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Nucleoside diphosphate kinases (NDPKs) are nucleotide metabolism enzymes that play different physiological functions in different species. However, the roles of NDPK in phytopathogen and mycotoxin production are not well understood. In this study, we showed that Fusarium graminearum FgNdpk is important for vegetative growth, conidiation, sexual development, and pathogenicity. Furthermore, FgNdpk is required for deoxynivalenol (DON) production; deletion of FgNDPK downregulates the expression of DON biosynthesis genes and disrupts the formation of FgTri4-GFP-labeled toxisomes, while overexpression of FgNDPK significantly increases DON production. Interestingly, FgNdpk colocalizes with the DON biosynthesis proteins FgTri1 and FgTri4 in the toxisome, and coimmunoprecipitation (Co-IP) assays show that FgNdpk associates with FgTri1 and FgTri4 in vivo and regulates their localizations and expressions, respectively. Taken together, these data demonstrate that FgNdpk is important for vegetative growth, conidiation, and pathogenicity and acts as a key protein that regulates toxisome formation and DON biosynthesis in F. graminearum .

The transcription factor FgHtf1 is important for conidiogenesis in Fusarium graminearum and it positively regulates the expression of the sporulation-related gene FgCON7. However, the regulatory mechanism underlying its functions is still unclear. The present study intends to uncover the functional mechanism of FgHtf1 in relation to FgCon7 in F. graminearum. We demonstrated that FgCON7 serves as a target gene for FgHtf1. Interestingly, FgCon7 also binds the promoter region of FgHTF1 to negatively regulate its expression, thus forming a negative-feedback loop. We demonstrated that FgHtf1 and FgCon7 have functional redundancy in fungal development. FgCon7 localizes in the nucleus and has transcriptional activation activity. Deletion of FgCON7 significantly reduces conidia production. 4444 genes were regulated by FgCon7 in ChIP-Seq, and RNA-Seq revealed 4430 differentially expressed genes in FgCON7 deletion mutant, with CCAAT serving as a consensus binding motif of FgCon7 to the target genes. FgCon7 directly binds the promoter regions of FgMSN2, FgABAA, FgVEA and FgSMT3 genes and regulates their expression. These genes were found to be important for conidiogenesis. To our knowledge, this is the first study that unveiled the mutual regulatory functions of FgCON7 and FgHTF1 to form a negative-feedback loop, and how the loop mediates sporulation in F. graminearum., Competing Interests: Declaration of competing interest We have declared that no competing interests exist., (Copyright © 2024 Elsevier B.V. All rights reserved.)

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) facilitate docking and fusion of vesicles with their target membranes, playing a crucial role in vesicle trafficking and exocytosis. However, the spatial assembly and roles of plasma membrane (PM)-associated SNAREs in phytopathogen development and pathogenicity are not clearly understood. In this study, we analysed the roles and molecular mechanisms of PM-associated SNARE complexes in the banana Fusarium wilt fungus Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4). Our findings demonstrate that FocSso1 is important for the fungal growth, conidiation, host penetration and colonization. Mechanistically, FocSso1 regulates protein secretion by mediating vesicle docking and fusion with the PM and hyphal apex. Interestingly, a FocSso1-FocSec9-FocSnc1 complex was observed to assemble not only at the fungal PM but also on the growing hyphal apex, facilitating exocytosis. FocSso2, a paralogue of FocSso1, was also found to form a ternary SNARE complex with FocSec9 and FocSnc1, but it mainly localizes to the PM in old hyphae. The functional analysis of this protein demonstrated that it is dispensable for the fungal growth but necessary for host penetration and colonization. The other subunits, FocSec9 and FocSnc1, are involved in the fungal development and facilitate host penetration. Furthermore, FocSso1 and FocSnc1 are functionally interdependent, as loss of FocSso1 leads to mis-sorting and degradation of FocSnc1 in the vacuole and vice versa. Overall, this study provides insight into the formation of two spatially and functionally distinct PM SNARE complexes and their involvement in vesicle exocytosis to regulate development and pathogenicity of FocTR4., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The RNA exosome complex is a conserved, multisubunit RNase complex that contributes to the processing and degradation of RNAs in mammalian cells. However, the roles of the RNA exosome in phytopathogenic fungi and how it relates to fungal development and pathogenicity remain unclear. Herein, we identified 12 components of the RNA exosome in the wheat fungal pathogen Fusarium graminearum. Live-cell imaging showed that all the components of the RNA exosome complex are localized in the nucleus. FgEXOSC1 and FgEXOSCA were successfully knocked out; they are both involved in the vegetative growth, sexual reproduction, and pathogenicity of F. graminearum. Moreover, deletion of FgEXOSC1 resulted in abnormal toxisomes, decreased deoxynivalenol (DON) production, and downregulation of the expression levels of DON biosynthesis genes. The RNA-binding domain and N-terminal region of FgExosc1 are required for its normal localization and functions. Transcriptome sequencing (RNA-seq) showed that the disruption of FgEXOSC1 resulted in differential expression of 3,439 genes. Genes involved in processing of noncoding RNA (ncRNA), rRNA and ncRNA metabolism, ribosome biogenesis, and ribonucleoprotein complex biogenesis were significantly upregulated. Furthermore, subcellular localization, green fluorescent protein (GFP) pulldown, and coimmunoprecipitation (co-IP) assays demonstrated that FgExosc1 associates with the other components of the RNA exosome to form the RNA exosome complex in F. graminearum. Deletion of FgEXOSC1 and FgEXOSCA reduced the relative expression of some of the other subunits of the RNA exosome. Deletion of FgEXOSC1 affected the localization of FgExosc4, FgExosc6, and FgExosc7. In summary, our study reveals that the RNA exosome is involved in vegetative growth, sexual reproduction, DON production, and pathogenicity of F. graminearum. IMPORTANCE The RNA exosome complex is the most versatile RNA degradation machinery in eukaryotes. However, little is known about how this complex regulates the development and pathogenicity of plant-pathogenic fungi. In this study, we systematically identified 12 components of the RNA exosome complex in Fusarium head blight fungus Fusarium graminearum and first unveiled their subcellular localizations and established their biological functions in relation to the fungal development and pathogenesis. All the RNA exosome components are localized in the nucleus. FgExosc1 and FgExoscA are both required for the vegetative growth, sexual reproduction, DON production and pathogenicity in F. graminearum. FgExosc1 is involved in ncRNA processing, rRNA and ncRNA metabolism process, ribosome biogenesis and ribonucleoprotein complex biogenesis. FgExosc1 associates with the other components of RNA exosome complex and form the exosome complex in F. graminearum. Our study provides new insights into the role of the RNA exosome in regulating RNA metabolism, which is associated with fungal development and pathogenicity., Competing Interests: The authors declare no conflict of interest.

The mitotic exit is a key step in cell cycle, but the mechanism of mitotic exit network in the wheat head blight fungus Fusarium graminearum remains unclear. F. graminearum infects wheat spikelets and colonizes the entire head by growing through the rachis node at the bottom of each spikelet. In this study, we found that a small GTPase FgTem1 plays an important role in F. graminearum pathogenicity and functions in regulating the formation of infection structures and invasive hyphal growth on wheat spikelets and wheat coleoptiles, but plays only little roles in vegetative growth and conidiation of the phytopathogen. FgTem1 localizes to both the inner nuclear periphery and the spindle pole bodies, and negatively regulates mitotic exit in F. graminearum. Furthermore, the regulatory mechanisms of FgTem1 have been further investigated by high-throughput co-immunoprecipitation and genetic strategies. The septins FgCdc10 and FgCdc11 were demonstrated to interact with the dominant negative form of FgTem1, and FgCdc11 was found to regulate the localization of FgTem1. The cell cycle arrest protein FgBub2-FgBfa1 complex was shown to act as the GTPase-activating protein (GAP) for FgTem1. We further demonstrated that a direct interaction exists between FgBub2 and FgBfa1 which crucially promotes conidiation, pathogenicity and DON production, and negatively regulates septum formation and nuclear division in F. graminearum. Deletion of FgBUB2 and FgBFA1 genes caused fewer perithecia and immature asci formations, and dramatically down-regulated trichothecene biosynthesis (TRI) gene expressions. Double deletion of FgBUB2/FgBFA1 genes showed that FgBUB2 and FgBFA1 have little functional redundancy in F. graminearum. In summary, we systemically demonstrated that FgTem1 and its GAP FgBub2-FgBfa1 complex are required for fungal development and pathogenicity in F. graminearum., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Miao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Calcium and manganese transporters play important roles in regulating Ca 2+ and Mn 2+ homeostasis in cells, which is necessary for the normal physiological activities of eukaryotes. Gdt1 and Pmr1 function as calcium/manganese transporters in the Golgi apparatus. However, the functions of Gdt1 and Pmr1 have not been previously characterized in the plant pathogenic fungus Fusarium graminearum. Here, we identified and characterized the biological functions of FgGdt1 and FgPmr1 in F. graminearum. Our study shows that FgGdt1 and FgPmr1 are both localized to the cis- and medial-Golgi. Disruption of FgGdt1 or FgPmr1 in F. graminearum caused serious defects in vegetative growth, conidiation, sexual development and significantly decreased virulence in wheat but increased deoxynivalenol (DON) production. Importantly, FgGdt1 is involved in Ca 2+ and Mn 2+ homeostasis and the severe phenotypic defects of the ΔFggdt1 mutant were largely due to loss of FgGdt1 function in Mn 2+ transportation. FgGdt1-mCherry colocalizes with FgPmr1-GFP at the Golgi, and FgGDT1 exerts its biological function upstream of FgPMR1. Taken together, our results collectively demonstrate that the cis- and medial-Golgi-localized proteins FgGdt1 and FgPmr1 regulate Ca 2+ and Mn 2+ homeostasis of the Golgi apparatus, and this function is important in modulating the growth, development, DON biosynthesis and pathogenicity of F. graminearum., (© 2022 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Rab GTPases are key regulators of membrane and intracellular vesicle transports. However, the biological functions of FgRab1 are still unclear in the devastating wheat pathogen Fusarium graminearum . In this study, we generated constitutively active (CA) and dominant-negative (DN) forms of FgRAB1 from the wild-type PH-1 background for functional analyses. Phenotypic analyses of these mutants showed that FgRab1 is important for vegetative growth, cell wall integrity and hyphal branching. Compared to the PH-1 strain, the number of spores produced by the Fgrab1DN strain was significantly reduced, with obviously abnormal conidial morphology. The number of septa in the conidia of the Fgrab1DN mutant was fewer than that observed in the PH-1 conidia. Fgrab1DN was dramatically reduced in its ability to cause Fusarium head blight symptoms on wheat heads. GFP-FgRab1 was observed to partly localize to the Golgi apparatus, endoplasmic reticulum and Spitzenkörper. Furthermore, we found that FgRab1 inactivation blocks not only the transport of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane but also the fusion of endocytic vesicles with their target membranes and general autophagy. In summary, our results indicate that FgRab1 plays vital roles in vegetative growth, conidiogenesis, pathogenicity, autophagy, vesicle fusion and trafficking in F. graminearum .

The Phox homology (PX) domain is a membrane recruitment module that binds to phosphoinositides (PI) mediating the selective sorting and transport of transmembrane proteins, lipids, and other critical cargo molecules via membrane trafficking processes. However, the mechanism of vesicular trafficking mediated by PX domain-containing proteins in phytopathogenic fungi and how this relates to the fungal development and pathogenicity remain unclear. Here, we systematically identified and characterized the functions of PX domain-containing proteins in the plant fungal pathogen Fusarium graminearum. Our data identified 14 PX domain-containing proteins in F. graminearum, all of which were required for plant infection and deoxynivalenol (DON) production, with the exception of FgMvp1 and FgYkr078. Furthermore, all the PX domain-containing proteins showed distinct localization patterns that were limited to the endosomes, vacuolar membrane, endoplasmic reticulum, cytoplasm, and hyphal septa/tips. Remarkably, among these proteins, FgBem1 targeted to surface crescent and septal pores and was retained at the septum pores even after actin constriction during septum development. Further analyses demonstrated that the surface crescent targeting of FgBem1 solely depended on its SH3 domains, while its septum and apex anchoring localization relied on its PX domain, which was also indispensable for reactive oxygen species (ROS) production, sexual development, and pathogenicity in F. graminearum. In summary, our study is the first detailed and comprehensive functional analysis of PX domain-containing proteins in filamentous fungi, and it provides new insight into the mechanism of FgBem1 involved in septum and apex anchorage mediated by its PX domain, which is necessary for sexual development and pathogenicity of F. graminearum. IMPORTANCE Fusarium head blight (FHB), caused predominantly by Fusarium graminearum, is an economically devastating disease of a wide range of cereal crops. Our previous study identified F. graminearum Vps17, Vps5, Snx41, and Snx4 as PX domain-containing proteins that were involved in membrane trafficking mediating the fungal development and pathogenicity, but the identity and biological roles of the remaining members of this protein family remain unknown in this model phytopathogen. In this study, we first unveiled all the PX domain-containing proteins in F. graminearum and then established their subcellular localizations and biological functions in relation to the fungal development and pathogenesis. We found 14 PX domain-containing proteins that localized to distinct subcellular organelles, including the endosomes, vacuolar membrane, endoplasmic reticulum, cytoplasm, and hyphal septa/tips. Of these proteins, FgBem1 was found to be essential for sexual development and virulence of F. graminearum. Further analyses showed that the PX domain of FgBem1 was indispensable for its functions in septum and apex anchorage, which, in turn, was necessary for ROS production and pathogenicity of F. graminearum. Our findings are important because it not only served as the first comprehensive characterization of the PX domain family proteins in a plant-pathogenic fungus but also uncovered the novel roles of the PX domain involved in septation and apex targeting, which could provide new fungicidal targets for controlling the devastating FHB disease.

In filamentous fungi, hyphal growth depends on the continuous delivery of vesicles to the growing tips. It is unclear how fast-growing hyphae coordinate simultaneous cell extension and expansion in the tip cells. We have functionally characterized 12 TBC (Tre-2/Bub2/Cdc16) domain-containing proteins in Fusarium graminearum. Among them, FgMsb3 is found to regulate hyphal tip expansion and to be required for pathogenicity. The regulatory mechanism of FgMsb3 has been further investigated by genetic, high-resolution microscopy and high-throughput co-immunoprecipitation strategies. The FgMsb3 protein localizes at the polarisome and the hyphal apical dome (HAD) where it acts as a GTPase-activating protein for FgRab8 which is required for apical secretion-mediated growth and pathogenicity. Deletion of FgMSB3 causes excessive polarized trafficking but blocks the fusion of FgSnc1-associated vesicles to the plasma membrane. Moreover, we establish that FgSpa2 interacts with FgMsb3, enabling FgMsb3 tethering to the polarisome. Loss of FgSpa2 or other polarisome components (FgBud6 and FgPea2) causes complete shifting of FgMsb3 to the HAD and this affects the polarized growth and pathogenicity of the fungus. In summary, we conclude that FgSpa2 regulates FgMsb3-FgRab8 cascade and this is crucial for creating a steady-state equilibrium that maintains continuous polarized growth and contributes to the pathogenicity of F. graminearum., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The membrane trafficking system is important for compartmentalization of the biosynthesis pathway and secretion of deoxynivalenol (DON) mycotoxin (a virulence factor) in Fusarium graminearum. Flippases are transmembrane lipid transporters and mediate a number of essential physiological steps of membrane trafficking, including vesicle budding, charging, and protein diffusion within the membrane. However, the roles of flippases in secondary metabolism remain unknown in filamentous fungi. Herein, we identified five flippases (FgDnfA, FgDnfB, FgDnfC1, FgDnfC2, and FgDnfD) in F. graminearum and established their specific and redundant functions in the development and pathogenicity of this phytopathogenic fungus. Our results demonstrate that FgDnfA is critical for normal vegetative growth while the other flippases are dispensable. FgDnfA and FgDnfD were found crucial for the fungal pathogenesis, and a remarkable reduction in DON production was observed in ΔFgDNFA and ΔFgDNFD. Deletion of the FgDNFB gene increased DON production to about 30 times that produced by the wild type. Further analysis showed that FgDnfA and FgDnfD have positive roles in the regulation of trichothecene (TRI) genes (TRI1, TRI4, TRI5, TRI6, TRI12, and TRI101) expression and toxisome reorganization, while FgDnfB acts as a negative regulator of DON synthesis. In addition, FgDnfB and FgDnfD have redundant functions in the regulation of phosphatidylcholine transport, and double deletion of FgDNFB and FgDNFD showed serious defects in fungal development, DON synthesis, and virulence. Collectively, our findings reveal the distinct and specific functions of flippase family members in F. graminearum and principally demonstrate that FgDnfA, FgDnfD, and FgDnfB have specific spatiotemporal roles during toxisome biogenesis., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) facilitate intracellular vesicle trafficking and membrane fusion in eukaryotic cells, and play a vital role in growth, development and pathogenicity of phytopathogens. Fusarium head blight (FHB) caused by F. graminearum is one of the most devastating diseases of wheat and barley worldwide. Sec22 is a member of the SNARE family of proteins and its homologues have been shown to have diverse biological roles in different organisms. However, the functions of this protein in the development and pathogenesis of F. graminearum are currently unknown. In this study, we employed integrated biochemical, microbiological and molecular genetic approaches to investigate the roles of FgSec22 in F. graminearum. Our data reveal that this SNARE protein is localized to endoplasmic reticulum (ER) and is indispensable for normal conidiation, conidial morphology and pathogenesis of this phytopathogenic fungus. Our biochemical assay of deoxynivalenol (DON) reveals the active involvement of this protein in the production of this mycotoxin in F. graminearum. This has further been confirmed by qRT-PCR analyses of trichothecene (TRI) genes' expression where the ΔFgsec22 deletion mutant demonstrated a significant down-regulation of these genes in comparison to the wild-type PH-1. Unlike the wild-type and the complemented strain, the mutant strain presents a remarkable defect in colony formation which reflects the critical role it plays in vegetative growth. Collectively, our data support that the SNARE protein FgSec22 is required for vegetative growth, pathogenesis and DON biosynthesis in F. graminearum.

Fusarium wilt of banana is caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. cubense . We generated two chromosome-level assemblies of F. oxysporum f. sp. cubense race 1 and tropical race 4 strains using single-molecule real-time sequencing. The F. oxysporum f. sp. cubense race 1 and tropical race 4 assemblies had 35 and 29 contigs with contig N50 lengths of 2.08 and 4.28 Mb, respectively. These two new references genomes represent a greater than 100-fold improvement over the contig N50 statistics of the previous short-read-based F. oxysporum f. sp. cubense assemblies. The two high-quality assemblies reported here will be a valuable resource for the comparative analysis of F. oxysporum f. sp. cubense races at the pathogenic level.

AP-2 complex is widely distributed in eukaryotes in the form of heterotetramer that functions in the uptake of membrane proteins during mammalian/plant clathrin-mediated endocytosis. However, its biological function remains mysterious in pathogenic fungi. In this study, the wheat scab fungus, Fusarium graminearum, was used to characterise the biological function of the AP-2 complex. Our study shows that FgAP-2 complex plays a critical role in the maintenance of hyphal polarity. Lack of any subunit (FgAP2 α , FgAP2 β , FgAP2 σ , and FgAP2 mu ) of the FgAP-2 complex significantly affects the fungal vegetative growth, conidial morphology, and germination. Remarkably, FgAP-2 complex is important for the fungal pathogenicity, especially during colonisation and extension after infecting the host. The FgAP-2 complex is expressed ubiquitously at all developmental stages but having more concentrated protein distribution at the subapical collar and septa in young growing hyphae. Although FgAP-2 complex displays similar dynamic behaviour to the actin patch components and accumulates at endocytic sites, it is dispensable for general endocytosis. We further demonstrated that FgAP-2 complex is required for polar localisation of the lipid flippases FgDnfA and FgDnfB, which led to the proposal that FgAP-2 functions as a cargo-specific adaptor that promotes polar growth and colonising ability of F. graminearum., (© 2019 John Wiley & Sons Ltd.)

SR protein-specific kinases (SRPKs) uniquely with a spacer region are important splicing factors from yeast to human. However, little is known about their biological functions in filamentous fungi. Therefore, we characterized a SRPK called SRK1 in wheat scab fungus Fusarium graminearum. Our data showed that Srk1 is required for vegetative growth, sexual reproduction and plant infection, and plays critical roles in pre-mRNA alternative splicing and gene expression. Remarkably, we found that Srk1 displayed dynamic shuttling between cytoplasm and the nucleus, which is regulated by the divergent spacer domain rather than its kinase activity, suggesting a regulatory mechanism for Srk1. Interestingly, Srk1-GFP also localized to the septal pores, indicating a possible role of Srk1 unrelated to mRNA processing. Although both K1 and K2 lobes of the kinase domain are essential for Srk1 functions, the K2 but not K1 lobe is responsible for the septal pore localization. Lastly, we established that Srk1 physically interacts with the two SR proteins, FgNpl3 and FgSrp1. Overall, our results indicated that SRK1 regulates fungal development, plant infection and mRNA processing by phosphorylation of other splicing factors including SR proteins, and the spacer domain regulates the functions of Srk1 by modulating its nucleocytoplasmic shuttling., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sec4/Rab8 is one of the well-studied members of the Rab GTPase family, previous studies have shown that Sec4/Rab8 crucially promotes the pathogenesis of phytopathogens, but the upstream regulators of Rab8 are still unknown. Here, we have identified two Sec2 homologues FgSec2A and FgSec2B in devastating fungal pathogen Fusarium graminearum and investigated their functions and interactions with FgRab8 by live-cell imaging, genetic and functional analyses. Yeast two-hybrid assay shows that FgSec2A specifically interacts with FgRab8DN(N123I) and itself. Importantly, FgSec2A is required for growth, conidiation, DON production and virulence of F. graminearum. Live-cell imaging shows that FgSec2A and FgSec2B are both localized to the tip region of hyphae and conidia. Both N-terminal region and Sec2 domain of FgSec2A are essential for its function, but not for localization, whereas the C-terminal region is important for its polarized localization. Furthermore, constitutively active FgRab8CA(Q69L) partially rescues the defects of ΔFgsec2A. Consistently, FgSec2A is required for the polarized localization of FgRab8. Finally, FgSec2A and FgSec2B show partial functions, but FgSec2A does not interact and co-localize with FgSec2B. Taken together, these results indicate that FgSec2A acts as a FgRab8 guanine nucleotide exchange factor and is necessary for polarized growth, DON production and pathogenicity in F. graminearum., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Fusarium graminearum is a fungal pathogen that causes Fusarium head blight (FHB) in wheat and barley. Autophagy is a highly conserved vacuolar degradation pathway essential for cellular homeostasis in which Atg9 serves as a multispanning membrane protein important for generating membranes for the formation of phagophore assembly site. However, the mechanism of autophagy or autophagosome formation in phytopathogens awaits further clarifications. In this study, we identified and characterized the Atg9 homolog (FgAtg9) in F. graminearum by live cell imaging, biochemical and genetic analyses. We find that GFP-FgAtg9 localizes to late endosomes and trans-Golgi network under both nutrient-rich and nitrogen starvation conditions and also show its dynamic actin-dependent trafficking in the cell. Further targeted gene deletion of FgATG9 demonstrates that it is important for growth, aerial hyphae development, and pathogenicity in F. graminearum. Furthermore, the deletion mutant (ΔFgatg9) shows severe defects in autophagy and lipid metabolism in response to carbon starvation. Interestingly, small GTPase FgRab7 is found to be required for the dynamic trafficking of FgAtg9, and co-immunoprecipitation (Co-IP) assays show that FgAtg9 associates with FgRab7 in vivo. Finally, heterologous complementation assay shows that Atg9 is functionally conserved in F. graminearum and Magnaporthe oryzae. Taken together, we conclude that FgAtg9 is essential for autophagy-dependent development and pathogenicity of F. graminearum, which may be regulated by the small GTPase FgRab7., Competing Interests: The authors have declared that no competing interests exist.

Endosomal sorting machineries regulate the transport of their cargoes among intracellular compartments. However, the molecular nature of such intracellular trafficking processes in pathogenic fungal development and pathogenicity remains unclear. Here, we dissect the roles and molecular mechanisms of two sorting nexin proteins and their cargoes in endosomal recycling in Fusarium graminearum using high-resolution microscopy and high-throughput co-immunoprecipitation strategies. We show that the sorting nexins, FgSnx41 and FgSnx4, interact with each other and assemble into a functionally interdependent heterodimer through their respective BAR domains. Further analyses demonstrate that the dimer localizes to the early endosomal membrane and coordinates endosomal sorting. The small GTPase FgRab5 regulates the correct localization of FgSnx41-FgSnx4 and is consequently required for its trafficking function. The protein FgSnc1 is a cargo of FgSnx41-FgSnx4 and regulates the fusion of secreted vesicles with the fungal growing apex and plasma membrane. In the absence of FgSnx41 or FgSnx4, FgSnc1 is mis-sorted and degraded in the vacuole, and null deletion of either component causes defects in the fungal polarized growth and virulence. Overall, for the first time, our results reveal the mechanism of FgSnc1 endosomal recycling by FgSnx41-FgSnx4 heterodimer which is essential for polarized growth and pathogenicity in F. graminearum., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

In eukaryotic organisms, the 5-oxoprolinase is one of the six key enzymes in the γ-glutamyl cycle that is involved in the biosynthetic pathway of glutathione (GSH, an antioxidative tripeptide counteracting the oxidative stress). To date, little is known about the biological functions of the 5-oxoprolinase in filamentous phytopathogenic fungi. In this study, we investigated the 5-oxoprolinase in Fusarium graminearum for the first time. In F. graminearum, two paralogous genes (FgOXP1 and FgOXP2) were identified to encode the 5-oxoprolinase while only one homologous gene encoding the 5-oxoprolinase could be found in other filamentous phytopathogenic fungi or Saccharomyces cerevisiae. Deletion of FgOXP1 or FgOXP2 in F. graminearum led to significant defects in its virulence on wheat. This is likely caused by an observed decreased deoxynivalenol (DON, a mycotoxin) production in the gene deletion mutant strains as DON is one of the best characterized virulence factors of F. graminearum. The FgOXP2 deletion mutant strains were also defective in conidiation and sexual reproduction while the FgOXP1 deletion mutant strains were normal for those phenotypes. Double deletion of FgOXP1 and FgOXP2 led to more severe defects in conidiation, DON production and virulence on plants, suggesting that both FgOXP1 and FgOXP2 play a role in fungal development and plant colonization. Although transformation of MoOXP1into ΔFgoxp1 was able to complement ΔFgoxp1, transformation of MoOXP1 into ΔFgoxp2 failed to restore its defects in sexual development, DON production and pathogenicity. Taken together, these results suggest that FgOXP1 and FgOXP2 are likely to have been functionally diversified and play significant roles in fungal development and full virulence in F. graminearum.

Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. The vacuolar protein sorting (Vps) protein Vps27 is a component of ESCRT-0 involved in the multivesicular body (MVB) sorting pathway during endocytosis. In this study, we investigated the function of FgVps27 using a gene replacement strategy. The FgVPS27 deletion mutant (ΔFgvps27) exhibited a reduction in growth rate, aerial hyphae formation and hydrophobicity. It also showed increased sensitivity to cell wall-damaging agents and to osmotic stresses. In addition, FgHog1, the critical component of high osmolarity glycerol response pathway, was mis-localized in the ΔFgvps27 mutant upon NaCl treatment. Furthermore, the ΔFgvps27 mutant was defective in conidial production and was unable to generate perithecium in sexual reproduction. The depletion of FgVPS27 also caused a significant reduction in virulence. Further analysis by domain-specific deletion revealed that the FYVE domain was essential for the FgVps27 function and was necessary for the proper localization of FgVps27-GFP and endocytosis. Another component of ESCRT-0, the FgVps27-interacting partner FgHse1, also played an important role in F. graminearum development and pathogenesis. Overall, our results indicate that ESCRT-0 components play critical roles in a variety of cellular and biological processes., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Septins are GTP-binding proteins that regulate cell polarity, cytokinesis and cell morphogenesis. Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating diseases worldwide. In this study, we have functionally characterized the core septins, Cdc3, Cdc10, Cdc11 and Cdc12 in F. graminearum. The loss of FgCdc3, FgCdc11, FgCdc12, but not FgCdc10, mutants showed significant reduction in growth, conidiation and virulence. Microscopic analyses revealed that all of them were involved in septum formation and nuclear division. Moreover, disruption of septin genes resulted in morphological defects in ascospores and conidia. Interestingly, conidia produced by ΔFgcdc3, ΔFgcdc11 and ΔFgcdc12 mutants exhibited deformation with interconnecting conidia in contrast to their parent wild-type strain PH-1 and the ΔFgcdc10 mutant that produced normal conidia. Using yeast two-hybrid assays, we determined the interactions among FgCdc3, FgCdc10, FgCdc11 and FgCdc12. Taken together, our results indicate that septins play important roles in the nuclear division, morphogenesis and pathogenicity in F. graminearum., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In eukaryotes, the retromer is an endosome-localized complex involved in protein retrograde transport. However, the role of such intracellular trafficking events in pathogenic fungal development and pathogenicity remains unclear. The role of the retromer complex in Fusarium graminearum was investigated using cell biological and genetic methods. We observed the retromer core component FgVps35 (Vacuolar Protein Sorting 35) in the cytoplasm as fast-moving puncta. FgVps35-GFP co-localized with both early and late endosomes, and associated with the trans-Golgi network (TGN), suggesting that FgVps35 functions at the donor endosome membrane to mediate TGN trafficking. Disruption of microtubules with nocodazole significantly restricted the transportation of FgVps35-GFP and resulted in severe germination and growth defects. Mutation of FgVPS35 not only mimicked growth defects induced by pharmacological treatment, but also affected conidiation, ascospore formation and pathogenicity. Using yeast two-hybrid assays, we determined the interactions among FgVps35, FgVps26, FgVps29, FgVps17 and FgVps5 which are analogous to the yeast retromer complex components. Deletion of any one of these genes resulted in similar phenotypic defects to those of the ΔFgvps35 mutant and disrupted the stability of the complex. Overall, our results provide the first clear evidence of linkage between the retrograde transport mediated by the retromer complex and virulence in F. graminearum., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Rab GTPases represent the largest subfamily of Ras-related small GTPases and regulate membrane trafficking. Vesicular transport is a general mechanism that governs intracellular membrane trafficking along the endocytic and exocytic pathways in all eukaryotic cells. Fusarium graminearum is a filamentous fungus and causes the devastating and economically important head blight of wheat and related species. The mechanism of vesicular transport is not well understood, and little is known about Rab GTPases in F. graminearum. In this study, we systematically characterized all eleven FgRabs by live cell imaging and genetic analysis. We find that FgRab51 and FgRab52 are important for the endocytosis, FgRab7 localizes to the vacuolar membrane and regulates the fusion of vacuoles and autophagosomes, and FgRab8 and FgRab11 are important for polarized growth and/or exocytosis. Furthermore, both endocytic and exocytic FgRabs are required for vegetative growth, conidiogenesis, sexual reproduction, as well as pathogenesis and deoxynivalenol metabolism in F. graminearum. Thus, we conclude that Rab GTPases are essential for membrane trafficking-dependent growth and pathogenicity in F. graminearum., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Rho GTPases have multiple cellular and metabolic functions, including vesicular trafficking and pathogenesis, as signaling molecules in fungi. Wheat scab, caused by Fusarium graminearum, is one of the most important wheat diseases worldwide, yet the mechanisms associated with making this fungus such a devastating pathogen remain largely ambiguous. In an effort to better understand F. graminearum virulence, we functionally characterized all six Rho GTPases in F. graminearum. FgRHO1 was determined to be essential for fungal survival, while FgRho3 demonstrated functions only in vegetative growth and conidiation. Other four Rho GTPases, FgRho2, FgRho4, FgCdc42 and FgRac1, were multifunctional and were involved in sexual development and pathogenesis. While FgRho2 and FgRho4 were both involved in cell wall integrity, only FgRho4 showed a role in nuclear division and septum formation. FgRho4, FgCdc42 and FgRac1 were also important for hyphal growth and conidiation. All single deletion mutants showed impaired growth, particularly in conidial morphology, when compared to the wild-type progenitor. FgRac1 deletion mutants displayed a precocious, multi-site germ tube formation as well as hyperbranching of hyphae. Significantly we learned that FgRac1 negatively controls DON production whereas FgRho4 plays a positive role. FgCla4 was identified as a downstream target of FgRac1, but was dispensable for sexual development. We determined that FgRho GTPases contribute diversely to growth, conidiogenesis, sexual reproduction, DON production and pathogenesis in F. graminearum., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Conidia are primary means of asexual reproduction and dispersal in a variety of pathogenic fungi, and it is widely recognized that they play a critical role in animal and plant disease epidemics. However, genetic mechanisms associated with conidiogenesis are complex and remain largely undefined in numerous pathogenic fungi. We previously showed that Htf1, a homeobox transcription factor, is required for conidiogenesis in the rice pathogen Magnaporthe oryzae. In this study, our aim was to characterize how Htf1 homolog regulates common and also distinctive conidiogenesis in three key Fusarium pathogens: F. graminearm, F. verticillioides, and F. oxysporum. When compared to wild-type progenitors, the gene-deletion mutants in Fusarium species failed to form conventional phialides. Rather, they formed clusters of aberrant phialides that resembled elongated hyphae segments, and it is conceivable that this led to the obstruction of conidiation in phialides. We also observed that mutants, as well as wild-type Fusaria, can initiate alternative macroconidia production directly from hyphae through budding-like mechanism albeit at low frequencies. Microscopic observations led us to conclude that proper basal cell division and subsequent foot cell development of macroconidia were negatively impacted in the mutants. In F. verticillioides and F. oxysporum, mutants exhibited a 2- to 5- microconidia complex at the apex of monophialides resulting in a floral petal-like shape. Also, prototypical microconidia chains were absent in F. verticillioides mutants. F. graminearum and F. verticillioides mutants were complemented by introducing its native HTF1 gene or homologs from other Fusarium species. These results suggest that Fusarium Htf1 is functionally conserved homeobox transcription factor that regulates phialide development and conidiogenesis via distinct signaling pathways yet to be characterized in fungi.

The AP1 complex is a highly conserved clathrin adaptor that plays important roles in regulating cargo protein sorting and intracellular vesicle trafficking in eukaryotes. However, the functions of the AP1 complex in the plant pathogenic fungi including the devastating wheat pathogen Fusarium graminearum are still unclear. In this study, we investigated the biological functions of FgAP1σ, a subunit of the AP1 complex in F. graminearum. Disruption of FgAP1σ causes seriously impaired fungal vegetative growth, conidiogenesis, sexual development, pathogenesis, and deoxynivalenol (DON) production. The ΔFgap1σ mutants were found to be less sensitive to KCl- and sorbitol-induced osmotic stresses but more sensitive to SDS-induced stress than the wild-type PH-1. Although the growth inhibition rate of the ΔFgap1σ mutants was not significantly changed under calcofluor white (CFW) and Congo red (CR) stresses, the protoplasts released from ΔFgap1σ hyphae were decreased compared with the wild-type PH-1, suggesting that FgAP1σ is necessary for cell wall integrity and osmotic stresses in F. graminearum. Subcellular localization assays showed that FgAP1σ was predominantly localized to endosomes and the Golgi apparatus. In addition, FgAP1β-GFP, FgAP1γ-GFP, and FgAP1μ-GFP also localize to the Golgi apparatus. FgAP1β interacts with FgAP1σ, FgAP1γ, and FgAP1μ, while FgAP1σ regulates the expression of FgAP1β, FgAP1γ, and FgAP1μ in F. graminearum. Furthermore, the loss of FgAP1σ blocks the transportation of the v-SNARE protein FgSnc1 from the Golgi to the plasma membrane and delays the internalization of FM4-64 dye into the vacuole. Taken together, our results demonstrate that FgAP1σ plays vital roles in vegetative growth, conidiogenesis, sexual reproduction, DON production, pathogenicity, cell wall integrity, osmotic stress, exocytosis, and endocytosis in F. graminearum. These findings unveil the functions of the AP1 complex in filamentous fungi, most notably in F. graminearum, and lay solid foundations for effective prevention and control of Fusarium head blight (FHB). [ABSTRACT FROM AUTHOR]

Calcium and manganese transporters play important roles in regulating Ca2+ and Mn2+ homeostasis in cells, which is necessary for the normal physiological activities of eukaryotes. Gdt1 and Pmr1 function as calcium/manganese transporters in the Golgi apparatus. However, the functions of Gdt1 and Pmr1 have not been previously characterized in the plant pathogenic fungus Fusarium graminearum. Here, we identified and characterized the biological functions of FgGdt1 and FgPmr1 in F. graminearum. Our study shows that FgGdt1 and FgPmr1 are both localized to the cis‐ and medial‐Golgi. Disruption of FgGdt1 or FgPmr1 in F. graminearum caused serious defects in vegetative growth, conidiation, sexual development and significantly decreased virulence in wheat but increased deoxynivalenol (DON) production. Importantly, FgGdt1 is involved in Ca2+ and Mn2+ homeostasis and the severe phenotypic defects of the ΔFggdt1 mutant were largely due to loss of FgGdt1 function in Mn2+ transportation. FgGdt1‐mCherry colocalizes with FgPmr1‐GFP at the Golgi, and FgGDT1 exerts its biological function upstream of FgPMR1. Taken together, our results collectively demonstrate that the cis‐ and medial‐Golgi‐localized proteins FgGdt1 and FgPmr1 regulate Ca2+ and Mn2+ homeostasis of the Golgi apparatus, and this function is important in modulating the growth, development, DON biosynthesis and pathogenicity of F. graminearum. [ABSTRACT FROM AUTHOR]

Apical secretion at hyphal tips is important for the growth and development of filamentous fungi. In this study, we analyzed the role of the Rab GTPases FoSec4 involved in the secretion of the banana wilt fungal pathogen Fusarium odoratissimum. We found that the deletion of FoSEC4 affects the activity of extracellular hydrolases and protein secretion, indicating that FoSec4 plays an important role in the regulation of protein secretion in F. odoratissimum. As a typical Rab GTPase, Sec4 participates in the Rab cycle through the conversion between the active GTP-bound state and the inactive GDP-bound state, which is regulated by guanine nucleate exchange factors (GEFs) and GTPase-activating proteins (GAPs). We further found that FoSec2 can interact with dominant-negative FoSec4 (GDP-bound and nucleotide-free form, FoSec4DN), and that FoGyp5 can interact with dominant active FoSec4 (GTP-bound and constitutively active form, FoSec4CA). We evaluated the biofunctions of FoSec4, FoSec2 and FoGyp5, and found that FoSec4 is involved in the regulation of vegetative growth, reproduction, pathogenicity and the environmental stress response of F. odoratissimum, and that FocSec2 and FoGyp5 perform biofunctions consistent with FoSec4, indicating that FoSec2 and FoGyp5 may work as the GEF and the GAP, respectively, of FoSec4 in F. odoratissimum. We further found that the amino-terminal region and Sec2 domain are essential for the biological functions of FoSec2, while the carboxyl-terminal region and Tre-2/Bub2/Cdc16 (TBC) domain are essential for the biological functions of FoGyp5. In addition, FoSec4 mainly accumulated at the hyphal tips and partially colocalized with Spitzenkörper; however, FoGyp5 accumulated at the periphery of Spitzenkörper, suggesting that FoGyp5 may recognize and inactivate FoSec4 at a specific location in hyphal tips. [ABSTRACT FROM AUTHOR]

Rab GTPases play an important role in vesicle-mediated membrane trafficking in eukaryotes. Previous studies have demonstrated that deletion of RAB5 / VPS21 reduces endocytosis and virulence of fungal phytopathogens in their host plants. However, Rab5 GTPase cycle regulators have not been characterized in Fusarium graminearum , the causal agent of Fusarium head blight (FHB) or head scab disease in cereal crops. In this study, we have identified and characterized a Rab5 guanine nucleotide exchange factor (GEF), the Vps9 homolog FgVps9, in F. graminearum. Yeast two hybrid (Y2H) assays have shown that FgVps9 specifically interacts with the guanosine diphosphate (GDP)-bound (inactive) forms of FgRab51 and FgRab52, the Rab5 isoforms in F. graminearum. Deletion of FgVPS9 shows impaired fungal growth and conidiation. Pathogenicity assays indicate that deletion of FgVPS9 can significantly decrease the virulence of F. graminearum in wheat. Cytological analyses have indicated that FgVps9 colocalizes with FgRab51 and FgRab52 on early endosomes and regulates endocytosis and autophagy processes. Gene expression and cytological examination have shown that FgVps9 and FgRab51 or FgRab52 function in concert to control deoxynivalenol (DON) biosynthesis by regulating the expression of trichothecene biosynthesis-related genes and toxisome biogenesis. Taken together, FgVps9 functions as a GEF for FgRab51 and FgRab52 to regulate endocytosis, which, as a basic cellular function, has significant impact on the vegetative growth, asexual development, autophagy, DON production, and plant infection in F. graminearum. [ABSTRACT FROM AUTHOR]

Multivesicular bodies (MVBs) are critical intermediates in the trafficking of ubiquitinated endocytosed surface proteins to the lysosome/vacuole for destruction. Recognizing and packaging ubiquitin modified cargoes to the MVB pathway require ESCRT (Endosomal sorting complexes required for transport) machinery, which consists of four core subcomplexes, ESCRT-0, ESCRT-I, ESCRT-II, and ESCRT-III. Fusarium graminearum is an important plant pathogen that causes head blight of major cereal crops. Our previous results showed that ESCRT-0 is essential for fungal development and pathogenicity in Fusarium graminearum. We then, in this study, systemically studied the protein-protein interactions within F. graminearum ESCRT-I, -II or -III complex, as well as between ESCRT-0 and ESCRT-I, ESCRT-I and ESCRT-II, and ESCRT-II and ESCRT-III complexes and found that loss of any ESCRT component resulted in abnormal function in endocytosis. In addition, ESCRT deletion mutants displayed severe defects in growth, deoxynivalenol (DON) production, virulence, sexual, and asexual reproduction. Importantly genetic complementation with corresponding ESCRT genes fully rescued all these defective phenotypes, indicating the essential role of ESCRT machinery in fungal development and plant infection in F. graminearum. Taken together, the protein-protein interactome and biological functions of the ESCRT machinery is first profoundly characterized in F. graminearum , providing a foundation for further exploration of ESCRT machinery in filamentous fungi. [ABSTRACT FROM AUTHOR]