A pharmacological approach to investigating effector translocation in rice-Magnaporthe oryzae interactions.

Fungal pathogens deliver effector proteins into living plant cells to suppress plant immunity and control plant processes that are needed for infection. During plant infection, the devastating rice blast fungus, Magnaporthe oryzae, forms the specialized biotrophic interfacial complex (BIC), which is essential for effector translocation. Cytoplasmic effectors are first focally secreted into BICs, and subsequently packaged into dynamic membranous effector compartments (MECs), then translocated via clathrin-mediated endocytosis (CME) into the host cytoplasm. This study demonstrates that clathrin-heavy chain inhibitors endosidin-9 (ES9) and endosidin-9-17 (ES9-17) blocked the internalization of the fluorescently labeled effectors Bas1 and Pwl2 in rice cells, leading to swollen BICs lacking MECs. In contrast, ES9-17 treatment had no impact on the localization pattern of the apoplastic effector Bas4. This study provides further evidence that cytoplasmic effector translocation occurs by CME in BICs, suggesting a potential role for M. oryzae effectors in co-opting plant endocytosis.

The CRZ1 transcription factor in plant fungi: regulation mechanism and impact on pathogenesis.

Calcium (Ca2+) is a universal signaling molecule that is tightly regulated, and a fleeting elevation in cytosolic concentration triggers a signal cascade within the cell, which is crucial for several processes such as growth, tolerance to stress conditions, and virulence in fungi. The link between calcium and calcium-dependent gene regulation in cells relies on the transcription factor Calcineurin-Responsive Zinc finger 1 (CRZ1). The direct regulation of approximately 300 genes in different stress pathways makes it a hot topic in host-pathogen interactions. Notably, CRZ1 can modulate several pathways and orchestrate cellular responses to different types of environmental insults such as osmotic stress, oxidative stress, and membrane disruptors. It is our belief that CRZ1 provides the means for tightly modulating and synchronizing several pathways allowing pathogenic fungi to install into the apoplast and eventually penetrate plant cells (i.e., ROS, antimicrobials, and quick pH variation). This review discusses the structure, function, regulation of CRZ1 in fungal physiology and its role in plant pathogen virulence.

Unveiling the A-to-I mRNA editing machinery and its regulation and evolution in fungi.

A-to-I mRNA editing in animals is mediated by ADARs, but the mechanism underlying sexual stage-specific A-to-I mRNA editing in fungi remains unknown. Here, we show that the eukaryotic tRNA-specific heterodimeric deaminase FgTad2-FgTad3 is responsible for A-to-I mRNA editing in Fusarium graminearum. This editing capacity relies on the interaction between FgTad3 and a sexual stage-specific protein called Ame1. Although Ame1 orthologs are widely distributed in fungi, the interaction originates in Sordariomycetes. We have identified key residues responsible for the FgTad3-Ame1 interaction. The expression and activity of FgTad2-FgTad3 are regulated through alternative promoters, alternative translation initiation, and post-translational modifications. Our study demonstrates that the FgTad2-FgTad3-Ame1 complex can efficiently edit mRNA in yeasts, bacteria, and human cells, with important implications for the development of base editors in therapy and agriculture. Overall, this study uncovers mechanisms, regulation, and evolution of RNA editing in fungi, highlighting the role of protein-protein interactions in modulating deaminase function.

Secreted Aspartic Proteinases: Key Factors in Candida Infections and Host-Pathogen Interactions.

Extracellular proteases are key factors contributing to the virulence of pathogenic fungi from the genus Candida. Their proteolytic activities are crucial for extracting nutrients from the external environment, degrading host defenses, and destabilizing the internal balance of the human organism. Currently, the enzymes most frequently described in this context are secreted aspartic proteases (Saps). This review comprehensively explores the multifaceted roles of Saps, highlighting their importance in biofilm formation, tissue invasion through the degradation of extracellular matrix proteins and components of the coagulation cascade, modulation of host immune responses via impairment of neutrophil and monocyte/macrophage functions, and their contribution to antifungal resistance. Additionally, the diagnostic challenges associated with Candida infections and the potential of Saps as biomarkers were discussed. Furthermore, we examined the prospects of developing vaccines based on Saps and the use of protease inhibitors as adjunctive therapies for candidiasis. Given the complex biology of Saps and their central role in Candida pathogenicity, a multidisciplinary approach may pave the way for innovative diagnostic strategies and open new opportunities for innovative clinical interventions against candidiasis.

A Structural In Silico Analysis of the Immunogenicity of L-Asparaginase from Penicillium cerradense.

L-asparaginase is an essential drug used to treat acute lymphoid leukemia (ALL), a cancer of high prevalence in children. Several adverse reactions associated with L-asparaginase have been observed, mainly caused by immunogenicity and allergenicity. Some strategies have been adopted, such as searching for new microorganisms that produce the enzyme and applying protein engineering. Therefore, this work aimed to elucidate the molecular structure and predict the immunogenic profile of L-asparaginase from Penicillium cerradense, recently revealed as a new fungus of the genus Penicillium and producer of the enzyme, as a motivation to search for alternatives to bacterial L-asparaginase. In the evolutionary relationship, L-asparaginase from P. cerradense closely matches Aspergillus species. Using in silico tools, we characterized the enzyme as a protein fragment of 378 amino acids (39 kDa), including a signal peptide containing 17 amino acids, and the isoelectric point at 5.13. The oligomeric state was predicted to be a homotetramer. Also, this L-asparaginase presented a similar immunogenicity response (T- and B-cell epitopes) compared to Escherichia coli and Dickeya chrysanthemi enzymes. These results suggest a potentially useful L-asparaginase, with insights that can drive strategies to improve enzyme production.

The phosphorylation landscape of infection-related development by the rice blast fungus.

Many of the world's most devastating crop diseases are caused by fungal pathogens that elaborate specialized infection structures to invade plant tissue. Here, we present a quantitative mass-spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins following germination on a hydrophobic surface, revealing major re-wiring of phosphorylation-based signaling cascades during appressorium development. Comparing phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring (PRM) to identify phosphoproteins regulated by the fungal Pmk1 MAPK that controls plant infection by M. oryzae. We define 32 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of regulator Vts1 is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for the control of plant diseases.

Genetic Mutations in FKS1 Gene Associated with Acquired Echinocandin Resistance in Candida parapsilosis Complex.

Candida parapsilosis complex has recently received special attention due to naturally occurring FKS1 polymorphism associated with high minimal inhibitory concentrations for echinocandin and the increase of clonal outbreaks of strains resistant to commonly used antifungals such as fluconazole. Despite the previous fact, little is known about the genetic mechanism associated with echinocandin resistance. Therefore, the present study was designed to investigate the mechanism of acquired echinocandin resistance in C. parapsilosis complex strains. A total of 15 clinical C. parapsilosis complex isolates were sub-cultured for 30 days at a low concentration of micafungin at ½ the lowest MIC value of the tested isolates (0.12 µg/ml). After culturing, all the isolates were checked phenotypically for antifungal resistance and genotypically for echinocandin resistance by checking FKS1 gene hot spot one (HS1) and HS2 mutations. In vitro induction of echinocandin resistance confirmed the rapid development of resistance at low concentration micafungin, with no difference among C. parapsilosis, C. metapsilosis, and C. orthopsilosis in the resistance development. For the first time we identified different FKS1 HS1 and or HS2 mutations responsible for echinocandin resistance such as R658S and L1376F in C. parapsilosis, S656X, R658X, R658T, W1370X, X1371I, V1371X, and R1373X (corresponding to their location in C. parapsilosis) in C. metapsilosis, and L648F and R1366H in C. orthopsilosis. Our results are of significant concern, since the rapid development of resistance may occur clinically after short-term exposure to antifungals as recently described in other fungal species with the potential of untreatable infections.

Multi-omics analysis revealed that the protein kinase MoKin1 affected the cellular response to endoplasmic reticulum stress in the rice blast fungus, Magnaporthe oryzae.

BACKGROUND: Previous studies have shown that protein kinase MoKin1 played an important role in the growth, conidiation, germination and pathogenicity in rice blast fungus, Magnaporthe oryzae. ΔMokin1 mutant showed significant phenotypic defects and significantly reduced pathogenicity. However, the internal mechanism of how MoKin1 affected the development of physiology and biochemistry remained unclear in M. oryzae. RESULT: This study adopted a multi-omics approach to comprehensively analyze MoKin1 function, and the results showed that MoKin1 affected the cellular response to endoplasmic reticulum stress (ER stress). Proteomic analysis revealed that the downregulated proteins in ΔMokin1 mutant were enriched mainly in the response to ER stress triggered by the unfolded protein. Loss of MoKin1 prevented the ER stress signal from reaching the nucleus. Therefore, the phosphorylation of various proteins regulating the transcription of ER stress-related genes and mRNA translation was significantly downregulated. The insensitivity to ER stress led to metabolic disorders, resulting in a significant shortage of carbohydrates and a low energy supply, which also resulted in severe phenotypic defects in ΔMokin1 mutant. Analysis of MoKin1-interacting proteins indicated that MoKin1 really took participate in the response to ER stress. CONCLUSION: Our results showed the important role of protein kinase MoKin1 in regulating cellular response to ER stress, providing a new research direction to reveal the mechanism of MoKin1 affecting pathogenic formation, and to provide theoretical support for the new biological target sites searching and bio-pesticides developing.

An effector SsCVNH promotes the virulence of Sclerotinia sclerotiorum through targeting class III peroxidase AtPRX71.

Many plant pathogens secrete effector proteins into the host plant to suppress host immunity and facilitate pathogen colonization. The necrotrophic pathogen Sclerotinia sclerotiorum causes severe plant diseases and results in enormous economic losses, in which secreted proteins play a crucial role. SsCVNH was previously reported as a secreted protein, and its expression is significantly upregulated at 3 h after inoculation on the host plant. Here, we further demonstrated that deletion of SsCVNH leads to attenuated virulence. Heterologous expression of SsCVNH in Arabidopsis enhanced pathogen infection, inhibited the host PAMP-triggered immunity (PTI) response and increased plant susceptibility to S. sclerotiorum. SsCVNH interacted with class III peroxidase AtPRX71, a positive regulator of innate immunity against plant pathogens. SsCVNH could also interact with other class III peroxidases, thus reducing peroxidase activity and suppressing plant immunity. Our results reveal a new infection strategy employed by S. sclerotiorum in which the fungus suppresses the function of class III peroxidases, the major component of PTI to promote its own infection.

Rational modification of melatonin for broad-spectrum antifungal agents discovery.

Natural products, known for their environmental safety, are regarded as a significant basis for the modification and advancement of fungicides. Melatonin, as a low-cost natural indole, exhibits diverse biological functions, including antifungal activity. However, its potential as an antifungal agent has not been fully explored. In this study, a series of melatonin derivatives targeting the mitogen-activated protein kinase (Mps1) protein of fungal pathogens were synthesized based on properties of melatonin, among which the trifluoromethyl-substituted derivative Mt-23 exhibited antifungal activity against seven plant pathogenic fungi, and effectively reduced the severity of crop diseases, including rice blast, Fusarium head blight of wheat and gray mold of tomato. In particular, its EC50 (5.4 µM) against the rice blast fungus Magnaporthe oryzae is only one-fourth that of isoprothiolane (22 µM), a commercial fungicide. Comparative analyzes revealed that Mt-23 simultaneously targets the conserved protein kinase Mps1 and lipid protein Cap20. Surface plasmon resonance assays showed that Mt-23 directly binds to Mps1 and Cap20. In this study, we provide a strategy for developing antifungal agents by modifying melatonin, and the resultant melatonin derivative Mt-23 is a commercially valuable, eco-friendly and broad-spectrum antifungal agent to combat crop disease.

A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

Control of Sclerotinia sclerotiorum via an RNA interference (RNAi)-mediated targeting of SsPac1 and SsSmk1.

MAIN CONCLUSION: The study evaluates the potential of Spray-Induced Gene Silencing and Host-Induced Gene Silencing for sustainable crop protection against the broad-spectrum necrotrophic fungus Sclerotinia sclerotiorum. Sclerotinia sclerotiorum (Lib.) de Bary, an aggressive ascomycete fungus causes white rot or cottony rot on a broad range of crops including Brassica juncea. The lack of sustainable control measures has necessitated biotechnological interventions such as RNA interference (RNAi) for effective pathogen control. Here we adopted two RNAi-based strategies-Spray-Induced Gene Silencing (SIGS) and Host-Induced Gene Silencing (HIGS) to control S. sclerotiorum. SIGS was successful in controlling white rot on Nicotiana benthamiana and B. juncea by targeting SsPac1, a pH-responsive transcription factor and SsSmk1, a MAP kinase involved in fungal development and pathogenesis. Topical application of dsRNA targeting SsPac1 and SsSmk1 delayed infection initiation and progression on B. juncea. Further, altered hyphal morphology and reduced radial growth were also observed following dsRNA application. We also explored the impact of stable dsRNA expression in A. thaliana against S. sclerotiorum. In this report, we highlight the utility of RNAi as a biofungicide and a tool for preliminary functional genomics.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

Metabolic engineering of Aspergillus niger for accelerated malic acid biosynthesis by improving NADPH availability.

Microbial production of L-malic acid from renewable carbon sources has attracted extensive attention. The reduced cofactor NADPH plays a key role in biotransformation because it participates in both biosynthetic reactions and cellular stress responses. In this study, NADPH or its precursors nicotinamide and nicotinic acid were added to the fermentation medium of Aspergillus niger RG0095, which significantly increased the yield of malic acid by 11%. To further improve the titer and productivity of L-malic acid, we increased the cytoplasmic NADPH levels of A. niger by upregulating the NAD kinases Utr1p and Yef1p. Biochemical analyses demonstrated that overexpression of Utr1p and Yef1p reduced oxidative stress, while also providing more NADPH to catalyze the conversion of glucose into malic acid. Notably, the strain overexpressing Utr1p reached a malate titer of 110.72 ± 1.91 g L-1 after 108 h, corresponding to a productivity of 1.03 ± 0.02 g L-1 h-1. Thus, the titer and productivity of malate were increased by 24.5% and 44.7%, respectively. The strategies developed in this study may also be useful for the metabolic engineering of fungi to produce other industrially relevant bulk chemicals.

First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

BACKGROUND: Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. OBJECTIVES: This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. MATERIALS AND METHODS: Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. RESULTS: Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. CONCLUSIONS: These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

Functional analysis of the mating type genes in Verticillium dahliae.

BACKGROUND: Populations of the plant pathogenic fungus Verticillium dahliae display a complex and rich genetic diversity, yet the existence of sexual reproduction in the fungus remains contested. As pivotal genes, MAT genes play a crucial role in regulating cell differentiation, morphological development, and mating of compatible cells. However, the functions of the two mating type genes in V. dahliae, VdMAT1-1-1, and VdMAT1-2-1, remain poorly understood. RESULTS: In this study, we confirmed that the MAT loci in V. dahliae are highly conserved, including both VdMAT1-1-1 and VdMAT1-2-1 which share high collinearity. The conserved core transcription factor encoded by the two MAT loci may facilitate the regulation of pheromone precursor and pheromone receptor genes by directly binding to their promoter regions. Additionally, peptide activity assays demonstrated that the signal peptide of the pheromone VdPpg1 possessed secretory activity, while VdPpg2, lacked a predicted signal peptide. Chemotactic growth assays revealed that V. dahliae senses and grows towards the pheromones FO-a and FO-α of Fusarium oxysporum, as well as towards VdPpg2 of V. dahliae, but not in response to VdPpg1. The findings herein also revealed that VdMAT1-1-1 and VdMAT1-2-1 regulate vegetative growth, carbon source utilization, and resistance to stressors in V. dahliae, while negatively regulating virulence. CONCLUSIONS: These findings underscore the potential roles of VdMAT1-1-1 and VdMAT1-2-1 in sexual reproduction and confirm their involvement in various asexual processes of V. dahliae, offering novel insights into the functions of mating type genes in this species.

Candidalysin: An unlikely aide for fungal gut commensalism.

Fungi colonize the mammalian gastrointestinal (GI) tract and can adopt both commensal and opportunistic lifestyles. In a recent issue of Nature, Liang et al. unraveled the complex interplay between Candida morphotypes and the gut bacterial microbiota and described a key role for candidalysin in gut colonization.1.

Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes. Identification by mass spectrometry revealed labeling of a wide range of active P450s, including six plant P450s, 40 mouse P450s and 13 P450s of the fungal wheat pathogen Zymoseptoria tritici. We next used transient expression of GFP-tagged P450s by agroinfiltration to show ER-targeting and NADPH-dependent, activity-based labeling of plant, mouse and fungal P450s. Both global profiling and transient expression can be used to detect a broad range of active P450s to study e.g. their regulation and discover selective inhibitors.

[Diagnosis of allergen components in fungi and microbial communities: patient sensitization characteristics and main sensitizing proteins].

The human body, as a highly complex ecosystem, harbors diverse microbial communities, with major factors triggering allergic reactions encompassing the skin microbiome and fungi. The global diversity of fungi is estimated to range from approximately 600 000 to 1 million species, and theoretically, IgE-mediated sensitization may occur to any fungal species. As of now, the World Health Organization/IUIS official database records 113 fungal allergens originating from 30 different fungi species, covering 42 allergen families. Regarding the skin microbiome, 14 distinct Malassezia allergens have been identified, all derived from three different Malassezia fungi species--M. furfur, M. sympodialis, and M. globosa. The conditions of patients with these allergies are exceptionally complex. This article extensively discusses the latest research advancements and clinical applications related to skin microbiome and fungal allergies from the European Academy of Allergy and Clinical Immunology (EAACI) publication, "Molecular Allergology User's Guide 2.0". Additionally, it compiles information on the sources of fungal allergens, characteristics of allergen component protein families, clinical relevance, and management strategies, both domestically and internationally. The aim is to enhance the profound understanding of allergen components among relevant professionals. Through the application of advanced allergen component diagnostic techniques, the goal is to achieve precise diagnosis and treatment of fungal allergy patients and explore the mechanisms underlying fungal sensitization and pathogenesis, laying the foundation for studying the fungal allergen protein sensitization spectrum in the Chinese population.