Cytoplasmic vesicles in germinating spores ofGilbertella persicaria

Summary Germ tube apices ofGilbertella persicaria contain cytoplasmic vesicles, similar to the secretory vesicles found at the tips of vegetative hyphae. The vesicles are present at all stages of development, from the time of germ tube initiation to the establishment of branched hyphae. In contrast to the abundant vesicles at tips of established hyphae, the germ tubes have only a few apical vesicles in a layer next to the plasma membrane. When germinated spores are treated by washing and centrifuging prior to fixation, the cytoplasm is often disrupted near the apex, and the clusters of apical vesicles disappear. The findings indicate the delicate nature of hyphal tips and the necessity of avoiding prefixation stresses in order to preserve the apical apparatus of growing hyphae.     

Endophyte differentiation inCasuarina actinorhizae

Summary This is an ultrastructural study of development of infected cells in nitrogen fixing root nodules ofCasuarina spp. While several aspects of development are similar to those found in many other actinorhizae, unusual aspects of development of the host cell and differentiation of the endophyte inCasuarina are correlated with unusual changes in the wall of the infected cell. Instead of vesicles the endophyte forms atypical hyphae in mature infected cells. These unusual hyphal forms are termed intracellular hyphae. Intracellular hyphae are nonseptate hyphae which originate and terminate within the same host cell, and have a varying diameter and a multidirectional growth and branching pattern. A laminate surface layer previously undescribed on hyphae ofFrankia is a feature common to mostCasuarina endophytic hyphae and is probably similar chemically to the laminae comprising the multilamellate envelope of endophytic vesicles in other actinorhizae.This paper is Florida Agricultural Experiment Station Journal Series No. 7350.     

Fine structure of actinorhiza of theRhamnaceae growing in Chile I.Talguenea quinquenervia (Gill et Hook)

Summary Root nodulesTalguenea quinquenervia Gill et Hook (Rhamnaceae) are restricted to the middle region of the root cortex. The root endophyte possesses hyphae which are septate and vesicles. The vesicles are spherical and are continuous with that of the hyphae. The endophyte fine structure is similar to otherFrankia-induced root nodules.     

Intravacuolar membrane lysis in Saccharomyces cerevisiae. Does vacuolar targeting of Cvt17/Aut5p affect its function?

The integral membrane protein Cvt17/Aut5p is a putative lipase essential for intravacuolar lysis of autophagic bodies. It is localized at the endoplasmic reticulum, from which it is targeted via the multivesicular body (MVB) pathway to intravacuolar MVB vesicles. Proteinase protection experiments now demonstrate that the Aut5 amino terminus is located in the cytosol, and the carboxyl terminus is located inside the ER lumen. In contrast to procarboxypeptidase S, targeting of Cvt17/Aut5p to MVB vesicles is not blocked in cells lacking the ubiquitin ligase Tul1p or the deubiquitinating enzyme Doa4p. Also, truncation of the amino-terminal cytosolic Cvt17/Aut5p domain does not inhibit its targeting to MVB vesicles. These findings suggest that similar to Sna3p sorting of Cvt17/Aut5p to MVB vesicles is independent of ubiquitination. By fusing the ER retention/retrieval signal HDEL to the carboxyl terminus of Cvt17/Aut5p, we generated a construct that is held back at the ER. Detailed analysis of this construct suggests an essential role of vacuolar targeting of Cvt17/Aut5p for its function. Consistently, aut5Delta cells are found impaired in vacuolar degradation of autophagocytosed peroxisomes. Importantly, biochemical and morphological data further suggest involvement of Cvt17/Aut5p in disintegration of intravacuolar MVB vesicles. This points to a general function of Cvt17/Aut5p in intravacuolar membrane breakdown.     

Fine Structures Associated with Nutrition of the Intracellular Parasite Eimeria auburnensis*

SYNOPSIS. In the nearly mature macrogametes of Eimeria auburnensis, the cell membrane is a unit membrane, with underlying and overlying osmiophilic layers usually present. Cup-shaped micropores were occasionally seen. Smaller, V-shaped invaginations were also found in considerable numbers at the surface. At the deepest point, these invaginations were bounded only by a unit membrane. Immediately adjacent to this point, vesicles with homogenous electron-pale contents bounded by a similar unit membrane, were frequently seen. Pinocytosis evidently occurs at the site of these invaginations. Numerous folds of the host cell membrane bordering the vacuole in which the parasite lay extended about 0.1–0.7 μ into the vacuole. These “intravacuolar folds” varied in depth and number in different specimens. In some, the majority of folds had apparently become disconnected from the host cell membrane. A highly developed smooth endoplasmic reticulum occurred in the adjacent host cell cytoplasm. The intravacuolar folds may assist in transfer of nutrients, including membrane material, from the host cell to the parasite. The evidence indicates that in this species of Eimeria nutrients are taken into the parasite primarily as fluids by pinocytosis and possibly other processes.     

Fine structure of the host-fungus interface in orchid mycorrhiza

Summary Electron microscopy of protocorms of Dactylorhiza purpurella infected with a symbiotic Rhizoctonia sp. showed that the intracellular hyphae examined did not penetrate the plasmalemma of the host cell. Walls of hyphae within cells bore many hemispherical protuberances over which the host plasmalemma was closely pressed. we estimate that these protuberances would increase the area of contact between hyphae and host plasmalemma by about 15%. They were not found on hyphae growing on agar. Except for these protuberances, and some vesicles or tubules which invaginated the fungus plasmalemma, no other structures were seen which could be suggested to be adaptations to transport across the living fungus-host interface.     

Electron microscopy of the endophyte ofAlnus glutinosa

Earlier light microscopic investigations have revealed that the endophyte ofAlnus glutinosa presents itself in three different forms. In the present study this is confirmed by electron microscopy; also, new data on the cytology of the endophyte have been obtained.The host cells are primarily infected by the hyphal form of the endophyte. A plant cell nucleus and mitochondria can be found in the infected host cells.In the majority of the infected cells, so-called vesicles develop at the tips of the hyphae. Electron micrographs show that these vesicles, as well as the hyphae, are surrounded by the host-cell cytoplasmic membrane. The endophyte cytoplasm inside the vesicles is divided in all directions by cross walls, many of which are incomplete. Plasmalemmosomes are conspicuous. Some vesicles look vigorous but others shrunken or nearly devoid of cytoplasm as if being digested.A minority of host cells situated between the vesicle-containing ones are completely filled by bacteria-like cells. These host cells, in contrast to the other ones, do not contain a nucleus nor mitochondria, nor are the endophyte cells in them enveloped by a host cell cytoplasmic membrane: these host cells are dead. Vesicles are not found in these cells.It is inferred that a living host cell exerts a stimulus on the endophyte to which the latter responds by forming vesicles at the tips of the hyphae. At a later stage the host cells digest the vesicles and the hyphae. On the other hand, if a host cell does not survive the infection, the hyphae divide into bacteria-like cells, which are not digested owing to the absence of host cytoplasm.According to the cytology of the hyphae, the endophyte is an actinomycete.The cytology of the endophyte needs further elucidation. Its plasmalemmosomes, or membranous bodies connected with the cytoplasmic membrane, are beautifully developed. The striated bodies described on p. 359 under 4) may be a new feature, which may turn up in other actinomycetes or bacteria.     

Arbuscular mycorrhizal structure and fungi associated with mosses

We investigated the colonization and diversity of arbuscular mycorrhizal (AM) fungi associated with 24 moss species belonging to 16 families in China. AM fungal structures, i.e. spores, vesicles, hyphal coils (including intracellular hyphae), or intercellular nonseptate hyphae, were found in 21 moss species. AM fungal structures (vesicles, hyphal coils, and intercellular nonseptate hyphae) were present in tissues of 14 moss species, and spores and nonseptate hyphae on the surface of gametophytes occurred in 15 species. AM fungal structures were present in 11 of the 12 saxicolous moss species and in six of the ten terricolous moss species, but absent in two epixylous moss species. AM fungal structures were only observed in moss stem and leaf tissues, but not in rhizoids. A total of 15 AM fungal taxa were isolated based on trap culture with clover, using 13 moss species as inocula. Of these AM fungi, 11 belonged to Glomus, two to Acaulospora, one to Gigaspora, and one to Paraglomus. Our results suggest that AM fungal structures commonly occur in most mosses and that diverse AM fungi, particularly Glomus species, are associated with mosses.     

Isolation and ultrastructural identification of membranes from the fungusGilbertella persicaria

Summary Methods are described for isolating and identifying subcellular membranes from walled hyphae ofGilbertella persicaria. Differences in thickness and symmetry of membranes and in contents of vesicles were used to distinguish different types of membranes. Mitochondria, vacuoles, plasma membrane, and vesicles with attached ribosomes from homogenized germlings equilibrated at the 1.2/1.4 M interface in discontinuous sucrose gradients. Accelerated flotation in centrifuged Ficol-sucrose gradients resulted in the additional separation of the mixed membranes into three fractions: one contained predominantly intact mitochondria, another was composed of vacuoles and vesicles coated with ribosomes, and a third was enriched in plasma membranes. Based upon morphometric analysis, these fractions contained 92% mitochondria, 53% vacuoles, and 89% plasma membranes, respectively. The source of vesicles coated with ribosomes was investigated since rapidly growing hyphae ofG. persicaria contained little rough endoplasmic reticulum as compared with other classes of membranes. Reconstruction from electron micrographs of mitochondrial fragmentation and vesiculation suggested that most of the ribosome-coated vesicles originated from disrupted mitochondria rather than from rough endoplasmic reticulum. The study demonstrates the utility of ultrastructural markers to identify membranesin vitro independent of, or as an adjunct to, cytochemical and biochemical markers.     

Ca Shuttling in Vesicles During Tip Growth in Neurospora crassa

Tip-growing organisms maintain an apparently essential tip-high gradient of cytoplasmic Ca²⁺. In the oomycete Saprolegnia ferax, in pollen tubes and root hairs, the gradient is produced by a tip-localized Ca²⁺ influx from the external medium. Such a gradient is normally dispensable for Neurospora crassa hyphae, which may maintain their Ca²⁺ gradient by some form of internal recycling. We localized Ca²⁺ in N. crassa hyphae at the ultrastructural level using two techniques (a) electron spectroscopic imaging of freeze-dried hyphae and (b) pyroantimoniate precipitation. The results of both methods support the presence of Ca²⁺ in the wall vesicles and Golgi body equivalents, providing a plausible mechanism for the generation and maintenance of the gradient by Ca²⁺ shuttling in vesicles to the apex, without exogenous Ca²⁺ influx. Ca²⁺ sequestration into the vesicles seems to be dependent on Ca²⁺–ATPases since cyclopiazonic acid, a specific inhibitor of Ca²⁺ pumps, eliminated all Ca²⁺ deposits from the vesicles of N. crassa.     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

Ultrastructural Studies of the Intercellular Hypha aud Haustorium of Puccinia recondita f.sp. tritici

The ultrastructure of intercellular hyphae and D-hati-storia of P. recondita f.sp. tritici, and the host response to haustorial invasion, was investigated. The intercellular hyphae share common characteristics with those of other uredial stage rust fungi. Anastomosis was observed between intercellular hyphae. Two nucleoli were frequently observed in a single nucleus in the haustorium, indicating possible nuclear fusion between the two nuclei in D-haustoria of this fungus. The close association of host organelles, such as the nucleus, Golgi bodies, endo-plasmic reticulum, vesicles and mitochondria, with the developing haustorium, is described.     

A morphological study of the mycorrhiza ofEntoloma clypeatum f.hybridum onRosa multiflora

Mycorrhizas ofEntoloma clypeatum f.hybridum onRosa multiflora in the field in Japan were studied by stereo, light and electron microscopy. In most mycorrhizas, the root cap, meristem, and apical region of the cortex disappeared, but in a few mycorrhizas, these tissues remained. Fungal hyphae of the mycorrhizas invaded root tissues and branched palmately. Hyphae in contact with cortical cells were larger than those far from the root cells and contained many mitochondria, cisternae of endoplasmic reticulum and transitional vesicles. Invading hyphae were undulate in the apical part of the mycorrhiza, and some of them lacked distinct organelles. Electron-dense granules accumulated in the root cells adjacent to the fungal hyphae. Both the remnants of the plant cells and the fungal hyphae were included in the amorphous materials on the tip of the stele. These observations suggest the destructive infection by fungal hyphae of the root cells and their collapse near the tip of the stele.     

Occlusion of the septal pores of damaged hyphae ofNeurospora crassa by hexagonal crystals

Summary Hyphae ofNeurospora crassa were damaged by flooding colonies with water or cutting them with a razor blade. Loss of cytoplasm from damaged hyphae was reduced by the rapid occlusion of septal pores by hexagonal crystals. Re-growth of these hyphae occurred by the formation of intra-hyphal branches from the plugged septa, the production of side branches just below the septa, or the production of multiple branches from the lateral walls. Small vesicles accumulated behind plugged septa prior to branch production.     

Identification of the endophypte ofDryas andRubus (Rosaceae)

Summary Root nodules ofDryas drummondii are of the coralloid type (Alnus type). The endophyte is present in the middle cortical cells of the root-nodule tissue. Transmission electron micrographs revealed an actinorhizal endophyte with septate hyphae and non-septate spherical or ovoid vesicles. Vesicles always possess at the base a septum; septa formation in the endophyte is always associated with the presence of mesosomes. Branching of the endophyte is not necessarily correlated with septum formation. Hyphal structures are more prominent in the apical part of the root nodule and vesicles are more numerous in a broad zone below this. In the middle and towards the base of the root nodule the endophytic structures appear in a stage of disintegration. Vesicles appear in a broad region near the periphery of the host cell and regularly show no strict orientation towards the host-cell wall. In the center of the host cells only hyphae occur. In the intercellular spaces between the host cells theFrankia endophyte produces spore-like structures although the outline of the sporangia is often faint.The coralloid root ofRubus ellipticus shows characteristically a basal rootlet initiated below the dichotomous branching of the nodular lobes, but extending beyond the root nodule. The endophyte is only present in the outer cortex of the root nodule in a 1–2 cell wide layer. This endophytic layer is bounded, internally as well as externally, with a 4–5 cell wide layer of tannin-filled host cells. The implications of this situation are discussed. Tannin-filled cells occur regularly inRubus species and their arrangement has been used for taxonomic purposes within the genus. TheRubus endophyte is aFrankia species with septate hyphae and distinctly septate spherical vesicles. The ultrastructure of the vesicles of theRubus endophyte is very similar to that of theAlnus endophyte.     

甜菊愈伤组织细胞中的液泡膜内突和液泡内囊泡

对生长在分化培养基上的甜菊愈伤组织分生区细胞的液泡膜内突和液泡内囊泡,进行了超微结构和酸性磷酸酶细胞化学研究。在不同液泡化时期的细胞中,都存在不同大小和形态的液泡膜内突,它们有的缺乏明显的内含物;有的含有许多小泡或复杂膜系;有的含有一个较大的具许多小泡或复杂膜系的膜束缚囊泡。在液泡内还存在一些游离的液泡内囊泡,它们通常具有两层紧贴的界膜或为多层同心膜,推测它们来自液泡膜内突。ＡｃＰａｓｅ定位结果显     

Fine cytochemical detection of acid mucopolysaccharides in growing hyphae of Neurospora crassa

Summary The presence of mucopolysaccharides (MP) in Neurospora crassa hyphae was detected in samples of cultures from 4 h to 5 days.Using a modification of the method of Rothman (1969), acid MP were identified by electron microscopy in vesicles located at the apex, septae, and throughout the cytoplasm of the hyphae. The size, number, and localization of these vesicles was related to the stage of growth of the fungus.The mechanisms of release and transport of these macromolecules are compared to the process of secretion in cells endowed with a better developed endoplasmic reticulum and Golgi system.The specificity of the method was evaluated histochemically. The structures displaying greater electron density corresponded to the Alcian blue positive areas.     

Effects of nocodazole and brefeldin A on microtubule cytoskeleton and membrane organization in the homobasidiomyceteSchizophyllum commune

Summary The effects of nocodazole and brefeldin A (BFA) on the growth of dikaryotic hyphae inSchizophyllum commune corresponded with the development of abnormal structures in the apical region of treated hyphae. Microtubules (MTs) were totally depolymerized after 1 h nocodazole treatment, which correlated with strong branch formation in the apical cells. One reason for branching could be the shift in the position of apical vesicles from the center to the side of the tip, observed in some nocodazole-treated hyphae. After 2 h growth in the presence of nocodazole the apical cells had malformed or swollen tips, or tips of normal shape but containing only a few apical vesicles. After 0.5 h treatment with BFA, almost all the leading hyphae had swollen apical parts in which the endoplasmic reticulum (ER) formed an interconnected network and perturbed Golgi particles were found. The orientation of MTs in the BFA-treated hyphae often followed that of the interconnected ER network, which suggested an association between MTs and ER. The results of the experiments with nocodazole suggest that, in filamentous homobasidiomycetes the subtle organization of cytoplasm necessary for the polar growth at the apex is maintained only in the presence of an intact MT cytoskeleton. The BFA experiments indicated that the secretion pathway inS. commune is sensitive to BFA. In addition rapid change in apical morphology in the BFA-treated hyphae emphasizes the importance of correct orientation of components of the secretory pathway for normal apical growth to continue.Abbreviations BFA brefeldin A - EM electron microscopy - ER endoplasmic reticulum - IIF indirect immunofluorescence - MBC methylbenzimidazole-2-ylcarbamate - MT microtubule - MVB multivesicular body - RER rough endoplasmic reticulum     

Anatomical Structures of the VA Mycorrhiza in the Apocynaceae (Gentianales)

Roots of 19 Apocynaceae species were studied anatomically with respect to their symbiosis with VAM-fungi. In plants collected from the field, VAM-fungi were established in the root cortex. Also, inoculations with different Glomus species on the cultured plants are very successful in the infection and colonization of the root cortex. After penetration of the rhizodermis, the special exodermal short cells become colonized by winding hyphae. Then, in the root cortex of many Apocynaceae species, the VAM-fungi produce intercellular running hyphae which leads to extensive colonization of the root. Arbuscules develop on intracellular running hyphae, whereas vesicles develop mainly on intercellular hyphae. Except for some special details, this is the most common type of colonization of VAM fungi in flowering plants. But in Amsonia tabernaemontana, Nerium oleander, and Thevetia peruviana, another type of colonization could be observed. In these species, the colonization of the hyphae within the root cortex is only possible by intracellular growth. Intercellular running hyphae in the root are lacking. Therefore, after penetration the colonization in the cortex is cluster-like and strictly limited. Only by additional penetrations from hyphae in the soil, will roots show heavy infestations. This type of growth of the VAM fungi in the root is well known from the Gentianaceae and was explained as a structural incompatibility. In Catharanthus roseus, Pachypodium lamerei, and Trachelospermum jasminoides, intermediate stages of both types of colonization could be described. The results are discussed in the search for possible stimulants for structural incompatibility.     

Ultrastructure of freeze-substituted hyphae of the basidiomyceteLaetisaria arvalis

Summary The ultrastructure of freeze-substituted (FS) hyphae ofLaetisaria arvalis is described and compared to that of similar hyphae preserved by conventional chemical fixation (CF). The outline of membrane-bound organelles as well as the plasma membrane was smooth in FS cells. In contrast, hyphae preserved by CF exhibited membrane profiles that were extremely irregular. Centers of presumed Golgi activity were best preserved by FS. Microvesicles, 27–45 nm diameter and hexagonal in transverse section, were observed most readily in FS cells. Filasomes (= microvesicles within a filamentous matrix) were only observed in FS cells. Apical vesicles, 70–120 nm diameter, associated with the centers of Golgi activity and within the Spitzenkörper region exhibited finely granular matrices in FS hyphae, whereas in CF hyphae the contents were coarsely fibrous and less electron-dense. Microvesicles were present at hyphal apices and regions of septa formation. Filasomes were also found at regions of septa formation as well as along lateral hyphal tip cell walls. Microvesicles, but not filasomes, were observed in membrane-bound vesicles (= multivesicular bodies) and in larger vacuoles. Filaments, 5.2–5.4 nm wide, were juxtaposed with centripetally developing septa. Cytoplasmic inclusions, 20–40 m in length, composed of bundles of 6.7–8.0 nm wide filaments were observed in both FS and CF hyphae.