Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus

Abstract

We have used the vital fluorescent dye, FM4-64, as a marker of membrane development during zoospore formation in living zoosporangia of Allomyces macrogynus. Membrane development was visualized and documented using standard epifluorescence and laser scanning confocal microscopy. Video-enhanced light microscopy and transmission electron microscopy, using cryopreparation methods, were also employed in this study. In the first 10-12 min after the induction of zoospore formation, only the plasma membrane labelled with FM4-64. During this time, nuclei were strictly located in the cortical cytoplasm with their associated centrosomes positioned immediately adjacent to the plasma membrane (Lowry & Roberson, 1997). Between 12 and 20 min post-induction, increased fluorescence appeared along regions of the plasma membrane adjacent to the nuclei. From these sites, membranes (i.e. cleavage elements) extended laterally within the cortex and then, in conjunction with nuclear migration, rapidly elongated into the sporangial cytoplasm. By 25-35 min post-induction, cleavage elements had ramified throughout the cytoplasm forming a complex, interconnected membranous network. Transmission electron microscopy revealed that cleavage elements were paired membrane sheets with a lumen consisting of an electron opaque, granular matrix. Cleavage elements developed into a highly ordered network by 35-40 min post-induction, which fully delimited zoospore initials into polyhedral-shaped cells. Zoospore discharge occurred between 40 and 50 min post-induction. Our results have shown that cleavage elements undergo four stages of development during zoospore formation in A. macrogynus: (i) development of membrane initials, (ii) cortical extension, (iii) cytoplasmic elongation and ramification and (iv) zoospore initial delimitation.

The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola

“…1b), there is no indication that the long lag-phase is caused by nuclear divisions. However, preliminary electron-microscopical studies of mature sporangia indicate that the nuclei are not separated by plasma membranes, consistent with findings in other oomycetes such as Phytophthora cinnamomi (Hyde and Hardham 1993) and Allomyces macrogynus (Fisher et al 2000). The sporangium therefore represents a syncytium, and during the period prior to zoospore release the individual zoospores have to be separated from each other.…”

Cytokinesis of the binucleate zoosporangia of Allomyces macrogynus

“…In a higher magnification, these membranes responsible for the cleavage of cytoplasm were observed as paired membranes (Fig. 1B) and morphologically equivalent to the cleavage membranes described in the study of the cytoplasmic cleavage of multinucleate zoosporangia of A. macrogynus (Fisher et al, 2000;Lowry et al, 2004). Paired membranes could be easily distinguished from other membranous structures even at low magnification by their thickness, dark staining, smooth surface, and resistance to ice damage.…”

“…It has been reported that the fluorescent, lipophilic styryl dye FM4-64 was highly specific in staining the plasma membrane and the cleavage elements during the development of multinucleate zoosporangia of A. macrogynus (Fisher et al, 2000). FM4-64 also stained the plasma membrane and the internal membranes of the binucleate zoosporangia (Fig.…”

“…Vesicles that might be responsible for the formation of cleavage membrane were not identified as reported in a study of the multinucleate zoosporangia (Fisher et al, 2000).…”

“…As one example, asexual reproduction in the phylum Blastocladiomycota, a group of true fungi (James et al, 2006), is achieved through the development of multinucleate zoosporangia which discharge uninucleate zoospores after cytoplasmic cleavage (Alexopoulos, 1962). Cleavage of cytoplasm within the zoosporangia has been studied intensively in Allomyces macrogynus, a member of Blastocladiomycota (Barron and Hill, 1974;Lowry and Roberson, 1997;Lowry et al, 1998Lowry et al, , 2004Fisher et al, 2000) and these studies revealed the behavior of nuclei, the patterns of the development of new membranes, and the role of cytoskeletons associated with the cleavage of cytoplasm. Inherent complexities associated with the multiple divisions of cytoplasm within the zoosporangia, however, made it difficult to understand when the division sites are specified and how the membrane develops at the division sites.…”