Fungal Pathways Research Articles

Abstract 1019 Discovery of New Structures, Enzymes and Functions from Fungal Biosynthetic Pathways

Abstract 1019 Discovery of New Structures, Enzymes and Functions from Fungal Biosynthetic Pathways

MicroRNA cross-talk between Monilinia fungal pathogens and peach host

The reciprocal targeting of microRNAs (miRNA) and micro-like-RNAs (milRNA) between hosts and pathogens is critical for understanding their interactions. In this study, reciprocal miRNA targets were explored in two Monilinia fungal pathogens, M. fructicola & M. laxa, and their peach host (Prunus presica). Using in silico analysis, 355 and 266 putative miRNAs were predicted for M. fructicola and M. laxa, respectively. Number of miRNAs and their targets differed based on host and pathogen species as 209 M. fructicola miRNAs target 98 peach genes and 128 M. laxa miRNAs target 338 peach genes. On the other hand, peach miRNAs showed the species-specific responses targeting fungal pathways to struggle with its pathogens. These findings indicate distinct strategies and species-specific interactions in this pathosystem. Besides, through the in vitro experimental designs, 166 and 124 expressed miRNAs by M. fructicola were detected in the host-mimicked and control environments, respectively. Additionally, novel miRNAs were discovered, six of which were in the mimicked environment and the seven in the controlled environment as highlighting dynamic and specialized miRNA expression in M. fructicola depending on the environmental conditions. In conclusion, this study provides the first insights into miRNA-mediated interactions between M. fructicola, M. laxa, and peach hosts. Unrevealing the cross talk through the miRNAs in host–pathogen interactions enhances the understanding of pathogenesis and host defense mechanisms. These findings have implications for disease management strategies and contribute to the fields of basic science and evolutionary biology.

Mapping microhabitats of lignocellulose decomposition by a microbial consortium.

The leaf-cutter ant fungal garden ecosystem is a naturally evolved model system for efficient plant biomass degradation. Degradation processes mediated by the symbiotic fungus Leucoagaricus gongylophorus are difficult to characterize due to dynamic metabolisms and spatial complexity of the system. Herein, we performed microscale imaging across 12-µm-thick adjacent sections of Atta cephalotes fungal gardens and applied a metabolome-informed proteome imaging approach to map lignin degradation. This approach combines two spatial multiomics mass spectrometry modalities that enabled us to visualize colocalized metabolites and proteins across and through the fungal garden. Spatially profiled metabolites revealed an accumulation of lignin-related products, outlining morphologically unique lignin microhabitats. Metaproteomic analyses of these microhabitats revealed carbohydrate-degrading enzymes, indicating a prominent fungal role in lignocellulose decomposition. Integration of metabolome-informed proteome imaging data provides a comprehensive view of underlying biological pathways to inform our understanding of metabolic fungal pathways in plant matter degradation within the micrometer-scale environment.

Fungal luminescence pathways: research and applications

The fungal bioluminescence pathway (FBP) is a metabolic pathway responsible for the generation of bioluminescence derived from fungi. This pathway utilizes caffeic acid as the substrate, generating a high-energy intermediate, and the decomposition of which yields green fluorescence with a wavelength of approximately 520 nm. The FBP is evolutionally conserved in luminescent fungal groups. Unlike other bioluminescent systems, the FBP is particularly suitable for engineering applications in eukaryotic organisms, especially in plants. Currently, metabolically engineered luminescent plants are able to emit visible light to illuminate its surroundings, which can be visualized clearly in the dark. The fungal bioluminescent system could be explored in various applications in molecular biology, biosensors and glowing ornamental plants, and even green lighting along city streets.

β-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal β-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface β-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a β-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble β-1,3-glucan substantially inhibited this adhesion, indicating the involvement of β-1,3-glucan recognition. Biotinylated β-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the β-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding β-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to β-1,3-glucans. These findings underscore the complexity of binding via β-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of β-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for β-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Predicting fungal secondary metabolite activity from biosynthetic gene cluster data using machine learning.

Fungal secondary metabolites (SMs) contribute to the diversity of fungal ecological communities, niches, and lifestyles. Many fungal SMs have one or more medically and industrially important activities (e.g., antifungal, antibacterial, and antitumor). The genes necessary for fungal SM biosynthesis are typically located right next to each other in the genome and are known as biosynthetic gene clusters (BGCs). However, whether fungal SM bioactivity can be predicted from specific attributes of genes in BGCs remains an open question. We adapted machine learning models that predicted SM bioactivity from bacterial BGC data with accuracies as high as 80% to fungal BGC data. We trained our models to predict the antibacterial, antifungal, and cytotoxic/antitumor bioactivity of fungal SMs on two data sets: (i) fungal BGCs (data set comprised of 314 BGCs) and (ii) fungal (314 BGCs) and bacterial BGCs (1,003 BGCs). We found that models trained on fungal BGCs had balanced accuracies between 51% and 68%, whereas training on bacterial and fungal BGCs had balanced accuracies between 56% and 68%. The low prediction accuracy of fungal SM bioactivities likely stems from the small size of the data set; this lack of data, coupled with our finding that including bacterial BGC data in the training data did not substantially change accuracies currently limits the application of machine learning approaches to fungal SM studies. With >15,000 characterized fungal SMs, millions of putative BGCs in fungal genomes, and increased demand for novel drugs, efforts that systematically link fungal SM bioactivity to BGCs are urgently needed.IMPORTANCEFungi are key sources of natural products and iconic drugs, including penicillin and statins. DNA sequencing has revealed that there are likely millions of biosynthetic pathways in fungal genomes, but the chemical structures and bioactivities of >99% of natural products produced by these pathways remain unknown. We used artificial intelligence to predict the bioactivities of diverse fungal biosynthetic pathways. We found that the accuracies of our predictions were generally low, between 51% and 68%, likely because the natural products and bioactivities of only very few fungal pathways are known. With >15,000 characterized fungal natural products, millions of putative biosynthetic pathways present in fungal genomes, and increased demand for novel drugs, our study suggests that there is an urgent need for efforts that systematically identify fungal biosynthetic pathways, their natural products, and their bioactivities.

Double-Stranded RNA Targeting White Mold Sclerotinia sclerotiorum Argonaute 2 for Disease Control via Spray-Induced Gene Silencing.

Sclerotinia sclerotiorum, the causal agent of white mold infection, is a cosmopolitan fungal pathogen that causes major yield losses in many economically important crops. Spray-induced gene silencing has recently been shown to be a promising alternative method for controlling plant diseases. Based on our prior research, we focused on developing a spray-induced gene silencing approach to control white mold by silencing S. sclerotiorum argonaute 2 (SsAgo2), a crucial part of the fungal small RNA pathway. We compared the lesion size as a result of targeting each ∼500-bp segment of SsAgo2 from the 5' to the 3' end and found that targeting the PIWI/RNaseH domain of SsAgo2 is most effective. External application of double-stranded RNA (dsRNA)-suppressed white mold infection using either in vitro or in vivo transcripts was determined at the rate of 800 ng/0.2 cm2 area with a downregulation of SsAgo2 from infected leaf tissue confirmed by RT-qPCR. Furthermore, magnesium/iron-layered double hydroxide nanosheets loaded with in vitro- and in vivo-transcribed dsRNA segments significantly reduced the rate of S. sclerotiorum lesion expansion. In vivo-produced dsRNA targeting the PIWI/RNaseH domain of the SsAgo2 transcript showed increased efficacy in reducing the white mold symptoms of S. sclerotiorum when combined with layered double hydroxide nanosheets. This approach is promising to produce a large scale of dsRNA that can be deployed as an environmentally friendly fungicide to manage white mold infections in the field.

Investigation of the fermentation filtrate from soapberry (Sapindus mukorossi Gaertn.) pericarp on improving the microbial diversity and composition of the human scalp.

Microorganisms as a component of scalp ecosystem play a vital role in human scalp health. Soapberry pericarp is employed in improving scalp health, and its content of active ingredients could be enhanced resulted in fermentation. This study aims to investigate the effect of the fermentation filtrate from soapberry pericarp on the diversity of scalp microorganisms. The components in fermentation filtrate from soapberry pericarp were analyzed by HPLC-Q-Orbitrap HRMS, and 16S and ITS sequences of 198 samples from three different used stages (Day 0, Day 7, Day 28) were sequenced using the Illumina Novaseq platform. Microbial diversity was assessed using alpha diversity (Chao1 and Shannon indexes) and beta diversity (weighted unifrac and unweighted unifrac). Principal coordinate analysis (PCoA) and linear discriminant analysis (LDA) effect size analysis (LEfSe) were used to visualize microbial variation among different stages. There were 22 components were identified in the fermentation filtrate from soapberry pericarp by HPLC-Q-Orbitrap HRMS. The alpha-diversity and beta-diversity analyses showed that scalp microbial diversity and composition were influenced by the fermentation filtrate of soapberry pericarp. Based on functional analysis, this study found an enrichment of healthy scalp-related bacterial pathways, such as amino acid, nucleoside, and nucleotide biosynthesis, while a decrease in fungal pathogenesis pathways, specifically saprotroph and symbiotroph pathways, was observed. The study described about the complex community dynamics of human scalp microorganisms during the stages of using the fermentation filtrate from soapberry pericarp. This result will help rationally utilize the fermentation filtrate from soapberry pericarp to keep or improve human scalp health.

Beach sand mycobiome: The silent threat of pathogenic fungi and toxic metal contamination for beachgoers

Emphasis is always placed on bacterial but not fungal pathogens in marine environments. We analysed the fungal diversity, functional predictions, and toxic metals and metalloids contamination in beach sand from different South African locations. Results revealed a diverse fungal community, with Ascomycota, Rozellomycota, and Basidiomycota being the dominant phyla. Functional predictions highlighted fungal metabolic pathways related to of carbohydrates, amino acids, and lipids, in different beach samples. Elevated concentrations of toxic metals and metalloids were detected in Central and Harbour beach sands, likely due to anthropogenic activities. Correlations among different elements were observed, suggesting complex interactions in the coastal environment. Fungal pathogens like Cladosporium, Fusarium, Aspergillus, and Candida in beach sands raise potential public health risk concerns. Therefore, monitoring fungal diversity (including pathogens) alongside bacterial contamination in beach environments is imperative. The results contribute to understanding fungal community dynamics, functional potential, toxic metal and metalloid contamination, and potential risks associated with beach sand ecosystems.

Antifungal activity and mechanism of chitosan against Fusarium solani caused ginger soft rot during postharvest storage

Postharvest rot caused by Fusarium solani (F. solani) results in enormous economic loss to the global vegetables industry. Chitosan has been shown to be effective in control postharvest diseases of vegetables, but the specific mechanism of chitosan on F. solani has not been studied. In this study, we evaluated antifungal activity of chitosan against F. solani caused soft rot in vitro and in vivo. Antifungal assay results showed that chitosan significantly inhibited the mycelial growth and spore germination of F. solani. Simultaneously, chitosan showed disruptive activity on the mycelial morphology and cell membrane, which were achieved by decreasing intracellular sugars, proteins and lipids content. Furthermore, chitosan treatment significantly decreased tricarboxylic acid (TCA)-related enzymes activity, reducing the number of metabolites in the TCA cycle. UPLC-Triple-TOF/MS-based metabolomics analysis showed that chitosan interferes with fungal metabolic pathways, mainly related to energy metabolism, lipid metabolism and sugar metabolism. Exogenous α-D-glucose and linoleic acid treatment promoted mycelia growth, verifying the sugar catabolism and TCA cycle was involved in the inhibition of chitosan against F. solani. The in vivo study showed that chitosan treatment effectively reduced disease rate and severity of postharvest soft rot through activating the biochemical defense response and modulating the expression of phytopathogenic responses LysM genes. These results contribute to explore the underlying mechanisms of chitosan against F. solani.

Network Pharmacology, Molecular Docking, and Molecular Dynamics Simulation to Elucidate the Molecular Targets and Potential Mechanism of Phoenix dactylifera (Ajwa Dates) against Candidiasis.

Candidiasis, caused by opportunistic fungal pathogens of the Candida genus, poses a significant threat to immunocompromised individuals. Natural compounds derived from medicinal plants have gained attention as potential sources of anti-fungal agents. Ajwa dates (Phoenix dactylifera L.) have been recognized for their diverse phytochemical composition and therapeutic potential. In this study, we employed a multi-faceted approach to explore the anti-candidiasis potential of Ajwa dates' phytochemicals. Utilizing network pharmacology, we constructed an interaction network to elucidate the intricate relationships between Ajwa dates phytoconstituents and the Candida-associated molecular targets of humans. Our analysis revealed key nodes in the network (STAT3, IL-2, PTPRC, STAT1, CASP1, ALB, TP53, TLR4, TNF and PPARG), suggesting the potential modulation of several crucial processes (the regulation of the response to a cytokine stimulus, regulation of the inflammatory response, positive regulation of cytokine production, cellular response to external stimulus, etc.) and fungal pathways (Th17 cell differentiation, the Toll-like receptor signaling pathway, the C-type lectin receptor signaling pathway and necroptosis). To validate these findings, molecular docking studies were conducted, revealing the binding affinities of the phytochemicals towards selected Candida protein targets of humans (ALB-rutin (-9.7 kJ/mol), STAT1-rutin (-9.2 kJ/mol), STAT3-isoquercetin (-8.7 kJ/mol), IL2-β-carotene (-8.5 kJ/mol), CASP1-β-carotene (-8.2 kJ/mol), TP53-isoquercetin (-8.8 kJ/mol), PPARG-luteolin (-8.3 kJ/mol), TNF-βcarotene (-7.7 kJ/mol), TLR4-rutin (-7.4 kJ/mol) and PTPRC-rutin (-7.0 kJ/mol)). Furthermore, molecular dynamics simulations of rutin-ALB and rutin-STAT1 complex were performed to gain insights into the stability and dynamics of the identified ligand-target complexes over time. Overall, the results not only contribute to the understanding of the molecular interactions underlying the anti-fungal potential of specific phytochemicals of Ajwa dates in humans but also provide a rational basis for the development of novel therapeutic strategies against candidiasis in humans. This study underscores the significance of network pharmacology, molecular docking and dynamics simulations in accelerating the discovery of natural products as effective anti-fungal agents. However, further experimental validation of the identified compounds is warranted to translate these findings into practical therapeutic applications.

Litter from Native Shrubs in the Senegal River Valley Induces Modifications in Nematode Communities and Enzyme Activities in Cultivated Sandy Soils

ABSTRACT To reduce plant-parasitic nematodes (PPNs), an alternative solution based on amendments from selected native shrub was used to favor mainly the increase of free-living nematodes (FLNs) that include high trophic (bacterivorous, fungivorous, omnivorous, and carnivorous) nematodes and influence soil microbial activities. The consequences of the subsequent reduce of PPNs of the soil were also determined. Experiments were performed in a shade house using six treatments consisting of shrub residues from Piliostigma reticulatum (Pil), Boscia senegalensis (Bos), and Crotalaria juncea (Cro); a Straw-based Compost (Cs); a Farmer practice (FP) using Nitrogen-Phosphorus-Potassium (NPK) fertilizer; and a control (C0, no amendments). Abundances of nematodes, trophic groups, and nematodes ecological indices were determined. Enzymatic activities (ß-glucosidase and acid phosphatase) and the fluorescein diacetate hydrolysis (FDA) were also determined. Soil amended by Bos and Cro increased the density of nematodes compared to control soil or amended with NPK fertilizer. Indices of taxonomic diversity (H') did not differ significantly between the treatments. However, soils amended with Bos and Cro had the highest enrichment index (EI), and the lowest Nematode Channel Ratio (NCR), indicating that these two shrub residues exhibited a higher food web in response to the available resources provided by the fungal decomposition pathway. Moreover, Bos favored omnivorous and increased enzymatic activities. Bos induce ‘bottom-up’ while Cro induce ‘top-down’ controls on nematode populations.

Transcriptomics and phytohormone metabolomics provide comprehensive insights into the response mechanism of tea against blister blight disease

Blister blight caused by obligate biotrophic pathogen Exobasidium vexans is a prevalent foliar disease in tea plants that results in significant losses in both yield and quality. To further understand the response mechanism of tea plants against E. vexans infection, transcriptomics and targeted hormone metabonomics were performed to investigate the gene expression and phytohormone signaling networks. Transcriptome analysis revealed that the most of differentially expressed genes (DEGs) were highly enriched in the pathways of “photosynthesis-antenna proteins”, “plant hormone signal transduction”, “plant-pathogen interaction”, “MAPK signaling pathway-plant”, and “phenylpropanoid biosynthesis”. The majority of disease resistance (R) genes (e.g., CDPK, PIK1, and PRS4) were activated in response to pathogen invasion in the fungal PAMP related pathway. The activation of infection-induced genes corresponds to transcription factors such as WRKY, MYB, bHLH and AP2/ERF. SA acted as a crucial role in defense activation of tea immunity against E. vexans infection, which substances (SA and SAG) were positively correlated with the expression levels of disease-resistant genes (NPR1, TGA, and PR1) in the SA signaling pathway. The biosynthesis of defense-associated hormone metabolites such as JA, ETH, and BRs might be involved in associated with the defense response of tea plants to E. vexans infection. The correlation analysis of DEGs and hormone metabolites exhibited that JA and JA-ILE were positively regulated by the DEGs (e.g., BSK, ERF1/2, ETR, SAUR, and DELLA), suggesting JA- and BR-, ETH-, AUX-signaling might synergistically activate the expression of some defense-related genes. This finding was further validated through quantitative real-time PCR, which displayed the involvement of defense-related gene expressions in the response of tea plant to the infection caused by E. vexans. Collectively, this study provides comprehensive insights into the response mechanism of tea defense against blister blight disease to act as a valuable resource for understanding plant immunity against diverse pathogen infection and other biotic stresses in tea plants.

A quantitative autonomous bioluminescence reporter system with a wide dynamic range for Plant Synthetic Biology.

Plant Synthetic Biology aims to enhance the capacities of plants by designing and integrating synthetic gene circuits (SGCs). Quantitative reporting solutions that can produce quick, rich datasets affordably are necessary for SGC optimization. In this paper, we present a new, low-cost, and high-throughput reporter system for the quantitative measurement of gene expression in plants based on autonomous bioluminescence. This method eliminates the need for an exogenous supply of luciferase substrate by exploiting the entire Neonothopanus nambi fungal bioluminescence cyclic pathway to build a self-sustained reporter. The HispS gene, the pathway's limiting step, was set up as the reporter's transcriptional entry point as part of the new system's design, which significantly improved the output's dynamic range and brought it on par with that of the gold standard FLuc/RLuc reporter. Additionally, transient ratiometric measurements in N. benthamiana were made possible by the addition of an enhanced GFP as a normalizer. The performance of new NeoLuc/eGFP system was extensively validated with SGCs previously described, including phytohormone and optogenetic sensors. Furthermore, we employed NeoLuc/eGFP in the optimization of challenging SGCs, including new configurations for an agrochemical (copper) switch, a new blue optogenetic sensor, and a dual copper/red-light switch for tight regulation of metabolic pathways.

Virulence Factors of the Fungal Pathogen Stagonospora nodorum Manipulate Hormonal Signaling Pathways in Triticum aestivum L. by Regulating Host Plant MicroRNA Expressions.

Currently, the role of microRNAs in plant immune responses is being actively studied. Thus, our aim was to research the effect of Stagonospora nodorum (Berk.) NEs SnToxA and SnTox3 on the expression of miRNAs involved in the wheat-S. nodorum interaction and to determine the role of phytohormones in this process. The expressions of nine conserved microRNAs were studied by quantitative real-time polymerase chain reaction in three different wheat genotypes of bread spring wheat (Triticum aestivum L.) infected with S. nodorum. Phytohormone treatments (trans-zeatin, 2-chloroethylphosphonic acid (etefone is the chemical precursor of ethylene), and salicylic acid) were applied. The results were compared with disease symptoms, the redox status of plants, and the expression of fungal necrotrophic effector (NE) genes of SnToxA and SnTox3 and genes of SnPf2, SnStuA, alongside SnCon7 transcription factors (TFs). Salicylic acid (SA) and cytokinins (CK) are involved in the development of defense reactions in wheat plants against S. nodorum, by regulating the expression of fungal NEs and TFs genes, inducing an oxidative burst in all three wheat genotypes. Moreover, ethylene enhanced the virulence of the pathogen by increasing the expression of fungal NE and TF genes, thereby resulting in a decrease in the generation of reactive oxygen species in all three cultivars. The nine miRNAs played a role in the development of wheat resistance against S. nodorum. NE SnTox3 mainly suppressed the expression of three miRNAs: miR159, miR393, and miR408, while NE SnToxA suppressed miR166 expression. Conversely, treatment with CK and SA increased the expression of miR159 and miR408; treatment with CK increased the expression of miR393 and miR166. Ethylene inhibited the expression of miR159, miR408, miR393, and miR166. Suppression of miP159 expression by NE SnTox3 was most likely associated with the activation of the ethylene signaling pathway. NEs SnToxA and SnTox3 suppressed the expression of miR408, whose role most likely consisted of inhibiting the catalase activity, via SA and CK regulation. In addition, NE SnToxA hijacked the SA signaling pathway and manipulated it for fungal growth and development. Fungal TFs SnPf2 and SnStuA could be involved in the regulation of these processes indirectly through the regulation of the expression of NE genes. The results of this work show, for the first time, the role of microRNAs in the development of wheat resistance against S. nodorum and the effect of S. nodorum NEs SnToxA and SnTox3 on the activity of plant microRNAs.

Identification of theenantiomeric nature of 2-keto-3-deoxy-galactonate in the catabolic pathway of3,6-anhydro-L-galactose.

A novel metabolic pathway of 3,6-anhydro-L-galactose (L-AHG), the main sugar component in red macroalgae, was first discovered in the marine bacterium Vibrio sp. EJY3. L-AHG is converted to 2-keto-3-deoxy-galactonate (KDGal) in two metabolic steps. Here, we identified the enantiomeric nature of KDGal in the L-AHG catabolic pathway via stereospecific enzymatic reactions accompanying the biosynthesis of enantiopure L-KDGal and D-KDGal. Enantiopure L-KDGal and D-KDGal were synthesized by enzymatic reactions derived from the fungal galacturonate and bacterial oxidative galactose pathways, respectively. KDGal, which is involved in the L-AHG pathway, was also prepared. The results obtained from the reactions with an L-KDGal aldolase, specifically acting on L-KDGal, showed that KDGal in the L-AHG pathway exists in an L-enantiomeric form. Notably, we demonstrated the utilization of L-KDGal by Escherichia coli for the first time. E. coli cannot utilize L-KDGal as the sole carbon source. However, when a mixture of L-KDGal and D-galacturonate was used, E. coli utilized both. Our study suggests a stereoselective method to determine the absolute configuration of a compound. In addition, our results can be used to explore the novel L-KDGal catabolic pathway in E. coli and to construct an engineered microbial platform that assimilates L-AHG or L-KDGal as substrates. KEY POINTS: • Stereospecific enzyme reactions were used to identify enantiomeric nature of KDGal • KDGal in the L-AHG catabolic pathway exists in anL-enantiomeric form • E. coli can utilize L-KDGal as a carbon source when supplied with D-galacturonate.

Cis-Decalin-containing tetramic acids as inhibitors of insect steroidogenic glutathione S-transferase Noppera-bo.

Decalin-containing tetramic acid is a bioactive scaffold primarily produced by filamentous fungi. The structural diversity of this group of compounds is generated by characteristic enzymes of fungal biosynthetic pathways, including polyketide synthase/nonribosomal peptide synthetase hybrid enzymes and decalin synthase, which are responsible for the construction of a linear polyenoyl tetramic acid structure and stereoselective decalin formation via the intramolecular Diels-Alder reaction, respectively. Compounds that differed only in the decalin configuration were collected from genetically engineered mutants derived from decalin-containing tetramic acid-producing fungi and used for a structure-activity relationship study. Our evaluation of biological activities, such as cytotoxicity against several cancer cell lines and antibacterial, antifungal, antimalarial, and mitochondrial inhibitory activities, demonstrated that the activity for each assay varies depending on the decalin configurations. In addition to these known biological activities, we revealed that the compounds showed inhibitory activity against the insect steroidogenic glutathione S-transferase Noppera-bo. Engineering the decalin configurations would be useful not only to find derivatives with better biological activities but also to discover overlooked biological activities.

Genomics-driven derivatization of the bioactive fungal sesterterpenoid variecolin: Creation of an unnatural analogue with improved anticancer properties

A biosynthetic gene cluster for the bioactive fungal sesterterpenoids variecolin (1) and variecolactone (2) was identified in Aspergillus aculeatus ATCC 16872. Heterologous production of 1 and 2 was achieved in Aspergillus oryzae by expressing the sesterterpene synthase VrcA and the cytochrome P450 VrcB. Intriguingly, the replacement of VrcB with homologous P450s from other fungal terpenoid pathways yielded three new variecolin analogues (5–7). Analysis of the compounds' anticancer activity in vitro and in vivo revealed that although 5 and 1 had comparable activities, 5 was associated with significantly reduced toxic side effects in cancer-bearing mice, indicating its potentially broader therapeutic window. Our study describes the first tests of variecolin and its analogues in animals and demonstrates the utility of synthetic biology for creating molecules with improved biological activities.

Highly efficient gene knockout system in the maize pathogen Colletotrichum graminicola using Agrobacterium tumefaciens-mediated transformation (ATMT)

Colletotrichum graminicola, a hemibiotrophic pathogenic fungus, is the causal agent of anthracnose of maize, which causes significant yield losses worldwide, especially in warm and humid maize production regions. An efficient targeted genes knockout protocol is crucial to explore molecular mechanisms of fungal virulence to the host. In this study, we established a gene knockout transformation system by employing Agrobacterium tumefaciens-mediated transformation to knockout genes in M 1.001 strain of C. graminicola. The conidia germination status, induction medium type, and ratio of Agrobacterium cell and conidia suspension were optimized for the knockout of CgBRN1(OR352905), a gene relating to the fungal melanin biosynthesis pathway. Additionally, CgPKS18 (OR352906) and CgCDC25 (OR352903) were knocked out to test the applicability of the gene knockout transformation system. In this established system, transformation efficiency was 176 transformants per 1 × 105 conidia and the homologous recombination efficiency was 53.3 to 75%. Furthermore, disease index, lesion number and lesion size caused by the three above-mentioned mutant strains were found to be reduced significantly compared to the wild-type strain, which indicated reduction in fungal virulence due to the lack of those genes.