A Light and Electron Microscope Study of the Interaction of Yellow ...

Ann. Bot. 43, 183-189, 1979

A Light and Electron Microscope Study of the Interaction of Yellow Rust (Puccinia striiformis) with a Susceptible Wheat Cultivar D. J. MARES* Department of Plant Biology, University of Hull, Hull HU6 7RX, U.K.

ABSTRACT The microscopy and ultrastructure of the interaction of Puccinia striiformis with a susceptible wheat cultivar was examined at intervals from the time of first haustorium formation to the onset of sporulation. At any particular point in the radially expanding area of infection a sequence of morphological changes occurred in the infected host cells and the fungus which were correlated with successive phases of active fungal growth, accumulation of reserves and finally export of reserves to the developing reproductive structures. The observations are compared with previous work on other host-rust interactions. Key words: yellow rust, Puccinia striiformis, wheat, host-pathogen interaction. INTRODUCTION

Microscopic and ultrastructural studies of host-pathogen interactions have provided much detailed information on the structural features of many host-pathogen combinations (Littlefield and Bracker, 1971; Bushnell, 1972; Bracker and Littlefield, 1973; and cited references) and on the different mechanisms involved in the resistance of some plants to fungal pathogens (Littlefield and Aronson, 1969; Zimmer, 1970; Heath and Heath, 1971; El-Gewely, Smith and Colotelo, 1972; Heath, 1974; Skipp, Harder and Samborski, 1974; Abu-Zinada, Cobb and Boulter, 1975). Several members of the rust family have been examined in detail but, to date, the yellow rust fungus {Puccinia striiformis) has received little attention. This report describes microscopic and ultrastructural changes which occur in both the fungal components and the infected host cells during the development of the rust infection. MATERIALS AND METHODS An isolate of yellow rust (physiologic race 104 El37) and a susceptible wheat cultivar (Nord Desprez) were maintained according to procedures outlined in a previous report (Mares and Cousen, 1977). The mid-sections of just fully expanded fourth leaves of Nord Desprez seedlings were inoculated with freshly collected uredospores of yellow rust. Leaf segments (approximately 1 cm2) were taken at 1-2 day intervals from the time of inoculation until the start of sporulation and fixed with 3 per cent glutaraldehyde in 0-2 M phosphate buffer, pH 7-2, for 24 h at 4 °C following vacuum infiltration. Areas (0-5-1 mm2) which contained isolated colonies or sections of isolated colonies were located with the aid of a light microscope and dissected for further treatment. The tissue was washed in buffer, post-fixed with 1 per cent OsO4 in phosphate buffer at 4 °C for * Present address: Institut fur Pflanzenernahrung, Universitat Hohenheim, 7000 Stuttgart, 70 West Germany. 0305-7364/79/020183 + 13 $02.00/0

© 1979 Annals of Botany Company

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Accepted: 15 March 1978

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Mares - Interaction of Yellow Rust with a Susceptible Wheat Cultivar

4 h and finally embedded in Spurr's resin (Spurr, 1969). For light and electron microscopy serial sections, cut parallel to the longitudinal axis of the leaf, were stained with alkaline toluidine blue and uranyl acetate/lead citrate respectively. OBSERVATIONS AND DISCUSSION

The haustorium-mother cell. Haustorium-mother cells were separated from the multinucleate intercellular mycelium by a septum (Plate 2E) and resembled closely the haustorium-mother cells of wheat-stem rust (Puccinia graminis) and flax rust {Melampsora lini) (Shaw and Manocha, 1965; Littlefield and Bracker, 1972). When first formed the haustorium mother cell contained two fungal nuclei and a dense cytoplasm. Subsequent to the penetration of the host cell wall the nuclei and cytoplasm moved into the body of the developing fungal haustorium leaving the haustorium-mother cell virtually devoid of cytoplasm (Plate 2E). There was no septum between the haustorium-mother cell and the haustorium. The wall of the haustorium-mother cell was similar to that of the intercellular hyphae except at the region of contact with a host cell where penetration had occurred. A wedge of electron-dense material, possibly serving an adhesive function and similar to the interstitial material found in the contact regions between several other plant hosts and fungal pathogens (Littlefield and Bracker, 1972), was found between the


Light and electron microscopic appearance of the components of the developing rust colony The substomatal vesicle. The sub-stomatal vesicle was formed 7-12 h after inoculation (Mares and Cousen, 1977) and by 36-48 h post-inoculation consisted of a thin-walled structure which contained dense cytoplasm and several fungal nuclei (Plate 1A and B). During subsequent development of the rust colony the vesicle became increasingly vacuolate and the wall was thickened by the deposition of several layers of material, some of which were densely stained (Plate 1 c and D). The composition and functional significance of these layers is unknown. The intercellular fungal hyphae. One to three infection hyphae, produced by the substomatal vesicle, gave rise to a branched mycelium which ramified in the intercellular space of the leaf. As the diameter of the infection increased so also did the density of intercellular hyphae in the more central areas of the colony. The hyphal wall was similar to that found initially at the surface of the sub-stomatal vesicle and did not alter during subsequent rust colony development (Plate 1E). The intercellular hyphae of young colonies ( < 5 day post-inoculation) and the peripheral areas of older colonies was characterized by dense cytoplasm, numerous mitochondria, endoplasmic reticulum, ribosomes and some small vacuoles (Plates 1E, F and 2E). These observations are consistent with fungal hyphae in a phase of extension growth. Hyphae in the more central areas of older colonies ( < 5 day post-inoculation) contained fewer organelles, larger vacuoles and increasing amounts of an irregularly shaped deposit, possibly fungal glycogen, which was stained pink by toluidine blue and which was shown by electron microscopy to consist of an aggregate of electron-translucent granules (Plates 1G and 2 A). Smaller, round lipid bodies were also present in large numbers (Plate 2B). The intercellular mycelium contained nuclei with prominent nucleoli (Plate 2 c and D) and it remained in a multinucleate condition (Plate 2 c and E) except at those points in the colony where pustule formation was initiated 11—12 d post-inoculation. At each of these points a mass of septate, binucleate hyphae was produced which filled the available intercellular spaces and formed uredospores. As pustule development and sporulation proceeded the septate hyphae became vacuolate and storage deposits disappeared from the intercellular hyphae immediately adjacent to the pustules. Possibly the aseptate nature of the rust mycelium facilitated the transport of metabolites, absorbed from the host cells, to the central areas of the colony where they were stored in a non-diffusible form until required for the production of uredospores.


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The ultrastructural appearance of the haustorium-host cell interface The wall of the haustorium body and the undulating profile of the invaginated host cell plasmamembrane were separated by a narrow region, the haustorial sheath, which contained little electron dense material (Plate 4 B). By contrast, the host cell plasmamembrane adjacent to the haustorial-neck was smooth and the sheath very narrow (Plate 4 c). The origin of the haustorial sheath is not completely clear but it appears as a structural feature, rather than a fixation artifact, in many host-rust combinations (Littlefield and Bracker, 1971; Bushnell, 1972; Bracker and Littlefield, 1973). Mengden and Heitefuss (1975) examined the distribution of radioactivity in bean leaves following infection with tritium-labelled uredospores of Uromyces phaseoli. The haustorial sheath and the infected cell remained free of label suggesting that the sheath matrix was secreted by the infected host. The sheath is an important structural feature in view of the proposed role of rust haustoria in the absorption of essential host metabolites (Shaw, 1967). There was no evidence of vesicular elements in the sheath matrix or of any morphological continuities between the protoplasts of the host and the fungus. Microscopy and ultrastructure of the infected host cells There was no evidence of physical damage at the site of penetration of host cells by fungal haustoria. Penetration appears to involve enzymic digestion or loosening of the host wall (Stavely, Pillai and Hanson, 1969; Rijo and Sargent, 1974) but the exact


walls of the host mesophyll cell and haustorium-mother cell. In addition the haustoriummother cell wall adjacent to the site of host cell penetration consisted of three distinct layers; a densely stained layer was sandwiched between two layers which had staining characteristics typical of fungal hyphal walls (Plate 3 A). Littlefield and Bracker (1972) concluded that in flax rust the deposition of the densely stained material, which formed a lens-shaped area around the neck of the haustorium, preceded host penetration and showed that it persisted throughout the establishment and development of the haustorium. Neither the composition nor the function of the dense layer is known. The haustorium. The haustorium of the yellow rust fungus, like the haustorium of wheat stem rust (Shaw and Manocha, 1965), flax rust (Littlefield and Bracker, 1972) and several other rusts which have been studied, penetrated the host cell wall and caused invagination but not penetration of the host plasmamembrane. Haustoria in the first penetrated cells, 36-48 h post-inoculation, and at the periphery of older colonies consisted of a narrow neck and a small, round to oval body which contained dense cytoplasm, fungal nuclei, mitochondria, endoplasmic reticulum and only small vacuoles (Plate 3 B and c). Haustoria in cells nearer the centre of rust colonies had a larger, sometimes multi-lobed body (Plate 3D) whilst in older colonies the haustoria contained fewer organelles, larger vacuoles, lipid bodies, occasional deposits of fungal storage material and lomasomes (Plates 3E, 4 A and B). The haustorial-neck wall was continuous with the wall of the haustorium-mother cell and was interrupted at a point approximately midway between the proximal and distal ends of the haustorial neck by a ring of dark-staining material (Plate 4 c). A similar region in the neck of the flax rust haustorium has been examined in detail by Littlefield and Bracker (1972). These workers used a periodate-chromate-phosphotungstate staining procedure to show that the neck ring deposit represented an abrupt transition from the wall of the penetration peg to the wall of the haustorium body. Following an investigation of the neck ring of the haustoria in several host-pathogen combinations, Heath (1976) concluded that the material in the neck ring prevented apoplastic solute transport and hence the possible loss of host secreted materials along the wall of the haustorial neck.

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GENERAL CONCLUSIONS The microscopic and ultrastructural appearance of the interaction of the yellow rust fungus with a susceptible host was very similar to that of several other rust-host combinations (Shaw and Manocha, 1965; Littlefield and Bracker, 1971; Coffey, Palevitz and


mechanism of penetration is not clear (Abu-Zinada et al. 1975). Infected host cells near the centre of 6-8 day colonies showed an apparent increase in the volume of cytoplasm (Plate 4E). Cytoplasm containing the host nucleus and chloroplasts was aggregated in the region of the haustorium body (Plate 4E). By contrast, the cytoplasm and organelles of adjacent non-infected cells (Plate 4E) and of penetrated cells of young colonies and the periphery of older colonies, was restricted to an attenuated layer adjacent to the host cell wall. Golgi bodies and vesicles were a prominent feature of the infected host-cell cytoplasm (Plate 5 A) but were not normally prominent in healthy leaf tissue. Some vesicles appeared to be fused with the host plasmamembrane and may be involved in the transport of host metabolites to the host-pathogen interface. The presence of increased cytoplasm and Golgi suggested that the metabolic activity of the host-cells was stimulated following haustorial penetration. Large starch granules were observed in the chloroplasts of host cells which contained haustoria and which were located near the centre of 8-10 day rust colonies (Plate 5 B) but starch granules were absent from all other host cells both within the area of infection and in the adjacent non-infected leaf tissue. Mesophyll cell chloroplasts in non-inoculated control leaves of a comparable age contained small starch granules (Plate 5 c). MacDonald and Strobel (1970) observed that the starch content of Puccinia striiformis infected wheat leaves increased from 9-12 days to twice that of healthy leaves. Their micrographs indicate that starch accumulated in the chloroplasts of host-cells adjacent to fungal hyphae. In the present investigation observations of serial sections indicated that starch accumulation in infected leaves was confined to cells which had been penetrated by haustoria. The reduced accumulation of starch in the cells of rust infected leaves, except at the centres of the infection loci, suggested that there was a flow of leaf photosynthate to the host cell-haustorium complexes at the centres of the colonies. The continuous utilization of soluble host sugars to supply fungal energy requirements and for the synthesis of fungal and host reserve carbohydrates could create a concentration gradient along which photosynthate could flow. By 12 day post-inoculation, the start of the phase of sporulation, host cells in the centre of the infected tissue had lost some of their cytoplasm and chloroplasts and haustoria were encased by only an attenuated layer of cytoplasm (Plate 5D). Chloroplasts which persisted in this tissue contained little starch, fewer thylakoid membranes and an increasing quantity of osmiophilic material (Plate 5E). The pustuled area of the infected leaf was surrounded by an annulus of infected cells which were rich in cytoplasm and starch-containing chloroplasts and intercellular rust hyphae which contained stored carbohydrate and lipid. Cells and fungal elements at the periphery of the radially expanding infection were similar to those in a 4 day infection. The haustorial-neck of some haustoria in older colonies was encased by a collar which consisted of an amorphous matrix with some fibrillar components, which varied in shape and which was continuous with, but distinct from, the host cell wall (Plates 3 A and 4c). The collar did not extend beyond the haustorial neck. A thin region of host cytoplasm separated the collar from the haustorial neck wall (Plate 4 c). The collar material has been shown to have staining characteristics which are similar to those of callose (Heath, 1971; Heath and Heath, 1971; Hardwick, Greenwood and Wood, 1971) and Heath (1971) has suggested that the collar formation results from the gradual development of some degree of host injury or other form of incompatibility between host and parasite.


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Allen, 1972; Heath, 1974; Abu-Zinada, Cobb and Boulter, 1975). Fungal components and infected host cells in a particular section of the leaf showed a series of morphological changes which correlated with a phase of active fungal growth, a phase of accumulation and storage of fungal and host reserve materials and a period of export of nutrients to the developing reproductive structures of the fungus. Similar phases have been described for the interaction of Uromyces fabae with Viciafaba L. (Abu-Zinada etal, 1975). Rust infection accelerated host tissue senescence, but apart from the presence of collars around the necks orsome haustoria there was little morphological evidence of host cell injury. ACKNOWLEDGEMENTS

L I T E R A T U R E CITED ABU-ZINADA, A.-A. H., COBB, A. and BOULTER, D., 1975. An electron-microscopic study of the effects

of parasite interaction between Viciafaba L. and Uromyces fabae. Physiol. PI. Path. 5, 113-18. BRACKER, C. E. and LITTLEFIELD, L. J., 1973. Structural concepts of host-pathogen interfaces. In Fungal Pathogenicity and the Plant's Response, ed. R. J. W. Byrde and C. V. Cutting. Academic Press, London and New York. BUSHNELL, W. R., 1972. Physiology of fungal haustoria. A. Rev. Phytopathol. 10, 151-76. COFFEY, M. D., PALEVITZ, B. A. and ALLEN, P. J., 1972. The fine structure of two rust fungi, Puccinia helianthi and Melampsora lini. Can. J. Bot. 50, 231-40. EL-GEWELY, M. R., SMITH, W. E. and COLOTELO, N., 1972. The reaction of near-isogenic lines of flax

to the rust fungus Melampsora lini. 1. Host parasite interface. Can. J. Genet. Cytol. 14, 743-51. HARDWICK, N. V., GREENWOOD, A. D. and WOOD, R. K. S., 1971. The fine structure of the haustorium

of Uromyces appendiculatus in Phaseolus vulgaris. Can. J. Bot. 49, 383-90. HEATH, M. C., 1971. Haustorial sheath formation in cowpea leaves immune to rust infection. Phytopathology 61, 383-8. 1974. Light and electron microscope studies of the interactions of host and non-host plants with cowpea rust - Uromyces phaseoli var. vignae. Physiol. PI. Path. 4, 403-14. and HEATH, I. B., 1971. Infrastructure of an immune and a susceptible reaction of cowpea leaves to rust infection. Ibid. 1, 277-87. LITTLEFIELD, L. J. and ARONSON, S. J., 1969. Histological studies of Melampsora lini in flax. Can. J. Bot. 47, 1713-17. and BRACKER, C. E., 1972. Ultrastructural specialization at the host-pathogen interface in rustinfected flax. Protoplasma 74, 271-305. MACDONALD, P. W. and STROBEL, G. A., 1970. Adenosine diphosphate-glucose pyrophosphorylase and control of starch accumulation in rust-infected wheat leaves. PI. Physiol. 46, 126-35. MARES, D. J. and COUSEN, S., 1977. The interaction of yellow rust (Puccinia striiformis) with winter wheat cultivars showing adult plant resistance: macroscopic and microscopic events associated with the resistant reaction. Physiol. PL Path. 10, 257-74. MENGDEN, K. and HEITEFUSS, R., 1975. Micro-autoradiographic studies on host-parasite interactions. I. The infection of Phaseolus vulgaris with tritum-labeled uredospores of Uromyces phaseoli. Arch. Microbiol. 105, 193-9. Ruo, L. and SARGENT, J. A., 1974. The fine-structure of the coffee leaf rust, Hemileria vastatrix. Can. J. Bot. 52, 1363-7. SHAW, M., 1967. Cell biological aspects of host-parasite relations of obligate fungal parasites. Ibid. 45, 1205-20. SHAW, M. and MANOCHA, M. S., 1965. The physiology of host-parasite relations. XV. Fine structure in rust infected wheat leaves. Ibid. 43, 1285-92. SKIPP, R. A., HARDER, D. E. and SAMBORSKI, D. J., 1974. Electron microscopy studies on infection of

resistant (Sr6 gene) and susceptible near-isogenic wheat lines by Puccinia graminis f.sp. tritici. Ibid. 52, 2615-20. SPURR, A. R., 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26, 31-43.


This work was supported by a grant from the Agricultural Research Council to Professor J. Friend and Dr J. R. Coley-Smith. Thanks are due to Professor J. Friend and Dr J. R. Coley-Smith for their encouragement, to Dr R. Johnson of the Plant Breeding Institute, Cambridge, for supplies of seed and yellow rust isolates and to Shelagh Cousen for technical assistance.

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STAVELY, J. R., PILLAI, A. and HANSON, E. W., 1969. Electron microscopy of the development of Erysiphe

polygoni in resistant and susceptible Trifolium pratense. Phytopathology 59, 1688-93. ZIMMER, D. E., 1970. Fine structure of Puccinia carthami and the ultrastructural nature of the exclusionary seedling-rust resistance of safflower. Ibid. 60, 1157-63.

EXPLANATION OF PLATES ABBREVIATIONS

c

m MC N NC PM R S Sv W

mitochondrion haustorial mother cell nucleolus non-infected host cell host cell plasma membrane differentially staining ring of fungal wall around the haustorial neck haustorial sheath sub-stomatal vesicle host cell wall

PLATE 1

A. Sub-stomatal vesicle (Sv) 2 days after inoculation. The thin-walled vesicle contains dense cytoplasm and several fungal nuclei with densely stained nucleoli (N) x 1760. B. Part of a sub-stomatal vesicle 2 days after inoculation. The vesicle consists of dense cytoplasm bounded by a simple wall consisting of one, or possibly two, indistinct layers, x 46500. c. Sub-stomatal vesicle (Sv) 8 days after inoculation. The wall of the vacuolate vesicle consists of several layers, x 1760. D. Sub-stomatal vesicle wall 8 days after inoculation. The wall apparently consists of several layers with different staining characteristics deposited on the exterior surface of the original wall, x 38760. E. Section of rust hypha from the periphery of a 4 days colony. The cytoplasm consists of ribosomes and elements of endoplasmic reticulum. The wall consists of one or possibly two layers, x 26200. F. Section of rust hypha from the periphery of a 4 days colony. The fungal cytoplasm contains numerous fungal mitochondria and profiles of endoplasmic reticulum (ER). x 24000. G. Section of rust hypha from the centre of a 6-8 days colony. The hypha contains irregularly shaped deposits of material which stained pink with toluidine blue (arrows). Two vacuolate haustoriummother cells are present (MC). x 1300. PLATE 2

A. Electron micrograph of the irregularly shaped hyphal material. The deposit has the appearance of an aggregate of small, electron-translucent granules embedded in amorphous material in the fungal cytoplasm, x 22750. B. Rust hyphal lipid bodies, x 34000. c. Rust hypha from the periphery of 6 days colony which contains dense cytoplasm and several nuclei, prominent nucleoli (arrows). Nuclei are not separated by septa, x 1200. D. Fungal hypha nucleus. The nucleolus (N) is more densely stained than the nuclear material, x 40500. E. Section of rust hypha which contains dense cytoplasm, small vacuoles, several fungal nuclei and at one point there is a vacuolate haustorium-mother cell (MC) which is separated from the hypha by a septum (arrow), x 1600. PLATE 3

A. Haustorium-mother cell (MC) in contact with a host mesophyll cell. The fungal wall consists of an electron-dense layer (DL) sandwiched between less dense layers. The adjacent infected host cell contains haustorium-collar material (C) which is distinct from the host cell wall (W) and contains some fibrillar deposits, x 30800. B. First penetrated host mesophyll cell 2 days after inoculation containing a haustorium body (H). x 1760. c. Haustorium from the periphery of a 4 days colony which contains dense cytoplasm, mitochondria (m) and endoplasmic reticulum. x 21700. D. Section through a multilobed haustorium from a 4 d colony, x 9450. E. Haustorium from the centre of a 6 days colony which contains a fungal nucleus and deposits 6f storage material (arrows), x 18750.


collar or wall-like encasement around the haustorial neck DL electron-dense layer of material in the haustorial mother cell wall ER endoplasmic reticulum fw fungal cell wall Golgi g H haustorium IC infected host cell lb lipid body

MARES

-Interaction of Yellow Rust with a Susceptible Wheat Cultivar


PLATE 1 Ann. Bol. 43, 183-189, 1979

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MARES-Interaction of Yellow Rust with a Susceptible Wheat Cultivar


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Ann. Bot. 43, 183-189, 1979

MARES

- Interaction of Yellow Rust with a Susceptible Wheat Cultivar


PLATE 3 Ann. Bot. 43, 183-189, 1979

MARES-Interaction of Yellow Rust with a Susceptible Wheat Cultivar


PLATE 4 Ann. Bol. 43, 183-189, 1979

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-Interaction of Yellow Rust with a Susceptible Wheat Cultivar


PLATE 5 Ann. Bot. 43, 183-189, 1979



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PLATE 4

A. Haustorium which contains lipid bodies (lb) and a section of a fungal nucleus with nuclear pores (arrows), x 17500. B. Haustorial lomasomes. x 39000. c. Haustorial neck (HN), haustorial sheath (S) and haustorial collar (C). There is a small ring of denselystained material (R) about midway between the haustorial body (H) and the host cell wall (not shown), x 27000. D. Haustorial sheath (S) which contains some electron-dense material and which separates the host cytoplasm and host plasmamembrane (PM) from the fungal cell wall (fw) and fungal cytoplasm, x 51000. E. Infected host cell (IC) at the centre of a 6 day colony. Host cytoplasm, nucleus and chloroplasts are aggregated in the vicinity of the haustorium (H). x 1400.

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PLATE 5

A. Host cell cytoplasm containing Golgi (g), vesicles and a mitochondrion. Vesicles appear to collect near the host plasmamembrane and fungal sheath (S). x 55000. B. Infected host cells (IC) at the centre of an 8 d colony. Chloroplasts of infected host cells contain prominent starch granules. Non-infected cells (NC) contain little starch, x 350. c. Non-inoculated leaf tissue. Mesophyll cell chloroplasts contain small starch grains, x 1000. D. Infected cell, beneath a sporulating pustule, which contains a haustorium (H) but little cytoplasm and few chloroplasts. x 1200. E. Chloroplast with dilated membrane and osmiophilic granules, x 27000.