Transcriptomic analysis of polyaromatic hydrocarbon degradation by the halophilic fungus Aspergillus sydowii at hypersaline conditions.

Polycyclic aromatic hydrocarbons (PAHs) are among the most persistent xenobiotic compounds, with high toxicity effects. Mycoremediation with halophilic Aspergillus sydowii was used for their removal from a hypersaline medium (1 M NaCl). A. sydowii metabolized PAHs as sole carbon sources, resulting in the removal of up to 90% for both PAHs [benzo [a] pyrene (BaP) and phenanthrene (Phe)] after 10 days. Elimination of Phe and BaP was almost exclusively due to biotransformation and not adsorption by dead mycelium and did not correlate with the activity of lignin modifying enzymes (LME). Transcriptomes of A. sydowii grown on PAHs, or on glucose as control, both at hypersaline conditions, revealed 170 upregulated and 76 downregulated genes. Upregulated genes were related to starvation, cell wall remodelling, degradation and metabolism of xenobiotics, DNA/RNA metabolism, energy generation, signalling and general stress responses. Changes of LME expression levels were not detected, while the chloroperoxidase gene, possibly related to detoxification processes in fungi, was strongly upregulated. We propose that two parallel metabolic pathways (mitochondrial and cytosolic) are involved in degradation and detoxification of PAHs in A. sydowii resulting in intracellular oxidation of PAHs. To the best of our knowledge, this is the most comprehensive transcriptomic analysis on fungal degradation of PAHs.

Serratia sp., an endophyte of Mimosa pudica nodules with nematicidal, antifungal activity and growth-promoting characteristics.

In the present study, the nematicidal activity of an isolated strain of Mimosa pudica nodules was evaluated against the Nacobbus aberrans (J2) phytonymatodes with a mortality of 88.8%, while against the gastrointestinal nematode Haemonchus contortus (L3) and free-living Panagrellus redivivus was 100%. The ability to inhibit the growth of phytopathogenic fungi Fusarium sp., and Alternaria solani, as well as the oomycete Phytophthora capsici, this antifungal activity may be related to the ability to produce cellulases, siderophores and chitinases by this bacterial strain. Another important finding was the detection of plant growth promoter characteristics, such as auxin production and phosphate solubilization. The strain identified by sequences of the 16S and rpoB genes as Serratia sp. is genetically related to Serratia marcescens and Serratia nematodiphila. The promoter activity of plant growth, antifungal and nematicide of the Serratia sp. strain makes it an alternative for the biocontrol of fungi and nematodes that affect both the livestock and agricultural sectors, likewise, candidate as a growth-promoting bacterium.

Identification of a Huperzine A-producing endophytic fungus from Phlegmariurus taxifolius.

Highly prized huperzine A (Hup A), a natural alkaloid formerly isolated from the Chinese medicinal plant Huperzia serrata, has been widely used for the treatment of Alzheimer disease, inspiring us to search for endophytic fungi that produce this compound. In this study, we obtained the C17 fungus isolate from the Mexican club moss Phlegmariurus taxifolius, which produced a yield of 3.2 µg/g Hup A in mycelial dry weight, when cultured in potato dextrose broth medium. The C17 isolate was identified as belonging to the genus Fusarium with reference to the colony´s morphological characteristics and the presence of macroconidia and microconidia structures; and this was confirmed by DNA-barcoding analysis, by amplifying and sequencing the ribosomal internal transcribed spacer (rITS).

Expression, purification, and characterization of a metagenomic thioesterase from activated sludge involved in the degradation of acylCoA-derivatives.

Metagenomic libraries are a novel and powerful approach to seek for pathways involved in xenobiotic degradation, since this technique abolishes the need for cultivating microorganisms that otherwise would be overlooked if they cannot grow on standard laboratory media and conditions. In this paper, we describe the expression, purification and characterization of a novel metagenomic thioesterase which was described to be involved in phenylacetic acid degradation (A. Sánchez-Reyes, R. Batista-García, G. Valdés-García E. Ortiz, L. Perezgasga, A. Zárate-Romero, N. Pastor, J. L. Folch-Mallol, A Family 13 thioesterase isolated from an activated sludge metagenome: insights into aromatic compounds metabolism, Proteins 85 (2017) 1222-1237). According to similarity and phylogenetic analyses, the enzyme seems to belong to an Actinobacterium. Nevertheless, after a process of denaturation and refolding, the protein expressed in E. coli was obtained in an active form. New data concerning the substrate preferences for this enzyme are presented which suggest that this thioesterase could be involved in breaking the ester bond in the CoA-linear acyl derivatives of the phenylacetic acetic pathway.

A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism.

Activated sludge is produced during the treatment of sewage and industrial wastewaters. Its diverse chemical composition allows growth of a large collection of microbial phylotypes with very different physiologic and metabolic profiles. Thus, activated sludge is considered as an excellent environment to discover novel enzymes through functional metagenomics, especially activities related with degradation of environmental pollutants. Metagenomic DNA was isolated and purified from an activated sludge sample. Metagenomic libraries were subsequently constructed in Escherichia coli. Using tributyrin hydrolysis, a screening by functional analysis was conducted and a clone that showed esterase activity was isolated. Blastx analysis of the sequence of the cloned DNA revealed, among others, an ORF that encodes a putative thioesterase with 47-64% identity to GenBank CDS reported genes, similar to those in the hotdog fold thioesterase superfamily. On the basis of its amino acid similarity and its homology-modelled structure we deduced that this gene encodes an enzyme (ThYest_ar) that belongs to family TE13, with a preference for aryl-CoA substrates and a novel catalytic residue constellation. Plasmid retransformation in E. coli confirmed the clone's phenotype, and functional complementation of a paaI E. coli mutant showed preference for phenylacetate over chlorobenzene as a carbon source. This work suggests a role for TE13 family thioesterases in swimming and degradation approaches for phenyl acetic acid. Proteins 2017; 85:1222-1237. © 2017 Wiley Periodicals, Inc.

Simple screening protocol for identification of potential mycoremediation tools for the elimination of polycyclic aromatic hydrocarbons and phenols from hyperalkalophile industrial effluents.

A number of fungal strains belonging to the ascomycota, basidiomycota and zygomycota genera were subjected to an in vitro screening regime to assess their ligninolytic activity potential, with a view to their potential use in mycoremediation-based strategies to remove phenolic compounds and polycyclic aromatic hydrocarbons (PAHs) from industrial wastewaters. All six basidiomycetes completely decolorized remazol brilliant blue R (RBBR), while also testing positive in both the guaiacol and gallic acid tests indicating good levels of lignolytic activity. All the fungi were capable of tolerating phenanthrene, benzo-α- pyrene, phenol and p-chlorophenol in agar medium at levels of 10 ppm. Six of the fungal strains, Pseudogymnoascus sp., Aspergillus caesiellus, Trametes hirsuta IBB 450, Phanerochate chrysosporium ATCC 787, Pleurotus ostreatus MTCC 1804 and Cadophora sp. produced both laccase and Mn peroxidase activity in the ranges of 200-560 U/L and 6-152 U/L, respectively, in liquid media under nitrogen limiting conditions. The levels of adsorption of the phenolic and PAHs were negligible with 99% biodegradation being observed in the case of benzo-α-pyrene, phenol and p-chlorophenol. The aforementioned six fungal strains were also found to be able to effectively treat highly alkaline industrial wastewater (pH 12.4). When this wastewater was supplemented with 0.1 mM glucose, all of the tested fungi, apart from A. caesiellus, displayed the capacity to remove both the phenolic and PAH compounds. Based on their biodegradative capacity we found T. hirsuta IBB 450 and Pseudogymnoascus sp., to have the greatest potential for further use in mycoremediation based strategies to treat wastestreams containing phenolics and PAHs.

Identification of a novel carbohydrate esterase from Bjerkandera adusta: structural and function predictions through bioinformatics analysis and molecular modeling.

A new gene from Bjerkandera adusta strain UAMH 8258 encoding a carbohydrate esterase (designated as BacesI) was isolated and expressed in Pichia pastoris. The gene had an open reading frame of 1410 bp encoding a polypeptide of 470 amino acid residues, the first 18 serving as a secretion signal peptide. Homology and phylogenetic analyses showed that BaCesI belongs to carbohydrate esterases family 4. Three-dimensional modeling of the protein and normal mode analysis revealed a breathing mode of the active site that could be relevant for esterase activity. Furthermore, the overall negative electrostatic potential of this enzyme suggests that it degrades neutral substrates and will not act on negative substrates such as peptidoglycan or p-nitrophenol derivatives. The enzyme shows a specific activity of 1.118 U mg(-1) protein on 2-naphthyl acetate. No activity was detected on p-nitrophenol derivatives as proposed from the electrostatic potential data. The deacetylation activity of the recombinant BaCesI was confirmed by measuring the release of acetic acid from several substrates, including oat xylan, shrimp shell chitin, N-acetylglucosamine, and natural substrates such as sugar cane bagasse and grass. This makes the protein very interesting for the biofuels production industry from lignocellulosic materials and for the production of chitosan from chitin.

A novel TctA citrate transporter from an activated sludge metagenome: structural and mechanistic predictions for the TTT family.

We isolated a putative citrate transporter of the tripartite tricarboxylate transporter (TTT) class from a metagenomic library of activated sludge from a sewage treatment plant. The transporter, dubbed TctA_ar, shares ∼50% sequence identity with TctA of Comamonas testosteroni (TctA_ct) and other ß-Proteobacteria, and contains two 20-amino acid repeat signature sequences, considered a hallmark of this particular transporter class. The structures for both TctA_ar and TctA_ct were modeled with I-TASSER and two possible structures for this transporter family were proposed. Docking assays with citrate resulted in the corresponding sets of proposed critical residues for function. These models suggest functions for the 20-amino acid repeats in the context of the two different architectures. This constitutes the first attempt at structure modeling of the TTT family, to the best of our knowledge, and could aid functional understanding of this little-studied family.

Exophiala chapopotensis sp. nov., an extremotolerant black yeast from an oil-polluted soil in Mexico; phylophenetic approach to species hypothesis in the Herpotrichiellaceae family.

Exophiala is a black fungi of the family Herpotrichiellaceae that can be found in a wide range of environments like soil, water and the human body as potential opportunistic pathogen. Some species are known to be extremophiles, thriving in harsh conditions such as deserts, glaciers, and polluted habitats. The identification of novel Exophiala species across diverse environments underlines the remarkable biodiversity within the genus. However, its classification using traditional phenotypic and phylogenetic analyses has posed a challenges. Here we describe a novel taxon, Exophiala chapopotensis sp. nov., strain LBMH1013, isolated from oil-polluted soil in Mexico, delimited according to combined morphological, molecular, evolutionary and statistics criteria. This species possesses the characteristic dark mycelia growing on PDA and tends to be darker in the presence of hydrocarbons. Its growth is dual with both yeast-like and hyphal forms. LBMH1013 differs from closely related species such as E. nidicola due to its larger aseptate conidia and could be distinguished from E. dermatitidis and E. heteromorpha by its inability to thrive above 37°C or 10% of NaCl. A comprehensive genomic analyses using up-to-date overall genome relatedness indices, several multigene phylogenies and molecular evolutionary analyzes using Bayesian speciation models, further validate its species-specific transition from all current Exophiala/Capronia species. Additionally, we applied the phylophenetic conceptual framework to delineate the species-specific hypothesis in order to incorporate this proposal within an integrative taxonomic framework. We believe that this approach to delimit fungal species will also be useful to our peers.

Optimization of methyl parathion biodegradation and detoxification by cells in suspension or immobilized on tezontle expressing the opd gene.

The goal of this study was to optimize methyl parathion (O,O-dimethyl-O-4-p-nitrophenyl phosphorothioate) degradation using a strain of Escherichia coli DH5α expressing the opd gene. Our results indicate that this strain had lower enzymatic activity compared to the Flavobacterium sp. ATCC 27551 strain from which the opd gene was derived. Both strains were assessed for their ability to degrade methyl parathion (MP) in a mineral salt medium with or without the addition of glucose either as suspended cells or immobilized on tezontle, a volcanic rock. MP was degraded by both strains with similar efficiencies, but immobilized cells degraded MP more efficiently than cells in suspension. However, the viability of E. coli cells was much higher than that of the Flavobacterium sp. We confirmed the decrease in toxicity from the treated effluents through acetylcholinesterase activity tests, indicating the potential of this method for the treatment of solutions containing MP.

Complete genome sequence dataset of enthomopathogenic Aspergillus flavus isolated from a natural infection of the cattle-tick Rhipicephalus microplus.

As the most important bovine ectoparasite, the southern cattle tick Rhipicephalus microplus transmits lethal cattle diseases such as babesiosis and anaplasmosis, costing the global livestock industry billions of dollars annually. To control cattle ticks, preventive treatment of cattle with pesticides is a common practice; however, after decades of chemical treatment, pesticide resistance has arisen in cattle ticks, rendering most formulations ineffective over time. Facing the perspective of running out of effective chemical treatments against R. microplus, research on biocontrol alternatives is necessary. Acaro-pathogenic microorganisms isolated from different developmental stages of R. microplus offer potential as biocontrol agents. Aspergillus flavus strain INIFAP-2021, isolated from naturally infected cattle ticks, produced high levels of mobility and mortality in the tick population during experimental infections. The whole genome of the fungi was sequenced using the DNBSEQ platform by BGI. The genome was assembled using SOAPaligner, and A. flavus NRRL3357 was used as the reference genome; the complete genome contained eight pairs of chromosomes and 36.9 Mb with a GC content of 48.03%, exhibiting 11482 protein-coding genes. The final genome assembly was deposited at GenBank as a bio project under accession number PRJNA758689, and supplementary material is accessible through Mendeley DOI: 10.17632/mt8yxch6mz.1.

Identification of Virulence Factors in Entomopathogenic Aspergillus flavus Isolated from Naturally Infected Rhipicephalus microplus.

Aspergillus flavus has been found to be an effective entomopathogenic fungus for various arthropods, including ticks. In particular, natural fungal infections in cattle ticks show promise for biocontrol of the Rhipicephalus (Boophilus) microplus tick, which is a major ectoparasite affecting cattle worldwide. Our study aimed to elucidate the specific entomopathogenic virulence factors encoded in the genome of an A. flavus strain isolated from naturally infected cattle ticks. We performed morphological and biochemical phenotyping alongside complete genome sequencing, which revealed that the isolated fungus was A. flavus related to the L morphotype, capable of producing a range of gene-coded entomopathogenic virulence factors, including ribotoxin, aflatoxin, kojic acid, chitinases, killer toxin, and satratoxin. To evaluate the efficacy of this A. flavus strain against ticks, we conducted experimental bioassays using healthy engorged female ticks. A morbidity rate of 90% was observed, starting at a concentration of 105 conidia/mL. At a concentration of 107 conidia/mL, we observed a 50% mortality rate and a 21.5% inhibition of oviposition. The highest levels of hatch inhibition (30.8%) and estimated reproduction inhibition (34.64%) were achieved at a concentration of 108 conidia/mL. Furthermore, the tick larval progeny that hatched from the infected tick egg masses showed evident symptoms of Aspergillus infection after incubation.

Aspergillus luchuensis, an Endophyte Fungus from the Metal Hyperaccumulator Plant Prosopis laevigata, Promotes Its Growth and Increases Metal Translocation.

Heavy metal pollution is a worldwide environmental and human health problem. Prosopis laevigata is a hyperaccumulator legume that bioaccumulates Pb, Cu and Zn. With interest in designing phytoremediation strategies for sites contaminated with heavy metals, we isolated and characterized endophytic fungi from the roots of P. laevigata growing on mine tailings located in Morelos, Mexico. Ten endophytic isolates were selected by morphological discrimination and a preliminary minimum inhibitory concentration was determined for zinc, lead and copper. A novel strain of Aspergillus closest to Aspergillus luchuensis was determined to be a metallophile and presented a marked tolerance to high concentrations of Cu, Zn and Pb, so it was further investigated for removal of metals and promotion of plant growth under greenhouse conditions. The control substrate with fungi promoted larger size characters in P. laevigata individuals in comparison with the other treatments, demonstrating that A. luchuensis strain C7 is a growth-promoting agent for P. laevigata individuals. The fungus favors the translocation of metals from roots to leaves in P. laevigata, promoting an increased Cu translocation. This new A. luchuensis strain showed endophytic character and plant growth-promotion activity, high metal tolerance, and an ability to increase copper translocation. We propose it as a novel, effective and sustainable bioremediation strategy for copper-polluted soils.

Soil Type Influences Novel "Milpa" Isolates of Trichoderma virens and Aspergillus tubingensis That Promote Solubilization, Mineralization, and Phytoabsorption of Phosphorus in Capsicum annuum L.

The Capsicum genus has significant economic importance since it is cultivated and consumed worldwide for its flavor and pungent properties. In 2021, Mexico produced 3.3 billion tons on 45,000 hectares which yielded USD 2 billion in exports to the USA, Canada, Japan, etc. Soil type has a dramatic effect on phosphorus (P) availability for plantsdue to its ion retention.In a previous study, novel fungal isolates were shown to solubilize and mineralize P in different kinds of soils with different P retention capacities. The aim of this work was to study the effects of the mineralogy of different kinds of "milpa" soils on the germination, biomass production, and P absorption of chili plants (Capsicum annuum). The germination percentage, the germination speed index, and the mean germination time were significantly increased in the plants treated with dual inoculation. Foliar phosphorus, growth variables, and plant biomass of chili plants grown in a greenhouse were enhanced in different soil types and with different inocula. Correlation studies suggested that the most significant performance in the foliar P concentration and in the growth response of plants was achieved in Vertisol with dual inoculation of 7 × 106 mL-1 spores per chili plant, suggesting this would be an appropriate approach to enhance chili cultivation depending on the soil type. This study stresses the importance of careful analysis of the effect of the soil type in the plant-microbe interactions.

Surviving in the Brine: A Multi-Omics Approach for Understanding the Physiology of the Halophile Fungus Aspergillus sydowii at Saturated NaCl Concentration.

Although various studies have investigated osmoadaptations of halophilic fungi to saline conditions, only few analyzed the fungal mechanisms occurring at saturated NaCl concentrations. Halophilic Aspergillus sydowii is a model organism for the study of molecular adaptations of filamentous fungi to hyperosmolarity. For the first time a multi-omics approach (i.e., transcriptomics and metabolomics) was used to compare A. sydowii at saturated concentration (5.13 M NaCl) to optimal salinity (1 M NaCl). Analysis revealed 1,842 genes differentially expressed of which 704 were overexpressed. Most differentially expressed genes were involved in metabolism and signal transduction. A gene ontology multi-scale network showed that ATP binding constituted the main network node with direct interactions to phosphorelay signal transduction, polysaccharide metabolism, and transferase activity. Free amino acids significantly decreased and amino acid metabolism was reprogrammed at 5.13 M NaCl. mRNA transcriptional analysis revealed upregulation of genes involved in methionine and cysteine biosynthesis at extreme water deprivation by NaCl. No modifications of membrane fatty acid composition occurred. Upregulated genes were involved in high-osmolarity glycerol signal transduction pathways, biosynthesis of ß-1,3-glucans, and cross-membrane ion transporters. Downregulated genes were related to the synthesis of chitin, mannose, cell wall proteins, starvation, pheromone synthesis, and cell cycle. Non-coding RNAs represented the 20% of the total transcripts with 7% classified as long non-coding RNAs (lncRNAs). The 42% and 69% of the total lncRNAs and RNAs encoding transcription factors, respectively, were differentially expressed. A network analysis showed that differentially expressed lncRNAs and RNAs coding transcriptional factors were mainly related to the regulation of metabolic processes, protein phosphorylation, protein kinase activity, and plasma membrane composition. Metabolomic analyses revealed more complex and unknown metabolites at saturated NaCl concentration than at optimal salinity. This study is the first attempt to unravel the molecular ecology of an ascomycetous fungus at extreme water deprivation by NaCl (5.13 M). This work also represents a pioneer study to investigate the importance of lncRNAs and transcriptional factors in the transcriptomic response to high NaCl stress in halophilic fungi.

Loosenin, a novel protein with cellulose-disrupting activity from Bjerkandera adusta.

BACKGROUND: Expansins and expansin-like proteins loosen cellulose microfibrils, possibly through the rupture of intramolecular hydrogen bonds. Together with the use of lignocellulolytic enzymes, these proteins are potential molecular tools to treat plant biomass to improve saccharification yields. RESULTS: Here we describe a new type of expansin-related fungal protein that we have called loosenin. Its corresponding gene, loos1, from the basidiomycete Bjerkandera adusta, was cloned and heterologously expressed in Saccharomyces cerevisiae. LOOS1 is distantly related to plant expansins through the shared presence of a DPBB domain, however domain II found in plant expansins is absent. LOOS1 binds tightly to cellulose and chitin, and we demonstrate that cotton fibers become susceptible to the action of a commercial cellulase following treatment with LOOS1. Natural fibers of Agave tequilana also become susceptible to hydrolysis by cellulases after loosenin treatment. CONCLUSIONS: LOOS1 is a new type of protein with disrupting activity on cellulose. LOOS1 binds polysaccharides, and given its enhancing properties on the action of hydrolytic enzymes, LOOS1 represents a potential additive in the production of fermentable sugars from lignocellulose.

Evaluation of different lignocellulosic substrates for the production of cellulases and xylanases by the basidiomycete fungi Bjerkandera adusta and Pycnoporus sanguineus.

Agricultural waste products are potential resources for the production of a number of industrial compounds, including biofuels. Basidiomycete fungi display a battery of hydrolytic enzymes with prospective use in lignocellulosic biomass transformation, however little work has been done regarding the characterization of such activities. Growth in several lignocellulosic substrates (oak and cedar sawdust, rice husk, corn stubble, wheat straw and Jatropha seed husk) and the production of cellulases and xylanases by two basidiomycete fungi: Bjerkandera adusta and Pycnoporus sanguineus were analyzed. Growth for P. sanguineus was best in rice husk while corn stubble supported the highest growth rate for B. adusta. Among the substrates tested, cedar sawdust produced the highest cellulolytic activities in both fungal species, followed by oak sawdust and wheat straw. Xylanolytic activity was best in oak and cedar sawdust for both species. We found no correlation between growth and enzyme production. Zymogram analysis of xylanases and cellulases showed that growth in different substrates produced particular combinations of protein bands with hydrolytic activity.

A Versatile Peroxidase from the Fungus Bjerkandera adusta Confers Abiotic Stress Tolerance in Transgenic Tobacco Plants.

White-rot fungi are efficient lignin degraders due to the secretion of lignin peroxidase, manganese peroxidase, laccase, and versatile peroxidase (VP) on decayed wood. The VP is a high-redox-potential enzyme and could be used to detoxify reactive oxygen species (ROS), which accumulate in plants during biotic and abiotic stresses. We cloned the VP gene and expressed it via the Agrobacterium transformation procedure in transgenic tobacco plants to assay their tolerance to different abiotic stress conditions. Thirty independent T2 transgenic VP lines overexpressing the fungal Bjerkandera adustaVP gene were selected on kanamycin. The VP22, VP24, and VP27 lines showed significant manganese peroxidase (MnP) activity. The highest was VP22, which showed 10.87-fold more manganese peroxidase activity than the wild-type plants and led to a 34% increase in plant height and 28% more biomass. The VP22, VP24, and VP27 lines showed enhanced tolerance to drought, 200 mM NaCl, and 400 mM sorbitol. Also, these transgenics displayed significant tolerance to methyl viologen, an active oxygen-generating compound. The present data indicate that overproducing the VP gene in plants increases significantly their biomass and the abiotic stress tolerance. The VP enzyme is an effective biotechnological tool to protect organisms against ROS. In transgenic tobacco plants, it improves drought, salt, and oxidative stress tolerance. Thus, the VP gene represents a great potential for obtaining stress-tolerant crops.

Physcomitrium patens Infection by Colletotrichum gloeosporioides: Understanding the Fungal-Bryophyte Interaction by Microscopy, Phenomics and RNA Sequencing.

Anthracnose caused by the hemibiotroph fungus Colletotrichum gloeosporioides is a devastating plant disease with an extensive impact on plant productivity. The process of colonization and disease progression of C. gloeosporioides has been studied in a number of angiosperm crops. To better understand the evolution of the plant response to pathogens, the study of this complex interaction has been extended to bryophytes. The model moss Physcomitrium patens Hedw. B&S (former Physcomitrella patens) is sensitive to known bacterial and fungal phytopathogens, including C. gloeosporioides, which cause infection and cell death. P. patens responses to these microorganisms resemble that of the angiosperms. However, the molecular events during the interaction of P. patens and C. gloeosporioides have not been explored. In this work, we present a comprehensive approach using microscopy, phenomics and RNA-seq analysis to explore the defense response of P. patens to C. gloeosporioides. Microscopy analysis showed that appressoria are already formed at 24 h after inoculation (hai) and tissue colonization and cell death occur at 24 hai and is massive at 48 hai. Consequently, the phenomics analysis showed progressing browning of moss tissues and impaired photosynthesis from 24 to 48 hai. The transcriptomic analysis revealed that more than 1200 P. patens genes were differentially expressed in response to Colletotrichum infection. The analysis of differentially expressed gene function showed that the C. gloeosporioides infection led to a transcription reprogramming in P. patens that upregulated the genes related to pathogen recognition, secondary metabolism, cell wall reinforcement and regulation of gene expression. In accordance with the observed phenomics results, some photosynthesis and chloroplast-related genes were repressed, indicating that, under attack, P. patens changes its transcription from primary metabolism to defend itself from the pathogen.

Tracking gene expression, metabolic profiles, and biochemical analysis in the halotolerant basidiomycetous yeast Rhodotorula mucilaginosa EXF-1630 during benzo[a]pyrene and phenanthrene biodegradation under hypersaline conditions.

Polyaromatic phenanthrene (Phe) and benzo[a]pyrene (BaP) are highly toxic, mutagenic, and carcinogenic contaminants widely dispersed in nature, including saline environments. Polyextremotolerant Rhodotorula mucilaginosa EXF-1630, isolated from Arctic sea ice, was grown on a huge concentration range -10 to 500 ppm- of Phe and BaP as sole carbon sources at hypersaline conditions (1 M NaCl). Selected polycyclic aromatic hydrocarbons (PAHs) supported growth as well as glucose, even at high PAH concentrations. Initially, up to 40% of Phe and BaP were adsorbed, followed by biodegradation, resulting in 80% removal in 10 days. While extracellular laccase, peroxidase, and un-specific peroxygenase activities were not detected, NADPH-cytochrome c reductase activity peaked at 4 days. The successful removal of PAHs and the absence of toxic metabolites were confirmed by toxicological tests on moss Physcomitrium patens, bacterium Aliivibrio fischeri, human erythrocytes, and pulmonary epithelial cells (A549). Metabolic profiles were determined at the midpoint of the biodegradation exponential phase, with added Phe and BaP (100 ppm) and 1 M NaCl. Different hydroxylated products were found in the culture medium, while the conjugative metabolite 1-phenanthryl-ß-D-glucopyranose was detected in the medium and in the cells. Transcriptome analysis resulted in 870 upregulated and 2,288 downregulated transcripts on PAHs, in comparison to glucose. Genomic mining of 61 available yeast genomes showed a widespread distribution of 31 xenobiotic degradation pathways in different yeast lineages. Two distributions with similar metabolic capacities included black yeasts and mainly members of the Sporidiobolaceae family (including EXF-1630), respectively. This is the first work describing a metabolic profile and transcriptomic analysis of PAH degradation by yeast.