Distinct actomyosin–septin coordination governs conidiation and septation in Verticillium dahliae
Juan Tian, Mengli Pu, Bin Chen, Xiaxia Zhang, Yanjun Yu, Chunli Li, Haiyun Wang, Zhaosheng Kong
TL;DR
The paper reveals how actomyosin and septins work together during cell division in the fungus Verticillium dahliae, showing unique coordination during reproduction and growth.
Contribution
The study identifies a novel apical budding process and distinct actomyosin–septin coordination during conidiation in a filamentous fungus.
Findings
Septins transition from hourglass to double ring at the bud neck during apical budding.
Disruption of septins causes defective nuclear segregation and delayed myosin II recruitment.
Actomyosin and septins coordinate differently during conidiation versus hyphal septation.
Abstract
Conidiation is the primary mode of reproduction in filamentous fungi and is essential for the dispersal of pathogenic species. However, the fundamental cellular mechanisms regulating conidiation in plant pathogenic fungi remain largely unexplored. Here, using Verticillium dahliae as a model, we investigated the dynamic assembly and function of the contractile actomyosin ring (CAR) and septins during conidiation through live‐cell imaging. We show that septins, visualized via VdCdc11‐GFP, first accumulate at the tip of budding hyphae during the transition from hyphal elongation to apical budding, and undergo an hourglass‐to‐double‐ring transition at the bud neck. Following mitosis, myosin II and actin assemble simultaneously into a contractile ring to drive cytokinesis. Disruption of core septin function results in defective nuclear segregation and aberrant nuclear migration during…
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Figure 9- —National Natural Science Foundation of China10.13039/501100001809
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Taxonomy
TopicsFungal and yeast genetics research · Plant-Microbe Interactions and Immunity · Bacterial Genetics and Biotechnology
INTRODUCTION
Asexual sporulation, or “conidiation,” is the predominant mode of reproduction in filamentous ascomycete fungi and plays a central role in their survival, dispersal, and pathogenicity. Model systems such as Aspergillus nidulans and Neurospora crassa have contributed significantly to elucidating the genetic regulation of asexual development1, 2, 3. However, these systems provide limited insights into the cellular and morphogenetic mechanisms underlying conidiation. As a result, the spatiotemporal dynamics of cytoskeletal remodeling, nuclear division, and cytokinesis during asexual development remain poorly understood in multicellular filamentous fungi.
In fungal, amoeboid, and animal cells, cytokinesis—the final step of cell division—is mediated by the assembly and constriction of a contractile actomyosin ring (CAR) at the division site. Septins, a conserved family of GTP‐binding proteins, are also central components of the cytokinetic machinery. In Saccharomyces cerevisiae, septins recruit CAR components and other cytokinesis regulators to the division site and undergo dynamic structural remodeling to facilitate cell division4, 5, 6, 7. In contrast to unicellular yeast, filamentous fungi are multicellular organisms that show distinct modes of cell division depending on the developmental stage8. Two fundamental types of cytokinesis in these fungi are hyphal septation and conidial separation. Accumulating genetic evidence suggests that the core components of cytokinesis, including CAR and septins, play essential roles in both processes. Disruption of myosin II in Penicillium marneffei 9, Fusarium graminearum 10, A. nidulans 11, 12, and Magnaporthe oryzae 13 impairs hyphal growth and conidiation, and causes defects in septum formation and nuclear division. Similarly, deletion of septin genes such as aspA (ortholog of S. cerevisiae CDC11), aspB (CDC3), and aspC (CDC12) in A. nidulans results in abnormal germ tube and branch emergence, irregular septation, and reduced conidiation14, 15, 16. In N. crassa, mutants lacking core septin genes (cdc‐3, cdc‐10, cdc‐11, and cdc‐12) also show reduced septation, defective conidiation, and excessive branching17. While hyphal septation involves the insertion of cross‐walls (septa) that partition hyphae during vegetative growth18, 19, conidial separation requires cell cleavage and detachment at the terminal end of the specialized conidiophore. This suggests that distinct, possibly specialized, modes of CAR–septin coordination govern conidiation in filamentous fungi. However, the mechanistic basis of this interplay remains largely unexplored and warrants further investigation.
The filamentous fungus Verticillium dahliae is a typical soil–borne pathogen that causes devastating vascular wilt disease in hundreds of dicotyledonous plant species, including economically important crops such as cotton, tobacco, tomato, and potato20, 21, 22. To date, no sexual stage has been identified in V. dahliae, making asexual sporulation its sole known mode of reproduction22, 23. Conidiation is critical for V. dahliae's ability to colonize and proliferate within host vascular tissues, and is a key determinant of its pathogenicity24, 25, 26. Our recent live‐cell imaging studies revealed that V. dahliae produces conidia through a continuous, budding‐like process at the hyphal tips, facilitating invasive proliferation within xylem vessels26, 27. Importantly, we observed that the conidiophore structure of V. dahliae is relatively simple and readily amenable to microscopic analysis compared with those of many other filamentous fungi. This simplicity makes V. dahliae a promising model system for elucidating cellular mechanisms underlying conidiation in plant pathogenic fungi, which remain incompletely understood.
In this study, we used live‐cell imaging to investigate the dynamic behaviors of the CAR and septins during conidiation in V. dahliae, revealing several previously unrecognized aspects of their recruitment and coordination. Our observations indicate apical budding as a specialized cytokinesis mode in V. dahliae. Furthermore, we showed that actomyosin and septins cooperate through distinct mechanisms during conidiation and hyphal septation. Together, these findings broaden our understanding of cytokinetic diversity in filamentous fungi and provide comparative insights into how conserved cellular machinery is adapted to support both reproductive and vegetative functions in multicellular fungal systems.
RESULTS
Deletion of VdMyo2 disrupts conidial separation and morphogenesis
In many eukaryotic organisms, including fungi and animals, cytokinesis is initiated by the assembly of a CAR at the future division site. Actin filaments and type II myosin are evolutionarily conserved core components of the CAR28. Through homology searches in the Verticillium genome, we identified a single gene encoding a type II myosin in V. dahliae (VDAG_01900), hereafter referred to as VdMyo2 (Figure S1A). To gain a comprehensive understanding of the cytokinesis pattern during conidiation, we examined the spatial and temporal dynamics of CAR formation. For this, we constructed a strain co‐expressing a VdMyo2‐GFP fusion protein, driven by its native promoter, together with a Histone H2A‐mCherry fusion protein as a nuclear marker. This strain was subjected to live‐cell imaging of mitosis and cytokinesis using spinning‐disk confocal microscopy.
As shown in Figure 1A and Movie S1 in Supporting Information, nuclear division occurred when the apical bud had grown to approximately the size of a mature conidium. The VdMyo2‐GFP signal appeared at the bud neck 5 min 2 s ± 59 s (n = 19) after nuclear division (taking the time point of daughter nucleus separation as t = 0), persisted for 8 min 39 s ± 34 s (n = 16), and then gradually contracted, before disappearing completely prior to the separation of the new conidium from the mother hypha. Due to the narrowness of the bud neck, the resolution of conventional confocal microscopy was insufficient to determine whether VdMyo2‐GFP formed a ring. To overcome this limitation, we used structured illumination microscopy (SIM), which revealed that VdMyo2‐GFP indeed forms a distinct ring at the bud neck during apical budding (Figure S2A). To visualize CAR formation, we co‐expressed VdMyo2‐GFP with Lifeact‐mCherry to label actin filaments. The two signals were strongly colocalized during apical budding (Figure S2B), indicating that VdMyo2 and actin co‐assemble into a contractile ring at the bud neck following mitosis. These observations suggest that cytokinesis during hyphal apical budding in V. dahliae is tightly coupled to mitotic progression.
VdMyo2 is essential for proper conidium separation in Verticillium dahliae. (A) Time‐lapse fluorescence microscopy illustrating the dynamic localization of VdMyo2‐GFP during hyphal apical budding. Nuclei were visualized by VdH2A‐mCherry expression. Yellow arrows indicate segregated nuclei following mitosis; blue arrows show the localization of VdMyo2‐GFP. Scale bar, 5 μm. (B) Bright‐field microscopy images showing conidiophore morphology in the wild‐type strain V592, the ΔVdMyo2 mutant, and the complemented strain VdMyo2‐GFP/ΔVdMyo2. Scale bar, 20 μm. (C) Cryo‐scanning electron microscopy images of conidial morphology in V592 and ΔVdMyo2. Scale bar, 5 μm. (D) Representative fluorescence images of conidia stained with Calcofluor White (CFW), showing the morphology and nuclear distribution in V592 and ΔVdMyo2. Scale bar, 5 μm. (E) Quantitative analysis of conidial morphology and nuclear status in V592 and ΔVdMyo2. Categories include single nucleus per conidium, multinucleate conidia (≥2 nuclei), multipolar conidia, adhered conidia, and anucleate conidia. Data are presented as percentages of total observed events. Counts are based on pooled results from three independent biological replicates (n = number of conidia analyzed).
To further assess the functional role of the CAR during conidiation, we generated a VdMyo2 deletion strain (ΔVdMyo2) by replacing the coding sequence with a hygromycin‐resistance cassette using standard homologous recombination methods29. Using RT‐PCR and genomic sequencing, we confirmed that the expression of the VdMyo2 gene was significantly reduced in the ΔVdMyo2 mutant and that the hygromycin resistance cassette had precisely replaced the target gene (Figure S3). The ΔVdMyo2 mutant showed reduced colony growth and a significant decrease in conidial production compared to the wild‐type strain (Figure S1B–D). These phenotypes were rescued by expressing VdMyo2‐GFP under its native promoter in the ΔVdMyo2 background (Figures 1B, S1B–D).
To further characterize the conidiation defects caused by CAR dysfunction, we examined conidial morphology and nuclear distribution in both the wild‐type V592 and ΔVdMyo2 strains using cryo‐scanning electron microscopy (SEM). In V592, conidia were produced as discrete, ellipsoidal structures at hyphal tips (Figure 1C). In contrast, ΔVdMyo2 mutants produced malformed conidia (Figure 1C). We quantified these abnormalities using strains expressing VdH2A‐mCherry and stained with Calcofluor White (CFW) after elution of conidia from colony edges. In the wild‐type V592‐VdH2A‐mCherry strain, conidia were uniformly oval in shape, each containing a single nucleus (n = 212). In contrast, approximately 75.2% of conidia from the ΔVdMyo2‐VdH2A‐mCherry strain (n = 419) showed aberrant morphology or abnormal nuclear content. We identified four main types of abnormalities in ΔVdMyo2 conidia: (i) multiple nuclei within a single conidium; (ii) multipolar‐shaped conidia; (iii) clusters of conidia abnormally adhered together; and (iv) anucleate conidia. Among these, the most prevalent phenotype was the formation of abnormally adhered conidial clusters (Figure 1D–E). These cellular abnormalities indicate that CAR dysfunction impairs the completion of cytokinesis during conidiation, ultimately preventing proper separation of daughter conidia from the mother hypha. Together, our spatiotemporal analysis of VdMyo2 and actin dynamics supports the conclusion that the CAR plays a conserved role in hyphal apical‐budding–mediated cytokinesis and is essential for normal conidium development in V. dahliae.
Septin assembly is dynamically regulated throughout the conidiation process in V. dahliae
Septins are another group of core components involved in cytokinesis. In the budding yeast S. cerevisiae, the core septins Cdc3, Cdc10, Cdc11, and Cdc12 form nonpolar heterooligomeric complexes that further assemble into higher‐order structures such as bars, rings, and gauzes6, 30. To identify the septin homologs in V. dahliae, we performed homology searches of the Verticillium genome using BLAST, with the core septin sequences from S. cerevisiae and A. nidulans as queries. Four septin‐encoding genes were identified—VDAG_00736, VDAG_07169, VDAG_04382, and VDAG_01474—and were designated as VdCdc3, VdCdc10, VdCdc11, and VdCdc12, respectively (Figure S4).
To investigate the dynamic assembly of septins during conidiation, we generated a strain expressing VdCdc11 fused to GFP under the control of its native promoter, either alone or in combination with VdH2A‐mCherry to label nuclei. Live‐cell imaging revealed that VdCdc11‐GFP predominantly formed a cylindrical collar in the subapical region of elongating hyphae (Figure 2A and Movie S2). Upon transition from hyphal elongation to apical budding, the hyphal tip contracted, and VdCdc11‐GFP signals concentrated at the budding apex. As the bud emerged, VdCdc11 assembled into an hourglass‐shaped collar at the bud neck. This structure spanned the entire neck, remained stable throughout bud development, and only began to weaken as the newly divided nucleus entered the nascent conidium. Strikingly, prior to conidial separation, the hourglass‐shaped septin structure split into two distinct rings (Figure 2B and Movie S3). These rings persisted at the division site in both the mother and daughter cells following cytokinesis. This hourglass‐to‐double‐ring remodeling is reminiscent of septin dynamics observed in budding yeast6, 30, but appears to be adapted to the filamentous growth and apical budding morphology of V. dahliae. These observations suggest that septins in V. dahliae are dynamically regulated throughout conidiation and play a conserved yet contextually specialized role in cytokinesis during asexual reproduction in filamentous fungi.
Dynamic assembly and remodeling of the septin ring during conidiation in V. dahliae. (A) Time‐lapse fluorescence microscopy illustrating the dynamic localization of VdCdc11‐GFP during the transition from hyphal elongation to apical budding. Yellow arrows indicate the initial accumulation of the VdCdc11‐GFP signal at the hyphal tip during budding initiation. Scale bar, 2 μm. (B) Time‐lapse images illustrating VdCdc11‐GFP dynamics during hyphal apical budding. Yellow arrows indicate nuclei following mitotic division, while blue arrows mark the transition of the septin collar into a double‐ring structure at the bud neck. Scale bar, 2 μm.
Septins regulate nuclear division and coordinate VdMyo2 recruitment during conidiation
To further investigate the functional role of septins during conidiation, we generated deletion mutants for the core septin genes VdCdc3, VdCdc10, VdCdc11, and VdCdc12 by replacing each gene with a hygromycin resistance cassette. Among these, the ΔVdCdc3, ΔVdCdc12, and double mutant ΔVdCdc3ΔVdCdc11 showed the most severe phenotypes, including significantly reduced colony growth and dramatically decreased conidiation (Figures S5–S7). CFW‐stained conidia from the wild‐type strain V592 consistently contained a single nucleus (100%, n = 258). In contrast, 9.5% (n = 633) of conidia from ΔVdCdc3 and 13.7% (n = 643) from ΔVdCdc3ΔVdCdc11 displayed multinucleation. Unlike the broad range of morphological abnormalities observed in ΔVdMyo2 mutants, the predominant defect in these septin mutants was the presence of multiple nuclei per conidium, while other morphological abnormalities were rare (Figure 3A,B).
*Septins are required for VdMyo2 localization at the bud neck and for proper nuclear division during conidiation. (A) Morphology and nuclear distribution of conidia in wild‐type V592, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11 mutants. Conidia were harvested from colony margins, stained with CFW, and visualized by fluorescence microscopy. Yellow arrows indicate multinucleate conidia observed in the septin‐deletion mutants. Scale bar, 5 μm. (B) Quantification of conidial morphology and nuclear distribution in V592, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11 strains. Categories include single‐nucleus per conidium, multinucleate conidia (≥2 nuclei), multipolar conidia, adhered conidia, and anucleate conidia. Data are presented as the percentage of total observed events. Counts are based on pooled results from three independent biological replicates (n = number of conidia analyzed). (C) Time‐lapse imaging of VdMyo2‐GFP dynamics during hyphal apical budding in the ΔVdCdc3 mutant. Yellow arrows indicate failed nuclear segregation post‐mitosis; blue arrows indicate undivided nuclei migrating into the emerging conidium; and red arrows indicate successfully divided nuclei. Orange arrowheads mark the VdMyo2‐GFP signal. Scale bar, 5 μm. (D) Quantitative analysis of the timing of VdMyo2‐GFP appearance at the bud neck in V592 and ΔVdCdc3 strains. Error bars represent the standard error of the mean (SEM). ***p < 0.0001, unpaired two‐tailed t‐test.
To explore the underlying cause of multinucleate conidia in the septin mutants, we performed live‐cell imaging to track nuclear division and cytokinesis in ΔVdCdc3. As shown in Figure 3D, the recruitment of VdMyo2‐GFP to the bud neck was significantly delayed in ΔVdCdc3 compared to the wild type following mitosis. Unexpectedly, we also observed frequent failures in nuclear segregation and aberrant nuclear migration during mitosis in the ΔVdCdc3 strain (Figure 3C and Movie S4). These findings suggest that core septins are not only required for timely CAR assembly but also play a critical role in ensuring proper nuclear division and positioning during conidiation. We conclude that septins act as upstream regulators that coordinate nuclear segregation and actomyosin ring assembly at the onset of cytokinesis in V. dahliae.
VdMyo2 deletion prevents septin structural transition at the bud neck during conidiation
To further investigate the co‐localization and cell cycle coordination of the CAR and septin ring during conidiation, we constructed a strain expressing VdCdc11 fused to mCherry under its native promoter, co‐expressed with VdMyo2‐GFP. As shown in Figure 4A and Movie S5, prior to the appearance of the VdMyo2‐GFP signal, the VdCdc11‐mCherry signal weakened. When the VdMyo2‐GFP ring began to contract, the VdCdc11‐mCherry collar split into two distinct rings; the average time difference between these two events was approximately 30 s (n = 8). Each septin ring remained localized at the division sites of both mother and daughter cells following cytokinesis. These observations indicate a coordinated interplay between septins and the CAR during conidiation progression.
*Deletion of VdMyo2 prevents the hourglass‐to‐double‐ring transition of septin during conidiation. (A) Time‐lapse imaging of VdCdc11‐mCherry and VdMyo2‐GFP during conidiation in wild‐type V592. VdCdc11‐mCherry (septin marker) and VdMyo2‐GFP (CAR marker) co‐localized at the bud neck. Yellow arrows indicate the transition of the VdCdc11‐mCherry hourglass‐shaped collar into two discrete rings. Scale bar, 2 μm. (B) Time‐lapse imaging showing co‐localization of Lifeact‐mCherry (actin, green) and VdCdc11‐GFP (magenta) in the ΔVdMyo2 mutant during conidiation. Blue arrows mark actin signal at the bud neck. Scale bar, 2 μm. (C) Quantification of the actin‐ring area at the bud neck in V592 and ΔVdMyo2 strains. Error bars represent SEM. ***p < 0.0001, unpaired two‐tailed t‐test. (D) Quantification of the hourglass‐to‐double‐ring transition of septin in V592 and ΔVdMyo2 strains. Percentages were calculated based on time‐lapse observations of apical budding events (n = number of events analyzed).
Given the septin‐dependent localization of VdMyo2 at the bud neck, we next investigated whether VdMyo2 reciprocally influences septin organization. To this end, we examined septin dynamics during conidiation in the ΔVdMyo2 mutant by co‐expressing Lifeact‐mCherry to label actin and VdCdc11‐GFP. While deletion of VdMyo2 did not affect the formation of the hourglass‐shaped septin collar, it significantly impaired actin accumulation at the bud neck (Figure 4B,C and Movie S6). Notably, in 82.1% of hyphal apical budding events (n = 28), the septin collar failed to undergo the characteristic hourglass‐to‐double‐ring transition in the ΔVdMyo2 mutant (Figure 4D). These results demonstrate that loss of VdMyo2 disrupts both CAR assembly and septin remodeling during conidiation. However, septin recruitment to the incipient bud site occurs independently of CAR function.
VdMyo2 is required for CAR constriction during hyphal septation
In addition to cell separation during conidiation, filamentous fungi such as V. dahliae also undergo a distinct mode of cell division: septation of hyphal cells. To explore potential differences in the roles of the CAR and septins during these two cytokinesis processes within the same organism, we examined the timing and localization of CAR formation during hyphal proliferative growth in V. dahliae. As shown in Figure 5A,B and Movie S7, VdMyo2‐GFP initially appeared as a diffuse cortical band approximately 4 min 22 s ± 33 s (n = 22) after nuclear division. This band gradually condensed longitudinally into a dense ring prior to the onset of constriction. The VdMyo2‐GFP ring then constricted centripetally at a rate of approximately 0.11 μm per minute (n = 31) (Figure 5A,C and Movie S7).
VdMyo2 is required for CAR constriction during hyphal septation in V. dahliae. (A) Time‐lapse imaging of VdMyo2‐GFP during septation of hyphal cells. The blue arrow indicates the initial diffuse cortical band of VdMyo2 at the prospective septation site. Yellow arrows mark nuclei following mitosis. The yellow dashed arrow denotes the direction along which the kymograph was generated in panel (B). Scale bar, 5 μm. (B) Kymograph showing the dynamics of nuclear division together with CAR assembly during hyphal septation. Scale bar, 5 μm. (C) Four‐dimensional (4D) imaging series depicting the centripetal constriction of the VdMyo2‐GFP ring at the septation site over time. (D) Time‐lapse images illustrating the co‐localization of VdMyo2‐GFP and Lifeact‐mCherry (actin marker) during septation. The yellow dashed arrow denotes the direction along which the kymograph was generated in panel (E). Scale bar, 5 μm. (E) Kymograph showing the dynamics of CAR constriction during hyphal septation. Scale bar, 2 μm.
Co‐expression with Lifeact‐mCherry to label actin revealed near‐perfect co‐localization with VdMyo2‐GFP throughout the transition from a diffuse cortical band to CAR constriction. Notably, the actin signal disappeared at the central septation site shortly after CAR constriction was completed, whereas the VdMyo2‐GFP signal persisted as a distinct dot at this site (Figure 5D,E and Movie S8). These findings indicate that CAR‐mediated cytokinesis is conserved during hyphal septation and remains tightly coordinated with mitotic nuclear division during hyphal growth.
The CAR constriction drives contraction of the inner perimeter of the septin ring at the septation site
To evaluate whether the regulatory relationship between the CAR and septins during hyphal septation parallels that observed during conidiation, we tracked the dynamic localization of VdMyo2‐GFP and VdCdc11‐mCherry by live‐cell imaging. As shown in Figure 6A and Movie S9, VdCdc11‐mCherry co‐localized with VdMyo2‐GFP starting from the “diffuse cortical band” stage and subsequently condensed into a dense ring. Notably, unlike uniform contraction, the septin ring's inner perimeter extended toward the center concurrently with the contraction of the VdMyo2 ring, ultimately forming a hollow, disc‐shaped structure spanning the hypha at the septation site (Figure 6A and Movie S9).
*CAR constriction drives contraction of the inner perimeter of the septin ring at the septation site. (A) Time‐lapse images showing the co‐localization dynamics of CAR and septins during hyphal septation in the wild‐type strain V592 and the ΔVdMyo2 mutant. Left: Co‐localization of VdMyo2‐GFP and VdCdc11‐mCherry in V592. Right: Co‐localization of Lifeact‐mCherry (actin/CAR marker, green) and VdCdc11‐GFP (magenta) in ΔVdMyo2. Dashed yellow arrows denote the direction used for generating kymographs (bottom). Scale bar, 2 μm. (B) Quantification of CAR compression time in V592, ΔVdMyo2, and ΔVdCdc3ΔVdCdc11 strains. (C) Quantification of the CAR constriction rate in V592, ΔVdMyo2, and ΔVdCdc3ΔVdCdc11 strains. Error bars in (B) and (C) represent the SEM. Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. ***p < 0.0001; ns , not significant. ANOVA, analysis of variance.
In the ΔVdMyo2 mutant, CAR dynamics were visualized using Lifeact‐mCherry. Compared to the wild type, the transition from a diffuse cortical band to a dense ring was significantly delayed, and actin accumulation appeared to be irregular (Figure 6A–C and Movies S9 and S10). Consequently, the ring failed to contract symmetrically toward the center. Quantitative analysis revealed that the compression time in ΔVdMyo2 was approximately twice that of the wild type (V592), and the CAR constriction rate was markedly reduced. Consistently, VdCdc11‐GFP localization dynamics in ΔVdMyo2 mirrored the aberrant actin behavior (Figure 6B,C). In contrast, the ΔVdCdc3ΔVdCdc11 mutant showed no significant differences in CAR compression time or constriction rate relative to V592 (Figure 6B,C and Movie S10). These findings indicate that, during hyphal septation, septin localization occurs postmitotically and is dependent on CAR dynamics at the septation site, showing a regulatory pattern opposite to that observed during conidiation.
To further test whether septin localization at the septation site requires the CAR, we treated strains co‐expressing Lifeact‐mCherry and VdCdc11‐GFP with Latrunculin B to depolymerize F‐actin. Following treatment, neither Lifeact‐mCherry nor VdCdc11‐GFP localized to the septation site. As a control, treatment with the solvent dimethyl sulfoxide (DMSO) alone did not affect their localization (Figure S8). Together, these data demonstrate that the CAR is essential for septin localization at the septation site and plays a critical role in driving the contraction of the septin ring's inner perimeter during hyphal septation.
VdMyo2 and septin are required for the initiation of cytokinesis during septation of the hyphal cell
As described above, both the CAR and septin initially appear as a diffuse cortical band at the septum during hyphal septation. To determine whether the initiation of cytokinesis during hyphal septation depends on VdMyo2 or septins, we quantified the number of nuclei per hyphal cell in the wild‐type strain V592, the ΔVdMyo2 mutant, and core septin deletion mutants. In V592, 98.5% of hyphal cells contained a single nucleus (n = 779). In contrast, 29.6% of hyphal cells in the ΔVdMyo2 mutant showed abnormal nuclear distribution, with 26.3% containing two nuclei and 3.4% lacking a nucleus altogether (n = 358). The proportion of hyphal cells with a single nucleus was slightly reduced in ΔVdCdc3 (95.6%, n = 478) and ΔVdCdc3ΔVdCdc11 mutants (96.6%, n = 783) compared to the wild type (Figure 7A,B). These data indicate that VdMyo2 plays a critical role in initiating cytokinesis during hyphal septation. Moreover, analysis of nuclear division and the timing of VdMyo2‐GFP signal appearance in the ΔVdCdc3 mutant revealed a significant delay compared to the wild type following nuclear division (Figure 7C), accompanied by abnormal nuclear division events (Figure S9). Together, these results demonstrate that septins contribute to the regulation of nuclear division and cytokinesis initiation during hyphal septation.
*VdMyo2 and septins are required for initiation of cytokinesis during hyphal septation in V. dahliae. (A) Fluorescence microscopy images showing nuclear distribution within individual hyphal cells of the wild‐type strain V592 and the deletion mutants ΔVdMyo2, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11. Yellow arrows indicate hyphal cells containing multiple nuclei in ΔVdMyo2 and red arrows indicate cells lacking a nucleus in ΔVdMyo2. Scale bar, 2 μm. (B) Quantification of nuclear distribution phenotypes in single hyphal cells of V592 and deletion mutants. The proportions of cells with one nucleus, two or more nuclei, or no nucleus were calculated. Data are presented as the percentage of total observed cells. Counts are based on pooled results from three independent biological replicates (n = number of cells analyzed). (C) Timing of VdMyo2‐GFP appearance at the septation site after nuclear division in V592 and ΔVdCdc3. Error bars represent SEM. Statistical significance was assessed using an unpaired two‐tailed t‐test. p < 0.05.
The CAR constriction determines septum integrity, while septin contributes to septum thickness
It is well established that CAR constriction is tightly coupled with septum formation in yeast31, 32. To further elucidate the distinct roles of the contractile ring and septins in septum formation in V. dahliae, we performed transmission electron microscopy (TEM) to compare septal ultrastructure in the wild‐type strain V592 and the deletion mutants ΔVdMyo2, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11. As shown in Figure 8A,B, V592 (n = 75) produced regular, complete septa with uniform composition spanning the entire hyphal width. In contrast, approximately 78.3% of septa in the ΔVdMyo2 mutant (n = 69) were irregular and distorted, with 46.4% showing lacunae of various sizes and 31.9% showing severe structural abnormalities. Conversely, the ΔVdCdc3 and ΔVdCdc3ΔVdCdc11 mutants formed intact septa; however, their septa were significantly thinner compared to V592 (Figure 8B,C). These findings indicate that CAR constriction is critical for maintaining septal integrity, whereas septins primarily contribute to septum thickness.
*CAR constriction ensures septum integrity, while septins regulate septum thickness in V. dahliae. (A) Transmission electron microscopy (TEM) images showing the ultrastructure of septa in the wild‐type strain V592 and deletion mutants ΔVdMyo2, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11. Septa in V592 show uniform thickness and continuous structure, whereas ΔVdMyo2 septa display irregular morphology, lacunae, and curvature. Scale bar, 1 μm. (B) Quantitative analysis of septal morphology in the strains shown in (A). Septa were classified into three categories: (i) normal; (ii) aberrant with lacunae; and (iii) curved or distorted. Data are presented as the percentage of total observed events. Counts are based on pooled results from three independent biological replicates (n = number of septa analyzed). (C) Measurement of septum thickness in V592, ΔVdCdc3, and ΔVdCdc3ΔVdCdc11. Error bars represent the SEM. Statistical comparisons were performed using one‐way ANOVA, followed by Tukey's multiple comparisons test. ***p < 0.001; ***p < 0.0001.
DISCUSSION
Cytokinesis, the final step of the cell cycle, physically divides segregated genomes into two daughter cells. While the core components of cytokinesis are conserved from yeast to humans, diverse organisms have evolved distinct regulatory mechanisms to orchestrate the assembly and function of the CAR. In this study, we dissected the cytokinesis mechanism underlying hyphal apical budding in the filamentous fungus V. dahliae using live‐cell imaging. Our results revealed that septins initially accumulate at the tip of the budding hypha as hyphal elongation transitions to apical budding, forming an hourglass‐shaped collar at the bud neck upon bud initiation (Figure 2 and Movies S2 and S3). Following mitosis and nuclear migration into the daughter cell, VdMyo2 and actin assemble at the bud neck to form the CAR, coinciding with the disassembly of the septin hourglass collar (Figures 1, 2, 4 and Movies S1, S3, S5). As the contractile ring constricts, the septin collar splits into two distinct rings at the bud neck (Figures 2, 4 and Movie S3, S5). To our knowledge, this dynamic sequence of septin remodeling, coupled with CAR assembly and contraction during hyphal apical budding, has not been previously described in filamentous fungi, establishing apical budding in V. dahliae as a novel cytokinesis mode. Our findings further reveal fundamental differences in how septins and the CAR are coordinated during conidiation versus hyphal septation. During conidiation, septins are recruited premitotically and their localization is largely actin‐independent. In contrast, during hyphal septation, septins are positioned postmitotically, and their organization depends on actin and CAR integrity at the septation site, functioning primarily in the late stages of cytokinesis (Figure 9).
Distinct coordination modes of actomyosin and septins during conidiation and hyphal septation in V. dahliae. Schematic summary illustrates the spatial and temporal interactions between CAR (VdMyo2 and actin) and septins during conidiation and hyphal septation.
Common and varied actions of cytokinesis components between model unicellular yeasts and multicellular filamentous fungi
The dynamic localization of septin ring and CAR formation during cytokinesis of hyphal apical budding shares several steps with the model budding yeast S. cerevisiae. However, there are some significant variances in the establishment of their cytokinesis machinery. One key difference is the timing of myosin localization at the division site. In budding yeast, Myo1 assembles into a ring at the presumptive bud site in late G1, shortly before bud emergence. The F‐actin ring then forms and merges with the Myo1 ring until the end of anaphase5, 33, 34. In the hyphal apical budding, however, actin and VdMyo2 co‐assembled into the contractile ring after mitosis (Figures 1 and S2 and Movie S1). Another key difference is the timing of the hourglass‐to‐double septin ring transition and the dependence of this transition on CAR. In S. cerevisiae, the septin hourglass‐shaped collar splits into two distinct rings that sandwich the constricting CAR at onset of cytokinesis6, 30, 35. The measurements by Tamborrini et al.36 indicated that septin ring splitting preceded CAR contraction by 4–5 min, and suggested that septin ring splitting relieved the physical constraint to CAR contraction, which is a prerequisite for onset of cytokinesis. However, it is not clear whether CAR contraction affects septin ring splitting. Early evidence indicated that the septin hourglass splits with normal dynamics in Cyk1 mutants in which CAR contraction failure occurs34, 37, 38. In this study, we found that the loss of VdMyo2 function in V. dahliae affected the hourglass‐to‐double‐ring transition of septin, although septin localization at the bud neck was independent of CAR in the initiation of budding (Figures 4 and S8 and Movie S5 and S6). The contraction of CAR at the bud neck was almost simultaneous with septin hourglass‐to‐double‐ring transition (Movie S5). Strikingly, we also found that septins accumulate at the hyphal apex during the transition from polar growth to apical budding—a process not observed in yeast. This early septin localization may reflect a role in initiating morphological reprogramming toward conidiation and appears specific to filamentous fungi. Thus, it seems that budding hyphae and budding yeast have adapted distinct strategies during cytokinesis. These spatiotemporal variations in the cytokinesis process may reflect the unicellular versus multicellular nature of the organisms, as well as the differing shapes of mother cells in the two systems.
The action pattern of septins during hyphae septation is similar to that in the fission yeast S. pombe, another model organism for cell division studies, in which septins function primarily in the final stages of cytokinesis. During septation, Spn1–4 first appear as a diffuse band at the division site late in mitosis and then compact into a tight ring by the time of CAR constriction, and finally develop into double rings39, 40. In S. pombe, septin mutants are viable but display a delay in cell separation, which leads to a chained cell phenotype40, 41, 42. The septin ring has also been shown to mediate the localization of hydrolytic glucanases Eng1 and Agn1, delivered by exocytosis during separation of the yeast cells39, 43, 44, 45. Unlike fission yeast, no cell separation occurs during cytokinesis of hyphal septation in filamentous fungi. Therefore, we speculate that septins may function as a scaffold to recruit specialized cell wall materials to form the cross‐wall septum in V. dahliae. Notably, the timing of septin recruitment in V. dahliae differs significantly from that in S. pombe. In S. pombe, septins are recruited after the CAR is fully assembled, whereas in hyphal septation of filamentous fungi, septins and CAR appear simultaneously at the septation site. These variations likely reflect the distinct outcomes of the two cytokinesis processes—septation without cell separation in filamentous hyphae versus fission with cell separation in fission yeast.
Distinct roles of septins during conidiation and vegetative septum formation in V. dahliae
Septins are conserved GTP‐binding proteins that form hetero‐oligomeric complexes and assemble into higher‐order structures such as rings, filaments, and collars. In fungi, they contribute to morphogenesis, cytokinesis, and spatial organization of the cytoskeleton4, 5, 6, 7. Studies in N. crassa and A. nidulans have shown that deletion of core septins leads to a range of developmental defects, including delayed septation, abnormal branching, reduced conidiation, and altered nuclear distribution15, 16, 17. However, the precise timing and roles of septins during different cytokinesis processes remain incompletely understood. Our study shows that V. dahliae uses distinct septin–actomyosin strategies in conidiation and hyphal septation. During conidiation, septins are recruited early, before mitosis and CAR assembly, forming a stable hourglass at the bud neck. After mitosis, the CAR assembles and drives contraction, during which the septin hourglass splits into two rings (Figures 1, 2, 4 and Movies S1–S5). This remodeling depends on CAR function, indicating that septins not only specify the division site but are actively remodeled during constriction. In hyphal septation, septins and CAR components appear simultaneously as a diffuse cortical band. Septin assembly requires F‐actin, and septins follow CAR dynamics, contracting along with the ring to form a disc‐like septum (Figures 6 and S8 and Movies S9 and S10). In this context, septins appear to follow CAR dynamics rather than initiate the process. What governs the switch between these two modes of septin action? While the precise mechanism remains unclear, it may involve developmental regulators that distinguish reproductive (conidiation) from vegetative (septation) programs. Such regulation could ensure that cytokinesis is adapted to the specific morphological and functional demands of each growth stage.
Despite these mechanistic differences, the role of septins in regulating nuclear division appears to be conserved in both division modes. In S. cerevisiae, septins—but not the CAR—are essential for the cortical capture and positioning of astral microtubules during spindle orientation46. Similarly, in animal cells, septins localize to spindle microtubules and centrosomes, and their depletion, leading to chromosome segregation defects and mitotic exit delays47. Our findings further demonstrate that septin deletion mutants show delayed nuclear division at both the bud neck and hyphal septation sites (Figures 3 and 7), supporting a conserved role of septins in regulating nuclear dynamics. We propose two nonmutually exclusive mechanisms: (i) septins may act as scaffolds to facilitate timely CAR assembly by recruiting or stabilizing essential components and (ii) septin‐mediated defects in nuclear division may indirectly delay the onset of cytokinesis. In addition, we observed that septins and the CAR appear simultaneously during hyphal septation, with septin localization depending on the CAR. In contrast, this coordination is absent during conidiation. These observations indicate that septin function in nuclear division may be the dominant determinant of cytokinetic timing during hyphal septation, whereas both nuclear division regulation and scaffold‐like roles of septins are critical during conidiation.
Formation of a hollow‐disc‐shaped septin ring is driven by CAR contraction during hyphal septation
A key finding of this study is that the CAR contraction drives the constriction of the inner perimeter of the septin ring at the septation site during hyphal growth in V. dahliae. During this process, VdMyo2 and F‐actin first appeared as a diffuse cortical band, marking the early stage of CAR assembly (Figure 5 and Movie S7). This configuration resembles the septal actomyosin “tangle” reported in A. nidulans 11, 12 and the actomyosin “strings” observed in N. crassa 48, 49. Subsequently, this diffuse band condensed into a tightly compacted cortical ring that underwent centripetal constriction. Our live‐cell imaging revealed that septins, marked by VdCdc11, initially colocalized with the actomyosin components during the diffuse band stage and remained associated throughout ring condensation. Notably, as the CAR underwent contraction, only the inner perimeter of the septin ring underwent constriction, giving rise to a characteristic hollow‐disc‐shaped septin structure spanning the septation site (Figure 6). This observation suggests a mechanical interplay in which CAR contraction directly drives the spatial remodeling of septin architecture.
This dynamic remodeling of septin organization during hyphal septation appears to be distinct from that observed in S. pombe, where septins initially form a diffuse band that matures into nonconstricting double rings flanking the CAR40, 42. In contrast, dynamic septin behaviors during septation in filamentous fungi have been poorly understood, as most previous studies relied on static imaging and reported variable septin conformations without addressing their real‐time dynamics15, 16, 17, 50. Our findings, therefore, provide novel insights into the cellular mechanics of septum formation in filamentous fungi. Specifically, they reveal a previously uncharacterized septin remodeling process that is mechanically driven by CAR constriction, highlighting a coordinated mechanism distinct from classical yeast models and enriching our understanding of cytokinesis diversity across fungal systems.
MATERIALS AND METHODS
Fungal strain and culture conditions
The wild‐type strain used in this study was V. dahliae V592. All strains were routinely cultured on potato dextrose agar (PDA; 200 g potato, 20 g glucose, 15 g agar per liter) at 26°C in the dark. For live‐cell imaging, conidia were collected from the colony edges and inoculated into liquid Czapek–Dox medium (2 g of NaNO₃, 1 g of K₂HPO₄, 1 g of MgSO₄·7H₂O, 1 g of KCl, 0.02 g of FeSO₄, and 30 g of sucrose per liter) and incubated at 26°C for 12–20 h.
Plasmid construction and fungal transformation
For gene deletion constructs (pGKO‐VdMyo2, pGKO‐VdCdc3, pGKO‐VdCdc10, pGKO‐VdCdc11, and pGKO‐VdCdc12), approximately 1 kb upstream and downstream flanking sequences of each gene were amplified from V592 genomic DNA51. The hygromycin resistance cassette was derived from pUC‐Hyg. Fragments were assembled into the linearized pGKO2 vector using the ClonExpress MultiS One Step Cloning Kit (Vazyme). For the ΔVdCdc3ΔVdCdc11 double mutant, flanking sequences and a G418 resistance cassette from pCOM were assembled into the linearized pGKO vector.
For complementation, the VdMyo2 open reading frame (ORF) along with its native promoter was PCR‐amplified and cloned into the pSULPH‐GFP vector. For septin expression, VdCdc11 ORF and its promoter were fused with either GFP or mCherry and inserted into the pSULPH or pCOM vector. Lifeact‐mCherry and Histone H2A‐mCherry fusion constructs were generated by inserting the respective coding sequences into the pSul‐TEF vector.
All constructs were introduced into V. dahliae via Agrobacterium tumefaciens‐mediated transformation (ATMT) following standard protocols29. Primers used for plasmid construction and strain verification are listed in Table S1.
Phenotypic characterization
To assess radial growth, 5‐mm mycelial plugs were placed at the center of PDA plates and colony diameters were measured every 2 days. For conidia quantification, four 5‐mm plugs taken from the colony edge were suspended in 2 ml of sterile water and vortexed. Conidial concentrations were determined using a hemocytometer. All assays were performed in triplicate.
RT‐qPCR and gene expression analysis
Total RNA was extracted using TRIzol reagent, and cDNA was synthesized with the HiScript II Q RT SuperMix (Vazyme). Quantitative PCR was performed using SYBR Premix Ex Taq II (TOYOBO) on a Bio‐Rad CFX96 system. VdTub1 was used as the internal control. Relative expression levels were calculated using the 2−∆∆Ct method, based on three biological and three technical replicates.
Bioinformatic analyses
Homologs of type II myosin (VdMyo2; VDAG_01900) and core septins (VdCdc3–Cdc12; VDAG_00736, VDAG_07169, VDAG_04382, VDAG_01474) were identified by BLAST searches using sequences from S. cerevisiae and A. nidulans as queries. Multiple sequence alignments were performed using ClustalX2.0. Phylogenetic trees were constructed using the Neighbor‐joining method in MEGA‐X, with 1000 bootstrap replicates.
Cryo‐SEM
For cryo‐SEM, 5 mm × 5 mm culture plugs were mounted onto specimen holders, flash‐frozen in liquid nitrogen, and transferred under vacuum to a cryo‐preparation chamber. Samples were examined with a Hitachi SU8010 SEM equipped with a Gatan Alto 2100 cryo‐system.
TEM
Hyphae were fixed in a buffer containing 4% glutaraldehyde and 2.5% formaldehyde in 0.1 M phosphate buffer (pH 6.0), followed by postfixation with 1% osmium tetroxide. Samples were dehydrated through a graded acetone series and embedded in Spurr's resin. Ultrathin sections (70 nm) were stained with uranyl acetate and observed using a JEM‐1400 transmission electron microscope.
Live‐cell imaging
Dynamic observations were performed using single optical sections acquired continuously with a spinning‐disk confocal microscope (UltraView VoX; PerkinElmer) equipped with a Yokogawa CSU‐X1 scanner, a Hamamatsu EMCCD 9100‐13 camera, and a Nikon TiE inverted microscope with Perfect Focus. Imaging was conducted using a 100×/1.49 NA oil‐immersion objective lens. Fluorescence was detected using the following excitation wavelengths: 488 nm for GFP, 561 nm for mCherry, and 405 nm for CFW. Image processing and analysis were performed using Volocity (PerkinElmer), ImageJ, and MetaMorph software.
SIM
SIM was performed using a Nikon N‐SIM system equipped with a 100×/1.35 NA silicone oil‐immersion objective lens. Excitation was provided by a 488 nm laser (for GFP). For each optical section, 15 raw images (3 illumination angles × 5 phase shifts) were captured. The z‐step size was set to 0.1 μm to meet Nyquist sampling requirements, and the total z‐stack covered the entire structure. Image reconstruction was performed using NIS‐Elements (Nikon) software with SIM reconstruction parameters.
Whole‐genome resequencing and variant mapping
Genomic DNA was extracted from each mutant and sequenced by OE Biotech Co., Ltd. using the Illumina HiSeq X Ten platform (~350 bp insert size). After quality filtering, clean reads were aligned to the reference genome using BWA (v0.7.12). Duplicate reads were marked with Picard (v4.1.0.0), and variant calling was performed using GATK (v4.1.0.0). The alignments were visualized using IGV to confirm gene disruptions.
AUTHOR CONTRIBUTIONS
Juan Tian: Funding acquisition; investigation; visualization; writing—original draft. Mengli Pu: Formal analysis; investigation. Bin Chen: Resources. Xiaxia Zhang: Project administration. Yanjun Yu: Project administration. Chunli Li: Methodology. Haiyun Wang: Methodology. Zhaosheng Kong: Project administration; resources; supervision; writing—review and editing.
ETHICS STATEMENT
This study did not involve any research on animals or human subjects.
CONFLICT OF INTERESTS
The authors declare no conflict of interests.
Supporting information
Supplementary Information‐Table S1.
Movie S1. Dynamic localization of VdMyo2 during hyphal apical budding in * V. dahliae. *
Movie S2. Dynamic localization of VdCdc11‐GFP during the transition from hyphal elongation to apical budding in * V. dahlia. *.
Movie S3. Dynamic localization of VdCdc11‐GFP during hyphal apical budding.
Movie S4. Dynamic localization of VdMyo2 during hyphal apical budding in the Δ * VdCdc3 * mutant.
Movie S5. Co‐localization of VdMyo2‐GFP and VdCdc11–mCherry during conidiation in * V. dahlia. *
Movie S6. Co‐localization of Lifeact‐mCherry and VdCdc11‐GFP during conidiation in the Δ * VdMyo2 * mutant.
Movie S7. Dynamic localization of VdMyo2‐GFP during hyphal septation.
Movie S8. Co‐localization of VdMyo2‐GFP and Lifeact‐mCherry during hyphal septation. Time‐lapse imaging shows VdMyo2‐GFP and Lifeact‐mCherry signals during hyphal septation. Scale bar, 2 μm.
Movie S9. Co‐localization of VdMyo2‐GFP and VdCdc11–mCherry during hyphal septation in * V. dahlia *.
Movie S10. Co‐localization of Lifeact‐mCherry and VdCdc11‐GFP during hyphal septation in the Δ * VdMyo2 * mutant.
Figure S1. Growth phenotypes of the Δ * VdMyo2 * mutant. Phylogenetic analysis of the VdMyo2 protein and its homologs from selected fungi. The tree was constructed using full‐length protein sequences with MEGA. Species included Aspergillus nidulans (An), Fusarium graminearum (Fg), Fusarium fujikuroi (Ff), Magnaporthe oryzae (Mo), Neurospora crassa (Nc), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Verticillium dahliae (Vd), and Ustilago maydis (Um). Colored bars indicate predicted domain architectures based on Pfam (http://pfam.xfam.org/). (B) Colony morphology of wild‐type V592, ΔVdMyo2, and complemented strain VdMyo2–GFP/ΔVdMyo2 after 14 days on PDA plates. (C) Hyphal growth measurements of wild‐type, ΔVdMyo2, and VdMyo2–GFP/ΔVdMyo2 strains. Data represent means ± SD from four biological replicates, across three independent experiments. Statistical significance at day 14 was assessed by one‐way ANOVA with Tukey's multiple comparisons test; P < 0.001, *P < 0.0001. (D) Conidial production of wild‐type, ΔVdMyo2, and complemented strains after 7 days on PDA plates. Four replicates per strain across three independent experiments. Error bars represent SD. Statistical significance was determined by one‐way ANOVA with Tukey's multiple comparisons test (P < 0.05; ns, not significant). Figure S2. VdMyo2 forms a contractile ring and co‐localization of actin during hyphal apical budding. (A) 3D‐SIM reconstruction showing that VdMyo2‐GFP forms a distinct ring at the bud neck during apical budding. (B) Time‐lapse images showing the co‐localization of VdMyo2‐GFP and actin (visualized by Lifeact–mCherry) during conidiation in V. dahliae. Scale bar, 2 μm. Figure S3 . Genetic identification of * VdMyo2 * deletion and complemented mutants. (A) Schematic representation of the gene knockout strategy, showing the positions of primers used for mutant screening. HPTbox indicates the hygromycin B phosphotransferase resistance gene. (B) PCR confirmation of the VdMyo2 deletion mutant using the four primer pairs illustrated in (A). M, DNA marker. (C) Relative expression levels of VdMyo2 in wild‐type, ΔVdMyo2, and complemented VdMyo2–GFP/ΔVdMyo2 strains analyzed by RT‐qPCR. Statistical significance is indicated by triple asterisks () and quadruple asterisks (**) for P < 0.001 and P < 0.0001, respectively, determined by one‐way ANOVA with Tukey's multiple comparisons test. (D, E) Whole‐genome resequencing confirming that the hygromycin resistance cassette precisely replaced the target gene without additional fragment insertions elsewhere in the genome. (D) Chromosomal location of VdMyo2 showing the insertion site of the hygromycin resistance cassette. (E) Read coverage at the insertion locus in the deletion mutant, demonstrating precise gene replacement. Figure S4. Phylogenetic relationship of core septin proteins and their homologs from various fungal species. The phylogenetic tree was constructed using MEGA software based on full‐length protein sequences from multiple fungal species, including Aspergillus nidulans (An), Fusarium graminearum (Fg), Magnaporthe oryzae (Mo), Neurospora crassa (Nc), Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), Verticillium dahliae (Vd), and Ustilago maydis (Um). Figure S5. Growth phenotypes of core septin mutants. (A) Colony morphology of wild‐type V592, ΔVdCdc3, ΔVdCdc10, ΔVdCdc11, ΔVdCdc12, and ΔVdCdc3ΔVdCdc11 mutants, and their respective complemented strains after 14 days of growth on PDA plates. (B) Hyphal growth measurements of wild‐type V592, ΔVdCdc3, ΔVdCdc10, ΔVdCdc11, ΔVdCdc12, and ΔVdCdc3ΔVdCdc11 mutants, and complemented strains. Error bars represent standard deviations. Statistical significance (**) indicates P < 0.0001 by one‐way ANOVA with Tukey's multiple comparisons test at day 14. (C) Conidial production of wild‐type V592, ΔVdCdc3, ΔVdCdc10, ΔVdCdc11, ΔVdCdc12, ΔVdCdc3ΔVdCdc11 mutants, and complemented strains. Experiments were performed in triplicate. Statistical significance is indicated by **** (P < 0.0001) based on one‐way ANOVA with Tukey's multiple comparisons test. ns, not significant. Figure S6. Genetic identification of core septin gene deletion and complemented mutants. (A) Schematic diagram of the gene knockout strategy and the primers used for mutant verification. (B) PCR confirmation of the ΔVdCdc3, ΔVdCdc10, ΔVdCdc11, ΔVdCdc12, and ΔVdCdc3ΔVdCdc11 deletion mutants using the four primer pairs indicated in (A). M indicates the DNA marker. (C) RT‐qPCR analysis of transcription levels of VdCdc3, VdCdc10, VdCdc11, and VdCdc12 in wild‐type, deletion mutants (ΔVdCdc3, ΔVdCdc10, ΔVdCdc11, ΔVdCdc12, ΔVdCdc3ΔVdCdc11), and complemented strains. Statistical significance is indicated as * (P < 0.05), ** (P < 0.01), and **** (P < 0.0001); ns indicates no significant difference, as determined by two‐way ANOVA with Tukey's multiple comparisons test. Figure S7. Whole‐genome resequencing analysis of septin deletion mutants. Whole‐genome resequencing confirms that the hygromycin/G418 resistance cassette precisely replaced the target genes without introducing additional fragment insertions elsewhere in the genome. (A) Chromosomal locations of VdCdc3, VdCdc10, VdCdc11, and VdCdc12 genes, showing the insertion sites of the hygromycin/G418 resistance cassette. (B) Read coverage at the insertion loci in each deletion mutant, demonstrating the precise replacement of the target genes. Figure S8. Latrunculin B treatment disrupts the localization of actin and septin at the septation site. Yellow arrows indicate Lifeact‐mCherry and VdCdc11‐GFP signals at the bud neck, while blue arrows highlight their signals at the septation site. Scale bar, 10 μm. Figure S9. The Δ * VdCdc3 * mutant shows defects in nuclear segregation during hyphal septation. Time‐lapse images showing the dynamic localization of VdMyo2‐GFP during hyphal septation in the ΔVdCdc3 mutant. Yellow arrows indicate nuclei that failed to divide after mitosis, red arrows indicate divided nuclei, and orange arrowheads mark VdMyo2‐GFP signals. Scale bar, 10 μm.
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