Ascomycota Research Articles

Checklist, typification details, and nomenclature status of ascomycetous fungi originally described in Sri Lanka

Despite being a biodiversity-rich country, Sri Lanka’s fungal diversity remains largely unexplored. In the 19th century, British mycologists conducted extensive research, leading to the identification of more than 1,800 fungal taxa, including ascomycetous and basidiomycetous species. However, the majority of these taxa have not been revisited since their initial description, and molecular evidence is lacking. Moreover, a significant number of fungal taxa have been deemed invalid or illegitimate, indicating the need for a nomenclatural revision. To address this knowledge gap, this review provides a comprehensive list of ascomycetous taxa (including both lichen-forming and non-lichenized) that were originally described in Sri Lanka. Names are listed according to the original names which were included in the protologue. In the cases where species have been transferred to other genera, the current names and new classifications are provided. The typification details, nomenclature status, and classification are also presented. This checklist will serve as a valuable resource for the future epitypification of old taxa, as much of the existing information is poorly documented and scattered.

A bioinformatic workflow for in silico secretome prediction with the lignocellulose degrading ascomycete fungus Parascedosporium putredinis NO1.

The increasing availability of microbial genome sequences provides a reservoir of information for the identification of new microbial enzymes. Genes encoding proteins engaged in extracellular processes are of particular interest as these mediate the interactions microbes have with their environments. However, proteomic analysis of secretomes is challenging and often captures intracellular proteins released through cell death and lysis. Secretome prediction workflows from sequence data are commonly used to filter proteins identified through proteomics but are often simplified to a single step and are not evaluated bioinformatically for their effectiveness. Here, a workflow to predict a fungal secretome was designed and applied to the coding regions of the Parascedosporium putredinis NO1 genome. This ascomycete fungus is an exceptional lignocellulose degrader from which a new lignin-degrading enzyme has previously been identified. The 'secretome isolation' workflow is based on two strategies of localisation prediction and secretion prediction each utilising multiple available tools. The workflow produced three final secretomes with increasing levels of stringency. All three secretomes showed increases in functional annotations for extracellular processes and reductions in annotations for intracellular processes. Multiple sequences isolated as part of the secretome lacked any functional annotation and made exciting candidates for novel enzyme discovery.

Deciphering the role of fungi in post-fire succession in Canada's Great Lakes–St. Lawrence forest region

Fungi are important drivers of forest health, as well as culturally important for many Indigenous communities. In Canada, increasingly intense and frequent wildfires affect fungal substrates and habitats, environmental conditions, and physiological responses of hosts with which fungi may interact. In Ontario, aggressive fire suppression raises the question of how fungal regeneration, and taxonomic compositions, may be affected when fires do appear on the landscape. This study investigates the fungal regrowth post-fire above and below-ground and the role fungal species play in post fire ecological succession in Southeastern Ontario. The study sites are two black oak savannas in restoration, previously prescribed a burn (Northumberland County, 2022 and 2023), and a recent wildfire burned mixed deciduous forest (Centennial Lake, 2023). We collected fruiting body and soil samples for fungal identification via observational and DNA methods. Above ground vegetation assays were made at 9 burned plots within these three sites and another 8 adjacent unburned (control) plots. We extracted soil DNA and will amplify fungal ITS2 amplicons by PCR. Then, we will sequence the amplified DNA using MiSeq to compare burned and unburned soil fungi. Lastly, we will use statistical methods to differentiate effects from fire and soil texture and type in burned and control plots. Thus far, we found that Pyropyxis rubra, a pyrophilous ascomycete fungus, is extremely abundant just weeks after the lightning-induced fire at Centennial Lake. Our combined observational and quantitative descriptions of above-and below ground soil fungi provide a deeper understanding of how fire affects species composition, fungal reproduction, and population dynamics in Ontario. This research may help inform other successional processes, and fungal harvesting of culturally important species.

Diversity of hyperparasitic fungi on Meliolales (Sordariomycetes, Ascomycota): new species, records, and molecular data from Benin and Panama

Meliolales (black mildews) is an order of plant parasitic ascomycetous fungi in the tropics and subtropics. They are frequently overgrown and parasitized by other fungi, known as hyperparasites. During the last few years, species of hyperparasitic fungi on Meliolales have been collected in Benin and Panama. A new species of Paranectria and seven new reports of hyperparasites of different systematic groups are presented here with detailed descriptions and illustrations, together with new data concerning fungal hosts and host plants. The new species is called Paranectria longiappendiculata, characterized by exceptionally long appendages carried by the ascospores. New records for Benin and Panama are Calloriopsis herpotricha, Dimerosporiella cephalosporii, Isthmospora glabra, Isthmospora trichophila, Malacaria meliolicola, Paranectriella hemileiae, and Paranectriella minuta. Calloriopsis herpotricha is recorded for Africa and D. cephalosporii and P. hemileiae for America for the first time, suggesting an apparently pantropical distribution. Findings show a blatant lack of investigation on hyperparasitic fungi in the tropics. The phylogenetic positions of three of these newly reported species, C. herpotricha, D. cephalosporii, and P. minuta, are shown based on the analysis of internal transcribed spacer (ITS), large subunit (LSU), and small subunit (SSU) rDNA sequences. These sequences were generated in the context of the present study for the first time.

Exploring the Diversity and Systematics of Phaeosphaeriaceae: Taxonomic Novelties from Ecologically Diverse Habitats and Their Phylogenetic Resolution.

The family Phaeosphaeriaceae is a diverse group of ascomycetous fungi that are commonly found in terrestrial habitats worldwide. In recent years, there has been increasing interest in the biodiversity of Phaeosphaeriaceae in China, particularly in Sichuan Province, which has not been fully explored for its high fungal diversity. In our study, we conducted extensive surveys in Sichuan Province to identify and describe new species of Ascomycota with diverse habitats. Here, we present a taxonomic revision of Phaeosphaeriaceae with taxonomic novelties from terrestrial habitats in Sichuan Province based on a multi-gene phylogenetic approach. Our study focuses on the description of four new species of Phaeosphaeriaceae, representing novel additions to the diversity of this fungal family. Using a combination of morphological and molecular data, we provide detailed descriptions of the new taxa and their placement within the family. Additionally, we discuss the phylogenetic relationships of these new taxa with other members of Phaeosphaeriaceae, providing insights into the correct taxonomic classification of the family. Our taxonomic revision contributes to understanding fungal diversity in China and provides a foundation for future studies investigating the taxonomy and ecological roles of Phaeosphaeriaceae fungi. Furthermore, our multi-gene phylogenetic approach provides increased resolution and accuracy in the delimitation of species boundaries within the family. Our study highlights the importance of continued exploration and taxonomic revision in order to fully understand the diversity and distribution of fungal species in China and beyond. New species:Paraloratospora sichuanensis, Phaeosphaeria chengduensis, P. sichuanensis, and Septoriella shoemakeri. New combinations:Paraloratospora breonadiae, P. fructigena, Septoriella ammophilicola, S. asparagicola, S. festucae, S. luzulae, and S. verrucispora. New names:Septoriella paradactylidis, and S. neomuriformis.

Helminthosporium paraoligosporum sp. nov., a new ascomycetous fungus from Ontario, Canada

A novel anamorphic ascomycetous fungus, Helminthosporium paraoligosporum (Pleosporales, Ascomycota), isolated from branches of linden trees is described and characterized. The species is morphologically distinct from other Helminthosporium spp. by shorter conidiophores and conidia, and culture characteristics. Phylogenetic analysis of a combined set of ITS and LSU sequence data also supports the establishment of a new species. H. paraoligosporum is clustered with H. caespitosum, H. oligosporum, and H. tiliae. The species in this clade have similar morphological characteristics and host affiliation. In particular, conidiophores of the species (except H. tiliae) can be reduced to elongated conidiogenous cells. H. caespitosum is associated with Betula spp., while the other species in this clade have been collected from Tilia spp. only.

The completed genome sequence of Pestalotiopsis versicolor, a pathogenic ascomycete fungus with implications for bayberry production

The pathogenic fungus Pestalotiopsis versicolor is a major etiological agent of fungal twig blight disease affecting bayberry trees. However, the lack of complete genome sequence information for this crucial pathogenic fungus hinders the molecular and genetic investigation of its pathogenic mechanism. To address this knowledge gap, we have generated the complete genome sequence of P. versicolor strain XJ27, employing a combination of Illumina, PacBio, and Hi-C sequencing technologies. This comprehensive genome sequence, comprising 7 chromosomes with an N50 contig size of 7,275,017 bp, a GC content ratio of 50.16%, and a total size of 50.80 Mb, encompasses 13,971 predicted coding genes. By performing comparative genomic analysis between P. versicolor and the genomes of eleven plant-pathogenic fungi, as well as three closely related fungi within the same group, we have gained initial insights into its evolutionary trajectory, particularly through gene family analysis. These findings shed light on the distinctive characteristics and evolutionary history of P. versicolor. Importantly, the availability of this high-quality genetic resource will serve as a foundational tool for investigating the biology, molecular pathogenesis, and virulence of P. versicolor. Furthermore, it will facilitate the development of more potent antifungal medications by uncovering potential vulnerabilities in its genetic makeup.

Engineering transcriptional regulation of pentose metabolism in Rhodosporidiumtoruloides for improved conversion of xylose to bioproducts

Efficient conversion of pentose sugars remains a significant barrier to the replacement of petroleum-derived chemicals with plant biomass-derived bioproducts. While the oleaginous yeast Rhodosporidiumtoruloides (also known as Rhodotorulatoruloides) has a relatively robust native metabolism of pentose sugars compared to other wild yeasts, faster assimilation of those sugars will be required for industrial utilization of pentoses. To increase the rate of pentose assimilation in R.toruloides, we leveraged previously reported high-throughput fitness data to identify potential regulators of pentose catabolism. Two genes were selected for further investigation, a putative transcription factor (RTO4_12978, Pnt1) and a homolog of a glucose transceptor involved in carbon catabolite repression (RTO4_11990). Overexpression of Pnt1 increased the specific growth rate approximately twofold early in cultures on xylose and increased the maximum specific growth by 18% while decreasing accumulation of arabitol and xylitol in fast-growing cultures. Improved growth dynamics on xylose translated to a 120% increase in the overall rate of xylose conversion to fatty alcohols in batch culture. Proteomic analysis confirmed that Pnt1 is a major regulator of pentose catabolism in R.toruloides. Deletion of RTO4_11990 increased the growth rate on xylose, but did not relieve carbon catabolite repression in the presence of glucose. Carbon catabolite repression signaling networks remain poorly characterized in R.toruloides and likely comprise a different set of proteins than those mainly characterized in ascomycete fungi.

First evidence of a horizontally-acquired GH-7 cellobiohydrolase from a longhorned beetle genome.

Xylophagous larvae of longhorned beetles (Coleoptera; Cerambycidae) efficiently break down polysaccharides of the plant cell wall, which make the bulk of their food, using a range of carbohydrate-active enzymes (CAZymes). In this study, we investigated the function and evolutionary history of the first identified example of insect-encoded members of glycoside hydrolase family 7 (GH7) derived from the Lamiinae Exocentrus adspersus. The genome of this beetle contained two genes encoding GH7 proteins located in tandem and flanked by transposable elements. Phylogenetic analysis revealed that the GH7 sequences of E. adspersus were closely related to those of Ascomycete fungi, suggesting that they were acquired through horizontal gene transfer (HGT) from fungi. However, they were more distantly related to those encoded by genomes of Crustacea and of protist symbionts of termites and cockroaches, supporting that the same enzyme family was recruited several times independently in Metazoa during the course of their evolution. The recombinant E. adspersus GH7 was found to primarily break down cellulose polysaccharides into cellobiose, indicating that it is a cellobiohydrolase, and could also use smaller cellulose oligomers as substrates. Additionally, the cellobiohydrolase activity was boosted by the presence of calcium chloride. Our findings suggest that the combination of GH7 cellobiohydrolases with other previously characterized endo-β-1,4-glucanases and β-glucosidases allows longhorned beetles like E. adspersus to efficiently break down cellulose into monomeric glucose.

Deciphering the kinetics and pathway of lindane biodegradation by novel soil ascomycete fungi for its implication in bioremediation

Lindane, an organochlorine pesticide, negatively affects living beings and the ecosystem. In this study, the potential of 9 Ascomycetes fungi, isolated from an hexachlorocyclohexane dumpsite soil, was tested for biodegradation of lindane. The strain Pleurostoma richardsiae (FN5) showed lindane biodegradation rate constant (K value) of 0.144 d-1 and a half-life of 4.8d. The formation of intermediate metabolites upon lindane degradation including γ-pentachlorocyclohexene, 2,4-dichlorophenol, phenol, benzene, 1,3- cyclohexadiene, and benzoic acid detected by GC–MS and the potential pathway adopted by the novel fungal strain FN5 for lindane biodegradation has been elucidated. The study of gene profiles with reference to linA and linB in strain FN5 confirmed the same protein family with the reported heterologs from other fungal strains in the NCBI database. This study for the first time provides a thorough understanding of lindane biodegradation by a novel soil-borne Ascomycota fungal strain for its possible application in field-scale bioremediation.

Pleomorphic Dematiomelanommayunnanense gen. et sp. nov. (Ascomycota, Melanommataceae) from grassland vegetation in Yunnan, China.

During a survey of microfungi associated with grasslands and related vegetation types from Yunnan Province in China, various ascomycetous and coelomycetous fungi were isolated. This study reports the discovery of four strains of ascomycetous and coelomycetous fungi from dead stalks of Hypericummonogynum L. (Hypericaceae) and Rubusparvifolius L. (Rosaceae) in the Zhaotong region of Yunnan Province, China. The isolates were characterized using multi-locus phylogenetic analyses and were found to represent a new monophyletic lineage in Melanommataceae (Pleosporales, Dothideomycetes). This new clade was named as Dematiomelanommayunnanense gen. et sp. nov. which consists of both sexual and asexual morphs. The sexual morph is characterized by globose to subglobose ascomata with a central ostiole, cylindrical asci with a pedicel and ocular chamber, and muriform, ellipsoidal to fusiform ascospores. The asexual morph has synanamorphs including both brown, muriform macroconidia and hyaline, round to oblong or ellipsoidal microconidia. These findings contribute to the understanding of fungal diversity in grasslands and related vegetation types in Yunnan Province, China.

Tackling fungal diversity in lichen symbioses: molecular and morphological data recognize new lineages in Chaetothyriales (Eurotiomycetes, Ascomycota)

Lichens have been reappraised as self-sustaining and long-living ecosystems in which a multiplicity of microorganisms are housed, in addition to the main symbiotic partners. Lichen-associated microfungi can frequently occur cryptically, and their species diversity has recently been more fully elucidated by DNA metabarcoding studies and culture isolations. These lichen-associated fungi represent a wide array of major lineages in ascomycetes and basidiomycetes, including both filamentous and yeast species. Thanks to culture isolations, the morphology of a subset of the lichen-associated microfungal diversity has been studied. Metabarcoding analyses have shown high diversity of ascomycetous lichen-associated fungi in the two cosmopolitan rock-inhabiting lichens – Rhizoplaca melanophthalma and Tephromela atra – and many of these taxa were successfully isolated in culture. Based on DNA sequence data and morphological analyses, two new lineages within Chaetothyriales are here recognized. Both occur in lichens from dry habitats and are described here as the new species Cladophialophora endolichena Cometto, de Hoog, Muggia and Paracladophialophora lichenicola Cometto, de Hoog, Muggia. Other strains are placed in Pleostigmataceae, Trichomeriaceae, Pleosporales, Mycosphaerellales, Coniochaetales and Hypocreales, further filling gaps of knowledge of the high fungal diversity residing in lichen thalli.

An Ustilaginoidea virens glycoside hydrolase 42 protein is an essential virulence factor and elicits plant immunity as a PAMP.

Rice false smut, caused by the ascomycete fungus Ustilaginoidea virens, which infects rice florets before heading, severely threatens rice grain yield and quality worldwide. The U. virens genome encodes a number of glycoside hydrolase (GH) proteins. So far, the functions of these GHs in U. virens are largely unknown. In this study, we identified a GH42 protein secreted by U. virens, named UvGHF1, that exhibits β-galactosidase activity. UvGHF1 not only functions as an essential virulence factor during U. virens infection, but also serves as a pathogen-associated molecular pattern (PAMP) in Nicotiana benthamiana and rice. The PAMP activity of UvGHF1 is independent of its β-galactosidase activity. Moreover, UvGHF1 triggers cell death in N. benthamiana in a BAK1-dependent manner. Ectopic expression of UvGHF1 in rice induces pattern-triggered immunity and enhances rice resistance to fungal and bacterial diseases. RNA-seq analysis revealed that UvGHF1 expression in rice not only activates expression of many defence-related genes encoding leucine-rich repeat receptor-like kinases and WRKY and ERF transcription factors, but also induces diterpenoid biosynthesis and phenylpropanoid biosynthesis pathways. Therefore, UvGHF1 contributes to U. virens virulence, but is also recognized by the rice surveillance system to trigger plant immunity.

Field evaluation of biological wound treatments for the management of almond, cherry, and grapevine fungal canker diseases

The ascomycete fungi that cause wood canker diseases in perennial, horticultural crops are often generalist pathogens able to infect multiple host plants. These pathogens frequently infect pruning wounds, and the protection of such wounds with chemicals or biological control agents (BCAs) is an essential component of disease management. This study aimed at assessing four commercial BCAs for their ability to prevent almond, cherry, and grapevine pruning wound infections by three canker pathogens under field conditions for two growing seasons: Calosphaeria pulchella in cherry, Neofusicoccum parvum in almond and grapevine, and Eutypa lata in all three hosts. The two bacterial BCAs tested, Bacillus amyloliquefasciens MBI 600 and Streptomyces lydicus WYEC 108, performed poorly overall as infection rates in treated wounds were not significantly different than in the water-treated wounds for most pathogens in all crops. Trichoderma atroviride SC1 provided the best protection against E. lata and N. parvum in grapevine, achieving superior or equivalent protection than the standard conventional chemical thiophanate-methyl. Field trials also demonstrated that Clonostachys rosea J1446 provided satisfactory protection of pruning wounds against E. lata and C. pulchella in cherry and against E. lata and N. parvum in almond trees. The identification of effective BCAs for use in multiple crops and against multiple pathogens is a great step towards a sustainable management of fungal canker diseases by mitigating the negative impacts of chemical fungicides.

Transcriptome Metabolic Characterization of Tuber borchii SP1-A New Spanish Strain for In Vitro Studies of the Bianchetto Truffle.

Truffles are ascomycete hypogeous fungi belonging to the Tuberaceae family of the Pezizales order that grow in ectomycorrhizal symbiosis with tree roots, and they are known for their peculiar aromas and flavors. The axenic culture of truffle mycelium is problematic because it is not possible in many cases, and the growth rate is meager when it is possible. This limitation has prompted searching and characterizing new strains that can be handled in laboratory conditions for basic and applied studies. In this work, a new strain of Tuber borchii (strain SP1) was isolated and cultured, and its transcriptome was analyzed under different in vitro culture conditions. The results showed that the highest growth of T. borchii SP1 was obtained using maltose-enriched cultures made with soft-agar and in static submerged cultures made at 22 °C. We analyzed the transcriptome of this strain cultured in different media to establish a framework for future comparative studies, paying particular attention to the central metabolic pathways, principal secondary metabolite gene clusters, and the genes involved in producing volatile aromatic compounds (VOCs). The results showed a transcription signal for around 80% of the annotated genes. In contrast, most of the transcription effort was concentrated on a limited number of genes (20% of genes account for 80% of the transcription), and the transcription profile of the central metabolism genes was similar in the different conditions analyzed. The gene expression profile suggests that T. borchii uses fermentative rather than respiratory metabolism in these cultures, even in aerobic conditions. Finally, there was a reduced expression of genes belonging to secondary metabolite clusters, whereas there was a significative transcription of those involved in producing volatile aromatic compounds.

Disruption of non-classically secreted protein (MoMtp) compromised conidiation, stress homeostasis, and pathogenesis of Magnaporthe oryzae

Blast disease, caused by the hemibiotrophic ascomycete fungus, Magnaporthe oryzae, is a significant threat to sustainable rice production worldwide. Studies have shown that the blast fungus secretes vast arrays of functionally diverse proteins into the host cell for a successful disease progression. However, the final destinations of these effector proteins inside the host cell and their role in advancing fungal pathogenesis remain a mystery. Here, we reported that a putative mitochondrial targeting non-classically secreted protein (MoMtp) positively regulates conidiogenesis and appressorium maturation in M. oryzae. Moreover, MoMTP gene deletion mutant strains triggered a hypersensitive response when inoculated on rice leaves displaying that MoMtp is essential for the virulence of M. oryzae. In addition, cell wall and oxidative stress results indicated that MoMtp is likely involved in the maintenance of the structural integrity of the fungus cell. Our study also demonstrates an upregulation in the expression pattern of the MoMTP gene at all stages of infection, indicating its possible regulatory role in host invasion and the infectious development of M. oryzae. Furthermore, Agrobacterium infiltration and sheath inoculation confirmed that MoMtp-GFP protein is predominantly localized in the host mitochondria of tobacco leaf and rice cells. Taken together, we conclude that MoMtp protein likely promotes the normal conidiation and pathogenesis of M. oryzae and might have a role in disturbing the proper functioning of the host mitochondria during pathogen invasion.

Microdochium sichuanense sp. nov. (Microdochiaceae, Xylariales), from a Poaceae host in Sichuan, China

During our investigation of ascomycetes fungi on Poaceae, a microdochium-like fungus was collected from dead grass leaves from a terrestrial habitat in Chengdu, Sichuan Province, China. Based on morphological characterization and phylogenetic analyses, we introduce this fungus as Microdochium sichuanense sp. nov. Maximum likelihood, and Bayesian analyses of combined genes (LSU, ITS and rpb2) were performed to clarify the phylogenetic affinities of the new species. Microdochium sichuanense is phylogenetically closely related to M. hainanense and morphologically it fits the generic characteristics of Microdochium species in having ostiolate ascomata, peridium cells of textura angularis to textura prismatica, unitunicate, clavate to oblong, 8-spored asci and hyaline, oval to fusiform ascospores. The new species was compared with phylogenetically and morphologically closely related species, and detailed descriptions, micrographs, and phylogenetic trees of the new species are provided.

A chromosome-scale genome assembly of the grape powdery mildew pathogen Erysiphe necator reveals its genomic architecture and previously unknown features of its biology.

Erysiphe necator is an obligate fungal pathogen that causes grape powdery mildew, globally the most important disease on grapevines. Previous attempts to obtain a quality genome assembly for this pathogen were hindered by its high repetitive DNA content. Here, chromatin conformation capture (Hi-C) with long-read PacBio sequencing was combined to obtain a chromosome-scale assembly and a high-quality annotation for E. necator isolate EnFRAME01. The resulting 81.1 Mb genome assembly is 98% complete and consists of 34 scaffolds, 11 of which represent complete chromosomes. All chromosomes contain large centromeric-like regions and lack synteny to the 11 chromosomes of the cereal PM pathogen Blumeria graminis. Further analysis of their composition showed that repeats and transposable elements (TEs) occupy 62.7% of their content. TEs were almost evenly interspersed outside centromeric and telomeric regions and massively overlapped with regions of annotated genes, suggesting that they could have a significant functional impact. Abundant gene duplicates were observed as well, particularly in genes encoding candidate secreted effector proteins. Moreover, younger in age gene duplicates exhibited more relaxed selection pressure and were more likely to be located physically close in the genome than older duplicates. A total of 122 genes with copy number variations among six isolates of E. necator were also identified and were enriched in genes that were duplicated in EnFRAME01, indicating they may reflect an adaptive variation. Taken together, our study illuminates higher-order genomic architectural features of E. necator and provides a valuable resource for studying genomic structural variations in this pathogen. IMPORTANCE Grape powdery mildew caused by the ascomycete fungus Erysiphe necator is economically the most important and recurrent disease in vineyards across the world. The obligate biotrophic nature of E. necator hinders the use of typical genetic methods to elucidate its pathogenicity and adaptation to adverse conditions, and thus comparative genomics has been a major method to study its genome biology. However, the current reference genome of E. necator isolate C-strain is highly fragmented with many non-coding regions left unassembled. This incompleteness prohibits in-depth comparative genomic analyses and the study of genomic structural variations (SVs) that are known to affect several aspects of microbial life, including fitness, virulence, and host adaptation. By obtaining a chromosome-scale genome assembly and a high-quality gene annotation for E. necator, we reveal the organization of its chromosomal content, unearth previously unknown features of its biology, and provide a reference for studying genomic SVs in this pathogen.

VmPacC-mediated pH regulation of Valsa mali confers to host acidification identified by comparative proteomics analysis

Apple valsa canker caused by the Ascomycete fungus Valsa mali is one of the most serious diseases of apple, resulting in huge economic losses in the apple-growing area of China. Previous study found that the pathogen could acidify the infected tissues to make lower ambient pH (from 6.0 to 3.5) for their successfully colonization. The pH signaling transcription factor VmPacC is required for acidification of its environment and for full virulence in V. mali. It is known that the functional cooperation of proteins secreted by V. mali plays pivotal role in its successful colonization of host plants. In this study, we used tandem mass tag (TMT) labeling coupled with LC-MS/MS-based quantitative proteomics to analyze the VmPacC-mediated pH regulation in V. mali, focusing on differentially expressed proteins (DEPs). We identified 222 DEPs specific to VmPacC deletion, and 921 DEPs specific to different pH conditions (pH 6.0 and 3.4). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that these DEPs were mainly involved in pathways associated with carbon metabolism, biosynthesis of antibiotics, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, glutathione metabolism, ribosomes, and pentose phosphate pathways. Additionally, we identified 119 DEPs that were shared among the VmPacC deletion mutant and different pH conditions, which were mainly related to energy metabolism pathways, providing the energy required for the hyphal growth and responses to environmental stresses. A protein-protein interaction (PPI) network analysis indicated that most of the shared proteins were mapped to an interaction network with a medium confidence score of 0.4. Notably, one uncharacterized protein (KUI69106.1), and two known proteins (heat shock protein 60 (KUI73579.1), aspartate aminotransferase (KUI73864.1)) located in the core of the network were highly connected (with ≥ 38 directed edges) with the other shared DEPs. Our results suggest that VmPacC participates in the pathogen’s regulation to ambient pH through the regulation of energy metabolism pathways such as the glycolysis/gluconeogenesis pathway and TCA cycle. Finally, we proposed a sophisticated molecular regulatory network to explain pH decrease in V. mali. Our study, by providing insights into V. mali regulating pH, helps to elucidate the mechanisms of host acidification during pathogen infection.

Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role.

Multicopper oxidases are promiscuous biocatalysts with great potential for the production of industrial compounds. This study is focused on the elucidation of the structure-function determinants of a novel laccase-like multicopper oxidase from the thermophilic fungus Thermothelomyces thermophila (TtLMCO1), which is capable of oxidizing both ascorbic acid and phenolic compounds and thus is functionally categorized between the ascorbate oxidases and fungal ascomycete laccases (asco-laccases). The crystal structure of TtLMCO1, determined using an AlphaFold2 model due to a lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations showed that the ability of TtLMCO1 to oxidize ortho-substituted phenols stems from the movement of two polar amino acids at the hydrophilic side of the substrate-binding region, providing structural evidence for the promiscuity of this enzyme.