The Slt2-Swi6 signaling pathway plays a pivotal role in regulating cell wall biosynthesis, secondary metabolite production, and the pathogenicity of pear fungal A. alternata

MAPKK Regulates Cell Wall Biogenesis in Cooperation with the Cek1-Pathway in Candida albicans

Dual regulatory roles of the zinc cluster transcription factor MrFTRP1 in development, stress tolerance, and virulence of Metarhizium robertsii.

The entomopathogenic fungus Metarhizium robertsii shows significant potential as a multifunctional biocontrol agent. The Zn(II)₂Cys₆ (C6) transcription factors (TFs) family is the largest class of transcription factors in M. robertsii, yet their functions are generally underexplored. In this study, we functionally characterized a putative C6 TF, designated MrFTRP1. Deletion of Mrftrp1 reduced conidial yield, which correlated with the downregulation of key central developmental regulators, including brlA, abaA, and wetA. Moreover, ΔMrftrp1 exhibited attenuated virulence against Galleria mellonella, a defect attributable to reduced conidial hydrophobicity, impaired adhesion, compromised appressorium formation, and decreased proliferation of yeast-like hyphal bodies in vivo. Conversely, the loss of Mrftrp1 significantly enhanced conidial tolerance to heat shock and UV-B irradiation, accompanied by the upregulation of stress-response and DNA repair-related genes. Collectively, our findings demonstrated that MrFTRP1 exerts a dual-regulatory functioning as a positive regulator of conidiation and pathogenesis but a negative regulator of environmental stress tolerance. This dual-functional role expands our understanding of the regulatory diversity within the C6 TF family and identifies MrFTRP1 as a promising target for the genetic improvement of mycoinsecticides.

Integrated transcriptomic and metabolomic analyses reveal TPS1 as a key regulator linking cell wall integrity to aflatoxin production in Aspergillus flavus.

As a widely distributed opportunistic pathogen, Aspergillus flavus poses a critical challenge to global food safety, principally through the contamination of staple crops with Aflatoxin B1 (AFB1), a highly potent carcinogen and hepatotoxin that accumulates in the food chain and gravely threatens human and animal health. Trehalose-6-phosphate synthase (TPS1) serves as a metabolic gatekeeper, yet its pleiotropic roles in A. flavus morphogenesis and secondary metabolism remain undefined. Here, we demonstrate that TPS1 is indispensable for sclerotial development, stress adaptation, and aflatoxin biosynthesis. Although the TPS1 deletion mutant (ΔTPS1) retained normal vegetative growth on solid media, it exhibited altered pellet morphology in liquid culture and completely lost the ability to produce sclerotia. Physiologically, TPS1 deficiency depleted intracellular trehalose-6-phosphate (T6P) and trehalose pools, leading to compromised cell wall integrity characterized by reduced β-1,3-glucan and chitin content, anomalous septation, and remodeled surface chemistry. Crucially, the mutant showed significantly diminished AFB1 production and attenuated virulence on host substrates (maize, peanut, and onion). Integrated transcriptomic and metabolomic analyses revealed that TPS1 deletion triggered a systemic metabolic rewiring: it disrupts glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid synthesis, while redirecting flux toward the pentose phosphate pathway. This metabolic shift correlated with the transcriptional downregulation of 23 genes within the AFB1 biosynthetic cluster. Collectively, our findings establish TPS1 as a key regulator linking carbon metabolism to morphogenesis, stress response, and virulence, highlighting its potential as a novel intervention target for controlling toxigenic fungi.

Autophagy-related protein 4 contributes to siderophore biosynthesis, toxin production, and virulence in Alternaria alternata.

The tangerine pathotype of Alternaria alternata infects multiple citrus cultivars, causing brown spot disease. The role of autophagy in toxin production and siderophore biosynthesis remains to be further confirmed. This study identifies autophagy-related protein 4 (AaAtg4) as a critical regulator of fungal growth, development, stress resistance, iron homeostasis, and virulence, as determined by genetic and biochemical analyses. Targeted deletion of the AaAtg4 gene using split-marker recombination generated two ΔAaAtg4 mutants, which displayed reduced growth on minimal medium, impaired conidiation, delayed germination, and diminished formation of appressorium-like structures compared with the wild-type strain. The mutants were hypersensitive to hydrogen peroxide and iron stress, highlighting the roles of AaAtg4 in oxidative stress tolerance and iron metabolism. ΔAaAtg4 mutant strains failed to produce detectable siderophores and exhibited downregulation of siderophore production-related genes (AaHapX, AaNps6, AaMirB) alongside upregulation of the AaSreA gene encoding a siderophore repressor. Toxin profiling further revealed altered host-selective toxin production, with distinct shifts in retention times in ΔAaAtg4 compared to the wild-type strain. However, AaAtg4, but not autophagy itself, was required for toxin production, as deletion of other autophagy-related genes had no effect. Reintroduction of functional AaAtg4 into a ΔAaAtg4 mutant rescues all defects, confirming that the loss of AaAtg4 function directly causes the observed phenotypes. These findings demonstrate that AaAtg4 is important for spore germination, siderophore biosynthesis, iron acquisition, oxidative stress resistance, and toxin production, thereby establishing its critical role in A. alternata virulence.

Aspergillus flavus bZIP-Type Transcription Factors as Promising Novel Targets for Future Aflatoxin Control Strategies

The bZIP type transcription factors (bZIPs) are global regulators governing vegetative growth, development, stress defense and secondary metabolism, including mycotoxin production in filamentous fungi. In this work, we constructed and phenotypically characterized gene deletion and complementation mutants of some bZIPs, including Afap1, AflatfA, LziP, AflatfB and bZIP6 in Aspergillus flavus. Environmental and fungicide stress responses, as well as aflatoxin production of the mutants in both surface cultures and infected maize kernels, were studied. Phenotypic characterization of the mutants revealed that Afap1 and AflatfA were involved in the oxidative (H2O2, menadione, tert-butyl hydroperoxide), cell wall integrity (Congo Red) and heavy metal (CdCl2) stress responses of A. flavus. In addition, the Afap1 and AflatfA gene deletions decreased the diamide and prothioconazole tolerances of the fungus, respectively. The ΔLziP strain showed increased growth in the presence of diamide, while reduced colony diameters were observed after exposure to CdCl2 and fludioxonil. The ΔAflatfB gene deletion mutant was also sensitive to tBOOH, while azoxystrobin and prothioconazole fungicides significantly inhibited the growth of ΔbZIP6. When aflatoxin (AFB1) production was measured in surface cultures, decreased AFB1 levels were detected only in the ΔAflatfA gene deletion mutant strain. However, in corn kernel infection assays, the ΔAfap1, ΔAflatfA, and ΔAflatfB mutants were characterized by significantly reduced aflatoxin production, while the deletion of bZIP6 almost completely abolished AFB1 biosynthesis. Our results suggest that Afap1 and AflatfA appear to be promising targets for the development of new antifungal agents, as their inhibition may increase the sensitivity of A. flavus to environmental stress, simultaneously reducing the aflatoxin production of the fungus and the use of azoles (AflatfA) in the antifungal protection of maize. In addition, bZIP6 may also be considered as an attractive target for further studies when aiming to eliminate aflatoxin production and minimize the use of azoxystrobin and prothioconazole in maize crop protection.

The Gti1/Pac2 family protein CFG1 controls fungal chlamydospore formation through orchestrating cell wall remodeling, lipid metabolism, and ribosome biogenesis

ABSTRACT The morphological transition of fungi from vegetative hyphae to thick-walled chlamydospores enhances their longevity in harsh environmental conditions. Owing to this resilience, pathogenic fungi that form chlamydospores are particularly difficult to control. Therefore, understanding the mechanisms of chlamydospore formation is critically important. Here, we show that the hyphae of the filamentous fungus Trichoderma guizhouense can differentiate into typical terminal and intercalary chlamydospores characterized by double-layered spherical or ellipsoidal cell walls with accumulated lipid bodies and nuclei. We found that during chlamydospore formation, ribosome biogenesis was gradually downregulated, indicating the entry of cells into dormancy. Comparative transcriptomic analyses across developmental stages and media identified the Gti1/Pac2 family protein CFG1 as an essential regulator, as the Δcfg1 strain failed to form chlamydospores under all inducing conditions. Lipidomic analysis showed its involvement in lipid metabolism, and mutants lacking lipid metabolism genes pdat or dgat produced fewer chlamydospores. Our work reveals the molecular mechanism of chlamydospore formation in T. guizhouense. IMPORTANCE In fungal biology, the morphological transition from vegetative hyphae to thick-walled, lipid-rich chlamydospores represents a fundamental developmental switch into dormancy, crucial for survival under environmental stress. Understanding the regulatory mechanisms behind this process is essential for deciphering the basic principles of fungal cell differentiation and adaptation. This study employs multi-omics approaches to systematically characterize chlamydospore formation and identifies the Gti1/Pac2 family protein CFG1 as a master regulator. Functional analysis reveals that CFG1 governs this transition by directly influencing lipid metabolism—a key pathway for spore maturation and structural integrity. These findings uncover a previously unknown molecular switch in fungal development and provide new insights into how filamentous fungi coordinate metabolic reprogramming with cellular differentiation to ensure long-term survival. In fungal biology, the morphological transition from vegetative hyphae to thick-walled, lipid-rich chlamydospores represents a fundamental developmental switch into dormancy, crucial for survival under environmental stress. Understanding the regulatory mechanisms behind this process is essential for deciphering the basic principles of fungal cell differentiation and adaptation. This study employs multi-omics approaches to systematically characterize chlamydospore formation and identifies the Gti1/Pac2 family protein CFG1 as a master regulator. Functional analysis reveals that CFG1 governs this transition by directly influencing lipid metabolism—a key pathway for spore maturation and structural integrity. These findings uncover a previously unknown molecular switch in fungal development and provide new insights into how filamentous fungi coordinate metabolic reprogramming with cellular differentiation to ensure long-term survival.