Protoplast filaments develop from filtered-Ms and conidia.

<p>(a–c) Three filament types developed from 0.8 µm-filtererd <i>Rhodotorula</i> mycosomes (AB/SIV stain): (a) Narrow reticulate filaments (<0.5 µm in diameter) produced large numbers of lipoid bodies, many pinching-off into individual spheroid-Ms. (b) Sheet-like fenestrated protoplasts may form by expansion of the PMO outer membrane. (c) <i>Rhodotorula</i> mycosomes filtered into rich media produced an AB-staining cytoplasm containing large lipoid-PMOs, some with a prominent inclusion. (d–e) Protoplast filaments from cultured <i>Mycosphaerella</i> conidia. (e) The conidia filaments presumably contain STYO 9-staining PMOs. (d) Filaments from (e) transferred to MsM-Soy over YM agar. Large numbers of SIV-staining PMOs developed within acytoplasmic (non-AB-staining) filaments, associated with an AB-staining filament mass. Bars  = 5 µm.</p

The <i>A. pullulans</i> promycosome organelle: light vs. electron microscopy.

<p>(a) Light microscopy; (b–h) electron microscopy. (a) The left hypha contains many presumptive budding PMOs (box and arrow), which express a lipoid body (arrow, center hypha) within the PMO inner membrane (IM). Walled endospores (right hypha) apparently develop within the PMO lipoid body. (b–h) First generation yeast sectioned for EM. (b) Electron-opaque budding organelles require image lightening to reveal internal bodies (white arrows) presumed to be mycosome initials. (c) PMOs are identified by invaginating membranes (arrows) and the electron-lucent space between the outer membrane and the opaque body; (see also b, f and g). (d) PMOs without lipoid content (asterisks) show numerous vesicles between the two membranes. PMOs lacking inner membrane expression may appear as vacuoles that contain electron-opaque bodies (double asterisk). (e) A mycosome-containing PMO with prominent membrane lamellae; see also (g), double arrowheads. (f) A lipoid PMO containing opaque budding mycosome initials; note invaginating membranes (arrow). (g) A lipoid PMO with a membranous infolding (short arrow) and a bud (double arrowhead) containing membrane lamellae and a putative opaque mycosome. Vacuole-like PMOs occur near the cell margin (arrowheads), some containing a putative mycosome initial (long arrows). (h) The fungal plasmalemma may bud to form periplasmic vesicles (long arrow), and may invaginate (short arrow) to form a vacuole-like PMO that contains a vesicle (arrowhead), similar to (g), long arrows. Photos (a and c) from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095266#pone.0095266-Atsatt1" target="_blank">[22]</a> with permission. Bars  = (a) 2.0 µm; (b–g) 1.0 µm; (h) 0.2 µm.</p

Protoplast filaments from <i>Psilotum</i> contain chloroplasts and form <i>A. pullulans</i> cells within PMOs.

<p>(a–b) Protoplast filaments develop from the margin of <i>Psilotum</i> chloroplasts; note that portions of the plastid envelope contain dark punctate bodies (also f, red arrows). (a) Vacuolate-PMOs (vPMO) enlarge within the filaments, and (b) pinch-off as spheroid vacuolate-Ms (vMs). (b) Filaments radiating from chloroplasts also contain small chloroplasts. (c) The filaments mature as a dense chloroplast-containing matrix; or (e), expand as fenestrated, chloroplast (cp)-containing filaments (AB stain). Walled cells (wc) occasionally develop within the ‘open’ areas. (d) vPMOs apparently develop from the AB-staining chloroplast margin (red arrow). (f–h) <i>Psilotum</i> chloroplasts release green Ms (black arrows) from outside a dark chloroplast envelope (f, red arrow), and from green unbounded membranes (g–h). (h) This Type II Ms contains a greenish body, similar to <i>Psilotum</i> lipoid-Ms that stain red (i) with SIV. (j) SYTO-9 staining bodies are present within the Type II protoplast. (k–n) Filaments from post-reproductive stems contain numerous vPMOs (k), some expressing a lipoid body (AB/SIV stain). (l–n) <i>Aureobasidium</i> walled cells (wc) develop within the PMO vacuole. (n) Note presence of condensed-Ms, enlarging vacuolate-PMOs and yeast cell release from a vacuolate-PMO. Bars  = 5.0 µm.</p

Mycosomes from egg yolk revert to walled cells.

<p>(a–b) Yolk-Ms show prolific reproduction within thin membrane sacs. (a) Phase contrast, (b) SYTO 9 stain. (c) IAA-treated Ms formed large membrane-sacs containing numerous spheroid protoplasts, each with a prominent central body. (d–e) Ms cultured in MsM flooded over Sabouraud dextrose agar. The variable-shaped lipoid bodies (d) contain (e) fluorescing PMOs or Ms (SYTO 9 stain). (f–k) Cell wall formation by <i>Rhodotorula glutinis</i> stained for chitin with Fungi-Fluor. (f and h) Yeast cells were present at 38 days within a diffuse protoplast matrix containing lipoid-Ms (phase contrast). (g and i) Ms and the protoplast matrix do not express chitin; only the mature cell (g) and thin walled yeast cells (i) fluoresce. (j–k) Sampled two weeks later, three walled cells and many enlarging Ms show variable cell wall fluorescence. Bars  = 2.0 µm.</p

Overview of mycosome structure: light microscopy.

<p><b>Left cartoon:</b> Condensed-Ms develop as spheroid or filamentous protoplasts that express one or more vacuole-like PMOs. Type I Ms develop as acytoplasmic (grey) or cytoplasmic (blue) protoplasts that express multiple PMOs. Type II Ms express a single PMO, bounded by a narrow budding protoplast. The PMO may appear vacuolate, or contain a lipoid compartment, a starch grain, or a walled parent-type cell. <b>Right: Examples of Type II lipoid-Ms.</b> (a) A lipoid-Ms cultured from <i>Cuscuta subinclusa</i> (yellow-pigmented) shows prolific budding (phase contrast and SYTO 9 fluorescence). (b) A <i>Rhodotorula</i> Type II Ms at two focal planes (AB/SIV stain). The red lipoid-body (left) is expressed within the non-staining vacuole-like PMO; condensed-Ms (right) are released from the crescent-shaped protoplast. (c–e) Viewed with phase contrast, refractive lipoid bodies (left) are bounded by a SYTO-9 staining envelope (right) that contains a fluorescing body. (c and e) <i>Saccharomyces</i>. (d) <i>Aureobasidium.</i> (f) An orange autofluorescing (chlorophyll-containing) lipid body from a <i>Psilotum</i> chloroplast contains two prominent DAPI-stained Ms (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095266#pone.0095266-Atsatt1" target="_blank">[22]</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095266#pone-0095266-g003" target="_blank">Fig. 3a–c</a>). (g) Budding yeast-like forms within the lipoid-body of a large <i>Filobasidium</i> Type II Ms (MR stain). Bars  = 5.0 µm. Use bar d for c and e.</p

Endophytic fungi isolated from plant anthers (An) fruit (Fr) and stems (St).

<p>All experimental fungi (bold type) produced the mycosome phase.</p

<i>A. pullulans</i> multivesicular bodies release budding Ms-vesicles.

<p>(a) Ms-vesicles are formed within multivesicular bodies (MVB). (b) Similar vesicles are present within the periplasmic space of a dividing yeast cell (tangential section). (c) Mature MVB contain large numbers of budding vesicles, which are apparently released into the periplasmic space (d). (d) Inset: An enlarged budding vesicle. (e) Similar budding vesicles are observed within the envelope of modified <i>Psilotum</i> chloroplasts that may be enclosed by a fused fungal plasmalemma (note double-thickness of the outer membrane in the enlarged inset (e), and the ‘eruptions’ from the outer membrane). An opaque vesicle associated with the plastid inner membrane (inset e, arrow) may be budding into the plastid stroma. (f) An infected plastid containing large numbers of dividing vesicles within the expanded envelope. Most of the plastid inner membrane is missing: a short segment (visible right) is lined with vesicles, two with electron lucent centers (asterisk). The vesicles enlarge as electron dense bodies (white arrow), or as vacuole-like forms (asterisks). Note budding from the ‘vacuole’ margin (3 arrows) and presence of internal electron-dense bodies (double-ended arrow). The enlarged vesicle (3f) box) was cultured from modified <i>Cuscuta subinclusa</i> plastids. Bars  = (a, b) 5.0 µm; (c) 100 nm; (d) 0.5 µm, inset 100 nm; (e) 5.0 µm, inset 100 nm; (f) 1.0 µm; boxed vesicle is 175 nm.</p

Starch grains develop within mycosomes from fungi and plants.

<p>(a–i) Starch grains from <i>Taphrina</i> and <i>Rhodotorula</i> mycosomes filtered 0.8 µm. (a–b) A <i>Taphrina</i> protoplast containing numerous starch grains and static yeast cells (MR stain). (b) An enlarged portion of (a) showing the yeast cells, some of which develop as division products of starch-Ms. (c–i) <i>Rhodotorula,</i> AB/SIV stain; (c) Cytoplasmic fungal protoplasts contain starch granules that (d) develop within the vacuole-like PMO. (e–f) A starch-Ms stained with AB (e), then with MR (f). (g) Starch-Ms often form lipoid-Ms from their boundary. (h–i) Large granules show the bounding Ms-membrane (arrows) and typical growth rings (arrowheads). (j) A fungal protoplast cultured from kiwifruit cell extract is packed with large and small Ms-starch grains (AB/SIV stain). (k) An enlargement of boxed area (j), showing chain-like division of Type II Ms-starch grains. (Inset l): A dividing Type II starch-Ms, photographed inside a kiwifruit cell (MR stain). Bars  = 10 µm. Use bar (b) for c through i.</p

Mycosomes produced by endophytic fungi. Left panel:

<p>(a–h) Ms are associated with, or released from modified cell walls. (a–b) <i>Cryptococcus victoriae</i>, MR stain. (c–d) <i>C. stepposus</i> stained with MR (c), and AB/SIV (d). (e) An IAA-treated <i>Taphrina</i> cell enclosed by a thin expanded wall associated with lipoid-Ms and one AB-staining Ms (arrow). (f) An IAA-treated <i>Rhodotorula</i> cell showing a single AB-staining Ms. (g–i) Ms released from <i>Penicillium</i> modified cell walls (g–h) reproduced in chain-like filaments (i). (j) Condensed-Ms (here <i>Cladosporium</i>) often enlarge as spheroid protoplasts that express a dark punctate body within an internal compartment. (k) Cultured in 2xT864 medium, <i>Penicillium</i> conidia cell walls appear to expand as Ms-forming protoplasts. (l) Young <i>Penicillium</i> filaments released Ms within membrane sacs. (m) <i>Taphrina</i> Ms enlarge as protoplasts that form numerous red-brown bodies within a light-yellow staining compartment (MR stain). Bars  = 2.0 µm. <b>Right panel:</b> Phase contrast. (n) SYTO 9-stained Ms are released through <i>Aureobasidium</i> cell walls. (o–p) <i>Rhodotorula</i> cells: (o) Refractive lipoid-PMOs surround a vacuole. (p) Presumptive PMOs often contain a Ms-like body. (q–r) <i>Saccharomyces</i> cells express presumptive vacuolate PMOs with SYTO 9-staining boundaries. Note Ms release through the cell wall (q) and non-staining bodies within the PMOs (r). (s–v) <i>Taphrina</i> cells: (s) Lipoid-Ms released from a degraded cell wall (AB/SIV stain); (t) Type I protoplasts contain numerous dividing bodies, possibly PMOs; (u–v) the presumptive PMOs show refractive lipids (u), and are apparently incorporated into buds that form Type II lipoid-Ms (v). Bars in (o) and (t)  = 2.0 µm in all photos.</p

In vitro mycosome-phase culture.

<p>(a) Protoplast filaments from <i>Psilotum nudum</i> cell extract contain condensed-Ms (3 small arrows) that enlarge as spheroid protoplasts (short arrow) and <i>Aureobasidium pullulans</i> conidia (long arrow). (b and c) <i>A. pullulans</i> hyphae cultured 14 months in distilled water + erythromycin (Ms longevity test) produced condensed-Ms that divide symmetrically or by budding, and enlarge as spheroid forms that express a central vacuole. (b₁) and (c₁) are enlargements of (b) and the boxed area of (c); the enlargements are artificially colored with a Photoshop filter sensitive to differences in stain density. The Ms-boundary, otherwise seen as an AB-staining wall-like structure (b, arrow), is actually a narrow protoplast bounded by vacuole and plasmalemma membranes (b₁, arrows). A walled cell potentially develops within the central vacuole-like organelle (c₁, arrows). Bars  = 2.0 µm. <b>Right: In vitro mycosome cycle.</b> (1) Cultured in liquid media, cell extract from macerated plant tissue yields walled fungus cells that develop from the mycosome (Ms) phase. (2) Fungus pure cultures are isolated on nutrient agar. (3) Induced in liquid media, fungus cells produce Ms. (4) Ms separated from parent cells (filtered 0.8 µm) are capable of reverting to walled cells.</p