The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

Overproduction of mycotoxin biosynthetic enzymes triggers Fusarium toxisome-shaped structure formation via endoplasmic reticulum remodeling.

Mycotoxin deoxynivalenol (DON) produced by the Fusarium graminearum complex is highly toxic to animal and human health. During DON synthesis, the endoplasmic reticulum (ER) of F. graminearum is intensively reorganized, from thin reticular structure to thickened spherical and crescent structure, which was referred to as "DON toxisome". However, the underlying mechanism of how the ER is reorganized into toxisome remains unknown. In this study, we discovered that overproduction of ER-localized DON biosynthetic enzyme Tri4 or Tri1, or intrinsic ER-resident membrane proteins FgHmr1 and FgCnx was sufficient to induce toxisome-shaped structure (TSS) formation under non-toxin-inducing conditions. Moreover, heterologous overexpression of Tri1 and Tri4 proteins in non-DON-producing fungi F. oxysporum f. sp. lycopersici and F. fujikuroi also led to TSS formation. In addition, we found that the high osmolarity glycerol (HOG), but not the unfolded protein response (UPR) signaling pathway was involved in the assembly of ER into TSS. By using toxisome as a biomarker, we screened and identified a novel chemical which exhibited high inhibitory activity against toxisome formation and DON biosynthesis, and inhibited Fusarium growth species-specifically. Taken together, this study demonstrated that the essence of ER remodeling into toxisome structure is a response to the overproduction of ER-localized DON biosynthetic enzymes, providing a novel pathway for management of mycotoxin contamination.

Discovery of a rapidly evolving yeast defense factor, KTD1, against the secreted killer toxin K28.

Secreted protein toxins are widely used weapons in conflicts between organisms. Elucidating how organisms genetically adapt to defend themselves against these toxins is fundamental to understanding the coevolutionary dynamics of competing organisms. Within yeast communities, "killer" toxins are secreted to kill nearby sensitive yeast, providing a fitness advantage in competitive growth environments. Natural yeast isolates vary in their sensitivity to these toxins, but to date, no polymorphic genetic factors contributing to defense have been identified. We investigated the variation in resistance to the killer toxin K28 across diverse natural isolates of the Saccharomyces cerevisiae population. Using large-scale linkage mapping, we discovered a novel defense factor, which we named KTD1. We identified many KTD1 alleles, which provided different levels of K28 resistance. KTD1 is a member of the DUP240 gene family of unknown function, which is rapidly evolving in a region spanning its two encoded transmembrane helices. We found that this domain is critical to KTD1's protective ability. Our findings implicate KTD1 as a key polymorphic factor in the defense against K28 toxin.

The Toll pathway mediates Drosophila resilience to Aspergillus mycotoxins through specific Bomanins.

Host defense against infections encompasses both resistance, which targets microorganisms for neutralization or elimination, and resilience/disease tolerance, which allows the host to withstand/tolerate pathogens and repair damages. In Drosophila, the Toll signaling pathway is thought to mediate resistance against fungal infections by regulating the secretion of antimicrobial peptides, potentially including Bomanins. We find that Aspergillus fumigatus kills Drosophila Toll pathway mutants without invasion because its dissemination is blocked by melanization, suggesting a role for Toll in host defense distinct from resistance. We report that mutants affecting the Toll pathway or the 55C Bomanin locus are susceptible to the injection of two Aspergillus mycotoxins, restrictocin and verruculogen. The vulnerability of 55C deletion mutants to these mycotoxins is rescued by the overexpression of Bomanins specific to each challenge. Mechanistically, flies in which BomS6 is expressed in the nervous system exhibit an enhanced recovery from the tremors induced by injected verruculogen and display improved survival. Thus, innate immunity also protects the host against the action of microbial toxins through secreted peptides and thereby increases its resilience to infection.

Profiling the Pyrenophora tritici-repentis secretome: The Pf2 transcription factor regulates the secretion of the effector proteins ToxA and ToxB.

The global wheat disease tan spot is caused by the necrotrophic fungal pathogen Pyrenophora tritici-repentis (Ptr) which secretes necrotrophic effectors to facilitate host plant colonization. We previously reported a role of the Zn2 Cys6 binuclear cluster transcription factor Pf2 in the regulation of the Ptr effector ToxA. Here, we show that Pf2 is also a positive regulator of ToxB, via targeted deletion of PtrPf2 which resulted in reduced ToxB expression and defects in conidiation and pathogenicity. To further investigate the function of Ptr Pf2 in regulating protein secretion, the secretome profiles of two Δptrpf2 mutants of two Ptr races (races 1 and 5) were evaluated using a SWATH-mass spectrometry (MS) quantitative approach. Analysis of the secretomes of the Δptrpf2 mutants from in vitro culture filtrate identified more than 500 secreted proteins, with 25% unique to each race. Of the identified proteins, less than 6% were significantly differentially regulated by Ptr Pf2. Among the downregulated proteins were ToxA and ToxB, specific to race 1 and race 5 respectively, demonstrating the role of Ptr Pf2 as a positive regulator of both effectors. Significant motif sequences identified in both ToxA and ToxB putative promoter regions were further explored via GFP reporter assays.

Role of ferroptosis in food-borne mycotoxin-induced toxicities.

Contamination by toxic substances is a major global food safety issue, which poses a serious threat to human health. Mycotoxins are major class of food contaminants, mainly including aflatoxins (AFs), zearalenone (ZON), deoxynivalenol (DON), ochratoxin A (OTA), fumonisins (FBs) and patulin (PAT). Ferroptosis is a newly identified iron-dependent form of programmed or regulated cell death, which has been found to be involved in diverse pathological conditions. Recently, a growing body of evidence has shown that ferroptosis is implicated in the toxicities induced by certain types of food-borne mycotoxins, which provides novel mechanistic insights into mycotoxin-induced toxicities and paves the way for developing ferroptosis-based strategy to combat against toxicities of mycotoxins. In this review article, we summarize the key findings on the involvement of ferroptosis in mycotoxin-induced toxicities and propose issues that need to be addressed in future studies for better utilization of ferroptosis-based approach to manage the toxic effects of mycotoxin contamination.

A Miniaturized and Highly Sensitive "Windmill" Three-Channel Fluorescence Detector for Simultaneous Detection of Various Mycotoxins.

A novel "windmill" three-channel light-emitting diode induced fluorescence detector (LED-IF) was proposed to maximize the excitation efficiency and fluorescence collection efficiency. Compared with the typical collinear arrangement, the fluorescence intensity of the three channels was increased by 7.85, 3.88, and 2.94 times, respectively. The compact shaping optical path was designed to obtain higher excitation efficiency and a lower background stray light effect caused by high divergence angle high-power ultraviolet (UV)-LEDs simultaneously, which increased the sensitivity of three channels by 4.6 to 5.7 times. It was found that using a photodiode (PD) with a flat window and a larger photosensitive surface can collect the Lambertian emission fluorescence in the flow cell more efficiently, increasing the signal-to-noise ratio of each channel 1.3 to 1.8 times. The limits of detection (LODs, 3 times peak-peak noise) of aflatoxin B2 (AFB2), ochratoxin (OTA), and zearalenone (ZEN) were 0.33, 1.80, and 28.2 ng/L, respectively. Finally, six mycotoxins were analyzed simultaneously by the detector coupling with HPLC. The results showed that the sensitivity of the detector was at the best level to date, which was better than that of the top commercial fluorescence detectors (FLDs). The developed detector has the advantages of having small volume, low cost, and long lifetime and being robust, which has wide application and market prospects.

Ultrasensitive Imaging Assay of Multiple Mycotoxins Using Cobalt DNA-Inorganic Hybrid Superstructure with High Chemiluminescence Catalytic Property.

A multiplex assay of mycotoxins in food and medicine is urgently needed and challenging due to synergistic hazards of trace mycotoxins and a lack of sensitive and user-friendly detection approaches. Herein, a cobalt DNA-inorganic hybrid superstructure (Co@DS) was developed through isothermal rolling circle amplification (RCA) for an ultrasensitive chemiluminescence (CL) imaging assay of multiple mycotoxins. Cobalt ions were enriched in the RCA product, endowing the Co@DS with a high CL catalytic property. Experimental studies elucidated the formation and CL catalytic mechanism of Co@DS. Co@DS was facilely integrated with biotinylated DNA to function as a universal platform and combined with a disposable immunosensor array chip. After a competitive immunoassay and biotin-avidin recognition, the CL signals of luminol and hydrogen peroxide, catalyzed by Co@DS captured on each testing zone of the array chip, were imaged simultaneously. Target mycotoxins can be quantitated by CL intensities. To validate the concept, the CL imaging approach was employed for joint determination of aflatoxin B1, ochratoxins A, and zearalenone. Under optimal conditions, it showed advantages including simple sample pretreatment, acceptable throughput, high accuracy, minimal sample consumption, broad linear ranges, and detection limits as low as 0.75, 0.62, and 0.61 pg mL-1, respectively. Furthermore, the approach was applied in analyzing real coix seed samples, showcasing excellent performance in effectively distinguishing qualified and contaminated medicine, revealing the great potential in managing the complex issue of mycotoxins cocontamination in food and medicine.

Highly Sensitive Microarray Immunoassay for Multiple Mycotoxins on Engineered 3D Porous Silicon SERS Substrate with Silver Nanoparticle Magnetron Sputtering.

A high-throughput, rapid, and highly sensitive surface-enhanced Raman spectroscopy (SERS) microarray for screening multiple mycotoxins has been developed on a three-dimensional silver nanoparticle porous silicon (3D AgNP-Psi) SERS substrate, which was easy to be engineered by electrochemical etching and magnetron sputtering technology. The etching current density, etching waveform, and target material for magnetron sputtering have been investigated to obtain an optimal 3D SERS substrate. The optimized 3D AgNP-Psi SERS substrate showed an enhancement factor of 2.3 × 107 at 400 mA/cm2 constant current density etching for 20 s and Ag target magnetron sputtering for 200 nm thickness on the surface of Psi. The simulation electric field distribution showed the near-field enhancement can reach 3× higher than that of AuNPs. A protein microarray has been designed to screen multiple mycotoxins by AuNP Raman tags and a competitive immunoassay protocol on the surface of the 3D SERS substrate. The SERS protein microarray displayed wide linear detection ranges of 0.001-100 ng/mL for ochratoxin A, 0.01-100 ng/mL for aflatoxin B1, 0.001-10 ng/mL for deoxynivalenol, along with pg/mL low limit of detection, good recovery rates, repeatability, and reproducibility. The 3D SERS protein microarray is easily engineered and has a great potential application in medicine, environment, and food industry fields.

Photonic Microbead Array Digital Time-Resolved Fluorescence Ultrasensitive Platform for Simultaneous Detection of Multiple Mycotoxins.

Limitations in the sensitivity, linear detection range, and cross-reaction of lateral flow immunoassays mainly hamper their application in rapid screening for multiple targets. In this work, we designed a new time-resolved fluorescence immunoassay (TRFIA) platform to overcome these limitations. This platform uses europium chelate polystyrene (PS@Eu) nanoparticles conjugated with monoclonal antibodies to sense multiple mycotoxins. We employed a competitive TRFIA protocol in which the conjugated PS@Eu was used on the surfaces of photonic microbead arrays (PMAs). The TRFIA signal of PMAs on the pad was recorded with the digital time-resolved fluorescence reader. The developed TRFIA shows wide detection linear ranges (0.01-1000 ng/mL for DON, 0.1-100 ng/mL for OTA, and 0.01-100 ng/mL for AFB1), low limits of detection (LODs) (7.9 pg/mL for DON, 18 pg/mL for OTA, and 7.7 pg/mL for AFB1), good specificity, good recovery ratios (76.68-117.26%), and good reproducibility in grain samples. The simulated fluorescence enhancement effect of PMA indicated that the electric field distribution on the surface of PS@Eu on PMA is twice higher than that on the surface of PS@Eu. The new TRFIA for three kinds of mycotoxins was 1000-fold more sensitive than the classical TRFIA, and it has great potential application in rapid screening for multiple targets.

A comparison between the role of enniatins and deoxynivalenol in Fusarium virulence on different tissues of common wheat.

BACKGROUND: Fusarium graminearum and Fusarium avenaceum are two of the most important causal agents of Fusarium head blight (FHB) of wheat. They can produce mycotoxins that accumulate in infected wheat heads, including deoxynivalenol (DON) and enniatins (ENNs), produced by F. graminearum and F. avenaceum, respectively. While the role of DON as a virulence factor in F. graminearum toward wheat is well known, ENNs in F. avenaceum has been poorly explored. Results obtained to-date indicate that ENNs may confer an advantage to F. avenaceum only on particular hosts. RESULTS: In this study, with the use of ENN-producing and ENN non-producing F. avenaceum strains, the role of ENNs on F. avenaceum virulence was investigated on the root, stem base and head of common wheat, and compared with the role of DON, using DON-producing and DON non-producing F. graminearum strains. The DON-producing F. graminearum strain showed a significantly higher ability to cause symptoms and colonise each of the tested tissues than the non-producing strain. On the other hand, the ability to produce ENNs increased initial symptoms of the disease and fungal biomass accumulation, measured by qPCR, only in wheat heads, and not in roots or stem bases. LC-MS/MS analysis was used to confirm the presence of ENNs and DON in the different strains, and results, both in vitro and in wheat heads, were consistent with the genetics of each strain. CONCLUSION: While the key role of DON on F. graminearum virulence towards three different wheat tissues was noticeable, ENNs seemed to have a role only in influencing F. avenaceum virulence on common wheat heads probably due to an initial delay in the appearance of symptoms.

Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Expression of endogenous UDP-glucosyltransferase in endophyte Phomopsis liquidambaris reduces deoxynivalenol contamination in wheat.

Fusarium head blight is a devastating disease that causes severe yield loses and mycotoxin contamination in wheat grain. Additionally, balancing the trade-off between wheat production and disease resistance has proved challenging. This study aimed to expand the genetic tools of the endophyte Phomopsis liquidambaris against Fusarium graminearum. Specifically, we engineered a UDP-glucosyltransferase-expressing P. liquidambaris strain (PL-UGT) using ADE1 as a selection marker and obtained a deletion mutant using an inducible promoter that drives Cas9 expression. Our PL-UGT strain converted deoxynivalenol (DON) into DON-3-G in vitro at a rate of 71.4 % after 36 h. DON inactivation can be used to confer tolerance in planta. Wheat seedlings inoculated with endophytic strain PL-UGT showed improved growth compared with those inoculated with wildtype P. liquidambaris. Strain PL-UGT inhibited the growth of Fusarium graminearum and reduced infection rate to 15.7 %. Consistent with this finding, DON levels in wheat grains decreased from 14.25 to 0.56 µg/g when the flowers were pre-inoculated with PL-UGT and then infected with F. graminearum. The expression of UGT in P. liquidambaris was nontoxic and did not inhibit plant growth. Endophytes do not enter the seeds nor induce plant disease, thereby representing a novel approach to fungal disease control.

Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

Mycobiota contaminating some market cake samples with reference to their toxin and enzyme.

Fungi can spoil the majority of baked products. Spoilage of cake during storage is commonly associated with fungi. Therefore, this study aimed to assess the quality of different types of cakes sold in the market. The most predominant fungal genera in the tested cake samples (14 samples) were Aspergillus spp., and Penicillium spp. On Potato Dextrose Agar (PDA), the medium fungal total count was 43.3 colonies /g. Aspergillus was the most dominant genus and was isolated from six samples of cake. Aspergillus was represented by 3 species namely, A. flavus, A. niger, and A. nidulans, represented by 13.32, 19.99, and 3.33 colonies /g respectively. On Malt Extract Agar (MEA) Medium, the fungal total count was 123.24 colonies / g. Aspergillus was the most dominant isolated genus from 11 samples of cake and was represented by 5 species, namely, A. flavus, A. niger, A. ochraceous, A. terreus, and A. versicolor (26. 65, 63.29, 3.33, 6.66, and 3.33 colonies / g , respectively). Twenty-four isolates (88.88 %) of the total tested twenty-seven filamentous fungi showed positive results for amylase production. Ten isolates (37.03%) of the total tested filamentous fungi showed positive results for lipase production, and finally eleven isolates (40.74 %) of the total fungal isolates showed positive results for protease production. Aflatoxins B1, B2, G1, G2, and ochratoxin A were not detected in fourteen collected samples of cake. In this study, clove oil was the best choice overpeppermint oil and olive oil for preventing mold development when natural agents were compared. It might be due to the presence of a varietyof bioactive chemical compounds in clove oil, whose major bioactive component is eugenol, which acts as an antifungal reagent. Therefore, freshly baked cake should be consumed within afew days to avoid individuals experiencing foodborne illnesses.

The Velvet transcription factor PnVeA regulates necrotrophic effectors and secondary metabolism in the wheat pathogen Parastagonospora nodorum.

The fungus Parastagonospora nodorum causes septoria nodorum blotch on wheat. The role of the fungal Velvet-family transcription factor VeA in P. nodorum development and virulence was investigated here. Deletion of the P. nodorum VeA ortholog, PnVeA, resulted in growth abnormalities including pigmentation, abolished asexual sporulation and highly reduced virulence on wheat. Comparative RNA-Seq and RT-PCR analyses revealed that the deletion of PnVeA also decoupled the expression of major necrotrophic effector genes. In addition, the deletion of PnVeA resulted in an up-regulation of four predicted secondary metabolite (SM) gene clusters. Using liquid-chromatography mass-spectrometry, it was observed that one of the SM gene clusters led to an accumulation of the mycotoxin alternariol. PnVeA is essential for asexual sporulation, full virulence, secondary metabolism and necrotrophic effector regulation.

Profiling of deubiquitinases that control virulence in the pathogenic plant fungus Fusarium graminearum.

Eukaryotes have evolved sophisticated post-translational modifications to regulate protein function and numerous biological processes, including ubiquitination controlled by the coordinated action of ubiquitin-conjugating enzymes and deubiquitinating enzymes (Dubs). However, the function of deubiquitination in pathogenic fungi is largely unknown. Here, the distribution of Dubs in the fungal kingdom was surveyed and their functions were systematically characterized using the phytopathogen Fusarium graminearum as the model species, which causes devastating diseases of all cereal species world-wide. Our findings demonstrate that Dubs are critical for fungal development and virulence, especially the ubiquitin-specific protease 15 (Ubp15). Global ubiquitome analysis and subsequent experiments identified three important substrates of Ubp15, including the autophagy-related protein Atg8, the mitogen-activated protein kinase Gpmk1, and the mycotoxin deoxynivalenol (DON) biosynthetic protein Tri4. Ubp15 regulates the deubiquitination of the Atg8, thereby impacting its subcellular localization and the autophagy process. Moreover, Ubp15 also modulates the deubiquitination of Gpmk1 and Tri4. This modulation subsequently influences their protein stabilities and further affects the formation of penetration structures and the biosynthetic process of DON, respectively. Collectively, our findings reveal a previously unknown regulatory pathway of a deubiquitinating enzyme for fungal virulence and highlight the potential of Ubp15 as a target for combating fungal diseases.

Nursery pigs fed with feed contaminated by aflatoxin B1 (Aspergillus flavus) and anti-mycotoxin blend: Pathogenesis and negative impact on animal health and weight gain.

The present study aimed to evaluate whether a moderate dose of aflatoxin B1 in pigs' diet interferes with pigs' growth and health in the nursery phase and whether an anti-mycotoxin mixture minimizes the adverse effects of the toxin. One blend with Saccharomyces cerevisiae lysate, zeolite, silicon dioxide, propylene glycol, Carduus marianus extract, soy lecithin, and carbonate was used as an anti-mycotoxin. Four treatments, with six repetitions per treatment and three pigs/pen: Afla0-AntiMyc0 - negative control (without aflatoxin); Afla500-AntiMyc0 - positive control (500 ppb of aflatoxin); Afla0-AntiMyc1000 - 1000 mg/kg of anti-mycotoxin blend; Afla500-AntiMyc1000 - 500 ppb aflatoxin +1000 mg/kg of anti-mycotoxin blend. It was observed that pigs in the positive control (Afla500-AntiMyc0) had lower body weight and weight gain when compared to the other treatments during the experimental period. Also, pigs from Afla500-AntiMyc0 had lower feed intake between days 1-20 and 1 to 30 than Afla0-AntiMyc0. The pigs from Afla500-AntiMyc0 had higher levels of liver enzymes aspartate aminotransferase and alanine aminotransferase compared to other treatments. The pigs from Afla500-AntiMyc0 had higher villus height than the other treatments, while the folded size was smaller in this treatment. Crypts were deeper in the intestines of pigs in both treatments that consumed aflatoxin. In general, it is concluded that the intake of aflatoxin B1 by nursery pigs has negative impacts on the health and, consequently, the animals' growth performance; however, the addition of the contaminated feed with an anti-mycotoxin blend was able to protect the pigs, minimizing the adverse effects caused by the mycotoxin.

Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.