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TRAnsport Protein Particle (TRAPP) is a conserved multi-subunit tethering complex known to be involved in intracellular movement of proteins. However, its components and molecular functions in filamentous fungi remain poorly characterized. Here, we identify four TRAPPIII-specific subunits (FgTrs85, TRAPPC11, TRAPPC12, and TRAPPC13) in the phytopathogenic fungus Fusarium graminearum . Genetic and functional analyses reveal that FgTrs85 serves as the core subunit, collaborating with auxiliary subunits TRAPPC11, TRAPPC12, and TRAPPC13 to orchestrate fungal perithecium formation, growth, and virulence. TRAPPIII localizes to the phagophore assembly site and promotes autophagosome biogenesis by recruiting FgAtg9 through interactions involving FgTrs85 and TRAPPC13. Notably, TRAPPIII mutants exhibit more severe growth defects than autophagy-deficient strains, suggesting that the roles of TRAPPIII extend beyond autophagy. TRAPPIII regulates ER-to-Golgi and endosome-to-Golgi transport by ensuring the proper localization of secretory regulators (FgSec22, FgRud3, FgSnc1). Moreover, the overexpression of FgRab1-GTP largely suppresses all phenotypic defects associated with perithecium formation, growth, and virulence in TRAPPIII mutants, suggesting the function of TRAPPIII as a guanine nucleotide exchange factor that activates FgRab1. Altogether, our results demonstrate that TRAPPIII coordinates autophagy and intracellular transport to regulate fungal development, growth and virulence in F. graminearum .

Enoyl-CoA hydratase (Ech) is an important and well-recognized enzyme that functions in the degradation of fatty acids by β-oxidation. However, its functions in plant pathogenic fungi are not well known. We characterized an Ech1 orthologue, FgEch1 , in Fusarium graminearum . The FgEch1 deletion mutant was defective in the utilization of short-chain fatty acids and conidiation, but not in hyphal growth on glucose-rich media or in perithecium formation. The FgEch1 deletion mutant showed reduced deoxynivalenol (DON) production and virulence in plants. Deletion of FgEch1 also led to increased production of lipid droplets and autophagy. FgEch1, which was localized in the mitochondrion, required the MTS domain for mitochondrial localization and function in F. graminearum . Taken together, these data indicate that mitochondrial FgEch1 is important for conidiation, DON production, and plant infection.

In the conserved autophagy pathway, the double-membrane autophagosome (AP) engulfs cellular components to be delivered for degradation in the lysosome. While only sealed AP can productively fuse with the lysosome, the molecular mechanism of AP closure is currently unknown. Rab GTPases, which regulate all intracellular trafficking pathways in eukaryotes, also regulate autophagy. Rabs function in GTPase modules together with their activators and downstream effectors. In yeast, an autophagy-specific Ypt1 GTPase module, together with a set of autophagy-related proteins (Atgs) and a phosphatidylinositol-3-phosphate (PI3P) kinase, regulates AP formation. Fusion of APs and endosomes with the vacuole (the yeast lysosome) requires the Ypt7 GTPase module. We have previously shown that the Rab5-related Vps21, within its endocytic GTPase module, regulates autophagy. However, it was not clear which autophagy step it regulates. Here, we show that this module, which includes the Vps9 activator, the Rab5-related Vps21, the CORVET tethering complex, and the Pep12 SNARE, functions after AP expansion and before AP closure. Whereas APs are not formed in mutant cells depleted for Atgs, sealed APs accumulate in cells depleted for the Ypt7 GTPase module members. Importantly, depletion of individual members of the Vps21 module results in a novel phenotype: accumulation of unsealed APs. In addition, we show that Vps21-regulated AP closure precedes another AP maturation step, the previously reported PI3P phosphatase-dependent Atg dissociation. Our results delineate three successive steps in the autophagy pathway regulated by Rabs, Ypt1, Vps21 and Ypt7, and provide the first insight into the upstream regulation of AP closure.

Mounting evidence indicates that viruses exploit elevated reactive oxygen species (ROS) levels to promote replication and pathogenesis, yet the mechanistic underpinnings of this viral strategy remain elusive for many viral systems. This study uncovers a sophisticated viral counter‐defense mechanism in the Cryphonectria hypovirus 1 (CHV1)‐Fusarium graminearum system, where the viral p29 protein subverts host redox homeostasis to overcome antiviral responses. That p29 directly interacts with and inhibits the enzymatic activity of fungal NAD(P)H‐dependent FMN reductase 1 (FMR1), leading to increased ROS accumulation and subsequent autophagy activation is demonstrated. Strikingly, this ROS‐induced autophagy selectively targets for degradation two core antiviral RNA silencing components against CHV1 in F. graminearum, Dicer‐like 2 (DCL2) and Argonaute‐like 1 (AGL1), thereby compromising the host's primary antiviral defense system. Genetic analysis confirms this coordinated hijacking of host machineries, as CHV1 shows enhanced accumulation in the FMR1 knockout and reduced accumulation in autophagy‐deficient fungal strains. This work reveals a tripartite interplay among oxidative stress, autophagy, and RNA silencing that CHV1 manipulates through p29 multifunctional activity. These findings provide a model for how viruses coordinately regulate distinct host defense systems to optimize infection. Understanding the virus‐host arms race has been a fascinating topic in virology; contributing significantly to the development of treatments and control strategies for viral diseases. CHV1 employs a sophisticated counter‐defense mechanism in its fungal host by utilizing a viral silencing suppressor to inhibit fungal FMN reductase; thereby modulating ROS levels. This alteration triggers autophagic degradation of DCL and AGL proteins; ultimately attenuating the antiviral RNA interference (RNAi) responses.

The rapid production of hydrogen peroxide (H2O2) is a hallmark of plants’ successful recognition of pathogen infection and plays a crucial role in innate immune signaling. Aquaporins (AQPs) are membrane channels that facilitate the transport of small molecular compounds across cell membranes. In plants, AQPs from the plasma membrane intrinsic protein (PIP) family are utilized for the transport of H2O2, thereby regulating various biological processes. Plants contain two PIP families, PIP1s and PIP2s. However, the specific functions and relationships between these subfamilies in plant growth and immunity remain largely unknown. In this study, we explore the synergistic role of AtPIP1;4 and AtPIP2;4 in regulating plant growth and disease resistance in Arabidopsis. We found that in plant cells treated with H2O2, AtPIP1;4 and AtPIP2;4 act as facilitators of H2O2 across membranes and the translocation of externally applied H2O2 from the apoplast to the cytoplasm. Moreover, AtPIP1;4 and AtPIP2;4 collaborate to transport bacterial pathogens and flg22-induced apoplastic H2O2 into the cytoplasm, leading to increased callose deposition and enhanced defense gene expression to strengthen immunity. These findings suggest that AtPIP1;4 and AtPIP2;4 cooperatively mediate H2O2 transport to regulate plant growth and immunity.

Fusarium graminearum is recognized as the pathogen responsible for wheat head blight. It produces deoxynivalenol (DON) during infection, which endangers human health. DON biosynthesis occurs within toxisomes in the endoplasmic reticulum (ER). In eukaryotes, the ER membrane protein complex (EMC) is critical for the ER’s normal operation. However, the specific role of the EMC in F. graminearum remains poorly understood. In this study, six EMC subunits (FgEmc1-6) were identified in F. graminearum, and all of them were localized to the toxisomes. Our results demonstrate that the EMC is indispensable for vegetative growth and asexual and sexual reproduction, which are the fundamental life processes of F. graminearum. Importantly, EMC deletion led to reduced virulence in wheat spikes and petioles. Further investigation revealed that in ΔFgemc1-6, the expression of trichothecene (TRI) genes is decreased, the biosynthesis of lipid droplets (LDs) is diminished, toxisome formation is impaired, and DON production is reduced. Additionally, defects in the formation of the infection cushion were observed in ΔFgemc1-6. In conclusion, the EMC is involved in regulating growth and virulence in F. graminearum. This study enhances our understanding of the EMC functions in F. graminearum and offers valuable insights into potential targets for managing wheat head blight.

Fusarium graminearum is a plant filamentous pathogenic fungi and the predominant causal agent of Fusarium head blight (FHB) in cereals worldwide. The regulators of the secretory pathway contribute significantly to fungal mycotoxin synthesis, development, and virulence. However, their roles in these processes in F. graminearum remain poorly understood. Here, we identified and functionally characterized the endoplasmic reticulum (ER) cargo receptor FgErv14 in F. graminearum. Firstly, it was observed that FgErv14 is mainly localized in the ER. Then, we constructed the FgErv14 deletion mutant (ΔFgerv14) and found that the absence of the FgErv14 caused a serious reduction in vegetative growth, significant defects in asexual and sexual reproduction, and severely impaired virulence. Furthermore, we found that the ΔFgerv14 mutant exhibited a reduced expression of TRI genes and defective toxisome generation, both of which are critical for deoxynivalenol (DON) biosynthesis. Importantly, we found the green fluorescent protein (GFP)-tagged FgRud3 was dispersed in the cytoplasm, whereas GFP-FgSnc1-PEM was partially trapped in the late Golgi in ΔFgerv14 mutant. These results demonstrate that FgErv14 mediates anterograde ER-to-Golgi transport as well as late secretory Golgi-to-Plasma membrane transport and is necessary for DON biosynthesis, asexual and sexual reproduction, vegetative growth, and pathogenicity in F. graminearum.

The environmentally friendly antibiotic phenazine-1-carboxylic acid (PCA) protects plants, mammals and humans effectively against various fungal pathogens. However, the mechanism by which PCA inhibits or kills fungal pathogens is not fully understood. We analyzed the effects of PCA on the growth of two fungal model organisms, Saccharomyces cerevisiae and Candida albicans , and found that PCA inhibited yeast growth in a dose-dependent manner which was inversely dependent on pH. In contrast, the commonly used antibiotic hygromycin B acted in a dose-dependent manner as pH increased. We then screened a yeast mutant library to identify genes whose mutation or deletion conferred resistance or sensitivity to PCA. We isolated 193 PCA-resistant or PCA-sensitive mutants in clusters, including vesicle-trafficking- and autophagy-defective mutants. Further analysis showed that unlike hygromycin B, PCA significantly altered intracellular vesicular trafficking under growth conditions and blocked autophagy under starvation conditions. These results suggest that PCA inhibits or kills pathogenic fungi in a complex way, in part by disrupting vesicular trafficking and autophagy.

Mitochondrial porin, the voltage-dependent anion-selective channel (VDAC), is the most abundant protein in the outer membrane, and is critical for the exchange of metabolites and phospholipids in yeast and mammals. However, the functions of porin in phytopathogenic fungi are not known. In this study, we characterized a yeast porin orthologue, Fgporin, in Fusarium graminearum. The deletion of Fgporin resulted in defects in hyphal growth, conidiation, and perithecia development. The Fgporin deletion mutant showed reduced virulence, deoxynivalenol production, and lipid droplet accumulation. In addition, the Fgporin deletion mutant exhibited morphological changes and the dysfunction of mitochondria, and also displayed impaired autophagy in the non-nitrogen medium compared to the wild type. Yeast two-hybrid and bimolecular fluorescence complementation assays indicated that Fgporin interacted with FgUps1/2, but not with FgMdm35. Taken together, these results suggest that Fgporin is involved in hyphal growth, asexual and sexual reproduction, virulence, and autophagy in F. graminearum.

Ypt/Rab are key regulators of intracellular trafficking in all eukaryotic cells. In yeast, Ypt1 is essential for endoplasmic reticulum (ER)-to-Golgi transport, whereas Ypt31/32 regulate Golgi-to-plasma membrane and endosome-to-Golgi transport. TRAPP is a multisubunit complex that acts as an activator of Ypt/Rab GTPases. Trs85 and Trs130 are two subunits specific for TRAPP III and TRAPP II, respectively. Whereas TRAPP III was shown to acts as a Ypt1 activator, it is still controversial whether TRAPP II acts as a Ypt1 or Ypt31/32 activator. Here, we use GFP-Snc1 as a tool to study transport in Ypt and TRAPP mutant cells. First, we show that expression of GFP-Snc1 in trs85Δ mutant cells results in temperature sensitivity. Second, we suggest that in ypt1ts and trs85Δ, but not in ypt31Δ/32ts and trs130ts mutant cells, GFP-Snc1 accumulates in the ER. Third, we show that overexpression of Ypt1, but not Ypt31/32, can suppress both the growth and GFP-Snc1 accumulation phenotypes of trs85Δ mutant cells. In contrast, overexpression of Ypt31, but not Ypt1, suppresses the growth and GFP-Snc1 transport phenotypes of trs130ts mutant cells. These results provide genetic support for functional grouping of Ypt1 with Trs85-containing TRAPP III and Ypt31/32 with Trs130-containing TRAPP II.