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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 missorted 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.

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 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. 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.

During sexual reproduction in flowering plants, haploid spores are formed from meiosis of spore mother cells. The spores then undergo mitosis, develop into female and male gametophytes, and give rise to seeds after fertilization. We identified a female sterile mutant ap1g2-4 from EMS mutagenesis, and analyses of two T-DNA insertion mutants, ap1g2-1 +/− and ap1g2-3 −/− , and detected a partial female and male sterility. The ap1g2 mutant gametophyte development was arrested at one nuclear stage. A complementation test using a genomic sequence of AP1G2 with its native promoter restored the function in the three ap1g2 mutant lines. Transcriptome profiling of ap1g2 ovules revealed that four genes encoding clathrin assembly proteins PICALM5A/B and PICALM9A/B, which were involved in endocytosis, were downregulated, which were confirmed to interact with AP1G2 through yeast two-hybrid assays and BIFC analysis. Our result also demonstrated that RALFL4-8-15-19-26 CML16 and several calcium-dependent protein kinases, including CPK14-16-17 , were all downregulated in the ovules of ap1g2-1 +/− . Moreover, Ca 2+ concentration was low in impaired gametophytes. Therefore, we proposed that through interaction with PICALM5A/B and PICALM9A/B, AP1G2 may mediate gametogenesis accompanied by Ca 2+ signaling in Arabidopsis. Our findings revealed a crucial role of AP1G2 in female and male gametogenesis in Arabidopsis and enhanced our understanding of the molecular mechanisms underpinning sexual reproduction in flowering plants.

Fusarium head blight (FHB) caused by the ascomycete fungus Fusarium graminearum can result in significant crop losses and render the crops harmful to human health due to contamination with mycotoxin. Although the pathogenesis of F. graminearum is widely investigated by molecular genetics approaches, detailed studies about its cellular and developmental processes at the initial stages of infection are very limited. We applied live-cell imaging approach to characterize the spatial and temporal development of growing hyphae and plant responses during F. graminearum and wheat coleoptile interactions. The present investigation demonstrates that F. graminearum uses two strategies to penetrate the host epidermal cells. The pathogen breaks through the host cell wall with appressoria-like structures derived from surficial hyphae, and also with the narrow pegs from thick and bulbous intracellular hyphae. Live cell imaging in the presence of the endocytic tracker FM4–64 showed that the plasma and intermembranes of the invaded wheat coleoptile cells were intact. Invasive hyphae exhibit branching, budding, pseudohyphae-like growth, cell-to-cell spreading ability, and were sealed within a plant membrane, indicating a biotrophic lifestyle of this fungus inside the invaded cells. Time-lapse imaging suggested that there were callose depositions at the plant cell walls in the form of continuous lines and also on the outer linings of the fungal invasive hyphae at colonization stage. In addition, our studies demonstrate that the activation of the toxisome-related gene ( TRI4 ) requires external stimuli and is spatio-temporally modulated. Generally, this study provides new insights into the colonization strategies and host response features during F. graminearum -plant interactions. Further tracing of cellular details will significantly contribute to our understanding of molecular mechanisms of F. graminearum -host interactions.

In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.

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. Two SNARE complexes Sso1–Sec9–Snc1 and Sso2–Sec9–Snc1 were identified in Fusarium oxysporum f. sp. cubense that coordinately facilitate docking and fusion of exocytic vesicles with the plasma membrane in a spatially specific way.

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 MoOXP1 into 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.

The retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A , ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum .

In nature, fungal endophytes often have facultative endohyphal bacteria (FEB). Can a model plant pathogenic fungus have them, and does it affect their phenotype? We constructed a growth system/microcosm to allow an F. graminearum isolate to grow through natural soil and then re-isolated it on a gentamicin-containing medium, allowing endohyphal growth of bacteria while killing other bacteria. F. graminearum PH-1 labelled with a His1mCherry gene staining the fungal nuclei fluorescent red was used to confirm the re-isolation of the fungus. Most new re-isolates contained about 10 16SrRNA genes per fungal mCherry gene determined by qPCR. The F. graminearum + FEB holobiont isolates containing the bacteria were sub-cultured several times, and their bacterial contents were stable. Sequencing the bacterial 16SrRNA gene from several Fg-FEB holobiont isolates revealed endophytic bacteria known to be capable of nitrogen fixation. We tested the pathogenicity of one common Fg-FEB holobiont association, F. graminearum + Stenatrophomonas maltophilia, and found increased pathogenicity. The 16SrRNA gene load per fungal His1mCherry gene inside the wheat stayed the same as previously found in vitro. Finally, strong evidence was found for Fg-S. maltophilia symbiotic nitrogen fixation benefitting the fungus.