The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.

Abstract. The penetration hypha of basidiospore-derived infection structures of the cowpea rust fungus (Uromyces vignae Barclay) in epidermal cells of the nonhost, broad bean (Viciafaba L.), was studied with the electron microscope after high-pressure freezing and freeze substitution. After fungal invasion of the epidermis, a plug in the penetration hypha separated the infection structures on the cuticle from the intraepidermal vesicle of the fungus. The plug and the fungal cell wall reacted with a polyclonal 1,3-13-glucan antibody. The plug in the haploid stage seems to have a task similar to the septum formed in the diploid stage of the fungus. Around the penetration hypha, the plant wall stained darkly and a papilla was deposited by the plant. In the papilla, 1,3-]3-glucans were labelled by a monoclonal and a polyclonal antibody. In the infected epidermal cell, clathrin-coated pits, coated vesicles, partially coated reticula and multivesicular bodies were found. The contents of the coated pits, coated vesicles, partially coated reticula and multivesicular bodies bound to monoclonal and polyclonal 1,3-f3-glucan antibodies. Accumulation and uptake of this paramural material into the plant cell by endocytosis is concentrated at the fungal penetration site. It may influence the host-parasite interaction.

SUMMARYLiving epidermal cells of cowpea (Vigna unguiculata (L.) Walp.) were examined by contrast-enhanced video microscopy during penetration by invasion hyphae of the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). In resistant or susceptible host cvs, the plant nucleus migrated to the penetration site before the fungus had fully penetrated the plant wall and, at sites of unsuccessful infection, remained during the formation of a callose-containing papilla. Nuclear migration was also induced by applying hemicellulase, but not HgOg, to localized sites of wall damage. Hemicellulase-induced migration was inhibited by calcium chelators and a protein kinase inhibitor, but not catalase. In both resistant and susceptible cvs, the plant nucleus migrated away fronn successful infection sites at about the time that the fungal penetration peg made contact wdth the plant plasma membrane, and the epidermal cell showed no further cytological responses to the growth of the fungal intraepidermal vesicle. In the susceptible cv., the nucleus migrated back to the fungus when the latter initiated tip growth. Inhibitors of transcription or translation did not affect this migration and only slightly reduced fungal growth. In the resistant cv. in which the invaded cell exhibited a hypersensitive response (HR), the plant nucleus changed its appearance before the cessation of cytoplasmic streaming and usually did not migrate to the fungus, even if the latter initiated tip growth. Nuclear DNA cleavage usually followed the subsequent cessation of cytoplasmic streaming. Treatments that delayed cell death and increased fungal growth also increased the frequency of nuclear migration to the fungus. It is argued that these and other data suggest that U. vignae negates nonspecific, penetration-induced, defence responses upon entering cells of both susceptible and resistant cultivars. The results also suggest that effects on the plant nucleus are one of the earliest signs of the HR in this system, often preceding the cessation of cytoplasmic streaming and detectable changes in plasma membrane permeability.

Basidiospore germlings of the cowpea rust fungus (Uromyces vignae) penetrate the epidermal cell wall of the nonhost plant Vicia faba. In order to characterize the wall structure of the penetration site, leaves were high pressure frozen, freeze substituted, and embedded in appropriate resins. With antibodies against epitopes present in pectin, polygalacturonic acid, xyloglucan, and callose, we studied the modification of these wall components during infection. The density of epitopes was determined at the penetration site and compared with noninfected areas of the epidermal wall. Along the fungal penetration hypha, a zone of the plant wall, 0.2 µm wide, exhibited a reduced density of pectin and xyloglucan epitopes. A similar reduction of epitope density was also found for xyloglucan after treatment of sections from noninoculated plants with cellulase and xylanase and for pectin after treatment with pectinase. The density of polygalacturonic acid epitopes remained unchanged in the outer layer of the epidermal wall, but increased over the inner layer. A high density of polygalacturonic acid epitopes was found over a collarlike wall apposition produced by the plant cell along the penetration hypha. These results indicate that the fungus degrades the plant cell wall at the penetration site and that the plant cell secretes new wall material into this area to form the wall apposition.

The infection process of basidiospores of the cowpea rust fungus (Uromyces vignae) was studied on living host (Vigna sinensis) and nonhost (Vicia faba) leaves using light microscopy with differential interference contrast optics. During the first 8 h, fungal development was similar on host and on nonhost leaves. Penetration and production of intraepidermal vesicles occurred in nonhost cells 4 h earlier than in host cells. Penetration frequency was also higher in nonhost leaves. These results suggest that the cuticle of the cowpea plant delays basidiospore infection. Both host and nonhost cells produced cytoplasmic aggregates during appressorium formation and again, a few hours later, during penetration of the epidermal cell wall. Cytoplasmic aggregates were also observed in cells adjacent to a collapsing cell. Papillae were observed at most infection sites in both host and nonhost cells. The nuclei of infected cells migrated towards the penetration site in both plant–pathogen combinations. Nuclear size increased considerably only in the nonhost epidermis and decreased again markedly after cell collapse. In the nonhost cells, three types of defence reactions occurred during or after formation of the intraepidermal vesicle. First, following the halt of cytoplasmic streaming, the cytoplasm of the invaded cell either contracted or disintegrated into granules. Alternatively, the cytoplasm continued streaming but darkly pigmented material encased the fungal infection structure. Key words: basidiospore, broad bean (Vicia faba), cowpea (Vigna sinensis), cowpea rust fungus (Uromyces vignae), hypersensitivity, nonhost resistance.