Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in MPMI's June 2015 issue.

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Secondary Metabolites of the Rice Blast Fungus Pyricularia oryzae: Biosynthesis and Biological Function

International Journal of Molecular Sciences ◽

10.3390/ijms21228698 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8698

Author(s):

Takayuki Motoyama

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Rice Blast ◽

Plant Pathogens ◽

Pathogenic Fungus ◽

Pathogenic Fungi ◽

Pyricularia Oryzae ◽

Blast Disease ◽

Plant Pathogenic Fungi ◽

Biosynthetic Genes

Plant pathogenic fungi produce a wide variety of secondary metabolites with unique and complex structures. However, most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Moreover, the relationship between pathogenicity and secondary metabolites remains unclear. To activate silent gene clusters in fungi, successful approaches such as epigenetic control, promoter exchange, and heterologous expression have been reported. Pyricularia oryzae, a well-characterized plant pathogenic fungus, is the causal pathogen of rice blast disease. P. oryzae is also rich in secondary metabolism genes. However, biosynthetic genes for only four groups of secondary metabolites have been well characterized in this fungus. Biosynthetic genes for two of the four groups of secondary metabolites have been identified by activating secondary metabolism. This review focuses on the biosynthesis and roles of the four groups of secondary metabolites produced by P. oryzae. These secondary metabolites include melanin, a polyketide compound required for rice infection; pyriculols, phytotoxic polyketide compounds; nectriapyrones, antibacterial polyketide compounds produced mainly by symbiotic fungi including endophytes and plant pathogens; and tenuazonic acid, a well-known mycotoxin produced by various plant pathogenic fungi and biosynthesized by a unique NRPS-PKS enzyme.

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IMG-ABC: A Knowledge Base To Fuel Discovery of Biosynthetic Gene Clusters and Novel Secondary Metabolites

mBio ◽

10.1128/mbio.00932-15 ◽

2015 ◽

Vol 6 (4) ◽

Cited By ~ 66

Author(s):

Michalis Hadjithomas ◽

I-Min Amy Chen ◽

Ken Chu ◽

Anna Ratner ◽

Krishna Palaniappan ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Genomic Data ◽

Gene Clusters ◽

Metagenomic Data ◽

Integrated Analysis ◽

Analysis Tool ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Analysis Tools

ABSTRACTIn the discovery of secondary metabolites, analysis of sequence data is a promising exploration path that remains largely underutilized due to the lack of computational platforms that enable such a systematic approach on a large scale. In this work, we present IMG-ABC (https://img.jgi.doe.gov/abc), an atlas of biosynthetic gene clusters within the Integrated Microbial Genomes (IMG) system, which is aimed at harnessing the power of “big” genomic data for discovering small molecules. IMG-ABC relies on IMG's comprehensive integrated structural and functional genomic data for the analysis of biosynthetic gene clusters (BCs) and associated secondary metabolites (SMs). SMs and BCs serve as the two main classes of objects in IMG-ABC, each with a rich collection of attributes. A unique feature of IMG-ABC is the incorporation of both experimentally validated and computationally predicted BCs in genomes as well as metagenomes, thus identifying BCs in uncultured populations and rare taxa. We demonstrate the strength of IMG-ABC's focused integrated analysis tools in enabling the exploration of microbial secondary metabolism on a global scale, through the discovery of phenazine-producing clusters for the first time inAlphaproteobacteria. IMG-ABC strives to fill the long-existent void of resources for computational exploration of the secondary metabolism universe; its underlying scalable framework enables traversal of uncovered phylogenetic and chemical structure space, serving as a doorway to a new era in the discovery of novel molecules.IMPORTANCEIMG-ABC is the largest publicly available database of predicted and experimental biosynthetic gene clusters and the secondary metabolites they produce. The system also includes powerful search and analysis tools that are integrated with IMG's extensive genomic/metagenomic data and analysis tool kits. As new research on biosynthetic gene clusters and secondary metabolites is published and more genomes are sequenced, IMG-ABC will continue to expand, with the goal of becoming an essential component of any bioinformatic exploration of the secondary metabolism world.

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Characterizing the Pathogenic, Genomic, and Chemical Traits ofAspergillus fischeri, a Close Relative of the Major Human Fungal PathogenAspergillus fumigatus

mSphere ◽

10.1128/msphere.00018-19 ◽

2019 ◽

Vol 4 (1) ◽

Cited By ~ 11

Author(s):

Matthew E. Mead ◽

Sonja L. Knowles ◽

Huzefa A. Raja ◽

Sarah R. Beattie ◽

Caitlin H. Kowalski ◽

...

Keyword(s):

Secondary Metabolites ◽

Aspergillus Fumigatus ◽

Secondary Metabolism ◽

Human Disease ◽

Chemical Characterization ◽

Gene Clusters ◽

Invasive Disease ◽

Close Relative ◽

Low Oxygen ◽

Content Type

ABSTRACTAspergillus fischeriis closely related toAspergillus fumigatus, the major cause of invasive mold infections. Even thoughA. fischeriis commonly found in diverse environments, including hospitals, it rarely causes invasive disease. WhyA. fischericauses less human disease thanA. fumigatusis unclear. A comparison ofA. fischeriandA. fumigatusfor pathogenic, genomic, and secondary metabolic traits revealed multiple differences in pathogenesis-related phenotypes. We observed thatA. fischeriNRRL 181 is less virulent thanA. fumigatusstrain CEA10 in multiple animal models of disease, grows slower in low-oxygen environments, and is more sensitive to oxidative stress. Strikingly, the observed differences for some traits are of the same order of magnitude as those previously reported betweenA. fumigatusstrains. In contrast, similar to what has previously been reported, the two species exhibit high genomic similarity; ∼90% of theA. fumigatusproteome is conserved inA. fischeri, including 48/49 genes known to be involved inA. fumigatusvirulence. However, only 10/33A. fumigatusbiosynthetic gene clusters (BGCs) likely involved in secondary metabolite production are conserved inA. fischeriand only 13/48A. fischeriBGCs are conserved inA. fumigatus. Detailed chemical characterization ofA. fischericultures grown on multiple substrates identified multiple secondary metabolites, including two new compounds and one never before isolated as a natural product. Additionally, anA. fischerideletion mutant oflaeA, a master regulator of secondary metabolism, produced fewer secondary metabolites and in lower quantities, suggesting that regulation of secondary metabolism is at least partially conserved. These results suggest that the nonpathogenicA. fischeripossesses many of the genes important forA. fumigatuspathogenicity but is divergent with respect to its ability to thrive under host-relevant conditions and its secondary metabolism.IMPORTANCEAspergillus fumigatusis the primary cause of aspergillosis, a devastating ensemble of diseases associated with severe morbidity and mortality worldwide.A. fischeriis a close relative ofA. fumigatusbut is not generally observed to cause human disease. To gain insights into the underlying causes of this remarkable difference in pathogenicity, we compared two representative strains (one from each species) for a range of pathogenesis-relevant biological and chemical characteristics. We found that disease progression in multipleA. fischerimouse models was slower and caused less mortality thanA. fumigatus. Remarkably, the observed differences betweenA. fischeriandA. fumigatusstrains examined here closely resembled those previously described for two commonly studiedA. fumigatusstrains, AF293 and CEA10.A. fischeriandA. fumigatusexhibited different growth profiles when placed in a range of stress-inducing conditions encountered during infection, such as low levels of oxygen and the presence of chemicals that induce the production of reactive oxygen species. We also found that the vast majority ofA. fumigatusgenes known to be involved in virulence are conserved inA. fischeri, whereas the two species differ significantly in their secondary metabolic pathways. These similarities and differences that we report here are the first step toward understanding the evolutionary origin of a major fungal pathogen.

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Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr

Applied and Environmental Microbiology ◽

10.1128/aem.00293-19 ◽

2019 ◽

Vol 85 (14) ◽

Cited By ~ 2

Author(s):

Xiao-Ting Shen ◽

Xu-Hua Mo ◽

Li-Ping Zhu ◽

Ling-Ling Tan ◽

Feng-Yu Du ◽

...

Keyword(s):

Network Analysis ◽

Secondary Metabolites ◽

Pleurotus Ostreatus ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Molecular Network ◽

Terpene Synthase ◽

High Morbidity ◽

Content Type ◽

Trametes Robiniophila

ABSTRACT Candida albicans and Cryptococcus neoformans, human-pathogenic fungi found worldwide, are receiving increasing attention due to high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by coculturing 16 wood-decaying basidiomycetes, among which coculture of Trametes robiniophila Murr and Pleurotus ostreatus was found to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis revealed that 44 features were either newly synthesized or produced at high levels in this coculture system and that 6 of the features that belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with MICs of 1 to 32 μg/ml against pathogenic fungi. Furthermore, dynamic 13C-labeling analysis revealed an association between induced features and the corresponding fungi. Unusual sesterterpenes were 13C labeled only in P. ostreatus in a time course after stimulation by the coculture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr. Sesterterpene compounds 1 to 3 were renamed postrediene A to C. Real-time reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that transcriptional levels of three genes encoding terpene synthase, farnesyl-diphosphate farnesyltransferase, and oxidase were found to be 8.2-fold, 88.7-fold, and 21.6-fold higher, respectively, in the coculture than in the monoculture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A to postrediene C was then proposed based on structures of sesterterpenes and molecular network analysis. IMPORTANCE A number of gene clusters involved in biosynthesis of secondary metabolites are presumably silent or expressed at low levels under conditions of standard laboratory cultivation, resulting in a large gap between the pool of discovered metabolites and genome capability. This work mimicked naturally occurring competition by construction of an artificial coculture of basidiomycete fungi for the identification of secondary metabolites with novel scaffolds and excellent bioactivity. Unusual linear sesterterpenes of postrediene A to C synthesized by P. ostreatus not only were promising lead drugs against human-pathogenic fungi but also highlighted a distinct pathway for sesterterpene biosynthesis in basidiomycetes. The current work provides an important basis for uncovering novel gene functions involved in sesterterpene synthesis and for gaining insights into the mechanism of silent gene activation in fungal defense.

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Fumigation of Fruit with Short-Chain Organic Acids to Reduce the Potential of Postharvest Decay

Plant Disease ◽

10.1094/pdis.1998.82.6.689 ◽

1998 ◽

Vol 82 (6) ◽

pp. 689-693 ◽

Cited By ~ 40

Author(s):

P. L. Sholberg

Keyword(s):

Formic Acid ◽

Fungal Pathogens ◽

Citrus Fruit ◽

Penicillium Expansum ◽

Monilinia Fructicola ◽

Rhizopus Stolonifer ◽

Pome Fruit ◽

Fruit Peel ◽

Postharvest Decay ◽

Fruit Decay

Vapors of acetic (1.9 or 2.5 μl/liter), formic (1.2 μl/liter), and propionic (2.5 μl/liter) acids were tested for postharvest decay control on 8 cherry, 14 pome, and 3 citrus fruit cultivars. Surfacesterilized fruit were inoculated with known fungal pathogens by drying 20-μl drops of spore suspension on marked locations on each fruit, placing at 10°C to equilibrate for approximately 24 h, and fumigating by evaporating the above acids in 12.7-liter airtight fumigation chambers for 30 min. Immediately after fumigation, the fruit were removed, aerated, aseptically injured, and placed at 20°C until decay occurred. All three fumigants controlled Monilinia fructicola, Penicillium expansum, and Rhizopus stolonifer on cherry. Formic acid increased fruit pitting on six of eight cultivars and was the only organic acid to increase blackening of cherry stems when compared to the control. Decay of pome fruit caused by P. expansum was reduced from 98% to 16, 4, or 8% by acetic, formic, and propionic acids, respectively, without injury to the fruit. Decay of citrus fruit by P. digitatum was reduced from 86 to 11% by all three acids, although browning of the fruit peel was observed on grapefruit and oranges fumigated with formic acid.

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Fumigation of Empty Fruit Bins with Carbon Dioxide to Control Diapausing Codling Moth Larvae and Penicillium expansum Link. ex Thom Spores

HortScience ◽

10.21273/hortsci.39.2.429 ◽

2004 ◽

Vol 39 (2) ◽

pp. 429-432 ◽

Cited By ~ 11

Author(s):

J.E. Cossentine ◽

P.L. Sholberg ◽

L.B.J. Jensen ◽

K.E. Bedford ◽

T.C. Shephard

Keyword(s):

Carbon Dioxide ◽

Plant Pathogens ◽

Cydia Pomonella ◽

Pathogenic Fungi ◽

Codling Moth ◽

Penicillium Expansum ◽

Late Instar ◽

Fumigation Chamber ◽

The Impact ◽

Fruit Pathogens

Wooden fruit bins are a source of diapausing codling moth and postharvest pathogenic fungi. The redistribution of codling moths within bins is a problem where codling moth populations are being controlled by areawide codling moth sterile release programs, mating disruption programs, or both. Laboratory and fumigation chamber trials were carried out to determine the impact of relatively low levels of carbon dioxide on late-instar codling moth (Cydia pomonella L.) and two postharvest fruit pathogens, Penicillium expansum Link ex Thom and Botrytis cinerea Pers. ex Fr. Fumigation of diapausing codling moth with 40% CO2 in laboratory trials resulted in over 60% mortality after only 6 days of exposure and mortality increased with time of exposure. Significant mortality (68%) of diapausing codling moth larvae occurred after 14 days of exposure in the laboratory to 13% CO2 and a mean of 88% mortality was recorded after fumigation for 20 days. A significant number of P. expansum (46%) spores failed to germinate after laboratory exposure to 13% CO2 for 12 and 18 days respectively. Close to 100% of the P. expansum spores failed to germinate by day 20. When diapausing codling moth larvae and spores from both plant pathogens were placed in wooden fruit bins and fumigated for 21 days at 13% CO2, 75% of the diapausing codling moths died and 80% of the P. expansum spores failed to germinate. No effect on B. cinerea was observed.

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Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay

PeerJ ◽

10.7717/peerj.6170 ◽

2019 ◽

Vol 7 ◽

pp. e6170 ◽

Cited By ~ 5

Author(s):

Guangxi Wu ◽

Wayne M. Jurick II ◽

Franz J. Lichtner ◽

Hui Peng ◽

Guohua Yin ◽

...

Keyword(s):

Gene Cluster ◽

Gene Clusters ◽

Apple Fruit ◽

Penicillium Expansum ◽

Whole Genome ◽

Blue Mold ◽

Pome Fruit ◽

Fruit Decay ◽

Postharvest Rot ◽

Fungal Interactions

Blue mold is a postharvest rot of pomaceous fruits caused by Penicillium expansum and a number of other Penicillium species. The genome of the highly aggressive P. expansum strain R19 was re-sequenced and analyzed together with the genome of the less aggressive P. solitum strain RS1. Whole genome scale similarities and differences were examined. A phylogenetic analysis of P. expansum, P. solitum, and several closely related Penicillium species revealed that the two pathogens isolated from decayed apple with blue mold symptoms are not each other’s closest relatives. Among a total of 10,560 and 10,672 protein coding sequences respectively, a comparative genomics analysis revealed 41 genes in P. expansum R19 and 43 genes in P. solitum RS1 that are unique to these two species. These genes may be associated with pome fruit–fungal interactions, subsequent decay processes, and mycotoxin accumulation. An intact patulin gene cluster consisting of 15 biosynthetic genes was identified in the patulin producing P. expansum strain R19, while only a remnant, seven-gene cluster was identified in the patulin-deficient P. solitum strain. However, P. solitum contained a large number of additional secondary metabolite gene clusters, indicating that this species has the potential capacity to produce an array of known as well as not-yet-identified products of possible toxicological or biotechnological interest.

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Conservation of a gene cluster reveals novel cercosporin biosynthetic mechanisms and extends production to the genus Colletotrichum

10.1101/100545 ◽

2017 ◽

Author(s):

Ronnie de Jonge ◽

Malaika K. Ebert ◽

Callie R. Huitt-Roehl ◽

Paramita Pal ◽

Jeffrey C. Suttle ◽

...

Keyword(s):

Sugar Beet ◽

Gene Cluster ◽

Fungal Pathogens ◽

Plant Pathogens ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Phylogenomic Analysis ◽

First Case ◽

Horizontal Transfers ◽

Fungal Genus

AbstractSpecies in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean and other major food crops. Here we sequenced the genome of the sugar beet pathogen C. beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae. Although cercosporin biosynthesis has been thought to-date to rely on an eight gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein (CFP) previously shown to be involved with cercosporin auto-resistance, and four additional genes required for cercosporin biosynthesis including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Finally, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide new insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.Significance StatementSpecies in the fungal genus Cercospora cause diseases in many important crops worldwide. Their success as pathogens is largely due to the secretion of cercosporin during infection. We report that the cercosporin toxin biosynthesis (CTB) cluster is ancient and was horizontally transferred to diverse fungal pathogens on an unprecedented scale. Since these analyses revealed genes adjacent to the established CTB cluster, we evaluated their role in C. beticola to show that four are necessary for cercosporin biosynthesis. Finally, we confirmed that the apple pathogen Colletotrichum fioriniae produces cercosporin, the first case outside the family Mycosphaerellaceae. Other Colletotrichum plant pathogens also harbor the CTB cluster, which points to a wider concern that this toxin may play in virulence and human health.

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Complex evolutionary origins of specialized metabolite gene cluster diversity among the plant pathogenic fungi of theFusarium graminearumspecies complex

10.1101/639641 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sabina Moser Tralamazza ◽

Liliana Oliveira Rocha ◽

Ursula Oggenfuss ◽

Benedito Corrêa ◽

Daniel Croll

Keyword(s):

Gene Cluster ◽

Plant Pathogens ◽

De Novo ◽

Chromosomal Rearrangements ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Death Process ◽

Head Blight ◽

Fungal Genomes ◽

Gain Loss

AbstractFungal genomes encode highly organized gene clusters that underlie the production of specialized (or secondary) metabolites. Gene clusters encode key functions to exploit plant hosts or environmental niches. Promiscuous exchange among species and frequent reconfigurations make gene clusters some of the most dynamic elements of fungal genomes. Despite evidence for high diversity in gene cluster content among closely related strains, the microevolutionary processes driving gene cluster gain, loss and neofunctionalization are largely unknown. We analyzed theFusarium graminearumspecies complex (FGSC) composed of plant pathogens producing potent mycotoxins and causing Fusarium head blight on cereals. Wede novoassembled genomes of previously uncharacterized FGSC members (two strains ofF. austroamericanum,F. cortaderiaeandF. meridionale). Our analyses of eight species of the FGSC in addition to 15 otherFusariumspecies identified a pangenome of 54 gene clusters within FGSC. We found that multiple independent losses were a key factor generating extant cluster diversity within the FGSC and theFusariumgenus. We identified a modular gene cluster conserved among distantly related fungi, which was likely reconfigured to encode different functions. We also found strong evidence that a rare cluster in FGSC was gained through an ancient horizontal transfer between bacteria and fungi. Chromosomal rearrangements underlying cluster loss were often complex and were likely facilitated by an enrichment in specific transposable elements. Our findings identify important transitory stages in the birth and death process of specialized metabolism gene clusters among very closely related species.

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A detailed in silico analysis of secondary metabolite biosynthesis clusters in the genome of the broad host range plant pathogenic fungus Sclerotinia sclerotiorum

10.21203/rs.2.11845/v2 ◽

2019 ◽

Author(s):

Carolyn Graham-Taylor ◽

Lars G Kamphuis ◽

Mark Derbyshire

Keyword(s):

Secondary Metabolites ◽

Host Range ◽

Secondary Metabolite ◽

Sclerotinia Sclerotiorum ◽

Plant Pathogens ◽

Pathogenic Fungus ◽

Gene Clusters ◽

Sequence Diversity ◽

Broad Host Range ◽

Data Set

Abstract Background The broad host range pathogen Sclerotinia sclerotiorum infects over 400 plant species and causes substantial yield losses in crops worldwide. Secondary metabolites are known to play important roles in the virulence of plant pathogens, but little is known about the secondary metabolite repertoire of S. sclerotiorum. In this study, we predicted secondary metabolite biosynthetic gene clusters in the genome of S. sclerotiorum and analysed their expression during infection of Brassica napus using an existing transcriptome data set. We also investigated their sequence diversity among a panel of 25 previously published S. sclerotiorum isolate genomes.Results We identified 80 putative secondary metabolite clusters. Over half of the clusters contained at least three transcriptionally coregulated genes. Comparative genomics revealed clusters homologous to clusters in the closely related plant pathogen Botrytis cinerea for production of carotenoids, hydroxamate siderophores, DHN melanin and botcinic acid. We also identified putative phytotoxin clusters that can potentially produce the polyketide sclerin and an epipolythiodioxopiperazine. Secondary metabolite clusters were enriched in subtelomeric genomic regions, and those containing paralogues showed a particularly strong association with repeats. The positional bias we identified was borne out by intraspecific comparisons that revealed putative secondary metabolite genes suffered more presence / absence polymorphisms and exhibited a significantly higher sequence diversity than other genes.Conclusions These data suggest that S. sclerotiorum produces numerous secondary metabolites during plant infection and that their gene clusters undergo enhanced rates of mutation, duplication and recombination in subtelomeric regions. The microevolutionary regimes leading to S. sclerotiorum secondary metabolite diversity have yet to be elucidated. Several potential phytotoxins documented in this study provide the basis for future functional analyses.

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