Fumonisin B1 Biosynthesis Research Articles

Volatilome of the maize phytopathogenic fungus Fusarium verticillioides: potential applications in diagnosis and biocontrol.

Fusarium verticillioides is a maize fungal phytopathogen and a producer of volatile organic compounds (VOCs) and fumonisin B1 (FB1). Our aim was to study the volatilome, conidial production, ergosterol and FB1 biosynthesis in maize cultures over a 30-day incubation period (5, 10, 15, 20, 25, 30 days post inoculation [DPI]). The effect of pure VOCs on the same parameters was then evaluated to study their potential role as biocontrol agents. In total, 91 VOCs were detected, with volatile profiles being more similar between 5 and 10 DPI compared with 15, 20, 25 and 30 DPI. Ergosterol content increased steadily with incubation time, and three growth stages were identified: a lag phase (0 to 15 DPI), an exponential phase (15 to 20 DPI) and a stationary phase (20 to 30 DPI). The maximum concentration of FB1 was detected at 25 (0.030 μg FB1/μg ergosterol) and 30 DPI (0.037 μg FB1/μg ergosterol), whereas conidial production showed a maximum value at 15 DPI (4.3 ± 0.2 × 105 conidia/μg ergosterol). Regarding pure VOCs, minimal inhibitory concentration values ranged from 0.3 mm for 4-hexen-3-one to 7.4 mm for 2-undecanone. Pure VOCs reduced radial growth, conidial production and ergosterol and FB1 biosynthesis. The marked resemblance between VOC profiles at 5 and 10 DPI suggests that they could act as early indicators of fungal contamination, particularly 4-ethylguaiacol, 4-ethyl-2-methoxyanisole, heptanol and heptyl acetate. On the other hand, their role as inhibitors of fungal growth and FB1 biosynthesis prove their great potential as safer alternatives to control phytopathogenic fungi. © 2024 Society of Chemical Industry.

Functional analysis of cutinase transcription factors in Fusarium verticillioides

Fusarium verticillioides is an important pathogen of maize and causes serious yield losses and food safety issues worldwide. F. verticillioides produces highly toxic mycotoxin Fumonisin B1 (FB1) in infested commodities which makes these food and feeds unsafe for humans and animals. For pathogenic fungi to successfully penetrate its plant hosts, the pathogen secretes hydrolytic enzymes that can facilitate penetration into the plant cutin layer. However, there is limited information on how cutinases transcriptionally regulated to impact F. verticillioides pathogenicity. In this study, our aim is to functionally characterize cutinase transcription factors that regulate key cutinase activities that are directly associated with F. verticillioides pathogenicity and FB1 biosynthesis. Gene deletion of cutinase transcription factor FvCTF1α did not affect the growth and morphology of the fungal mycelia on CMII medium, whereas the conidiation, utilization of sodium acetate and sodium oleate, stress tolerance against cell wall interfering agent, and the cutinase and pectinase activities in the ΔFvctf1α mutant were negatively impacted. FvCtf1α regulates the expression of induced cutinase genes FvCUT1 and FvCUT4 by binding to their GC-rich promoters. In addition, FvCtf1α, containing a novel function in regulating FB1, interacts with the promoter of FvFUM1 and FvFUM6 to down-regulate the expression of FvFUM1 and FvFUM6, resulting in decreased production of FB1 in the ΔFvctf1α strain. ΔFvctf1α exhibited decreased pathogenicity in maize due to the down-regulation of pathogenicity-related genes as well as key downstream cutinase genes FvCUT3 and FvCUT4 in F. verticillioides. We also demonstrated that FvCtf1α regulated FvCUT3 and FvCUT4 differently; FvCUT4 via direct regulation while FvCUT3 via indirect regulation by interacting with FvFarB, a homologous protein of FvCtf1α. Moreover, RNA-seq analysis showed that FvCtf1α was associated with many pathways, such as fatty acid metabolism, carbon source utilization, cell wall integrity, oxidative stress, and fumonisin synthesis in F. verticillioides. Our study demonstrated that FvCtf1α was not only involved in the regulation of cutinases but also a broad spectrum of pathways that ultimately affect F. verticillioides virulence and mycotoxin biosynthesis.

Validamycin A Inhibited FB1 Biosynthesis by the Target FvNth in Fusarium verticillioides.

Validamycin A (VMA) is an antifungal antibiotic derived from Streptomyces hygroscopicus commonly used in plant disease management. Surprisingly, VMA was discovered to impede the production of fumonisin B1 (FB1) in agricultural settings. However, the specific target of VMA in Fusarium verticillioides remained unclear. To unravel the molecular mechanism of VMA, ultrastructural observations unveiled damage to mitochondrial membranes. Trehalase (FvNth) was pinpointed as the target of VMA by utilizing a 3D-printed surface plasmon resonance sensor. Molecular docking identified Trp285, Arg447, Asp452, and Phe665 as the binding sites between VMA and FvNth. A ΔFvnth mutant lacking amino acids 250-670 was engineered through homologous recombination. Transcriptome analysis indicated that samples treated with VMA and ΔFvnth displayed similar expression patterns, particularly in the suppression of the FUM gene cluster. VMA treatment resulted in reduced trehalase and ATPase activity as well as diminished production of glucose, pyruvic acid, and acetyl-CoA. Conversely, these effects were absent in samples treated with ΔFvnth. This research proposes that VMA hinders acetyl-CoA synthesis by trehalase, thereby suppressing the FB1 biosynthesis. These findings present a novel target for the development of mycotoxin control agents.

The MAP kinase FvHog1 regulates FB1 synthesis and Ca2+ homeostasis in Fusarium verticillioides

The high osmolarity glycerol 1 mitogen-activated protein kinase (Hog1-MAPK) cascade genes are important for diverse biological processes. The activated Hog1 upon multiple environmental stress stimuli enters into the nucleus where it directly phosphorylates transcription factors to regulate various physiological processes in phytopathogenic fungi. However, their roles have not been well-characterized in Fusarium verticillioides. In this study, FvHog1 is identified and functionally analyzed. The findings reveal that the phosphorylation level and nuclear localization of FvHog1 are increased in Fumonisin B1 (FB1)-inducing condition to regulate the expression of FB1 biosynthesis FUM genes. More importantly, the deletion mutants of Hog1-MAPK pathway show increased sensitivity to Ca2+ stress and elevated intracellular Ca2+ content. The phosphorylation level and nuclear localization of FvHog1 are increased with Ca2+ treatment. Furthermore, our results show that FvHog1 can directly phosphorylate Ca2+-responsive zinc finger transcription factor 1 (FvCrz1) to regulate Ca2+ homeostasis. In conclusion, our findings indicate that FvHog1 is required for FB1 biosynthesis, pathogenicity and Ca2+ homeostasis in F. verticillioides. It provides a theoretical basis for effective prevention and control maize ear and stalk rot disease.

FvKex2 is required for development, virulence, and mycotoxin production in Fusarium verticillioides

Fusarium verticillioides is one of the most important fungal pathogens causing maize ear and stalk rots, thereby undermining global food security. Infected seeds are usually unhealthy for consumption due to contamination with fumonisin B1 (FB1) mycotoxin produced by the fungus as a virulence factor. Unveiling the molecular factors that determine fungal development and pathogenesis will help in the control and management of the diseases. Kex2 is a kexin-like Golgi-resident proprotein convertase that is involved in the activation of some important proproteins. Herein, we identified and functionally characterized FvKex2 in relation to F. verticillioides development and virulence by bioinformatics and functional genomics approaches. We found that FvKex2 is required for the fungal normal vegetative growth, because the growth of the ∆Fvkex2 mutant was significantly reduced on culture media compared to the wild-type and complemented strains. The mutant also produced very few conidia with morphologically abnormal shapes when compared with those from the wild type. However, the kexin-like protein was dispensable for the male role in sexual reproduction in F. verticillioides. In contrast, pathogenicity was nearly abolished on wounded maize stalks and sugarcane leaves in the absence of FvKEX2 gene, suggesting an essential role of Fvkex2 in the virulence of F. verticillioides. Furthermore, high-performance liquid chromatography analysis revealed that the ∆Fvkex2 mutant produced a significantly lower level of FB1 mycotoxin compared to the wild-type and complemented strains, consistent with the loss of virulence observed in the mutant. Taken together, our results indicate that FvKex2 is critical for vegetative growth, FB1 biosynthesis, and virulence, but dispensable for sexual reproduction in F. verticillioides. The study presents the kexin-like protein as a potential drug target for the management of the devastating maize ear and stalk rot diseases. Further studies should aim at uncovering the link between FvKex2 activity and FB1 biosynthesis genes.Key points•The kexin-like protein FvKex2 contributes significantly to the vegetative growth of Fusarium verticillioides.•The conserved protein is required for fungal conidiation and conidial morphology, but dispensable for sexual reproduction.•Deletion of FvKEX2 greatly attenuates the virulence and mycotoxin production potential of F. verticillioides.

Characterization of Fusarium verticillioides Med1 LxxLL Motif Involved in Fumonisin Biosynthesis.

The Med1 transcriptional coactivator is a crucial component of the Mediator middle complex, which regulates the expression of specific genes involved in cell development, differentiation, reproduction, and homeostasis. The Med1 LxxLL motif, a five-amino-acid peptide sequence, is essential for Med1-mediated gene expression. Our previous study revealed that the disruption of the Med1 subunit leads to a significant increase in fumonisin B1 (FB1) production in the maize pathogen Fusarium verticillioides. However, our understanding of how Med1 regulates FB1 biosynthesis in F. verticillioides, particularly through the Med1 LxxLL motifs, remains limited. To characterize the role of LxxLL motifs, we generated a series of Med1 LxxLL deletion and amino acid substitution mutants. These mutants exhibited impaired mycelial growth and conidia germination while demonstrating enhanced conidia production and virulence. Similar to the Med1 deletion mutant, Med1 LxxLL motif mutants also exhibited increased FB1 biosynthesis in F. verticillioides. Proteomic profiling revealed that the Med1 LxxLL motif regulated the biosynthesis of several key substances that affected FB1 production, including starch and carotenoid. Subsequent studies demonstrated that the production of amylopectin, which is strongly linked to FB1 biosynthesis, was significantly increased in Med1 LxxLL motif mutants. In addition, the disruption of carotenoid metabolic genes decreased carotenoid content, thus stimulating FB1 biosynthesis in F. verticillioides. Taken together, our results provide valuable insights into how the Med1 LxxLL motif regulates FB1 biosynthesis in the mycotoxigenic fungus F. verticillioides.

Fumonisin B1 Biosynthesis Is Associated with Oxidative Stress and Plays an Important Role in Fusarium proliferatum Infection on Banana Fruit.

Fungal response to oxidative stress during infection on postharvest fruit is largely unknown. Here, we found that hydrogen peroxide (H2O2) treatment inhibited the growth of Fusarium proliferatum causing crown rot of banana fruit, confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation. H2O2 exposure increased endogenous reactive oxygen species (ROS) and fumonisin B1 (FB1) production in F. proliferatum, possibly by modulating FUM or ROS-related gene expression. Importantly, H2O2 treatment inhibited F. proliferatum growth in vivo but induced FB1 accumulation in banana peel. Finally, we constructed the FpFUM21 deletion mutant (ΔFpfum21) of F. proliferatum that was attenuated in FB1 biosynthesis and less tolerant to oxidative stress. Moreover, the ΔFpfum21 strain was less virulent compared to the wild type (WT) due to the inability to induce FB1 production in the banana host. These results suggested that FB1 biosynthesis is associated with oxidative stress in F. proliferatum and contributes to fungal infection on banana fruit.

Vacuole Proteins with Optimized Microtubule Assembly Is Required for Fum1 Protein Localization and Fumonisin Biosynthesis in Mycotoxigenic Fungus Fusarium verticillioides.

Fumonisin contamination of corn caused by Fusarium verticillioides is a major concern worldwide. While key genes involved in fumonisin biosynthesis are known, the location within the fungal cell where this process occurs has yet to be fully characterized. In this study, three key enzymes, i.e., Fum1, Fum8, and Fum6, associated with early steps of fumonisin biosynthesis pathway, were tagged with GFP, and we examined their cellular localization. Results showed that these three proteins co-localized with the vacuole. To further understand the role of the vacuole in fumonisin B1 (FB1) biosynthesis, we disrupted two predicted vacuole associated proteins, FvRab7 and FvVam7, resulting in a significant reduction of FB1 biosynthesis and a lack of Fum1-GFP fluorescence signal. Furthermore, we used the microtubule-targeting drug carbendazim to show that proper microtubule assembly is critical for proper Fum1 protein localization and FB1 biosynthesis. Additionally, we found that α1 tubulin is a negative regulator in FB1 biosynthesis. We concluded that vacuole proteins with optimized microtubule assembly play a crucial role in proper Fum1 protein localization and fumonisin production in F. verticillioides.

Fusarium verticillioides Pex7/20 mediates peroxisomal PTS2 pathway import, pathogenicity, and fumonisin B1 biosynthesis.

Fusarium verticillioides, a well-known fungal pathogen that causes severe disease in maize and contaminates the grains with fumonisin B1 (FB1) mycotoxin, affects the yield and quality of maize worldwide. The intrinsic roles of peroxisome targeting signal (PTS)-containing proteins in phytopathogens remain elusive. We therefore explored the regulatory role and other biological functions of the components of PTS2 receptor complex, FvPex7 and FvPex20, in F. verticillioides. We found that FvPex7 directly interacts with the carboxyl terminus of FvPex20 in F. verticillioides. PTS2-containing proteins are recognized and bound by the FvPex7 receptor or the FvPex7-Pex20 receptor complex in the cytoplasm, but the peroxisome localization of the PTS2-Pex7-Pex20 complex is only determined by Pex20 in F. verticillioides. However, we observed that some putative PTS2 proteins that interact with Pex7 are not transported into the peroxisomes, but a PTS1 protein that interacts with Pex5 was detected in the peroxisomes. Furthermore, ΔFvpex7pex20 as well as ΔFvpex7pex5 double mutants exhibited reduced pathogenicity and FB1 biosynthesis, along with defects in conidiation. The PTS2 receptor complex mutants (ΔFvpex7pex20) grew slowly on minimal media and showed reduced sensitivity to cell wall and cell membrane stress-inducing agents compared to the wild type. Taken together, we conclude that the PTS2 receptor complex mediates peroxisome matrix proteins import and contributes to pathogenicity and FB1 biosynthesis in F. verticillioides. KEY POINTS: • FvPex7 directly interacts with FvPex20 in F. verticillioides. • vThe PTS2 receptor complex is essential for the importation of PTS2-containing matrix protein into peroxisomes in F. verticillioides. • Fvpex7/pex20 is involved in pathogenicity and FB1 biosynthesis in F. verticillioides.

Sphingolipid synthesis inhibitor fumonisin B1 causes verticillium wilt in cotton

Verticillium wilt caused by Verticillium dahliae is a major disease of cotton. Acidic protein-lipopolysaccharide complexes are thought to be the toxins responsible for its symptoms. Here, we determined that the sphingolipid biosynthesis inhibitor fumonisin B1 (FB1) acts as a toxin and phenocopies the symptoms induced by V. dahliae. Knocking out genes required for FB1 biosynthesis reduced V. dahliae pathogenicity. Moreover, we showed that overexpression of a FB1 and V. dahliae both downregulated gene, GhIQD10, enhanced verticillium wilt resistance by promoting the expression of brassinosteroid and anti-pathogen genes. Our results provide a new strategy for preventing verticillium wilt in cotton.

The peroxisomal matrix shuttling receptor Pex5 plays a role in FB1 production and virulence in Fusarium verticillioides

The peroxisomal matrix oxidase, catalase and peroxidase are imported peroxisomes through the shuttling receptors, which regulates the cellular oxidative homeostasis and function. Here, we report that PTS1 shuttling receptor FvPex5 is involved in the localization of PTS1, utilization of carbon sources and lipids, elimination ROS, cell wall stress, conidiation, fumonisin B1 (FB1) production, and virulence in maize pathogen Fusarium verticillioides. Significantly, differential expression of PTS1-, PTS2-, PEX- and FB1 toxin-related genes in wild type and ΔFvpex5 mutant were examined by RNA-Seq analyses and confirmed by RT-PCR assay. In addition, different expression of PTS1 and PTS2 genes of the ΔFvpex5 mutant were enriched in diverse biochemical pathways, such as carbon metabolism, nitrogen metabolism, lipid metabolism and the oxidation balance by combining GO and KEGG annotations. Overall, we showed that FvPex5 is involved in the regulation of genes associated with PTS, thereby affecting the oxidation balance, FB1 and virulence in F. verticillioides. The results help to clarify the functional divergence of Pex5 orthologs, and may provide a possible target for controlling F. verticillioides infections and FB1 biosynthesis.

Glycosyltransferase FvCpsA Regulates Fumonisin Biosynthesis and Virulence in Fusarium verticillioides

Fusarium verticillioides is the major maize pathogen associated with ear rot and stalk rot worldwide. Fumonisin B1 (FB1) produced by F. verticillioides, poses a serious threat to human and animal health. However, our understanding of FB1 synthesis and virulence mechanism in this fungus is still very limited. Glycosylation catalyzed by glycosyltransferases (GTs) has been identified as contributing to fungal infection and secondary metabolism synthesis. In this study, a family 2 glycosyltransferase, FvCpsA, was identified and characterized in F. verticillioides. ΔFvcpsA exhibited significant defects in vegetative growth. Moreover, ΔFvcpsA also increased resistance to osmotic and cell wall stress agents. In addition, expression levels of FUM genes involved in FB1 production were greatly up-regulated in ΔFvcpsA. HPLC (high performance liquid chromatography) analysis revealed that ΔFvcpsA significantly increased FB1 production. Interestingly, we found that the deletion of FvCPSA showed penetration defects on cellophane membrane, and thus led to obvious defects in pathogenicity. Characterization of FvCpsA domain experiments showed that conserved DXD and QXXRW domains were vital for the biological functions of FvCpsA. Taken together, our results indicate that FvCpsA is critical for fungal growth, FB1 biosynthesis and virulence in F. verticillioides.

Fusarium verticillioides FvPex8 Is a Key Component of the Peroxisomal Docking/Translocation Module That Serves Important Roles in Fumonisin Biosynthesis but Not in Virulence.

Peroxisomes are ubiquitous organelles in eukaryotes that fulfill various important metabolic functions. In this study, we investigated the role of docking/translocation module (DTM) peroxins, mainly FvPex8, FvPex13, FvPex14, and FvPex33, in Fusarium verticillioides development, virulence, and fumonisin B1 (FB1) biosynthesis. Protein interaction experiments suggested that FvPex13 serves as the central DTM subunit in F. verticillioides. Notably, FvPex8 and FvPex14 did not show direct interaction in our experiments. We generated gene-deletion mutants (ΔFvpex8, ΔFvpex13, ΔFvpex14, ΔFvpex33, ΔFvpex33/14) and further examined the functional role of these peroxins. Deletion mutants exhibited disparity in carbon nutrient utilization and defect in cell-wall integrity when stress agents were applied. Under nutrient starvation, mutants also showed higher levels of lipid droplet accumulation. Particularly, ΔFvpex8 mutant showed significant FB1 reduction and altered expression of key FB1 biosynthesis genes. However, FvPex13 was primarily responsible for asexual conidia reproduction and virulence, while the ΔFvpex33/14 double mutant also showed a virulence defect. In summary, our study suggests that FvPex13 is the central component of DTM, with direct physical interaction with other DTM peroxins, and regulates peroxisome membrane biogenesis as well as PTS1- and PTS2-mediated transmembrane cargo transportation. Importantly, we also characterized FvPex8 as a key component in F. verticillioides DTM that affects peroxisome function and FB1 biosynthesis.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Effects of the volatile organic compounds produced by Enterococcus spp. strains isolated from maize grain silos on Fusarium verticillioides growth and fumonisin B1 production

Fusarium verticillioides is a phytopathogenic fungus that can contaminate maize grain silos and result in important losses in the post-harvest product. The objective of this work was to investigate the effects of volatile organic compounds produced by four lactic acid bacterial strains isolated from maize grain silos on F. verticillioides M3125 growth and fumonisin B1 (FB1) production. The bacterial isolates 55 and 49 were identified as Enterococcus faecium and M4A and M4G as Enterococcus casseliflavus. The fungal growth was inhibited by 33.33% by the volatiles released by the M4A strain and by approximately 10% by the volatiles emitted by the 55 and 49 strains. The volatiles produced by the M4A strain also significantly reduced (88.75%) FB1 biosynthesis. The gas chromatography–mass spectrometer analysis identified 21 volatile organic compounds, with diacetyl, acetic acid and acetoin being the main volatiles emitted by the four bacterial strains. Acetoin was the volatile produced in the highest proportion by the four strains, with M4A generating the highest proportion of diacetyl (35.11%). Diacetyl and acetic acid completely inhibited fungal growth at concentrations of 0.3 and 1 mM, respectively, while acetoin promoted fungal growth. Only acetoin significantly reduced FB1 production. These results showed that diacetyl was the main compound involved in fungal inhibition, while the effect on FB1 production could have been due to the combination of the volatile organic compounds produced by the M4A strain. In conclusion, the volatiles emitted by the E. casseliflavus M4A strain could be a promising tool for the biocontrol of F. verticillioides in storage maize grain silos.

Two regulators of G-protein signaling (RGS) proteins FlbA1 and FlbA2 differentially regulate fumonisin B1 biosynthesis in Fusarium verticillioides.

Fumonisins are a group of mycotoxins produced by maize pathogen Fusarium verticillioides that pose health concerns to humans and animals. Yet we still lack a clear understanding of the mechanism of fumonisins regulation during pathogenesis. The heterotrimeric G protein complex, which consists of canonical subunits and various regulators of G-protein signaling (RGS) proteins, plays an important role in transducing signals under environmental stress. Earlier studies demonstrated that Gα and Gβ subunits are positive regulators of fumonisin B1 (FB1) biosynthesis and that two RGS genes, FvFlbA1 and FvFlbA2, were highly upregulated in Gβ deletion mutant ∆Fvgbb1. Notably, FvFlbA2 has a negative role in FB1 regulation. While many fungi contain a single copy of FlbA, F. verticillioides harbors two putative FvFlbA paralogs, FvFlbA1 and FvFlbA2. In this study, we further characterized functional roles of FvFlbA1 and FvFlbA2. While ∆FvflbA1 deletion mutant exhibited no significant defects, ∆FvflbA2 and ∆FvflbA2/A1 mutants showed thinner aerial hyphal growth while promoting FB1 production. FvFlbA2 is required for proper expression of key conidia regulation genes, including putative FvBRLA, FvWETA, and FvABAA, while suppressing FUM21, FUM1, and FUM8 expression. Split luciferase assays determined that FvFlbA paralogs interact with key heterotrimeric G protein components, which in turn will lead altered G-protein-mediated signaling pathways that regulate FB1 production and asexual development in F. verticillioides.

Comparison of the In Vitro Antifungal and Anti-fumonigenic Activities of Copper and Silver Nanoparticles Against Fusarium verticillioides

The in vitro antifungal and anti-fumonigenic activities of copper nanoparticles (CuNPs) and silver nanoparticles (AgNPs) synthesized through chemical reduction were investigated. The formation of metal NPs was confirmed by UV–Vis spectroscopy, spectrofluorometry, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The surface charge and the concentration of metal NPs was evaluated by means of Z potential (ζ) and nanoparticle tracking analysis (NTA), respectively. Fumonisin B1 (FB1) was quantified by high pressure liquid chromatography (HPLC). In general, the synthesis protocol produced highly stable and monodispersed CuNPs and AgNPs with a narrow size distribution with average sizes of 2.5 ± 0.3 nm and 17 ± 1.5 nm, respectively. Both metal NPs exhibited satisfactory antifungal and anti-fumonigenic activities towards F. verticillioides. The minimum inhibitory concentration (MIC) was recorded at 125 mg/L (equivalent to 1.9 × 1010 particles/mL medium) and at 75 mg/L (equivalent to 2.8 × 1010 particles/mL medium) for CuNPs and AgNPs, respectively. Furthermore, CuNPs at ≥ 100 mg/L (equivalent to 1.6 × 1010 particles/mL medium) completely inhibited FB1 production, whereas AgNPs suppressed FB1 biosynthesis at ≥ 20 mg/L (equivalent to 8.0 × 109 particles/mL medium). From these results, it is concluded that AgNPs have important antifungal but significantly enhanced anti-fumonigenic activity against F. verticillioides under in vitro conditions.

A Rab GTPase protein FvSec4 is necessary for fumonisin B1 biosynthesis and virulence in Fusarium verticillioides.

Rab GTPases are responsible for a variety of membrane trafficking and vesicular transportation in fungi. But the role of Rab GTPases in Fusarium verticillioides, one of the key corn pathogens worldwide, remains elusive. These Small GTPases in fungi, particularly those homologous to Saccharomyces cerevisiae Sec4, are known to be associated with protein secretion, vesicular trafficking, secondary metabolism and pathogenicity. In this study, our aim was to investigate the molecular functions of FvSec4 in F. verticillioides associated with physiology and virulence. Interestingly, the FvSec4 null mutation did not impair the expression of key conidiation-related genes. Also, the mutant did not show any defect in sexual development, including perithecia production. Meanwhile, GFP-FvSec4 localized to growing hyphal tips and raised the possibility that FvSec4 is involved in protein trafficking and endocytosis. The mutant exhibited defect in corn stalk rot virulence and also significant alteration of fumonisin B1 production. The mutation led to higher sensitivity to oxidative and cell wall stress agents, and defects in carbon utilization. Gene complementation fully restored the defects in the mutant demonstrating that FvSec4 plays important roles in these functions. Taken together, our data indicate that FvSec4 is critical in F. verticillioides hyphal development, virulence, mycotoxin production and stress responses.

Effect of γ-irradiation on fumonisin producing Fusarium associated with animal and poultry feed mixtures.

Contamination of animal and poultry feeds by Fusarium and the mycotoxin Fumonisin B1 is frequent in the feed supply chain. The present study evaluated the prevalence of fumonisin B1 producing Fusarium among irradiated and non-irradiated animal and poultry feed mixtures. Further, the efficiency of γ-rays (2.5, 5.0, 7.5, and 10.0kGy) to minimize Fusarium growth and biosynthesis of fumonisin B1 in artificially inoculated feed was evaluated. A total of 108 feed samples were collected in which 45.37% of feed mixtures were contaminated with Fusarium species. Among the contaminated samples, the frequency levels of F. verticillioides and F. proliferatum were 42.59 and 24.07%, respectively. Out of the 98 Fusarium isolates from feed samples, 84.7% of F. verticillioides and 64.28% of F. proliferatum were positive for FUM1 set of primers. Fumonisin B1 biosynthesis by the FUM1 positive isolates in feed was confirmed by LC/MS which recorded 0.1-45µg/g of feed. Fungal growth and viable count of Fusarium in PDA medium and feed decreased with increasing irradiation dosage. Interestingly, fumonisin content was 11µg/g of feed in 2.5kGy irradiated sample as compared to 5µg/g of feed in non-irradiated control. Ionizing radiation at 7.5kGy was found lethal for fungal growth and fumonisin production. Our findings suggest that γ-radiation above 7.5kGy effectively prevented fungal growth in feed mixtures and minimized the exposure of animal and human life to the potential risk of mycotoxin. Also it is necessary to maintain proper storage system for feeds until consumption.

Involvement of FvSet1 in Fumonisin B1 Biosynthesis, Vegetative Growth, Fungal Virulence, and Environmental Stress Responses in Fusarium verticillioides.

Fusarium verticillioides (teleomorph, Gibberella moniliformis) is an important plant pathogen that causes seedling blight, stalk rot, and ear rot in maize (Zea mays). During infection, F. verticillioides produce fumonsins B1 (FB1) that pose a serious threat to human and animal health. Recent studies showed that Set1, a methyltransferase of H3K4, was responsible for toxin biosynthesis in filamentous fungi. However, to date, the regulation of FvSet1 on FB1 biosynthesis remains unclear. In the current study, we identified only one Set1 ortholog in F. verticillioides (FvSet1) and found that the deletion of FvSET1 led to various defects in fungal growth and pathogenicity. More interestingly, the FvSET1 deletion mutant (ΔFvSet1) showed a significant defect in FB1 biosynthesis and lower expression levels of FUM genes. FvSet1 was also found to play an important role in the responses of F. verticillioides to multiple environmental stresses via regulating the phosphorylation of FvMgv1 and FvHog1. Taken together, these results indicate that FvSet1 plays essential roles in the regulation of FB1 biosynthesis, fungal growth and virulence, as well as various stress responses in F. verticillioides.

Multi-Homologous Recombination-Based Gene Manipulation in the Rice Pathogen Fusarium fujikuroi.

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1 ), which is associated with fumonisin B1 biosynthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.