Novel symbiotic protoplasts formed by endophytic fungi explain their hidden existence, lifestyle switching, and diversity within the plant kingdom

Abstract

Diverse fungi live all or part of their life cycle inside plants as asymptomatic endophytes. While endophytic fungi are increasingly recognized as significant components of plant fitness, it is unclear how they interact with plant cells; why they occur throughout the fungal kingdom; and why they are associated with most fungal lifestyles. Here we evaluate the diversity of endophytic fungi that are able to form novel protoplasts called mycosomes. We found that mycosomes cultured from plants and phylogenetically diverse endophytic fungi have common morphological characteristics, express similar developmental patterns, and can revert back to the free-living walled state. Observed with electron microscopy, mycosome ontogeny within Aureobasidium pullulans may involve two organelles: double membrane-bounded promycosome organelles (PMOs) that form mycosomes, and multivesicular bodies that may form plastid-infecting vesicles. Cultured mycosomes also contain a double membrane-bounded organelle, which may be homologous to the A. pullulans PMO. The mycosome PMO is often expressed as a vacuole-like organelle, which alternatively may contain a lipoid body or a starch grain. Mycosome reversion to walled cells occurs within the PMO, and by budding from lipid or starch-containing mycosomes. Mycosomes discovered in chicken egg yolk provided a plant-independent source for analysis: they formed typical protoplast stages, contained fungal ITS sequences and reverted to walled cells, suggesting mycosome symbiosis with animals as well as plants. Our results suggest that diverse endophytic fungi express a novel protoplast phase that can explain their hidden existence, lifestyle switching, and diversity within the plant kingdom. Importantly, our findings outline "what, where, when and how", opening the way for cell and organelle-specific tests using in situ DNA hybridization and fluorescent labels. We discuss developmental, ecological and evolutionary contexts that provide a robust framework for continued tests of the mycosome phase hypothesis.

Figure 1. In vitro mycosome-phase culture.

(a) Protoplast filaments from Psilotum nudum cell extract contain condensed-Ms (3 small arrows) that enlarge as spheroid protoplasts (short arrow) and Aureobasidium pullulans conidia (long arrow). (b and c) A. pullulans 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. Right: In vitro mycosome cycle. (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.

Figure 2. The A. pullulans promycosome organelle: light vs. electron microscopy.

(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 with permission. Bars  = (a) 2.0 µm; (b–g) 1.0 µm; (h) 0.2 µm.

Figure 3. A. pullulans multivesicular bodies release budding Ms-vesicles.

(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 Psilotum 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 Cuscuta subinclusa 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.

Figure 4. Overview of mycosome structure: light microscopy.

Left cartoon: 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. Right: Examples of Type II lipoid-Ms. (a) A lipoid-Ms cultured from Cuscuta subinclusa (yellow-pigmented) shows prolific budding (phase contrast and SYTO 9 fluorescence). (b) A Rhodotorula 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) Saccharomyces. (d) Aureobasidium. (f) An orange autofluorescing (chlorophyll-containing) lipid body from a Psilotum chloroplast contains two prominent DAPI-stained Ms (see also [22]; Fig. 3a–c). (g) Budding yeast-like forms within the lipoid-body of a large Filobasidium Type II Ms (MR stain). Bars  = 5.0 µm. Use bar d for c and e.

Figure 5. Mycosomes produced by endophytic fungi. Left panel:

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

Figure 6. Protoplast filaments develop from filtered-Ms and conidia.

(a–c) Three filament types developed from 0.8 µm-filtererd Rhodotorula 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) Rhodotorula 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 Mycosphaerella 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.

Figure 7. Starch grains develop within mycosomes from fungi and plants.

(a–i) Starch grains from Taphrina and Rhodotorula mycosomes filtered 0.8 µm. (a–b) A Taphrina 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) Rhodotorula, 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.

Figure 8. Protoplast filaments from Psilotum contain chloroplasts and form A. pullulans cells within PMOs.

(a–b) Protoplast filaments develop from the margin of Psilotum 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) Psilotum 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 Psilotum 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) Aureobasidium 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.

Figure 9. Mycosome reversion to walled cells.

(a–d) Type II lipoid-Ms often contain budding yeast-like bodies that do not revert to parent cells. (a) Taphrina, MR stain. (b) Penicillium, AB stain. (c) Fusarium, AB/SIV stain. (d) Rhodotorula, AB/SIV stain. (e–f) Ms filtered from IAA-treated Rhodotorula formed walled yeast cells within spheroid (e) and filamentous (f) protoplasts. (g) IAA-treated Taphrina cells produced narrow filaments that formed lipoid-Ms and yeast cells, AB/SIV stain. (h–j) Walled cells develop from the margin of Type II lipoid-Ms: (h) Filobasidium, MR stain; (i) Rhodotorula and (j) Wickerhamomyces, AB/SIV stain. (k) Trichoderma conidia developed within lipoid-Ms, AB/SIV stain. (l) Taphrina yeast cells originating within a cluster of lipoid-Ms, AB/SIV stain. (m–n) Walled cells develop as division products of starch-producing Ms, MR stain. (m) A Penicillium conidium observed within an apple cell, MR stain. (n) Multipolar budding of Wickerhamomyces yeast from starch-Ms within a kiwifruit cell. (o–q) Cladosporium conidia germinate within mycosome PMOs. Bars  = 5.0 µm.

Figure 10. Mycosomes from egg yolk revert to walled cells.

(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 Rhodotorula glutinis 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.