Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.

Living 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 H2O2, 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 from successful infection sites at about the time that the fungal penetration peg made contact with the plant plasma membrane, and the epidermal cell showed no further cytological responses to the growth of the fungal intra-epidermal 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.