Redundant and Distinct Roles of Two 14-3-3 Proteins in Fusarium sacchari, Pathogen of Sugarcane Pokkah Boeng Disease

Fusarium sacchari, a key pathogen of sugarcane, is responsible for the Pokkah boeng disease (PBD) in China. The 14-3-3 proteins have been implicated in critical developmental processes, including dimorphic transition, signal transduction, and carbon metabolism in various phytopathogenic fungi. However, their roles are poorly understood in F. sacchari. This study focused on the characterization of two 14-3-3 protein-encoding genes, FsBmh1 and FsBmh2, within F. sacchari. Both genes were found to be expressed during the vegetative growth stage, yet FsBmh1 was repressed at the sporulation stage in vitro. To elucidate the functions of these genes, the deletion mutants ΔFsBmh1 and ΔFsBmh2 were generated. The ΔFsBmh2 exhibited more pronounced phenotypic defects, such as impaired hyphal branching, septation, conidiation, spore germination, and colony growth, compared to the ΔFsBmh1. Notably, both knockout mutants showed a reduction in virulence, with transcriptome analysis revealing changes associated with the observed phenotypes. To further investigate the functional interplay between FsBmh1 and FsBmh2, we constructed and analyzed mutants with combined deletion and silencing (ΔFsBmh/siFsBmh) as well as overexpression (O-FsBmh). The combinations of ΔFsBmh1/siFsBmh2 or ΔFsBmh2/siFsBmh1 displayed more severe phenotypes than those with single allele deletions, suggesting a functional redundancy between the two 14-3-3 proteins. Yeast two-hybrid (Y2H) assays identified 20 proteins with pivotal roles in primary metabolism or diverse biological functions, 12 of which interacted with both FsBmh1 and FsBmh2. Three proteins were specifically associated with FsBmh1, while five interacted exclusively with FsBmh2. In summary, this research provides novel insights into the roles of FsBmh1 and FsBmh2 in F. sacchari and highlights potential targets for PBD management through the modulation of FsBmh functions.


Introduction
Sugarcane Pokkah boeng disease (PBD) is a pervasive airborne fungal disease caused by the Fusarium species complex (FSC) [1,2].The disease is characterized by the dissemination of fungal spores via air and rain, which infect the sugarcane shoot tips, leading to symptoms such as leaf yellowing, crinkling, and twisting, with severe cases resulting in tip rot [3].In China, F. sacchari emerges as the predominant pathogen responsible for PBD [4].Despite extensive research encompassing pathogen identification, germplasm resistance, environmental factor influences, and pathogen effectors [5][6][7], the pathogenic mechanisms of F. sacchari are still far from clear.The 14-3-3 protein family, with a low molecular weight of approximately 30 kDa, is ubiquitously present in eukaryotic organisms [8,9].These proteins exert their influence by binding to target proteins, thereby modulating their activity, localization, and presentation, to modulate a wide variety of cellular processes [10][11][12][13].Within the cell, 14-3-3 proteins typically exist as homodimers or heterodimers [14].Isoforms are prevalent within this family, with examples including 15 isoforms in the plant Arabidopsis and seven in mammals [15], while fungi typically exhibit a lesser number, such as one or two [16,17].Despite high conservation among isoforms, distinct isoforms may exhibit varying affinities for their preferred targets, thus executing different functions [18].
In Saccharomyces cerevisiae, the 14-3-3-encoding genes Bmh1 and Bmh2 are pivotal in regulating vegetative growth, and the knockout of both genes results in a lethal phenotype [19].Similarly, in the human pathogen Candida albicans, Bmh1 is crucial for growth, morphogenesis, and pathogenesis [20].In the plant pathogen Ustilago maydis, Pdc1, which encodes a homolog of Bmh1 from yeast, plays a role in the regulation of mycelial growth and virulence [21].In Fusarium graminearum, two 14-3-3 proteins encoding genes have partially overlapping roles in mediating nitrogen catabolite repression, yet only FgBmh2 is implicated in the regulation of reproduction and virulence [22].However, the functional role of 14-3-3 proteins in F. sacchari has remained unexplored.This study delves into the functions of the 14-3-3 protein-encoding genes FsBmh1 and FsBmh2 in F. sacchari using a comprehensive approach.The findings reveal that both genes are essential for virulence and exhibit redundancy in many functions, with FsBmh2 playing a predominant role in the regulation of mycelial growth and conidiation in F. sacchari.

Fungal Strains and Culturing Conditions
The wild-type strain CNO-1 of F. sacchari and mutant strains were stored in 25% (v/v) glycerin at −80 • C. The phenotypic characteristics, including the colony diameter and pigmentation, were monitored on potato dextrose agar (PDA) medium at 28 • C for 7 days [23].

Generation of Mutant Strains
To knockout FsBmh1 and FsBmh2, deletion fragments were amplified using fusion PCR with the primer pairs FsBmh1-1F/R, FsBmh2-1F/R, FsBmh1-2F/R, FsBmh2-2F/R, Hph-F/R, and G418-F/R (Table S1).The hygromycin resistance gene (hph) and the geneticin resistance gene (NeoR) were employed as selection markers [24].The amplified fragments were transformed into protoplasts of the wild-type strain CNO-1 using a polyethylene glycol (PEG)-mediated method, resulting in the creation of ∆FsBmh1 and ∆FsBmh2 mutants, respectively [25].For the complementation mutant strains, full-length FsBmh1 and FsBmh2 fragments, including their promoter sequences, were amplified using the primer pairs C-FsBmh1-F/R and C-FsBmh2-F/R (Table S1).These fragments were cloned into the pCPXG418 or pCPXHY2 vector using the pEASY-Basic Seamless Cloning and Assembly Kit (Transgen Biotech, Beijing, China) and then transformed into the protoplasts of ∆FsBmh1 or ∆FsBmh2 mutants.
RNA interference was utilized to silence FsBmh1 in the ∆FsBmh2 background or vice versa [26].The interference fragments for FsBmh1 and FsBmh2, 520 bp and 519 bp, respectively, were amplified from the genomic DNA of the wild-type strain and cloned into the pCPXG418 or pCPXHY2 vector using the pEASY-Basic Seamless Cloning and Assembly Kit.The overexpression strains were developed by introducing additional copies of FsBmh1 or FsBmh2 into the CNO-1 protoplasts, which were then selected on media supplemented with the appropriate antibiotics.

RNA Extraction and Quantitative Real-Time RT-PCR
The total RNA from each strain was extracted using the RNA extraction kit (TaKaRa, Beijing, China) according to the manufacturer's protocol.First-strand cDNA synthesis was performed using the FastQuant RT Kit (TaKaRa, Beijing, China).Quantitative real-time PCR was conducted using the SuperReal PreMix Plus (SYBR Green) (TaKaRa, Beijing, China) with the target gene primer pairs (Table S2) and 18S rRNA as an internal control.The relative expression levels were calculated using the 2 −∆∆CT method [27].The data are presented as the mean ± standard deviation from three independent biological replicates, each with three technical repeats.

Induction of Macrospores
Carnation leaf agar is often used to induce macrospore production in Fusarium spp.CLA medium was prepared by aseptically placing sterile carnation leaf pieces, 3-5 mm 2 , into a dish and adding sterile water agar (20 g agar in 1 L sterile water) [28].Macroconidia were induced by inoculating mycelial plugs on carnation leaf agar for 7 days.

Microscopy
Microconidia from each strain were harvested from PDA plates or macroconidia from carnation leaf agar plates using 10 mL of sterile water.Images were captured using a differential-interference contrast microscope.
The hyphae and spores were stained with calcofluor white (CFW) for septa observation [29].The sugarcane tissue samples were stained with 0.05% of aniline blue, which specifically binds the fungal cell to allow it to be visualized in a blue color to trace the development of mycelium [30].The samples were viewed under an Olympus DP70 microscope (Olympus, Tokyo, Japan).

Pathogenicity Assays
Susceptible sugarcane seedlings (Zhongzhe 9, ZZ9) were grown in a greenhouse until the 5-leaf stage.F. sacchari conidia from 7-days-old PDA cultures were adjusted to 1 × 10 4 conidia/mL.For inoculation, a volume of 300 µL of spore suspension was injected into the sugarcane stem around the tip meristem using a sterile needle.The disease severity was evaluated 21 days post-inoculation (dpi), and the disease severity index (DSI) was calculated using a symptom severity scale (Table S3).The DSI was determined using the formula DSI = 100 × (Σ score/5N), where N is the number of observed seedlings (N = 100).Statistical analysis was performed using one-way ANOVA with SPSS 23.0 software, and each assay was replicated three times.

Transcriptomic Analysis
The strains of interest were cultured in potato dextrose broth (PDB) medium at 28 • C and 200 rpm for 3 days.The mycelium was harvested and washed, and the total RNA was extracted using TRIzol ® Reagent (Invitrogen, San Diego, CA, USA) with genomic DNA removal via DNase I (TaKaRa, Beijing, China) treatment.Transcriptome libraries were constructed using the TruSeq TM RNA sample preparation Kit (Illumina, San Diego, CA, USA) and sequenced on the Illumina NovaSeq 6000 platform.The raw reads were filtered and mapped to the F. sacchari CNO-1 genome (unpublished data) using TopHat2 v2.1.1.Differentially expressed genes (DEGs) were identified based on a minimum twofold change in expression (log2 ratio ≤ −1 or ≥1) at a false discovery rate of p = 0.05 or less [31].DEGs were annotated using the NCBI protein databases for Fusarium spp., and KEGG pathway enrichment analysis was performed for all the DEGs with a corrected p-adjust < 0.05 [32].Phenotype-associated gene transcripts were verified using cDNA from transcriptome samples and the fungal 18S RNA gene as an internal standard.Data were obtained from three biological replicates.

Yeast Two-Hybrid Assays
For Y2H analysis, the coding sequences of the genes under investigation were amplified from CNO-1 cDNA using the primer pairs listed in Table S12.These fragments were cloned into the yeast GAL4-binding domain vector pGBKT7 and GAL4 activation vector pGADT7 (TaKaRa, Beijing, China).The Y2H constructs were co-transformed into S. cerevisiae Y2H gold (Coolaber, Beijing, China) following the lithium acetate/single-stranded DNA/polyethylene glycol transformation protocol [33].Positive and negative controls were included, and the transformants were cultured on synthetic medium lacking Leu and Trp, followed by transfer to medium lacking His, Ade, Leu, and Trp.The Y2H assay results were confirmed using three independent experiments.

Statistical Analysis
Statistical analysis was conducted using one-way ANOVA with the SPSS statistical package version 23.0 (IBM, Amonk, NY, USA).The Student-Newman-Keuls test was employed for inter-treatment comparisons at a significance level of p < 0.05.

Conservation of 14-3-3 Proteins in Fusarium Species
Two distinct genes (FsBmh1 and FsBmh2) both encoding 14-3-3 proteins were identified in the CNO-1 genome by blast of the F. sacchari genome database (our unpublished data) using the S. cerevisiae Bmh1 and Bmh2 as queries.The cDNAs of FsBmh1 (FVER_07211, NCBI accession No. MH999450.1)and FsBmh2 (FVER_01000, NCBI accession No. MH999451.1)were 807 bp and 849 bp, encoding 268 and 276 amino acids, respectively.These proteins possess the characteristic functional domain of the 14-3-3 protein superfamily and exhibit 69.6% identity and 83.4% similarity at the amino acid level.Notably, the amino acid identity within the Fusarium genus (F.graminearum, F. fujikuroi, and F. oxysporum) ranged from 91.4% to 97%, whereas the identity between different fungal genera was significantly lower, such as S. cerevisiae (62.4%) and C. albicans (73.1%) (Figure S1A).Phylogenetic analysis revealed that Bmh1 and Bmh2 segregate into distinct clusters among fungal species, with the F. sacchari-derived Bmh1 and Bmh2 showing the closest phylogenetic relationship to their counterparts in F. fujikuroi and F. oxysporum, respectively (Figure S1B).

Expression Patterns of FsBmh1 and FsBmh2 during Infection and Vegetative Growth
Sugarcane seedlings were inoculated with the spore suspension, and the invasion of the plant by hyphae was observed within 24 h post-inoculation (hpi).Hyphal branching, plant cell disintegration, and necrotic spots on the leaf surface were evident at 48 hpi, with severe tissue necrosis, hyphae proliferation within infected cells, and new spore formation observed at 96 hpi (Figure 1A,B).In concert with these developmental stages, the transcript level of FsBmh1 increased by 2-3-fold at 24 and 48 hpi and sharply lifted by 41-fold at 96 hpi compared to 0 hpi; the FsBmh2 transcript levels remained unchanged during the initial 24 hpi but increased by approximately 2-fold at 48 hpi and 15-fold at 96 hpi (Figure 1C).Although both FsBmh1 and FsBmh2 exhibited transcriptional responses during host colonization, FsBmh1 responded earlier and at a significantly higher level at the later stage (96 hpi) in planta compared to FsBmh2.
To explore the correlation between the developmental timeline and transcriptional dynamics of FsBmh in a saprophytic context, F. sacchari spores were inoculated into potato dextrose broth (PDB) medium.Initial hyphal growth was observed at 10 h, branching at 14 h, and new spore formation at 22 h (Figure 1D).Transcript level analysis indicated that FsBmh1 was up-regulated by 3.3-fold at 10 h, followed by a decline; in contrast, FsBmh2 was up-regulated by 4.3-fold at 10 h and maintained a relatively stable level thereafter (Figure 1E).A comparative analysis of the transcription profiles (Figure 1C vs. Figure 1E) revealed distinct transcriptional dynamics for FsBmh1 and FsBmh2 during plant infection and vegetative growth in the medium.revealed distinct transcriptional dynamics for FsBmh1 and FsBmh2 during plant infection and vegetative growth in the medium.

The Expression of FsBmh1 and FsBmh2 Are Mutually Compensated in Cells
To elucidate the distinct functions of two 14-3-3 proteins, we generated the gene deletion mutants ΔFsBmh1 and ΔFsBmh2 by homologous recombination (Figures S2 and  S3).Intriguingly, the deletion of FsBmh1 resulted in the elevated expression of FsBmh2 (4.5-fold higher), and the deletion of FsBmh2 resulted in the elevated expression of FsBmh1 (2.2-fold higher).However, the reintroduction of a wild copy of FsBmh1 or FsBmh2 into the corresponding mutants (C-ΔFsBmh1 and C-ΔFsBmh2) abolished this expression compensation (Figure 2).

The Expression of FsBmh1 and FsBmh2 Are Mutually Compensated in Cells
To elucidate the distinct functions of two 14-3-3 proteins, we generated the gene deletion mutants ∆FsBmh1 and ∆FsBmh2 by homologous recombination (Figures S2 and S3).Intriguingly, the deletion of FsBmh1 resulted in the elevated expression of FsBmh2 (4.5-fold higher), and the deletion of FsBmh2 resulted in the elevated expression of FsBmh1 (2.2-fold higher).However, the reintroduction of a wild copy of FsBmh1 or FsBmh2 into the corresponding mutants (C-∆FsBmh1 and C-∆FsBmh2) abolished this expression compensation (Figure 2).

The Expression of FsBmh1 and FsBmh2 Are Mutually Compensated in Cells
To elucidate the distinct functions of two 14-3-3 proteins, we generated the gene deletion mutants ΔFsBmh1 and ΔFsBmh2 by homologous recombination (Figures S2 and  S3).Intriguingly, the deletion of FsBmh1 resulted in the elevated expression of FsBmh2 (4.5-fold higher), and the deletion of FsBmh2 resulted in the elevated expression of FsBmh1 (2.2-fold higher).However, the reintroduction of a wild copy of FsBmh1 or FsBmh2 into the corresponding mutants (C-ΔFsBmh1 and C-ΔFsBmh2) abolished this expression compensation (Figure 2).

Diverse Contributions of FsBmh1 and FsBmh2 to Fungal Phenotypes
Relative to the wild-type strain, the ∆FsBmh1 and ∆FsBmh2 mutants displayed a loss of light brown pigmentation and a reduction in growth by approximately 10% and 30%, respectively.The deletion of FsBmh1 resulted in longer, thinner microspores, while the deletion of FsBmh2 led to an increased number of hyphal branches, significantly shorter mycelial cells, and smaller microspores (Figure 3A and Tables S4 and S5).These findings indicate that both FsBmh1 and FsBmh2 are involved in regulating fungal cell architecture, albeit in distinct manners within F. sacchari.

Diverse Contributions of FsBmh1 and FsBmh2 to Fungal Phenotypes
Relative to the wild-type strain, the ΔFsBmh1 and ΔFsBmh2 mutants displayed a loss of light brown pigmentation and a reduction in growth by approximately 10% and 30%, respectively.The deletion of FsBmh1 resulted in longer, thinner microspores, while the deletion of FsBmh2 led to an increased number of hyphal branches, significantly shorter mycelial cells, and smaller microspores (Figure 3A and Tables S4 and S5).These findings indicate that both FsBmh1 and FsBmh2 are involved in regulating fungal cell architecture, albeit in distinct manners within F. sacchari.
Microconidia quantification revealed that both FsBmh1 and FsBmh2 contribute to sporulation, with FsBmh2 having a more pronounced impact.For instance, the deletion of FsBmh1 reduced spore production by 43%, while the deletion of FsBmh2 led to an 80% reduction (Figure 3B).Furthermore, the ΔFsBmh2 spores exhibited a lower germination rate (67%) compared to the wild-type, whereas ΔFsBmh1 did not significantly affect germination (Figure 3C).The reintroduction of wild-type copies of FsBmh1 into ΔFsBmh1 (C-ΔFsBmh1) or FsBmh2 into ΔFsBmh2 (C-ΔFsBmh2) fully restored all the observed defects.Microconidia quantification revealed that both FsBmh1 and FsBmh2 contribute to sporulation, with FsBmh2 having a more pronounced impact.For instance, the deletion of FsBmh1 reduced spore production by 43%, while the deletion of FsBmh2 led to an 80% reduction (Figure 3B).Furthermore, the ∆FsBmh2 spores exhibited a lower germination rate (67%) compared to the wild-type, whereas ∆FsBmh1 did not significantly affect germination (Figure 3C).The reintroduction of wild-type copies of FsBmh1 into ∆FsBmh1 (C-∆FsBmh1) or FsBmh2 into ∆FsBmh2 (C-∆FsBmh2) fully restored all the observed defects.

FsBmh1 and FsBmh2
Interactively Regulate Spore Morphology in F. sacchari Attempts to simultaneously knockout both FsBmh1 and FsBmh2 in a single strain were unsuccessful, suggesting that the simultaneous deletion of these genes may result in a lethal phenotype in F. sacchari.Alternatively, we constructed strains with silenced FsBmh2 in the ∆FsBmh1 background (∆FsBmh1/siFsBmh2) and silenced FsBmh1 in the ∆FsBmh2 background (∆FsBmh2/siFsBmh1), with expression levels reduced to 15.3% and 11.1% of the wild-type strain, respectively (Figure 4A).At the colony level, silencing FsBmh2 in ∆FsBmh1 further inhibited mycelial growth compared to ∆FsBmh1 alone, which was similar to ∆FsBmh2; however, silencing FsBmh1 in ∆FsBmh2 did not significantly affect the growth (Figure 4B and Table S4).Examination of the hyphae revealed that the cell length of ∆FsBmh2 and ∆FsBmh1/siFsBmh2 was significantly shorter than that of the wild-type or ∆FsBmh1 (Figure 4B).The deletion of FsBmh1 and FsBmh2 reduced the spore yield by approximately 40% and 80%, respectively, indicating that FsBmh2 plays a more critical role in sporulation.This was further supported by the intermediate spore yield observed in ∆FsBmh1/siFsBmh2 (Figure 4C).Conversely, the deletion of FsBmh1 did not affect the spore germination rate, but the deletion of FsBmh2 reduced the germination rate by about 30% (Figure 4D).

FsBmh1 and FsBmh2 Interactively Regulate Spore Morphology in F. sacchari
Attempts to simultaneously knockout both FsBmh1 and FsBmh2 in a single strain were unsuccessful, suggesting that the simultaneous deletion of these genes may result in a lethal phenotype in F. sacchari.Alternatively, we constructed strains with silenced FsBmh2 in the ΔFsBmh1 background (ΔFsBmh1/siFsBmh2) and silenced FsBmh1 in the ΔFsBmh2 background (ΔFsBmh2/siFsBmh1), with expression levels reduced to 15.3% and 11.1% of the wild-type strain, respectively (Figure 4A).At the colony level, silencing FsBmh2 in ΔFsBmh1 further inhibited mycelial growth compared to ΔFsBmh1 alone, which was similar to ΔFsBmh2; however, silencing FsBmh1 in ΔFsBmh2 did not significantly affect the growth (Figure 4B and Table S4).Examination of the hyphae revealed that the cell length of ΔFsBmh2 and ΔFsBmh1/siFsBmh2 was significantly shorter than that of the wild-type or ΔFsBmh1 (Figure 4B).The deletion of FsBmh1 and FsBmh2 reduced the spore yield by approximately 40% and 80%, respectively, indicating that FsBmh2 plays a more critical role in sporulation.This was further supported by the intermediate spore yield observed in ΔFsBmh1/siFsBmh2 (Figure 4C).Conversely, the deletion of FsBmh1 did not affect the spore germination rate, but the deletion of FsBmh2 reduced the germination rate by about 30% (Figure 4D).On carnation leaf agar medium, a distribution of 35% microspores (mini and normal) and 65% macrospores (2-4 cells) was observed in the wild-type strain CNO-1.In contrast, the deletion of FsBmh1 increased the proportion of macrospores to 83%, while the deletion of FsBmh2 increased the microspores to 67%.The silencing of FsBmh2 in ∆FsBmh1 completely eliminated the increase in macrospores, whereas the silencing of FsBmh1 in ∆FsBmh2 did not alter the spore type distribution.The reintroduction of a wild-type copy of FsBmh1 into ∆FsBmh1 or FsBmh2 into ∆FsBmh2 fully restored the spore types and proportions.The overexpression of FsBmh1 or FsBmh2 did not significantly alter the sporulation pattern (Figure 5).
were stained with CFW and examined under a fluorescent microscope, scale bar = 10 µm .To minimize the error of comparing cells of different ages, only the fourth cells from the tip of a hypha were measured.The septa were marked by yellow arrows.Spores were photographed by a differentialinterference contrast microscope, scale bar = 10 µm.(C) Statistics of conidial yield of strains.Conidia were harvested from colonies of 7-days-old PDA plates.(D) Conidial germination rate in PDB at 28 °C with rotation of 150 rpm for 6 h.Different letters indicate significant differences at p < 0.05.
On carnation leaf agar medium, a distribution of 35% microspores (mini and normal) and 65% macrospores (2-4 cells) was observed in the wild-type strain CNO-1.In contrast, the deletion of FsBmh1 increased the proportion of macrospores to 83%, while the deletion of FsBmh2 increased the microspores to 67%.The silencing of FsBmh2 in ΔFsBmh1 completely eliminated the increase in macrospores, whereas the silencing of FsBmh1 in ΔFsBmh2 did not alter the spore type distribution.The reintroduction of a wild-type copy of FsBmh1 into ΔFsBmh1 or FsBmh2 into ΔFsBmh2 fully restored the spore types and proportions.The overexpression of FsBmh1 or FsBmh2 did not significantly alter the sporulation pattern (Figure 5).

Overexpression of FsBmh Does Not Alter the Phenotype of F. sacchari
We developed overexpression strains for FsBmh1 and FsBmh2, which exhibited a 20.7-fold and 9.6-fold increase in expression, respectively.No significant phenotypic differences, including hyphal growth, branching, cell length, sporulation, spore morphology, and germination rate, were observed between the overexpression strains (O-FsBmh) and the wild-type strain (Figure S6).

FsBmh1 and FsBmh2 Are Essential for F. sacchari Virulence
To assess the impact of FsBmh1 and FsBmh2 on the virulence of F. sacchari, we inoculated sugarcane plants with conidia from the wild-type and mutant strains.The plants inoculated with the wild-type strain displayed typical PBD symptoms by 14 days postinoculation (dpi), whereas those inoculated with ∆FsBmh1, ∆FsBmh2, ∆FsBmh1/siFsBmh2, or ∆FsBmh2/siFsBmh1 exhibited significantly milder symptoms by 21 dpi.The virulence of FsBmh2 was fully restored by reintroducing a wild-type copy, while only about 60% of the virulence was restored in the FsBmh1 deletion mutant.However, the overexpression of FsBmh1 resulted in a significant increase in virulence, whereas the overexpression of FsBmh2 did not (Figure 6).
different types of spores (n = 100).The images were taken using a differential-interference contrast microscope.

Overexpression of FsBmh Does Not Alter the Phenotype of F. sacchari
We developed overexpression strains for FsBmh1 and FsBmh2, which exhibited a 20.7fold and 9.6-fold increase in expression, respectively.No significant phenotypic differences, including hyphal growth, branching, cell length, sporulation, spore morphology, and germination rate, were observed between the overexpression strains (O-FsBmh) and the wild-type strain (Figure S6).

FsBmh1 and FsBmh2 Are Essential for F. sacchari Virulence
To assess the impact of FsBmh1 and FsBmh2 on the virulence of F. sacchari, we inoculated sugarcane plants with conidia from the wild-type and mutant strains.The plants inoculated with the wild-type strain displayed typical PBD symptoms by 14 days postinoculation (dpi), whereas those inoculated with ΔFsBmh1, ΔFsBmh2, ΔFsBmh1/siFsBmh2, or ΔFsBmh2/siFsBmh1 exhibited significantly milder symptoms by 21 dpi.The virulence of FsBmh2 was fully restored by reintroducing a wild-type copy, while only about 60% of the virulence was restored in the FsBmh1 deletion mutant.However, the overexpression of FsBmh1 resulted in a significant increase in virulence, whereas the overexpression of FsBmh2 did not (Figure 6).

Transcriptomic Analysis Reveals the Functional Basis of FsBmh1 and FsBmh2
To understand the mechanisms by which FsBmh1 and FsBmh2 regulate fungal phenotypes, we performed comparative transcriptomic analysis on ΔFsBmh1, ΔFsBmh2, and the wild-type (WT) strain.A total of 4296 and 4921 differentially expressed genes (DEGs) were identified between ΔFsBmh1 and WT and between ΔFsBmh2 and WT, respectively.Among these, 3804 DEGs were shared by both mutants (Figure 7A).Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the up-regulated genes

Transcriptomic Analysis Reveals the Functional Basis of FsBmh1 and FsBmh2
To understand the mechanisms by which FsBmh1 and FsBmh2 regulate fungal phenotypes, we performed comparative transcriptomic analysis on ∆FsBmh1, ∆FsBmh2, and the wild-type (WT) strain.A total of 4296 and 4921 differentially expressed genes (DEGs) were identified between ∆FsBmh1 and WT and between ∆FsBmh2 and WT, respectively.Among these, 3804 DEGs were shared by both mutants (Figure 7A).Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the up-regulated genes in ∆FsBmh1 and ∆FsBmh2 were primarily enriched in pathways related to ribosome biogenesis, RNA polymerase, RNA transport, mismatch repair, and DNA replication.Specifically, the up-regulated DEGs in ∆FsBmh2 were enriched in amino acid and pantothenate metabolism pathways.Conversely, the down-regulated DEGs in both mutants were mainly involved in carbohydrate, amino acid, lipid, and energy metabolism (Figure 7B).Quantitative real-time PCR (qRT-PCR) validation of 15 randomly selected DEGs confirmed the consistency with the transcriptomic data (Figure S7 and Table S2).
in ΔFsBmh1 and ΔFsBmh2 were primarily enriched in pathways related to ribosome biogenesis, RNA polymerase, RNA transport, mismatch repair, and DNA replication.Specifically, the up-regulated DEGs in ΔFsBmh2 were enriched in amino acid and pantothenate metabolism pathways.Conversely, the down-regulated DEGs in both mutants were mainly involved in carbohydrate, amino acid, lipid, and energy metabolism (Figure 7B).Quantitative real-time PCR (qRT-PCR) validation of 15 randomly selected DEGs confirmed the consistency with the transcriptomic data (Figure S7 and Table S2).Hierarchical clustering analysis of DEGs enriched in KEGG pathways identified 981 genes (Table S11) that could be grouped into 10 clusters (Figure 8A).These genes encode proteins crucial for various cellular processes.Cluster VI, with the highest number of genes (450), contained DEGs up-regulated in both ΔFsBmh1 and ΔFsBmh2, with ΔFsBmh2 showing higher expression levels.Most of these genes were involved in DNA repair, mismatch repair, DNA metabolic processes, and stress response, as indicated by Hierarchical clustering analysis of DEGs enriched in KEGG pathways identified 981 genes (Table S11) that could be grouped into 10 clusters (Figure 8A).These genes encode proteins crucial for various cellular processes.Cluster VI, with the highest number of genes (450), contained DEGs up-regulated in both ∆FsBmh1 and ∆FsBmh2, with ∆FsBmh2 showing higher expression levels.Most of these genes were involved in DNA repair, mismatch repair, DNA metabolic processes, and stress response, as indicated by gene ontology (GO) enrichment.Notably, these included genes encoding heat shock protein SSB1 (FVER_06187), cell division control protein 45 (FVER_01583), and DNA mismatch repair protein MSH3 (FVER_14666), which align with the stressed phenotypes observed in ∆FsBmh2.Cluster V, consisting of 16 genes, were primarily involved in cysteine and methionine metabolism, with expression most suppressed in ∆FsBmh1 compared to WT and ∆FsBmh1.Cluster X, containing 33 genes with catalytic activity, such as chitin synthase (FVER_07617), gamma-glutamyl transpeptidase (FVER_11123), and malate dehydrogenase (FVER_13093), was specifically down-regulated in ∆FsBmh2.Clusters I through III, com-prising 14, 87, and 182 genes, respectively, contained genes that were highly expressed in the WT, including FVER_14638 (1,3-beta-glucosidase), pyruvate kinase (FVER_02565), low-affinity hexose transporter HXT3 (FVER_03150), vesicle-associated membrane protein 7 (Vam7, FVER_01791), and protein transport membrane glycoprotein Sec20 (FVER_03801) (Table S11).

Identification of Direct Targets of FsBmh1 and FsBmh2
Given the modulatory role of 14-3-3 proteins in cellular processes through their interactions with target proteins, we utilized the yeast two-hybrid (Y2H) system to identify the direct interactors of FsBmh1 and FsBmh2.Our analysis revealed a total of 15 and 17 proteins interacting with FsBmh1 and FsBmh2, respectively.Among these, 12 proteins were common targets for both FsBmh1 and FsBmh2, while 3 were specific to FsBmh1 and

Identification of Direct Targets of FsBmh1 and FsBmh2
Given the modulatory role of 14-3-3 proteins in cellular processes through their interactions with target proteins, we utilized the yeast two-hybrid (Y2H) system to identify the direct interactors of FsBmh1 and FsBmh2.Our analysis revealed a total of 15 and 17 proteins interacting with FsBmh1 and FsBmh2, respectively.Among these, 12 proteins were common targets for both FsBmh1 and FsBmh2, while 3 were specific to FsBmh1 and 5 to FsBmh2.The shared targets are predominantly involved in primary metabolic pathways, including starch and sucrose metabolism, glycolysis, protein and nucleotide synthesis, and energy generation, which are crucial for fungal growth and development.The Fs-Bmh1 or FsBmh2 specific target proteins are implicated in processes such as glycolysis, the tricarboxylic acid cycle, oxidoreduction, meiosis, and cell membrane stability (Table 1).Notably, FsBmh2 was found to interact with a hsp70-like molecular chaperone and the meiosis-related protein Mei5.If the functionality of these proteins is contingent upon their interaction with FsBmh2, the deletion of FsBmh2 could potentially have a profound effect on the growth and developmental processes of the fungus.

Discussion
This study elucidates the pivotal roles of the 14-3-3 protein genes FsBmh1 and FsBmh2 in the development, growth, conidiation, and virulence of F. sacchari, the causative agent of sugarcane Pokkah boeng disease.The observed developmental defects, including pigment loss, growth reduction, and attenuated virulence in the deletion mutants, align with previous findings that 14-3-3 proteins are integral to the regulation of cellular processes in various fungi, such as vegetative development, metabolism, stress response, signaling pathways, transcription factor activity, and pathogenicity [34][35][36][37][38].
The redundancy of essential genes, often present in multiple copies within a genome, ensures functional redundancy [39].Our findings indicate that while FsBmh1 or FsBmh2 can be singly dispensable for growth, their combined deletion is lethal, underscoring their essential roles in F. sacchari.The mutual compensation at the transcriptional level observed for FsBmh1 and FsBmh2 (Figures 2 and S6A) is consistent with findings in Beauveria bassiana and Ganoderma lucidum [40,41].
In F. graminearum, FgBmh1 and FgBmh2 co-regulate nitrogen sensing, but FgBmh2 is uniquely required for conidiation and virulence.In contrast, FsBmh1 and FsBmh2 exhibit redundant roles in growth, conidiation, and pathogenicity, yet they diverge in the regulation of other phenotypic traits.For instance, the knockdown of FsBmh2 alone leads to reduced spore germination, shorter hyphal cells, and increased branching (Figures 3 and 4).This contrasts with B. bassiana, where conidial germination is accelerated in both 14-3-3 genes deletion mutants, and G. lucidum, where silencing either GlBmh1 or GlBmh2 results in increased hyphal branching.Notably, FsBmh1 and FsBmh2 differentially regulate spore morphology; FsBmh1 knockdown results in slender spores, while FsBmh2 knockdown produces thicker, shorter spores under nutrient-rich conditions (Figures 3 and 4).However, on carnation leaf agar medium, FsBmh1 knockdown enhances macrospore production (Figure 5), a phenomenon not previously reported in filamentous fungi.
Furthermore, divergence in functions of FsBmh1 and FsBmh2 in germination, hyphal cell length could be linked to clusters of DEGs.For example, Chs1 (encoding chitin synthase 1) and BgIB (β-glucosidase), reported to regulate conidial germination through cell wall remodeling in Aspergillus fumigatus and A. flavus [52,53], were uniquely decreased in ∆FsBmh2; specific downregulation of Mae1, encoding a malate dehydrogenase which has been shown to regulate hyphal septa, cell length, and the number of nuclei in Arthrobotrys oligospora [54], was found only in ∆FsBmh2 (Figure 8, cluster X).On the other hand, decrease in aerial hyphae in ∆FsBmh1 was accompanied by the down regulation of expression of Mon1, vps39, and Met17, (Figure 8, cluster V), as these genes have been shown to be critical for aerial hyphae formation and virulence in F. graminearum and Alternaria alternata [55,56].Since both FsBmh1 and FsBmh2 locate in the cytoplasm and most of their potential targets also locate in the cytoplasm, these may form a spatial basis for their diversified functions.The discrepancy in target specificity between FsBmh1 and FsBmh2 may constitute the molecular basis for the non-redundant functions of the two 14-3-3 proteins in F. sacchari.

Figure 1 .
Figure 1.Expression patterns of FsBmh1 and FsBmh2 at different developmental stages in vivo and in vitro.(A) Symptom development.Sugarcane seedlings were inoculated with CNO-1.(B) Development of infection.Tissue sections were stained with aniline blue to trace fungal hyphae development.Samples were observed under a light microscope, scale bar = 50 µm.(C) Relative expression of FsBmh1 and FsBmh2 during infection.(D) Development of hyphae and asexual spores on liquid medium.The spore suspension of CNO-1 was inoculated in PDB medium, and representative pictures were photographed at 10 h, 14 h, and 22 h by microscope, scale bar = 50 µm.(E) Relative expression of FsBmh1 and FsBmh2 during saprophytic stages.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −ΔΔCT method) with 18S rRNA as internal reference.The transcript levels of 0 h post-inoculation were set to a value of 1.0 and indicated by the dotted line.Different letters indicate significant differences at p < 0.05.

Figure 1 .
Figure 1.Expression patterns of FsBmh1 and FsBmh2 at different developmental stages in vivo and in vitro.(A) Symptom development.Sugarcane seedlings were inoculated with CNO-1.(B) Development of infection.Tissue sections were stained with aniline blue to trace fungal hyphae development.Samples were observed under a light microscope, scale bar = 50 µm.(C) Relative expression of FsBmh1 and FsBmh2 during infection.(D) Development of hyphae and asexual spores on liquid medium.The spore suspension of CNO-1 was inoculated in PDB medium, and representative pictures were photographed at 10 h, 14 h, and 22 h by microscope, scale bar = 50 µm.(E) Relative expression of FsBmh1 and FsBmh2 during saprophytic stages.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −∆∆CT method) with 18S rRNA as internal reference.The transcript levels of 0 h post-inoculation were set to a value of 1.0 and indicated by the dotted line.Different letters indicate significant differences at p < 0.05.

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. Fungi 2024, 10, x FOR PEER REVIEW 5 of 18 revealed distinct transcriptional dynamics for FsBmh1 and FsBmh2 during plant infection and vegetative growth in the medium.

Figure 1 .
Figure 1.Expression patterns of FsBmh1 and FsBmh2 at different developmental stages in vivo and in vitro.(A) Symptom development.Sugarcane seedlings were inoculated with CNO-1.(B) Development of infection.Tissue sections were stained with aniline blue to trace fungal hyphae development.Samples were observed under a light microscope, scale bar = 50 µm.(C) Relative expression of FsBmh1 and FsBmh2 during infection.(D) Development of hyphae and asexual spores on liquid medium.The spore suspension of CNO-1 was inoculated in PDB medium, and representative pictures were photographed at 10 h, 14 h, and 22 h by microscope, scale bar = 50 µm.(E) Relative expression of FsBmh1 and FsBmh2 during saprophytic stages.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −ΔΔCT method) with 18S rRNA as internal reference.The transcript levels of 0 h post-inoculation were set to a value of 1.0 and indicated by the dotted line.Different letters indicate significant differences at p < 0.05.

Figure 2 .
Figure 2. Validation of FsBmh1 and FsBmh2 expression using qRT-PCR in FsBmh deletion and complementation mutants.Relative expression of FsBmh genes in FsBmh-deletion and complementation mutants were determined by qRT-PCR.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −∆∆CT method) with 18S rRNA as internal reference.The transcript levels of WT were set to a value of 1.0 and indicated by the dotted line.Different letters indicate significant differences at p < 0.05.

Figure 3 .Figure 3 .
Figure 3. Phenotypes of FsBmh-deletion mutants and complementation mutants.(A) Hyphal and conidial morphology.Colonies were photographed on PDA plates on day 7 after inoculation.Hyphal branches were photographed on PDA plates on day 3 after inoculation using a microscope, scale bar = 50 µm.To visualize septa, hyphae were stained with CFW and examined under a fluorescent microscope, scale bar = 10 µm.To minimize the error of comparing cells of different ages,Figure 3. Phenotypes of FsBmh-deletion mutants and complementation mutants.(A) Hyphal and conidial morphology.Colonies were photographed on PDA plates on day 7 after inoculation.Hyphal branches were photographed on PDA plates on day 3 after inoculation using a microscope, scale bar = 50 µm.To visualize septa, hyphae were stained with CFW and examined under a fluorescent microscope, scale bar = 10 µm.To minimize the error of comparing cells of different ages, only the fourth cells from the tip of a hypha were measured.The septa were marked by yellow arrows.Spore characteristics were photographed by a differential-interference contrast microscope, scale bar = 10 µm.(B) Statistics of conidial yield of strains.Conidia were harvested from colonies of 7-days-old PDA plates.(C) Conidial germination rate in PDB at 28 • C with rotation of 150 rpm for 6 h.Different letters indicate significant differences at p < 0.05.

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. Fungi 2024, 10, x FOR PEER REVIEW 7 of 18 only the fourth cells from the tip of a hypha were measured.The septa were marked by yellow arrows.Spore characteristics were photographed by a differential-interference contrast microscope, scale bar = 10 µm.(B) Statistics of conidial yield of strains.Conidia were harvested from colonies of 7-days-old PDA plates.(C) Conidial germination rate in PDB at 28 °C with rotation of 150 rpm for 6 h.Different letters indicate significant differences at p < 0.05.

Figure 4 .
Figure 4. Phenotypes of FsBmh-deletion mutants and FsBmh-silenced mutants.(A) Validation of FsBmh1 and FsBmh2 expression using qRT-PCR in ΔFsBmh1/siFsBmh2 and ΔFsBmh2/siFsBmh1.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −ΔΔCT method) with 18S rRNA as internal reference.The transcript levels of WT were set to a value of 1.0 and indicated by the dotted line.(B) Hyphal and conidial morphology.Colonies were photographed on PDA plates on day 7 after inoculation.Hyphal branches were photographed on PDA plates on day 3 after inoculation using a microscope, scale bar = 50 µm.To visualize septa, hyphae

Figure 4 .
Figure 4. Phenotypes of FsBmh-deletion mutants and FsBmh-silenced mutants.(A) Validation of FsBmh1 and FsBmh2 expression using qRT-PCR in ∆FsBmh1/siFsBmh2 and ∆FsBmh2/siFsBmh1.The expression of FsBmh1 and FsBmh2 transcripts was measured by quantitative real-time RT-PCR (2 −∆∆CT method) with 18S rRNA as internal reference.The transcript levels of WT were set to a value of 1.0 and indicated by the dotted line.(B) Hyphal and conidial morphology.Colonies were photographed on PDA plates on day 7 after inoculation.Hyphal branches were photographed on PDA plates on day 3 after inoculation using a microscope, scale bar = 50 µm.To visualize septa, hyphae were stained with CFW and examined under a fluorescent microscope, scale bar = 10 µm.To minimize the error of comparing cells of different ages, only the fourth cells from the tip of a hypha were measured.The septa were marked by yellow arrows.Spores were photographed by a differential-interference contrast microscope, scale bar = 10 µm.(C) Statistics of conidial yield of strains.Conidia were harvested from colonies of 7-days-old PDA plates.(D) Conidial germination rate in PDB at 28 • C with rotation of 150 rpm for 6 h.Different letters indicate significant differences at p < 0.05.

Figure 6 .
Figure 6.FsBmh-deletion mutants are defective in virulence.Each of the plants was injected with 300 µL of spore suspension at concentration of 1 × 10 4 conidia/mL.(A) Symptoms on sugarcane seedlings.Photographs were taken 21 dpi.(B) Quantification of disease severity.The disease severity index was determined using 100 seedlings per treatment with 3 replicates.Different letters indicate significant difference at p < 0.05.

Figure 6 .
Figure 6.FsBmh-deletion mutants are defective in virulence.Each of the plants was injected with 300 µL of spore suspension at concentration of 1 × 10 4 conidia/mL.(A) Symptoms on sugarcane seedlings.Photographs were taken 21 dpi.(B) Quantification of disease severity.The disease severity index was determined using 100 seedlings per treatment with 3 replicates.Different letters indicate significant difference at p < 0.05.

Figure 7 .
Figure 7. Distribution of DEGs (≥two-fold) in ΔFsBmh1 and ΔFsBmh2 versus WT. (A) Venn diagrams showing the overlapped counts of the genes up-regulated or down-regulated in both ΔFsBmh1 and ΔFsBmh2 versus WT. (B) KEGG pathway enrichment of genes up-regulated or downregulated in ΔFsBmh1 and ΔFsBmh2 versus WT.

Figure 7 .
Figure 7. Distribution of DEGs (≥two-fold) in ∆FsBmh1 and ∆FsBmh2 versus WT. (A) Venn diagrams showing the overlapped counts of the genes up-regulated or down-regulated in both ∆FsBmh1 and ∆FsBmh2 versus WT. (B) KEGG pathway enrichment of genes up-regulated or down-regulated in ∆FsBmh1 and ∆FsBmh2 versus WT.

Figure 8 .
Figure 8. Hierarchical clustering of DEGs among WT, ΔFsBmh1, and ΔFsBmh2.(A) A total of 981 DEGs that were enriched in KEGG pathways (ΔFsBmh1 and ΔFsBmh2 versus WT) were clustered into 10 clusters.Red indicates high expression, blue indicates low expression.(B) Gene expression values on selected clusters.The dotted line indicates the average expression level of all DEGs within the cluster and its value was set as 0.Color scale shows the level of gene expression of log10(FPKM+ 1) .

Figure 8 .
Figure 8. Hierarchical clustering of DEGs among WT, ∆FsBmh1, and ∆FsBmh2.(A) A total of 981 DEGs that were enriched in KEGG pathways (∆FsBmh1 and ∆FsBmh2 versus WT) were clustered into 10 clusters.Red indicates high expression, blue indicates low expression.(B) Gene expression values on selected clusters.The dotted line indicates the average expression level of all DEGs within the cluster and its value was set as 0.Color scale shows the level of gene expression of log10(FPKM+ 1) .