Sporangial Cytoplasm Research Articles

Ultrastructure of the intracellular parasite Parvilucifera sinerae (Alveolata, Myzozoa) infecting the marine toxic planktonic dinoflagellate Alexandrium minutum (Dinophyceae)

This ultrastructural study follows the infection by a parasite, Parvilucifera sinerae, of the marine toxic dinoflagellate Alexandrium minutum. In clonal cultures, infective zoospores, which are the infectious stage, entered host cells where they developed into sporangia. Mature, dark-colored sporangia were released from the dead dinoflagellate cells by the break-up of the host theca. The sporangial cytoplasm ultimately differentiated into many minute zoospores that escaped one host cell and subsequently infected healthy cells. The zoospores are 1.2–1.8 μm in diameter and possess an anterior (transverse) and shorter posterior flagellum in addition to alveoli, a refractile body, a mitochondrion with tubular cristae, micronemes, rhoptries, and a pseudoconoid. As in the other Parvilucifera species, the zoospore has a heteromorphic pair of central microtubules in the anterior axoneme and a reduced pseudoconoid. The micronemes have bulbous posterior ends and, as in other known species of the genus, there are (pseudo)conoid-associated micronemes. A comparison of these ultrastructural data with those available from the other two Parvilucifera species showed that, as in Parvilucifera infectans, the periphery of the nucleus is filled with thin fibers, in contrast to the syndinean-like nucleus of Parvilucifera prorocentri. Also, as in P. infectans, the transition region of the flagellar axoneme contains a dense structure, and the basal body a black globule; however, both structures are absent in P. prorocentri. There are no morphological differences of the zoospores from P. sinerae and P. infectans. The main difference between P. infectans and P. sinerae relay on the sporangium wall and ornamentation. The sporangium wall in P. sinerae consisted of three layers instead of two described layers in P. infectans. Therefore, morphologically, P. sinerae shows greater similarity to P. infectans than to P. prorocentri.

Novel protein kinase induced during sporangial cleavage in the oomycete Phytophthora infestans.

A study of the effect of inhibitors on zoospore development in Phytophthora infestans demonstrated the involvement of protein kinases and calcium and led to the discovery of a gene induced during zoosporogenesis that encoded a protein resembling Ca2+- and calmodulin-regulated serine/threonine protein kinases. The calcium channel blocker verapamil and the calmodulin antagonist trifluoroperazine inhibited zoosporogenesis and encystment. The protein kinase inhibitors K-252a and KN-93 inhibited zoospore release, encystment, and cyst germination, and K-252a reduced zoospore viability. In contrast, the inhibitors had minor or no effects on sporangia directly germinating in media. Spurred by these findings, a survey of putative protein kinase genes was performed to identify any that were up-regulated during zoosporogenesis. A kinase-encoding gene was identified for which mRNA accumulation was first detected soon after chilling sporangia in water, conditions that induce sporangial cytoplasm to cleave and release zoospores. The transcript persisted in motile zoospores and in germinated cysts but was not detected in other tissues, including hyphae, hyphae placed in water, or directly germinating sporangia. The structure of the predicted protein was novel, as its C-terminal region, which binds calmodulin in related proteins, was unusually short. Concentrations of actinomycin D previously used in experiments that suggested that de novo transcription was not needed for zoosporogenesis or encystment only partially inhibited transcription of the kinase gene, probably due to poor uptake into sporangia.

Cytoplasmic cleavage in living zoosporangia of Allomyces macrogynus.

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.

Microtubule organization during zoosporogenesis inAllomyces macrogynus

The microtubule cytoskeleton and cytoplasmic organization ofAllomyces macrogynus during zoosporogenesis was studied using light and electron microscopy. Indirect immunofluorescence methods revealed that the microtubule cytoskeleton progressed through three distinct stages of cytoplasmic distribution during zoospore development. During the first 10 minutes of zoosporogenesis, nuclei were strictly located in the periphery of the cytoplasm, and their associated centrosomes were positioned immediately adjacent to the plasma membrane. Microtubules emanated from centrosomes into the surrounding cytoplasm. Within 20 to 30 min after the induction of zoosporangial cleavage, nuclei migrated to new positions throughout the sporangial cytoplasm and microtubule arrays were primarily organized at and emanated from nuclear surfaces. During the final stage of zoosporogenesis, nuclear envelope-associated microtubules were not observed. Instead, primary organization of cytoplasmic microtubules returned to centrosomes (i.e., basal bodies) and flagella formation was evident. The MPM-2 antibody, which recognizes phosphorylated epitopes of several proteins associated with microtubule nucleation, stained centrosome regions throughout zoosporogenesis but did not stain nuclear envelopes.

Ultrastructure of Sporulating Bacillus larvae in a Broth Medium

An electron microscopic study of sporulation of Bacillus larvae, a honeybee pathogen, in TMYGP broth (D. W. Dingman and D. P. Stahly, Appl. Environ. Microbiol. 46:860-869, 1983) was conducted. No parasporal structures were evident in the sporangial cytoplasm. The stages of sporulation were similar to those observed in other sporeformers. A rather unusual inner coat layer consisting of seven lamellae was apparent.

Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.

Sporulating cells of Bacillus sphaericus 9602 containing fully engulfed forespores at different stages of maturity were broken by ultrasonic disruption, followed by grinding with alumina. In this way soluble enzymes derived mainly from the sporangial or from the forespore cytoplasms were obtained. Diaminopimelate ligase activity is required exclusively for cortical peptidoglycan synthesis, is absent during vegetative growth, and is synthesized during forespore maturation. It is found exclusively in the sporangial cytoplasm. L-lysine ligase is required for vegetative cell wall peptidoglycan synthesis but not for cortex synthesis. It is found in both fractions, but it has a fourfold higher specific activity in the forespore cytoplasm. Other enzymes that are required for synthesis of the nucleotide-pentapeptide precursors of both cortical and vegetative cell wall peptidoglycans are found in similar specific activities in both compartments. Mature spores, free of any residual sporangial material, have specific activities of all of these enzymes and of L-lysine ligase similar to those in forespores and in vegetative cells and are devoid of diaminopimelate ligase activity. Thus, the differential expression of at least one gene required for spore cortex synthesis in B. sphaericus occurs exclusively in the sporangial cytoplasm.

An ultrastructural study of zoosporogenesis inPythium proliferum de Bary

Zoosporogenesis in the oomycete,Pythium proliferum de Bary initially involves a condensation of cytoplasm at certain hyphal tips and the subsequent enlargement of these hyphal tips to form sporangia. Deposition of a septum at the base of the sporangium and initiation of an apical papilla are followed by cleavage of the sporangial cytoplasm. Packets of presumptive mastigonemes as well as flagella are recognized in the cytoplasm at this time. Subsequently, cleavage vesicles at the periphery of the sporogenic cytoplasm fuse with the plasmalemma thereby emptying their fibrous contents into the space between the sporogenic cytoplasm and the sporangial wall. It is felt that this fibrous material is instrumental in developing the internal pressure necessary within the sporangium to cause discharge of the sporogenic cytoplasm into an evanescent vesicle wherein delimitation of the zoospores is completed. The formation of the spore vesicle from the multilayered apical cap of the papilla is described here for the first time in the Oomycetes and a new term, “vesiculogen”, is suggested for this structure. Aspects of centriole replication within vegetative hyphae, papilla formation, and morphogenesis of various vesicular inclusions are also described in this study.

Nuclear degeneration in the myxomycetePhysarella oblonga

Nuclear degeneration is a distinct and normal process occurring during sporogenesis in the myxomycete,Physarella oblonga. About 30% of the nuclei present in the developing sporangium abort. Nuclei that undergo degeneration are first surrounded by vacuoles. The membranes of the vacuoles and nuclear envelope are 75 Ā thick. As degeneration progresses the vacuoles fuse resulting in a nucleus surrounded by a single, narrow vacuole. The membrane next to the nucleus degenerates while the membrane adjacent to the sporangial cytoplasm thickens to 100 Ā. Lysosomes then fuse with the vacuole, and digestion of the nucleus begins. The undigested remains of nuclei (residual bodies) are excreted from the protoplast. Acid phosphatase is localized in the lysosomes and degenerating nuclei.

Some Effects of Streptomycin on Pseudoperonospora humuli, the Downy Mildew of Hop

SUMMARY: Two sprays of streptomycin (1000 μg./ml.) considerably reduce sporulation of the hop downy mildew fungus, Pseudoperonospora humuli, on leaves of primary systemically infected shoots. Streptomycin markedly inhibited germination of its sporangia in vitro. Toxicity was dependent on sporangial concentration, incubation temperature and the cleanliness of the suspension but was independent of sporangial age. There were no marked differences in sensitivity to streptomycin between sporangia from five different isolates of Ps. humuli. Two commercial formulations of streptomycin were as inhibitory to sporangial germination as pure streptomycin sulphate. Sublethal concentrations of streptomycin interfered with cleavage of sporangial cytoplasm and increased the proportion of multiflagellate zoospores.The proportion of multiflagellate multinucleate zoospores liberated was dependent on streptomycin concentration. Germination of multiflagellate zoospores was observed. Streptomycin was very inhibitory to zoospore germination but had little effect on zoospore motility and germ-tube growth at concentrations which caused a high incidence of cyst lysis. Streptomycin was as toxic to sporangia of Phytophthora infestans as to those of Ps. humuli, whereas sporangia of Plasmopara viticola and Peronospora parasitica were less sensitive to the antibiotic.

Electron microscopy of the sporangium of Phytophthora capsici

This paper reports results of a study on the ultrastructural cytology of sporangia of Phytophthora capsici Leonian with emphasis on flagellum formation, general sporangial structure during zoospore cleavage, and the presence, structure, and transition of cytoplasmic organelles and inclusions during these processes.Non-cleaving sporangial cytoplasm contains a high concentration of ribosomes, mitochondria, vacuoles, lipid inclusions, and endoplasmic reticular cisternae scattered throughout the cytoplasm. Nuclei in mature sporangia are located at the periphery of the cell, with their narrow poles aligned towards the plasma membrane. The apical papilla measures 4–5 × 10 μ, and is initiated as a fibrous third layer under the two-layered cell wall several microns from the apex. The outer wall layer surrounds the papilla while the inner wall narrows and disappears near the crown. The basal septal plug is a combination of the inner wall layer and slime substances.One of the first structural changes in the cytoplasm during zoospore cleavage is the genesis of the flagellar apparatus. Paired centrioles next to the narrow poles of the nuclei elongate to form kinetosomes which extend through the cytoplasm toward receptive axonemal vesicles. Axonemes then form in the axonemal vesicles. The terminal plate and its prisms account for the appearance of the axoneme when it forms above the terminal plate in the axonemal vesicle. The axonemal cylinder has a typical 9 + 2 morphology and the axonemal sheath is continuous with the axonemal vesicle tonoplast. The nucleus is an integral part of the flagellar apparatus and appears to be connected to the kinetid (axoneme + kinetosome) base via microtubules. Golgi dictyosomes occur in the sporangia during all stages of growth and may be responsible for elaboration of needed membranes during zoospore production. Osmiophilic droplets (liposomes), located within vacuoles, are a predominant feature of precleavage cytoplasm. These globules are probably lipid in nature. As cleavage begins, the liposomes become less opaque at the margins, and striations appear, eventually encompassing all or most of the liposomes at the time of cleavage. The liposomes then become less spherical and expand, filling the vacuoles. Electron-transparent regions eventually appear throughout the liposomes and the vacuolar membrane may disappear.Cleavage of the cytoplasm into zoospores occurs by the alignment and fusion of cleavage vesicles around individual nuclei. During this period organelles migrate to these centers. The cleavage vesicles coalesce with each other and the axonemal membranes, eventually becoming the plasma membranes of the daughter zoospores.

ULTRASTRUCTURAL CHANGES IN DIRECTLY GERMINATING SPORANGIA OF PHYTOPHTHORA PARASITICA

Flagella and cleavage vesicles form during the initial stages of direct germination in the same manner as they do in indirect germination. Later, however, the flagella degenerate and cleavage of the cytoplasm is not complete. Instead, a new wall layer is deposited onto the existing sporangial wall, and this germination wall extends with the germ tube and forms the hyphal wall. Material for the germination wall first appears around vesicular aggregates concentrated at the periphery of the sporangial cytoplasm. As the wall forms the vesicular aggregates become encased in it, and the wall eventually consists of irregular deposits of wall material interspersed with the vesicular aggregates. These are clearly cytoplasmic in nature and different from lomasomes, since they are associated with ribosomes and other small elements of the cytoplasm. Likely to contribute to the wall formation are the endoplasmic reticulum, microbodies, dictyosome‐derived vesicles, and possibly polyvesicular bodies and larger vacuoles with fibrillar contents. The basal plug of the sporangium forms the same way as the germination wall and also contains numerous vesicular inclusions. Fewer vesicular inclusions are found in the sporangial wall, and none have been observed in the walls of the growing hyphae. This difference in wall structure can be explained on the basis of their different growth patterns. Penetration of the sporangial plug is concomitant with prominent lomasomal activity and the cytoplasm at the hyphal tip is characterized by the presence of numerous vesicles, which are probably derived from dictyosomes.

Ultrastructural Changes in Directly Germinating Sporangia of Phytophthor parasitica

Flagella and cleavage vesicles form during the initial stages of direct germination in the same manner as they do in indirect germination. Later, however, the flagella degenerate and cleavage of the cytoplasm is not complete. Instead, a new wall layer is deposited onto the existing sporangial wall, and this germination wall extends with the germ tube and forms the hyphal wall. Material for the germination wall first appears around vesicular aggregates concentrated at the periphery of the sporangial cytoplasm. As the wall forms the vesicular aggregates become encased in it, and the wall eventually consists of irregular deposits of wall material interspersed with the vesicular aggregates. These are clearly cytoplasmic in nature and different from lomasomes, since they are associated with ribosomes and other small elements of the cytoplasm. Likely to contribute to the wall formation are the endoplasmic reticulum, microbodies, dictyosome-derived vesicles, and possibly polyvesicular bodies and larger vacuoles with fibrillar contents. The basal plug of the sporangium forms the same way as the germination wall and also contains numerous vesicular inclusions. Fewer vesicular inclusions are found in the sporangial wall, and none have been observed in the walls of the growing hyphae. This difference in wall structure can be explained on the basis of their different growth patterns. Penetration of the sporangial plug is concomitant with prominent lomasomal activity and the cytoplasm at the hyphal tip is characterized by the presence of numerous vesicles, which are probably derived from dictyosomes.

Changes in the ultrastructure of sporangia of Phytophthora infestans associated with indirect germination and ageing

The ultrastructure of sporangia of Phytophthora infestons (Mont.) de Bary has been studied using young sporangia before and during indirect germination as well as old sporangia incapable of germinating indirectly. The sporangial wall is single-layered, having an apical pore blocked by a papilla into which the sporangial wall is inserted. The papilla is laminated and on the basis of enzyme studies it is suggested that it is composed of protein and pectin compounds. The sporangial cytoplasm contains structures such as have been recorded for other fungi. Storage vesicles and vesicles containing flagella are included, the latter being present before the induction of indirect germination. When indirect germination of sporangia is induced by chilling, the sporangial wall remains unaltered, but a new granular and vesicular layer appears between the papilla and the plasma membrane. It is suggested that this layer contains enzymes which degrade the papilla before zoospores are discharged. Degradation of the papilla is preceded by a rapid cleavage process, the lines of cleavage being composed of membranes and small vesicles. Old sporangia which are unable to germinate indirectly possess organelles similar to those of young sporangia. With ageing, a large vacuole appears and in some old sporangia a dense, non-vesiculated region occurs around the vacuole. Mitochondria may accumulate near the boundary between vesiculated and non-vesiculated regions. Ageing is also associated with an increase in osmo-philic particles. Flagella were never observed in old sporangia.

ULTRASTRUCTURAL CHANGES DURING ZOOSPORE FORMATION IN PHYTOPHTHORA PARASITICA

Cleavage of the multinucleate sporangial cytoplasm begins by a rearrangement and subsequent fusion of randomly dispersed cleavage vesicles. The vesicles line up in planes equidistant from neighboring nuclei and along the sporangial wall. In addition, they contribute to the enlargement of the central vacuole. Fusion of these vesicles with themselves and with the central vacuole cleaves the cytoplasm into uninucleate zoospores, each with two flagella. The sporangial wall consists of two layers, an outer thin one which is continuous over the plug of the discharge pore and an inner thick one which tapers off near the plug. The plug consists of fibrillar material and is ejected upon release of the zoospores. A plug‐like structure separating the forming sporangium from the hypha has a homogeneous matrix pervaded with an anastomosing network of fine electron‐dense channels. In addition, glycogen‐like granules occur within mitochondria and paired structures are interpreted as procentrioles.

芽胞形成菌におけるジピコリン酸生合成部位に関する研究

The participation of sporangial cytoplasm in dipicolinic acid biosynthesis was studied. When lysozyme was added to sporulating culture of B. subtilis at various stages, the further synthesis of dipicolinic acid was immediately and almost completely inhibited. It was also found that, when isolated forespores were incubated in culure supernatant obtained from sporulating culture, dipicolinic acid was not synthesized.The changes in the distribution of the diketopimelate oxidation system in cells were studied. The oxidative activity was found in the soluble fraction, and not in the residue fraction during all stages of sporulation. The activity was not detected in mechanical disintegrates of forespores and also in lysozyme lysates of the vegetative cells, and the activity suddenly emerged just before the onset of sporulation. The active substance was supposed to be low molecular organic substance by qualitative analyses. From the above results, it was concluded that the cytoplasm surrounding the developing spore is required for dipicolinic acid synthesis.