Nuclear dynamics during germination, conidiation, and hyphal fusion of Fusarium oxysporum

Abstract

In many fungal pathogens, infection is initiated by conidial germination. Subsequent stages involve germ tube elongation, conidiation, and vegetative hyphal fusion (anastomosis). Here, we used live-cell fluorescence to study the dynamics of green fluorescent protein (GFP)- and cherry fluorescent protein (ChFP)-labeled nuclei in the plant pathogen Fusarium oxysporum. Hyphae of F. oxysporum have uninucleated cells and exhibit an acropetal nuclear pedigree, where only the nucleus in the apical compartment is mitotically active. In contrast, conidiation follows a basopetal pattern, whereby mononucleated microconidia are generated by repeated mitotic cycles of the subapical nucleus in the phialide, followed by septation and cell abscission. Vegetative hyphal fusion is preceded by directed growth of the fusion hypha toward the receptor hypha and followed by a series of postfusion nuclear events, including mitosis of the apical nucleus of the fusion hypha, migration of a daughter nucleus into the receptor hypha, and degradation of the resident nucleus. These previously unreported patterns of nuclear dynamics in F. oxysporum could be intimately related to its pathogenic lifestyle.

Fig. 1.

(A and B) Scanning electron micrographs of tomato roots 24 h after inoculation with F. oxysporum f. sp. lycopersici isolate 4287. (C) Light microscopy image of isolate 4287 grown for 15 h in PDB diluted 1:50 in 20 mM glutamic acid. c, conidium; g, germ tube; r, root surface; s, septum. The arrows indicate conidial anastomosis tubes.

Fig. 2.

Live-cell imaging of mitoses after conidial germination. (A) First nuclear division in a germling of a FoH1::GFP strain. Images were recorded consecutively for GF and Nomarski optics (DIC) (see Materials and Methods). D/GF, merged images for DIC and GF. Conidial (c) and hyphal (h) compartments are indicated. Mitotic phases are indicated as follows: P, prophase, M, metaphase, A anaphase, and T, telophase. After karyokinesis, the formation of a septum was visualized (arrows). Scale bar, 5 μm. The time in minutes at which each micrograph was taken (see Movie S2 in the supplemental material) and the average times for mitosis and cytokinesis are indicated below the images. (B) Second nuclear division in a FoH1::GFP strain. The upper row shows fluorescence (GF), DIC, and merged images of a cell with two compartments at G₂ stage. Nuclei are indicated as n₁ and n₂. Note that while n₁ remains interphasic, n₂ undergoes mitosis. Samples were grown and imaged as described for panel A. Mitotic phases (MIT) and cell type are indicated. Scale bar, 5 μm.

Fig. 3.

Patterns of nuclear division in F. oxysporum. (A) Series of images of a developing hypha from Movie S3 in the supplemental material showing 4 consecutive mitoses, each followed by formation of a septum. The time in minutes (seconds in decimal scale) is documented in Movie S3 in the supplemental material. The images were acquired using an Axioplan2 microscope equipped with appropriate filters and a CoolSnap HQ camera at room temperature (RT) every 90 s for 15 h simultaneously for GFP and ChFP fluorescence and Nomarski optics, and merged for DIC and green and red fluorescence. (B) Kymograph of nuclear fluorescence (inverted image), using the Metamorph software package, from a selected hypha. Note that mitosis occurs exclusively in the apical compartment of a vegetatively growing hypha, generating one mitotically active daughter nucleus (shown in green in the diagram in panel C) and one mitotically dormant daughter nucleus (in white in the diagram in panel C). The mitotically active nucleus is located in the apical compartment (indicated as n_xb, where x is the mitosis number) and the dormant nucleus in the subapical compartment (indicated as n_xa, where x is the mitosis number). The speed of the apical nucleus after mitosis and apical-compartment elongation (v_x) is indicated in nm min⁻¹. (C) Model of the process. Filled circles, active nuclei; empty circles, dormant nuclei.

Fig. 4.

(A) Series of images from Movie S4 in the supplemental material showing the formation of microconidia. The time in minutes (seconds in decimal scale) is documented in Movie S4 in the supplemental material. (B) During conidiation, the apical daughter nucleus (located inside the new microconidium) is mitotically dormant, while the subapical nucleus (located inside the phialide) remains mitotically active for several additional divisions. Filled circles, active nuclei; empty circles, dormant nuclei; Hy, hypha; Ph, phialide; Mic, microconidium.

Fig. 5.

Examples of tip-to-tip and tip-to-side vegetative hyphal fusions. (A) Series of images from Movie S5 in the supplemental material showing an example of tip-to-side fusion between a GFP-tagged hyphal compartment (black arrow) and a ChFP-tagged germinating conidium (white arrow). After fusion, the ChFP-tagged nucleus divides, and one daughter nucleus (white arrowheads) migrates through the fusion bridge into the neighboring cell, moving past a GFP-tagged nucleus (black arrowheads), whose fluorescence disappears after 25 min. (B) Series of images from Movies S6 and S7 in the supplemental material showing an example of tip-to-tip fusion. Two apical cells home toward each other (black arrowheads), fuse, and converge into a single hyphal compartment led by the mitotically active, GFP-tagged nucleus (white arrowheads). Note that after fusion, both a GFP- and a ChFP-tagged nucleus coexist inside the same cell for approximately 429 min, followed by degradation of the ChFP-tagged nucleus. s, septum.

Fig. 6.

Series of images from Movie S8 in the supplemental material showing 2 quasisynchronous fusion events (1 and 2) involving three different hyphae. During fusion event 1 between the two FoH1::GFP hyphae (tip to side), a green fluorescent nucleus (black arrow) divides, and one of the daughter nuclei (white arrow) migrates through the fusion bridge to invade the neighboring cell, while the other daughter nucleus (yellow arrow) invades the underlying hyphal cell. After an extended period (156 min), the nucleus that invaded the neighboring hypha moves back through the fusion bridge to its original cell compartment, while the nuclei in the underlying cell compartment are visible as a single fluorescent spot throughout the rest of the movie. Note that the nucleus inside the neighboring recipient cell (red arrows) divides again and that one of the daughter nuclei is immediately degraded. During fusion event 2 between the FoH1-GFP and the FoH1-ChFP hyphae (tip-to-tip VHF), a red fluorescent nucleus (black arrowhead) divides and one of the daughter nuclei (white arrowheads) invades the adjacent FoH1-GFP hypha. After 95 min, the resident green fluorescent nucleus (yellow arrowhead) is degraded and the red fluorescent nucleus persists inside the cell and subsequently divides while the hypha continues to grow.