RESUMO
The mycotoxigenic fungus Fusarium verticillioides is a common pathogen of grain and medicine that contaminates the host with fumonisin B1 (FB1) mycotoxin, poses serious threats to human and animal health. Therefore, it is crucial to unravel the regulatory mechanisms of growth, and pathogenicity of F. verticillioides. Mbp1 is a component of the MluI cell cycle box binding factor complex and acts as an APSES-type transcription factor that regulates cell cycle progression. However, no information is available regarding its role in F. verticillioides. In this study, we demonstrate that FvMbp1 interacts with FvSwi6 that acts as the cell cycle transcription factor, to form the heteromeric transcription factor complexes in F. verticillioides. Our results show that ΔFvMbp1 and ΔFvSwi6 both cause a severe reduction of vegetative growth, conidiation, and increase tolerance to diverse environmental stresses. Moreover, ΔFvMbp1 and ΔFvSwi6 dramatically decrease the virulence of the pathogen on the stalk and ear of maize. Transcriptome profiling show that FvMbp1-Swi6 complex co-regulates the expression of genes associated with multiple stress responses. These results indicate the functional importance of the FvMbp1-Swi6 complex in the filamentous fungi F. verticillioides and reveal a potential target for the effective prevention and control of Fusarium diseases.
Assuntos
Proteínas Fúngicas , Fusarium , Fatores de Transcrição , Zea mays , Fusarium/metabolismo , Fusarium/patogenicidade , Fusarium/genética , Fusarium/crescimento & desenvolvimento , Virulência , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Zea mays/microbiologia , Estresse Fisiológico , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologiaRESUMO
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.
Assuntos
Cálcio , Fumonisinas , Proteínas Fúngicas , Fusarium , Homeostase , Proteínas Quinases Ativadas por Mitógeno , Fusarium/metabolismo , Fusarium/genética , Cálcio/metabolismo , Fumonisinas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Fosforilação , Regulação Fúngica da Expressão GênicaRESUMO
Bivalent histone modifications regulate gene expression during development, but little is known about their function in plant-microbe interactions. In a recent report, Zhao et al. showed that expression of bivalent chromatin-marked gene 1 (BCG1), containing a pathogen-associated molecular pattern (PAMP) motif, is epigenetically regulated by trimethylation of lysine 4 (H3K4me3) and lysine 27 (H3K27me3) of histone H3 to evade plant immunity.
RESUMO
BACKGROUND: Bromo-adjacent homology-plant homeodomain domain containing protein 1 (BP1) is a reader of histone post-translational modifications in fungi. BP1 recognizes trimethylation of lysine 27 in histone H3 (H3K27me3), an epigenetic hallmark of gene silencing. However, whether and how BP1 participates in transcriptional repression remains poorly understood. RESULTS: We report that BP1 forms phase-separated liquid condensates to modulate its biological function in Fusarium graminearum. Deletion assays reveal that intrinsically disordered region 2 (IDR2) of BP1 mediates its liquid-liquid phase separation. The phase separation of BP1 is indispensable for its interaction with suppressor of Zeste 12, a component of polycomb repressive complex 2. Furthermore, IDR2 deletion abolishes BP1-H3K27me3 binding and alleviates the transcriptional repression of secondary metabolism-related genes, especially deoxynivalenol mycotoxin biosynthesis genes. CONCLUSIONS: BP1 maintains transcriptional repression by forming liquid-liquid phase-separated condensates, expanding our understanding of the relationship between post-translational modifications and liquid-liquid phase separation.
Assuntos
Histonas , Separação de Fases , Histonas/metabolismo , Expressão Gênica , Complexo Repressor Polycomb 2/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Putrescine, spermidine, and spermine are the most common natural polyamines. Polyamines are ubiquitous organic cations of low molecular weight and have been well characterized for the cell function and development processes of organisms. However, the physiological functions of polyamines remain largely obscure in plant pathogenic fungi. Fusarium graminearum causes Fusarium head blight (FHB) and leads to devastating yield losses and quality reduction by producing various kinds of mycotoxins. Herein, we genetically analyzed the gene function of the polyamine biosynthesis pathway and evaluated the role of the endogenous polyamines in the growth, development, and virulence of F. graminearum. Our results found that deletion of spermidine biosynthesis gene FgSPE3 caused serious growth defects, reduced asexual and sexual reproduction, and increased sensitivity to various stresses. More importantly, ΔFgspe3 exhibited significantly decreased mycotoxin deoxynivalenol (DON) production and weak virulence in host plants. Additionally, the growth and virulence defects of ΔFgspe3 could be rescued by exogenous application of 5 mM spermidine. Furthermore, RNA-seq displayed that FgSpe3 participated in many essential biological pathways including DNA, RNA, and ribosome synthetic process. To our knowledge, these results indicate that spermidine is essential for growth, development, DON production, and virulence in Fusarium species, which provides a potential target to control FHB.
RESUMO
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.
Assuntos
Fumonisinas/metabolismo , Fusarium/metabolismo , Glicosiltransferases/isolamento & purificação , Virulência , Fusarium/enzimologia , Fusarium/genética , Fusarium/patogenicidade , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Glicosiltransferases/genética , Doenças das Plantas/microbiologia , Zea mays/microbiologiaRESUMO
Histone H3 lysine 27 methylation catalyzed by polycomb repressive complex 2 (PRC2) is conserved from fungi to humans and represses gene transcription. However, the mechanism for recognition of methylated H3K27 remains unclear, especially in fungi. Here, we found that the bromo-adjacent homology (BAH)-plant homeodomain (PHD) domain containing protein BAH-PHD protein 1 (BP1) is a reader of H3K27 methylation in the cereal fungal pathogen Fusarium graminearum. BP1 interacts with the core PRC2 component Suz12 and directly binds methylated H3K27. BP1 is distributed in a subset of genomic regions marked by H3K27me3 and co-represses gene transcription. The BP1 deletion mutant shows identical phenotypes on mycelial growth and virulence, as well as similar expression profiles of secondary metabolite genes to the strain lacking the H3K27 methyltransferase Kmt6. More importantly, BP1 can directly bind DNA through its PHD finger, which might increase nucleosome residence and subsequently reinforce transcriptional repression in H3K27me3-marked target regions. A phylogenetic analysis showed that BP1 orthologs are mainly conserved in fungi. Overall, our findings provide novel insights into the mechanism by which PRC2 mediates gene repression in fungi, which is distinct from the PRC1-PRC2 system in plants and mammals.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/genética , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , DNA/metabolismo , Proteínas Fúngicas/química , Fusarium/metabolismo , Histonas/química , Lisina/metabolismo , Proteínas Repressoras/metabolismo , Transcrição GênicaRESUMO
Phenamacril is a Fusarium-specific fungicide used for Fusarium head blight management. The target of phenamacril is FgMyo1, the sole class I myosin in Fusarium graminearum. The point mutation S217L in FgMyo1 is responsible for the high resistance of F. graminearum to phenamacril. Recent structural studies have shown that phenamacril binds to the 50 kDa cleft of the FgMyo1 motor domain, forming extensive interactions, including a hydrogen bond between the cyano group of phenamacril and the hydroxyl group of S217. Here, we produced FgMyo1IQ2, a truncated FgMyo1 composed of the motor domain and two IQ motifs complexed with the F. graminearum calmodulin in insect Sf9 cells. Phenamacril potently inhibited both the basal and the actin-activated ATPase activities of FgMyo1IQ2, with an IC50 in a micromolar range. S217 mutations of FgMyo1IQ2 substantially increased the IC50 of phenamacril. S217T or S217L each increased the IC50 of phenamacril for â¼60-fold, while S217A only increased the IC50 for â¼4-fold. These results indicate that the hydroxyl group of S217 plays an important, but nonessential role in phenamacril binding and that the bulky side chain at the position 217 sterically hinders phenamacril binding. On the other hand, S217P, which might alter the local conformation of the phenamacril-binding site, completely abolished the phenamacril inhibition. Because the cyano group of phenamacril does not form discernible interactions with FgMyo1 other than the nonessential hydrogen bond with the S217 hydroxyl group, we propose the cyano group of phenamacril as a key modification site for the development of novel fungicides.
RESUMO
BACKGROUND: The cyanoacrylate fungicide phenamacril targeting fungal myosin I has been widely used for controlling Fusarium head blight (FHB) of wheat caused by the pathogenic fungus Fusarium graminearum worldwide. Therefore, there is great interest in the discovery and development of novel FgMyo1 inhibitors through structure-based drug design for the treatment of FHB. RESULTS: In this study, the binding mechanism of phenamacril with FgMyo1 was predicted by an integrated molecular modeling strategy. The predicted key phenamacril-binding residues of FgMyo1 were further experimentally validated by point mutagenesis and phenamacril sensitivity assessment. Four novel key residues responsible for phenamacril binding were identified, highlighting the reliability of the theoretical predictions. The subsequent optimization of phenamacril derivatives led to the discovery of a novel compound (10) which shows better activity than phenamacril against conidial germination of F. graminearum, but not against other fungal species. Moreover, 10 also inhibits conidial germination of phenamacril-resistant strains effectively. Further experiments illustrated that application of 10 could dramatically inhibit deoxynivalenol biosynthesis. CONCLUSION: Overall, our results further optimize and develop the binding model of phenamacril-myosin I. Furthermore, 10 was found and has the potential to be developed as a species-specific fungicide for management of FHB. © 2020 Society of Chemical Industry.
Assuntos
Fungicidas Industriais , Fusarium , Antifúngicos/farmacologia , Fungicidas Industriais/farmacologia , Doenças das Plantas , Reprodutibilidade dos TestesRESUMO
Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Fusarium/patogenicidade , Citoesqueleto de Actina/metabolismo , Endocitose/fisiologia , Doenças das Plantas/microbiologia , Tricotecenos/metabolismo , VirulênciaRESUMO
AP2 is a heterotetrameric clathrin adaptor complex that owns important roles in vesicle generation and cargo recognition. Cell-wall integrity (CWI) pathway is essential for fungal development, virulence, and adaptation to environment stresses. To date, the relationship between AP2 and CWI is largely unknown in phytopathogenic fungi. In this study, we identified the adaptor complex FgAP2 in Fusarium graminearum. The biological function analysis showed that FgAP2 complex contains FgAP2α, FgAP2ß, FgAP2σ, and FgAP2µ, and the subunit FgAP2µ, which is required for hyphal growth, conidiation, CWI, and virulence. Yeast two-hybrid showed that FgAP2µ interacts with the CWI sensor FgWsc2B. Consistently, western blotting analysis revealed that FgWsc2B positively regulates phosphorylation of FgMgv1, the MAP kinase of CWI. Moreover, the FgWsc2B deletion mutant exhibited defects in hyphal growth, virulence, and response to CWI damaging agents. Taken together, our data indicated that FgAP2µ is involved in CWI and virulence via interacting with FgWsc2B in F. graminearum.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Parede Celular/genética , Endocitose/genética , Fusarium/genética , Parede Celular/microbiologia , Proteínas Fúngicas/genética , Fusarium/patogenicidade , Regulação Fúngica da Expressão Gênica , Hifas/genética , Hifas/patogenicidade , Pressão Osmótica , Fosforilação , Esporos Fúngicos/genética , Esporos Fúngicos/patogenicidade , Estresse Fisiológico/genética , Virulência/genéticaRESUMO
BACKGROUND: Fusarium head blight (FHB) is a devastating disease of cereal crops worldwide mainly caused by Fusarium graminearum. Due to the unavailability of FHB-resistant wheat cultivars, chemical fungicide application is currently the most effective approach for controlling FHB now. In the last few years, a novel cyanoacrylate fungicide, phenamacril, has been widely used in China for FHB disease management. In previous studies, we identified that myosin I (FgMyo1) is the target of phenamacril and is essential for mycotoxin deoxynivalenol (DON) biosynthesis and fungal growth. However, the regulation of FgMYO1 gene expression is still largely unknown. RESULTS: In this study, we identified a b-ZIP transcription factor, FgTfmI, which regulates the mRNA expression of FgMYO1 upon phenamacril treatment. The FgTfmI directly binds to the promoter region of FgMYO1, and is required for the upregulation of FgMYO1 in response to phenamacril treatment. The deletion mutant of FgTFMI (ΔFgTfmI) displayed a slight growth defect, while it showed hypersensitivity to phenamacril, but not to other tested fungicides. FgTfmI also contributed to DON biosynthesis and the infection process in planta. CONCLUSIONS: The transcription factor FgTfmI plays an important role in regulating transcription of the genes involved in phenamacril tolerance, DON biosynthesis and virulence in F. graminearum. © 2019 Society of Chemical Industry.
Assuntos
Cianoacrilatos/farmacologia , Resistência a Medicamentos/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Fusarium/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fusarium/efeitos dos fármacos , Fusarium/fisiologia , Doenças das Plantas/microbiologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
Peroxisomal proliferation is highly stimulated during the biosynthesis of mycotoxins and plant infection by Fusarium graminearum. Currently, the functions of the peroxisome in these cellular processes are poorly understood. In this study, we applied genetic, cell biological and biochemical analyses to investigate the functions of the peroxisomes. We constructed targeted deletion of docking machinery components, including FgPex13, FgPex14 and the filamentous fungal specific peroxin FgPex33. Our results indicated that peroxisome dysfunction resulted in a shortage of acetyl-CoA, the precursor of trichothecene biosynthesis, and subsequently decreased deoxynivalenol (DON) production. Deletion mutants of ΔFgPex13, ΔFgPex14 or ΔFgPex33 showed an increased accumulation of endogenous reactive oxygen species (ROS) and reduced phosphorylation of MAP (Mitogen-Activated Protein) kinase FgMgv1. In addition, mutants of the docking peroxin exhibited increased sensitivity toward host oxidative bursts and cell wall integrity stress agents and reduced virulence on host plants. More importantly, we found for the first time that FgPex14 is required for pexophagy in F. graminearum. Overall, our study suggests that peroxisomes play critical roles in DON biosynthesis and virulence in F. graminearum.
Assuntos
Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Micotoxinas/toxicidade , Peroxissomos/fisiologia , Tricotecenos/metabolismo , Tricotecenos/toxicidade , Parede Celular/metabolismo , Proteínas Fúngicas/genética , Fusarium/genética , Deleção de Genes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Micotoxinas/biossíntese , Micotoxinas/genética , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Sementes/microbiologia , Triticum/microbiologia , Virulência/genéticaRESUMO
Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.
Assuntos
Fusarium , Micotoxinas/biossíntese , Miosina Tipo I/fisiologia , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Micotoxinas/genética , Micotoxinas/metabolismo , Organismos Geneticamente Modificados , Metabolismo Secundário/genéticaRESUMO
Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS-generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper-EF-hand-containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial-produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.