Frontiers in fungal biology最新文献

The feruloyl esterase B gene (faeB) is specifically induced by hydroxycinnamic acids (e.g. ferulic acid, caffeic acid and coumaric acid) but the transcriptional regulation network involved in faeB induction and ferulic acid metabolism has only been partially addressed. To identify transcription factors involved in ferulic acid metabolism we constructed and screened a transcription factor knockout library of 239 Aspergillus niger strains for mutants unable to utilize ferulic acid as a carbon source. The ΔfarA transcription factor mutant, already known to be involved in fatty acid metabolism, could not utilize ferulic acid and other hydroxycinnamic acids. In addition to screening the transcription factor mutant collection, a forward genetic screen was performed to isolate mutants unable to express faeB. For this screen a PfaeB-amdS and PfaeB-lux₆₁₃ dual reporter strain was engineered. The rationale of the screen is that in this reporter strain ferulic acid induces amdS (acetamidase) expression via the faeB promoter resulting in lethality on fluoro-acetamide. Conidia of this reporter strain were UV-mutagenized and plated on fluoro-acetamide medium in the presence of ferulic acid. Mutants unable to induce faeB are expected to be fluoro-acetamide resistant and can be positively selected for. Using this screen, six fluoro-acetamide resistant mutants were obtained and phenotypically characterized. Three mutants had a phenotype identical to the farA mutant and sequencing the farA gene in these mutants indeed showed mutations in FarA which resulted in inability to growth on ferulic acid as well as on short and long chain fatty acids. The growth phenotype of the other three mutants was similar to the farA mutants in terms of the inability to grow on ferulic acid, but these mutants grew normally on short and long chain fatty acids. The genomes of these three mutants were sequenced and allelic mutations in one particular gene (NRRL3_09145) were found. The protein encoded by NRRL3_09145 shows similarity to the FarA and FarB transcription factors. However, whereas FarA and FarB contain both the Zn(II)₂Cys₆ domain and a fungal-specific transcription factor domain, the protein encoded by NRRL3_09145 (FarD) lacks the canonical Zn(II)₂Cys₆ domain and possesses only the fungal specific transcription factor domain.

Aspergillus fungi produce mycotoxins that are detrimental to human and animal health. Two sections of aspergilli are of particular importance to cereal food crops such as corn and barley. Aspergillus section Flavi species like A. flavus and A. parasiticus produce aflatoxins, while section Circumdati species like A. ochraceus and A. sclerotiorum produce ochratoxin A. Mitigating these toxins in food and feed is a critical and ongoing worldwide effort. We have previously investigated biosynthetic gene clusters in Aspergillus flavus that are linked to fungal virulence in corn. We found that one such cluster, asa, is responsible for the production of aspergillic acid, an iron-binding, hydroxamic acid-containing pyrazinone metabolite. Furthermore, we found that the asa gene cluster is present in many other aflatoxin- and ochratoxin-producing aspergilli. The core gene in the asa cluster encodes the small nonribosomal peptide synthetase-like (NRPS-like) protein AsaC. We have swapped the asaC ortholog from A. sclerotiorum into A. flavus, replacing its native copy, and have also cloned both asaC orthologs into Saccharomyces cerevisiae. We show that AsaC orthologs in section Flavi and section Circumdati, while only containing adenylation-thiolation-reductase (ATR) domains, can selectively biosynthesize distinct pyrazinone natural products: deoxyaspergillic acid and flavacol, respectively. Because pyrazinone natural products and the gene clusters responsible for their production are implicated in a variety of important microbe-host interactions, uncovering the function and selectivity of the enzymes involved could lead to strategies that ultimately benefit human health.

Autophagy (macroautophagy) is a survival and virulence mechanism of different eukaryotic pathogens. Autophagosomes sequester cytosolic material and organelles, then fuse with or enter into the vacuole or lysosome (the lytic compartment of most fungal/plant cells and many animal cells, respectively). Subsequent degradation of cargoes delivered to the vacuole via autophagy and endocytosis maintains cellular homeostasis and survival in conditions of stress, cellular differentiation, and development. PrA and PrB are vacuolar aspartyl and serine endoproteases, respectively, that participate in the autophagy of fungi and contribute to the pathogenicity of phytopathogens. Whereas the levels of vacuolar proteases are regulated by the expression of the genes encoding them (e.g., PEP4 for PrA and PRB1 for PrB), their activity is governed by endogenous inhibitors. The aim of the current contribution is to review the main characteristics, regulation, and role of vacuolar soluble endoproteases and Atg proteins in the process of autophagy and the pathogenesis of three fungal phytopathogens: Ustilago maydis, Magnaporthe oryzae, and Alternaria alternata. Aspartyl and serine proteases are known to participate in autophagy in these fungi by degrading autophagic bodies. However, the gene responsible for encoding the vacuolar serine protease of U. maydis has yet to be identified. Based on in silico analysis, this U. maydis gene is proposed to be orthologous to the Saccharomyces cerevisiae genes PRB1 and PBI2, known to encode the principal protease involved in the degradation of autophagic bodies and its inhibitor, respectively. In fungi that interact with plants, whether phytopathogenic or mycorrhizal, autophagy is a conserved cellular degradation process regulated through the TOR, PKA, and SNF1 pathways by ATG proteins and vacuolar proteases. Autophagy plays a preponderant role in the recycling of cell components as well as in the fungus-plant interaction.

Coevolution is an important biological process that shapes interacting proteins - may it be physically interacting proteins or consecutive enzymes in a metabolic pathway, such as the biosynthetic pathways for secondary metabolites. Previously, we developed FunOrder, a semi-automated method for the detection of co-evolved genes, and demonstrated that FunOrder can be used to identify essential genes in biosynthetic gene clusters from different ascomycetes. A major drawback of this original method was the need for a manual assessment, which may create a user bias and prevents a high-throughput application. Here we present a fully automated version of this method termed FunOrder 2.0. In the improved version, we use several mathematical indices to determine the optimal number of clusters in the FunOrder output, and a subsequent k-means clustering based on the first three principal components of a principal component analysis of the FunOrder output to automatically detect co-evolved genes. Further, we replaced the BLAST tool with the DIAMOND tool as a prerequisite for using larger proteome databases. Potentially, FunOrder 2.0 may be used for the assessment of complete genomes, which has not been attempted yet. However, the introduced changes slightly decreased the sensitivity of this method, which is outweighed by enhanced overall speed and specificity.

Candida parapsilosis is a leading cause of invasive candidiasis in southern Europe, Latin America and Asia. C. parapsilosis has been mostly considered susceptible to triazoles, but fluconazole resistance is on the rise in some countries. The main mechanism related to fluconazole resistance is the presence of ERG11p substitutions, dominated by the Y132F amino acid substitution. Isolates harbouring this substitution mimic C. auris given that they may cause hospital outbreaks, become endemic, and emerge simultaneously in distant areas around the world. At the moment, Spain is experiencing a brusque emergence of fluconazole resistance in C. parapsilosis; isolates harbouring the Y132F substitution were detected for the first time in 2019. A recent study on Candida spp isolates from blood cultures collected in 16 hospitals located in the Madrid metropolitan area (2019 to 2021) reported that fluconazole resistance in C. parapsilosis reached as high as 13.6%. Resistance rates rose significantly during those three years: 3.8% in 2019, 5.7% in 2020, and 29.1% in 2021; resistant isolates harboured either the dominant Y132F substitution (a single clone found in four hospitals) or G458S (another clone found in a fifth hospital). The COVID-19 pandemic may have increased the number of candidaemia cases. The reason for such an increase might be a consequence of uncontrolled intra-hospital patient-to-patient transmission in some hospitals, as an increase not only in C. parapsilosis candidaemia episodes but also in the spread of clonal fluconazole-resistant isolates might have occurred in other hospitals during the pandemic period. Patients affected with fluconazole-resistant C. parapsilosis harbouring the Y132F substitution presented a mortality rate ranging from 9% to 78%, were mainly admitted to intensive care wards but did not have differential risk factors compared to those infected by susceptible isolates. With scarce exceptions, few patients (≤20%) infected with fluconazole-resistant isolates had previously received fluconazole, thus supporting the fact that, although fluconazole might have been a key factor to promote resistance, the main driver promoting the spread of fluconazole-resistant isolates was patient-to-patient transmission.

Mucoralean fungi from the genus Rhizopus are common inhabitants of terrestrial ecosystems, being some pathogens of animals and plants. In this study, we analyzed the symbiotic and toxinogenic potential of Rhizopus species derived from agricultural soils dedicated to the production of papaya (Carica papaya L.) in Mexico. Four representative strains of soil-derived Rhizopus spp. were analyzed employing molecular, microscopic, and metabolic methods. The ITS phylogenies identified the fungi as Rhizopus microsporus HP499, Rhizopus delemar HP475 and HP479, and Rhizopus homothallicus HP487. We discovered that R. microsporus HP499 and R. delemar HP475 harbor similar endofungal bacterial symbionts that belong to the genus Mycetohabitans (Burkholderia sensu lato) and that none of the four fungi were associated with Narnavirus RmNV-20S and RmNV-23S. Intriguingly, the interaction between R. delemar - Mycetohabitans showed different phenotypes from known R. microsporus - Mycetohabitans symbioses. Elimination of bacteria in R. delemar HP475 did not cause a detrimental effect on fungal growth or asexual reproduction. Moreover, metabolic and molecular analyses confirmed that, unlike symbiotic R. microsporus HP499, R. delemar HP475 does not produce rhizoxin, one of the best-characterized toxins produced by Mycetohabitans spp. The rhizoxin (rhi) biosynthetic gene cluster seems absent in this symbiotic bacterium. Our study highlights that the symbioses between Rhizopus and Mycetohabitans are more diverse than anticipated. Our findings contribute to expanding our understanding of the role bacterial symbionts have in the pathogenicity, biology and evolution of Mucorales.

This study assess the population diversity and temporal variability of caused by Fusarium oxysporum f. sp. vasinfectum (FOV) races/genotypes infecting cotton cultivars with either FOV or Meloidogyne incognita resistance. All plants sampled demonstrated typical symptoms of FOV including wilting, chlorosis and necrosis of the leaves, and discoloration of the vascular tissue in the stem. A diverse population of FOV was characterized. Eight races/genotypes of FOV were collected throughout the three site years. FOV race 1 was the most predominant in all tests (AUDPC=101.1); statistically higher numbers of isolates from LA-108 (AUDPC=59.9), race 8 (AUDPC=47.5), and race 2 (AUDPC=38.6) were also found compared to other races and genotypes collected. FOV race 1, race 2, race 8, and 108 were the most virulent races identified. The genotypes MDS-12, LA-110, and LA-127/140 were found in all tests but at a low incidence, and LA-112 was only found in trace amounts. MDS-12, LA-110, LA-112, and LA-127/140 produced less disease pressure. FOV race 4 which is highly virulent and present in California and Texas was not found in Alabama. A positive correlation was observed between the accumulation of growing degree days and FOV race 1, race 2, race 8, LA-108, and LA-110. Later symptom expression influenced by seasonal heat partially mitigates damage allowing cotton to produce bolls though they may be reduced in number and lint quality. Plant resistance to the FOV as expressed in these cultivars appears to provide better protection than M. incognita resistance. PhytoGen 72, which is resistant to FOV races/genotypes had low levels of FOV infection even though it sustained a high level of M. incognita root population density. The M. incognita resistant cultivars Deltapine 1558NR B2RF and PhytoGen 480 W3FE supported a lower nematode population density, however, FOV disease incidence was not reduced. FOV races/genotypes did not vary significantly between the nematode resistant and nematode susceptible cultivars.

Ustilago maydis is a biotrophic phytopathogenic fungus that causes corn smut disease. As a well-established model system, U. maydis is genetically fully accessible with large omics datasets available and subject to various biological questions ranging from DNA-repair, RNA-transport, and protein secretion to disease biology. For many genetic approaches, tight control of transgene regulation is important. Here we established an optimised version of the Tetracycline-ON (TetON) system for U. maydis. We demonstrate the Tetracycline concentration-dependent expression of fluorescent protein transgenes and the system's suitability for the induced expression of the toxic protein BCL2 Associated X-1 (Bax1). The Golden Gate compatible vector system contains a native minimal promoter from the mating factor a-1 encoding gene, mfa with ten copies of the tet-regulated operator (tetO) and a codon optimised Tet-repressor (tetR*) which is translationally fused to the native transcriptional corepressor Mql1 (UMAG_05501). The metabolism-independent transcriptional regulator system is functional both, in liquid culture as well as on solid media in the presence of the inducer and can become a useful tool for toxin-antitoxin studies, identification of antifungal proteins, and to study functions of toxic gene products in Ustilago maydis.