other tissues as a result of chemical per- turbation (Hughes and Gunning 1980; Galway and McCully 1987).

In light of the common occurrence of callose in response to microbial or other sources of disturbance, it is surprising that callose is not detected at the sites of successful Frankia infec- tion. Several mechanisms of regulation of callose deposition

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808 CAN. J . BOT. VOL. 68, 1990

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Page 4: Callose-containing deposits in relation to root-hair infections of               Alnus rubra               by               Frankia

BERRY AND MCCULLY 80 1

in plants have been delineated experimentally (Kauss 1987; see also Aist 1983), all of which modulate the influx of external calcium in a circumscribed location. Calcium activates or increases 1,3-P-glucan synthase activity by a direct effect on the enzyme. At the site of successful infection, a mechanism may operate to bind or block influx of external calcium or to stabilize membranes in the vicinity of the infection so that cal- cium-triggered callose deposition does not occur. Alterna- tively, auxin-dependent interactions may be implicated in the dissolution of callose deposits, as has been reported recently for dormancy callose in pl-lloem of Vitis (Aloni et al. 1989).

It is evident in the present study that Frankia infections are initiated in several root hairs following inoculation, but that many such infections are arrested. Individual differences in cellular differentiation may regulate successful infection, or some type of competitive interaction or other intercellular sig- nal may prevent multiple successful root-hair infections.

The authors express their thanks to L. McIntyre and T. Sage for technical assistance and to L. Sunell for discussions. The research was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada to M. E. McCully and by California Agricultural Experiment Station Project CA-B-4500.

AIST, J. R. 1977. Mechanically induced wall appositions of plant cells can prevent penetration by a parasitic fungus. Science (Washington, D.C.), 197: 568-570.

1983. Structural responses as resistance mechanisms. In The dynamics of host defence. Edited by J. A. Bailey and B . J. Dev- erall. Academic Press, Sydney. pp. 33-70.

AIST, J. R., and WILLIAMS, P. H. 197 1. The cytology and kinetics of cabbage root hair penetration by Plasmodiophora brassicas. Can. J . Bot. 49: 2023-2034.

AIST, J. R . , KUNOH, H., and ISRAEL, H. W. 1979. Challenge appres- soria of Erysiphe graminis fail to breach preformed papillae of a compatible barley cultivar. Bhytopathology, 69: 1245-1250.

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FIGS. 1-3. Wall papillae in root hairs of nodulated roots of Alnus rubra. Root cross sections, stained with toluidine blue. Bright-field optics. 790 x . Fig. 1. Cross section of a nodulated root of A. rubra, 200-300 pm proximal to the infected root hair. Epidermal cells (e) are thin walled; a papilla (p) is evident within a root-hair lobe (rh). a , amyloplasts. Fig. 2. Infected root hair (irh) and portion of the prenodule. Wall of the infected hair is thick relative to other hairs. Note dark-staining wall ingrowths in the zone of infection (is), occurring in a deeply folded region of the wall. A section of the encapsulated Frankia hypha within the hair is evident at the upper part of the infection zone (h). There is a lightly staining papilla (p) along the lower wall of the root-hair lobe. ( 1 , amyloplasts. Fig. 3. Portion of a root hair (rh) that is highly branched and lobed. The subtending root-hair base, not seen, is perpendicular to the plane gf section. A limited zone of wall penetration can be noted in the upper part of the hair lobe, in an indented region of the wall. The papilla formed around the infection site is very lightly staining (p). Figs. 4-6. Fluorescence micrographs of nodulated roots of A. rubra stained with aniline blue, all at 750 x . Sections in Figs. 4- 6 are adjacent or nearly adjacent to sections 1-3, respectively. Fig. 4. Root hair papilla of Fig. 1 exhibits bright aniline-blue specific fluorescence (p). Walls of nearby hairs fluoresce relatively brightly, as do amyloplasts (a) in the cortical tissue. Small discrete dots of bright fluorescence, probably representing callose in primary pit fields (pf) occur in the cortical cell walls. Fluorescence of amyloplasts may indicate a degree of nonspecificity in the fluorochrome binding (Smith and McCully 1978). 30-s exposure. Fig. 5. Successfully infected root hair exhibits no aniline-blue fluorescence in penetration zone (is, cf. Fig. 2). A bright fluorescent region (p) corresponds to the papilla seen in Fig. 2 and in Fig. 8. 45-s exposure. a , amyloplasts. Fig. 6. Aniline-blue fluorescent papilla (p) is located at the site of arrested infection noted in Fig. 3 (arrow). 45-s exposure. Fig. 7. Inherent autofluorescence, prior to aniline-blue staining, of section shown in Fig. 4. Epidermal cell walls and papilla are weakly autofluorescent. Amyloplasts in the cortex are also faintly autofluorescent. 5-min exposure. Fig. 8. Electron micrograph of characteristic wall proliferation at the site of successful Frankia penetration (see Fig. 2). Wall ingrowths are finely fibrillar, electron-dense structures resembling transfer-cell wall (Berry et al. 1986). Infecting Frankia hypha (h) is located within folded host root hair wall. The cytoplasm does not appear senescent. 12 500 x . Fig. 9. Electron micrograph of the callose-containing papilla in an arrested root-hair infection, shown in Figs. 3 and 6. Hyphal segment at right (h) is highly electron dense, and is surrounded by a fibrillar zone of the host wall permeated with electron-dense material (ed). Toward the cell interior, bordered by a thin fibrillar inner wall layer, the papilla is more electron lucent (EL). The host cytoplasm is degraded (cyt). 45 000 x . Fig. 10. Arrested infection with associated papilla (p) located between two root hair lobes. Hyphae (h) are present in the folded region between the two lobes. The inner portion of the papilla is electron lucent; the outer portion is highly electron dense. The cytoplasm (cyt) of the root hair lobe on the right is dense, lacking recognizable organelles. 8000 x . Fig. 11. Electron micrograph of callosic papilla in a successfully infected root hair, also shown in Figs. 2 and 5. The papilla occurs between the outer, primary celi wall and the lamellar, finely fibrillar inner wall. Electron-lucent zones alternate with electron-dense fibrillar patches (small arrows) and other material. The cytoplasm (cyt) of this root hair appears normal. 45 000 x .

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