Ultrastructure and immunocytochemistry of the host–pathogen interface in poinsettia leaves infected with powdery mildew

Transmission electron microscopy of high-pressure frozen and freeze-substituted samples was used to examine details of the host–pathogen interface in poinsettia leaf epidermal cells infected by the powdery mildew fungus Oidium sp. Cryofixation provided excellent preservation of both host cells and fungal haustoria. Each haustorium possessed a slender neck and an expanded body with numerous slender, aseptate, coiled lobes. The extrahaustorial membrane that separated each haustorium from the cytoplasm of an invaded host cell appeared thicker than other cellular membranes and was highly convoluted, particularly near the distal end of the haustorium neck. The neck of each haustorium was encased by a papilla that formed in response to the development of a haustorium. Immunogold labeling was used to elucidate the chemical nature of these papillae. Epitopes of callose, xyloglucan, and arabinogalactan proteins were localized in three separate regions of papillae. The localization sites for these compounds in poinsettia pa pillae differed somewhat from their reported locations in papillae of various other plants infected by either true fungi or oomycetes. In the current study, no labeling with the antibodies used was observed on the extrahaustorial matrix, the extrahaustorial membrane, the fungal plasma membrane, or the host plasma membrane.Key words: Oidium sp., Euphorbia pulcherrima, host–pathogen interactions, papillae, haustoria.

Immunogold localization of callose and other plant cell wall components in soybean roots infected with the oomycete Phytophthora sojae

Immunolabeling and transmission electron microscopic techniques were used to investigate the chemical nature of wall appositions in roots of susceptible and resistant soybean plants inoculated with Phytophthora sojae race 2. The extrahaustorial matrix associated with the haustorium of Phytophthora sojae also was examined. Antibodies against (1 → 3)-β-glucan, a terminal α-fucosyl-containing epitope present in xyloglucan and rhamnogalacturonan I, and an arabinosylated (1 → 6)-β-galactan epitope present in arabinogalactan proteins were used. (1 → 3)-β-Glucan (callose), xyloglucan, and arabinogalactan proteins were found to be localized in all wall appositions regardless of how long after inoculation the appositions developed or whether plants were susceptible or resistant to Phytophthora sojae. (1 → 3)-β-Glucan also was found in fungal walls and at host cell plasmodesmata. None of the four antibodies labeled the extrahaustorial matrix. The antibody against arabinogalactan protein recognized the host plasma membrane, but not the invaginated host plasma membrane associated with the extrahaustorial matrix. This result indicates that the properties or the composition of the host plasma membrane may change locally once it becomes an extrahaustorial membrane. Key words: Phytophthora sojae, Glycine max, callose, immunolabeling, wall appositions, papillae.

Ultrastructure of the host–parasite interface in the fern rusts Milesia, Uredinopsis, and Hyalopsora (Pucciniastraceae, Uredinales)

Species of the genera Milesia, Uredinopsis, and Hyalopsora possessed D-haustoria the necks of which were sheathed by an extension of the extrahaustorial matrix. Haustoria of the investigated species of Milesia and Uredinopsis were botryose with a haustorial body that formed wormlike protuberances. The Hyalopsora spp. were characterized by a vesicular haustorium. In the species of Milesia and associated Uredo spp. the haustorial neck was frequently differentiated into areas of variable electron density and structure; in Milesia blechni, Milesia miyabei, and Uredo RB 2537 a second neckband was formed in many haustoria. The penetration channels were usually characterized by short, perpendicularly oriented fibrillar elements that extended into the surrounding host cell wall. The haustoria of Uredinopsis filicina, Hyalopsora polypodii, and Hyalopsora aspidiotus were different from those of the investigated species of Milesia and Uredo. Their necks were accompanied by short extensions of the extrahaustorial matrix and the neck walls stained more or less homogeneously proximal to the neckband. Key words: Milesia, Uredinopsis, Hyalopsora, D-haustoria, ultrastructure, systematics.

In vitro formation of haustoria of the cowpea rust fungus Uromyces vignae in the absence of a living plant cell. II. Electron microscopy

The ultrastructure of infection hyphae, haustorial mother cells, and haustoria of Uromyces vignae formed on collodion membranes is described after conventional preparation and after treatment with periodic acid – thiocarbonhydrazide – silver proteinate or periodic acid – chromic acid – phosphotungstic acid treatments. Infection hyphae and haustorial mother cells developed normally in vitro but lacked an outer wall layer present in older infections in the host plant. Haustorium formation in vitro was accompanied by the development of elaborations of the plasmalemma along the infection hypha side of the haustorial mother cell septum, but their contents did not show identical staining responses to the haustorial neck wall as reported for other rust fungus species. Haustorial necks, and to a lesser extent haustorial bodies, were coated with a fibrillar material, the staining characteristics of which were similar to material normally considered part of the extrahaustorial matrix in infected plants. The restriction of this material to the haustorium suggests that it may play an important role in the interaction between the haustorium and the plant cell.

Ultrastructure of the haustorium of the peanut late leaf spot fungus Cercosporidium personatum

Data from scanning and transmission electron microscopic observations support light microscopic reports of the production of haustoria by the hemibiotrophic fungus Cercosporidium personatum. The trunklike base of the haustorium extended a short distance into the host cell where it formed three to five slightly thinner primary branches. These branches terminated in multiple, smaller, mostly opposite branch tips that gave the end of the haustorium a coralloid appearance. The morphology of this haustorium was distinctly different from the more extensively studied haustoria of various biotrophic fungi. Haustoria of C. personatum were observed in both living and dead host cells. In living cells an extrahaustorial matrix and extrahaustorial membrane separated the haustorium wall from the host cell protoplast. In dead cells the extrahaustorial membrane was absent. Haustoria in dead cells remained intact and appeared healthy.

Cytochemical studies on Puccinia graminis f. sp. tritici in a compatible wheat host. II. Haustorium mother cell walls at the host cell penetration site, haustorial walls, and the extrahaustorial matrix

Various cytochemical tests on the wheat stem rust fungus were used to determine differences in components of the walls of the haustorium mother cell at the host cell penetration site and the haustorial neck and body and to describe some of the chemical properties of the extrahaustorial matrix. There were two transition zones with respect to wall composition. The first was at the host cell penetration site; chitin, present in haustorium mother cell walls, was not detected in haustorial neck walls. The second transition zone was at the neck ring; compared with walls of the proximal neck region, those distal to the neck ring contained more protein and lost much of their periodate – thiocarbohydrazide – silver proteinate reactive material and all concanavalin A binding material after treatment with protease. The two wall layers of the distal part of the haustorial neck were continuous with those of the haustorium; the wall layers of young haustorial bodies shared their staining properties and lectin affinities with those of the distal part of the haustorial necks, reflecting their common origin. As the haustoria matured, their body walls bound wheat germ lectin, but the neck walls did not. Tests indicated that polysaccharide and glycoprotein were present in the extrahaustorial matrix.

An ultrastructural study of the late-blight fungus Phytophthora infestans and its interaction with the foliage of two potato cultivars possessing different levels of general (field) resistance

In the susceptible reaction of foliage of the cultivar Majestic there was extensive colonization of the host tissue prior to the onset of necrosis. The development of intracellular hyphae was confined to the initially parasitized palisade cells. Thereafter the fungus grew as intercellular hyphae, which penetrated host cells and formed haustoria. Haustorial morphology was highly variable, ranging in type from small spherical to much larger digitlike structures. Haustorial formation was preceded by the laying down of a moderately electron-dense penetration matrix bounded by the host plasmalemma. This material probably constituted the extrahaustorial matrix once haustorial development had taken place. The matrical material stained with silver proteinate reagent, and this reaction was blocked by dimedone, indicating that it was carbohydrate in nature. In the resistant reaction of foliage of the cultivar Shamrock, the epidermal cells rapidly became necrotic. Quantities of exceedingly electron-dense granules appeared in the necrotic host cells. Host organelles were no longer recognizable, but the fungal cytoplasm remained intact. In adjoining host cells, thin cell wall appositions were formed, which had a heterogeneous composition. On occasions when the fungus attempted to invade an underlying mesophyll cell, papillae usually formed in that cell at the site of incipient penetration.

Cytochemical specialization at the haustorial interface of a biotrophic fungal parasite, Albugo Candida

The haustorial apparatus of Albugo candida consisted of a narrow neck attached to a spherical body, which was packed with mitochondria. Covering the haustorial body was an electron-dense matrix which was bounded by the extrahaustorial membrane. This membrane, a continuation of the host cell plasmalemma, was highly convoluted with many short tentacles extending into the surrounding host cytoplasm. Single cisternae of host rough endoplasmic reticulum were always closely associated with the distal portion of the haustorial neck. Silver proteinate staining indicated that both the penetration jacket, a distinct zone surrounding the proximal part of the haustorial neck, and the extrahaustorial matrix were polysaccharide in nature, possibly complexed to other macromolecules such as proteins. The haustorial cell wall reacted only slightly with the silver proteinate, and in the distal region of the neck, there was no reaction. The fungal plasmalemma reacted strongly with the stain, except in the distal neck region, where there was no staining. With PACP, a staining procedure believed specific for the plasmalemma, the extrahaustorial membrane did not stain, whilst the host plasmalemma continuous with it did stain. The distal region of the haustorial neck wall also stained intensely with PACP clearly differentiating it cytochemically from the much lighter staining proximal neck and haustorial body walls. The penetration jacket stained more intensely than the extrahaustorial matrix with PACP, which might suggest a different chemical composition. Following incubation in 0.8 M sucrose osmoticum, the extrahaustorial membrane pulled away from the proximal neck wall, but remained tightly affixed to the distal neck wall, indicating that a tight junction existed between that membrane and the fungal cell wall in that region. Enzymic digestion with either cellulase or an endo-β-1,3-glucanase removed neither the haustorial body wall nor the extrahaustorial matrix. The significance of this finding is briefly discussed.

Etude ultrastructurale des relations hôte–parasite au cours de l'infection des pommes par le Phytophthora cactorum

An electron microscopic study was performed on haustoria of Phytophthora cactorum (L. et C.) Schroeter developed in tissues of two cultivars of apple fruits: a susceptible variety ('Golden delicious') and a resistant one ('Belle de Boskoop'). Ultrastructure of intercellular hyphae and some aspects of their penetration between contiguous host cells were described. A light dissolution of the host cell walls was observed. Ontogenic investigations indicated that in the susceptible host, the wall of the fungal haustoria was covered with a dense-stained extrahaustorial matrix. Its origin and its polysaccharide nature were demonstrated. On the other hand, the resistant host developed, immediately after the inoculation, a papilla which gave rise, later on, to a sheath enclosing adult haustoria. The role of these callosic structures in the phenomenon of resistance was discussed.