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

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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Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1712798115 ◽

2018 ◽

Vol 115 (24) ◽

pp. E5459-E5466 ◽

Cited By ~ 27

Author(s):

Ronnie de Jonge ◽

Malaika K. Ebert ◽

Callie R. Huitt-Roehl ◽

Paramita Pal ◽

Jeffrey C. Suttle ◽

...

Keyword(s):

Sugar Beet ◽

Gene Cluster ◽

Plant Pathogens ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Cercospora Beticola ◽

Phylogenomic Analysis ◽

Food Crops ◽

Horizontal Transfers ◽

Insight Into

Species 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 Cercospora 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 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, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide 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.

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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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Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

10.1101/379305 ◽

2018 ◽

Author(s):

Malaika K. Ebert ◽

Rebecca E. Spanner ◽

Ronnie de Jonge ◽

David J. Smith ◽

Jason Holthusen ◽

...

Keyword(s):

Gene Cluster ◽

Plant Pathogens ◽

Phylogenetic Analyses ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Host Cells ◽

Plant Pathogenic Fungi ◽

Biosynthetic Pathways ◽

Biosynthetic Gene ◽

Melanin Production

SummaryPerylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease establishment. The sugar beet pathogenCercospora beticolasecretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis(CTB)gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs ofElsinoё fawcettiiandCladosporium phlei,respectively, based on gene cluster conservation with theCTBand hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identifiedC. beticola, E. fawcettii,andC. phleimelanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs inC. beticolaandE. fawcettiicoupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.Originality and significance statementGenes involved in secondary metabolite (SM) production are often clustered together to form biosynthetic pathways. These pathways frequently have highly conserved keystone enzymes which can complicate allocation of a biosynthetic gene cluster (BGC) to the cognate SM. In our study, we utilized a combination of comparative genomics, phylogenetic analyses and biochemical approaches to reliably identify BGCs for perylenequinone toxins and DHN-melanin in multiple plant pathogenic fungi. Furthermore, we show that earlier studies that aimed to identify these perylenequinone pathways were misdirected and actually reported DHN-melanin biosynthetic pathways. Our study outlines a reliable approach to successfully identify fungal SM pathways.

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The Ustilago hordei–Barley Interaction is a Versatile System for Characterization of Fungal Effectors

Journal of Fungi ◽

10.3390/jof7020086 ◽

2021 ◽

Vol 7 (2) ◽

pp. 86

Author(s):

Bilal Ökmen ◽

Daniela Schwammbach ◽

Guus Bakkeren ◽

Ulla Neumann ◽

Gunther Doehlemann

Keyword(s):

Fungal Pathogens ◽

Plant Pathogens ◽

Expression System ◽

Pathogenic Fungi ◽

Effector Proteins ◽

Mating Partner ◽

Fungal Virulence ◽

Functional Studies ◽

Infection Structures ◽

Ustilago Hordei

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.

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Fungal Pathogens Affecting the Production and Quality of Medical Cannabis in Israel

Plants ◽

10.3390/plants9070882 ◽

2020 ◽

Vol 9 (7) ◽

pp. 882

Author(s):

Shachar Jerushalmi ◽

Marcel Maymon ◽

Aviv Dombrovsky ◽

Stanley Freeman

Keyword(s):

Fungal Pathogens ◽

Plant Pathogens ◽

Pathogenic Fungi ◽

Plant Diseases ◽

Soilborne Pathogens ◽

Medical Cannabis ◽

Plant Parts ◽

Commercial Cultivation

The use of and research on medical cannabis (MC) is becoming more common, yet there are still many challenges regarding plant diseases of this crop. For example, there is a lack of formal and professional knowledge regarding fungi that infect MC plants, and practical and effective methods for managing the casual agents of disease are limited. The purpose of this study was to identify foliar, stem, and soilborne pathogens affecting MC under commercial cultivation in Israel. The predominant major foliage pathogens were identified as Alternaria alternata and Botrytis cinerea, while the common stem and soilborne pathogens were identified as Fusarium oxysporum and F. solani. Other important fungi that were isolated from foliage were those producing various mycotoxins that can directly harm patients, such as Aspergillus spp. and Penicillium spp. The sampling and characterization of potential pathogenic fungi were conducted from infected MC plant parts that exhibited various disease symptoms. Koch postulates were conducted by inoculating healthy MC tissues and intact plants with fungi isolated from infected commercially cultivated symptomatic plants. In this study, we report on the major and most common plant pathogens of MC found in Israel, and determine the seasonal outbreak of each fungus.

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A Rare Thioquinolobactin Siderophore Present in a Bioactive Pseudomonas sp. DTU12.1

Genome Biology and Evolution ◽

10.1093/gbe/evz267 ◽

2019 ◽

Vol 11 (12) ◽

pp. 3529-3533

Author(s):

Pavelas Sazinas ◽

Morten Lindqvist Hansen ◽

May Iren Aune ◽

Marie Højmark Fischer ◽

Lars Jelsbak

Keyword(s):

Gene Cluster ◽

Secondary Metabolite ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Complete Genome ◽

Plant Pathogens ◽

Gene Clusters ◽

Fungal Species ◽

Antagonistic Activity ◽

Evolutionary Origins

Abstract Many of the soil-dwelling Pseudomonas species are known to produce secondary metabolite compounds, which can have antagonistic activity against other microorganisms, including important plant pathogens. It is thus of importance to isolate new strains of Pseudomonas and discover novel or rare gene clusters encoding bioactive products. In an effort to accomplish this, we have isolated a bioactive Pseudomonas strain DTU12.1 from leaf-covered soil in Denmark. Following genome sequencing with Illumina and Oxford Nanopore technologies, we generated a complete genome sequence with the length of 5,943,629 base pairs. The DTU12.1 strain contained a complete gene cluster for a rare thioquinolobactin siderophore, which was previously described as possessing bioactivity against oomycetes and several fungal species. We placed the DTU12.1 strain within Pseudomonas gessardii subgroup of fluorescent pseudomonads, where it formed a distinct clade with other Pseudomonas strains, most of which also contained a complete thioquinolobactin gene cluster. Only two other Pseudomonas strains were found to contain the gene cluster, though they were present in a different phylogenetic clade and were missing a transcriptional regulator of the whole cluster. We show that having the complete genome sequence and establishing phylogenetic relationships with other strains can enable us to start evaluating the distribution and evolutionary origins of secondary metabolite clusters.

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At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Applied and Environmental Microbiology ◽

10.1128/aem.00507-07 ◽

2007 ◽

Vol 73 (16) ◽

pp. 5162-5172 ◽

Cited By ~ 96

Author(s):

Wei-Jen Chen ◽

François Delmotte ◽

Sylvie Richard Cervera ◽

Lisette Douence ◽

Charles Greif ◽

...

Keyword(s):

Plant Pathogen ◽

Fungicide Resistance ◽

Fungal Pathogens ◽

Plant Pathogens ◽

Plasmopara Viticola ◽

Isolated Populations ◽

Grapevine Downy Mildew ◽

First Case ◽

Wide Range ◽

Pathogen Populations

ABSTRACT Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc 1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.

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Extracellular hydrolytic enzymes in the fungal genus Verticillium: adaptations for pathogenesis

Canadian Journal of Microbiology ◽

10.1139/w99-085 ◽

1999 ◽

Vol 45 (10) ◽

pp. 856-864 ◽

Cited By ~ 10

Author(s):

Michael J Bidochka ◽

Susan Burke ◽

Luna Ng

Keyword(s):

Plant Pathogen ◽

Plant Pathogens ◽

Plant Cell Wall ◽

Extracellular Enzymes ◽

Hydrolytic Enzymes ◽

Pathogenic Fungi ◽

Verticillium Lecanii ◽

Opportunistic Pathogens ◽

Broad Array ◽

Fungal Genus

The insect and plant pathogens within the fungal genus Verticillium showed enzymatic adaptation (production and regulation) directed to the degradation of some of the polymers found in the integument of their respective hosts. For example, the facultative plant pathogens (V. albo-atrum and V. dahliae) produced greater levels of cellulase and xylanase than the facultative insect pathogen (V. lecanii). Verticillium lecanii produced extracellular subtilisin-like protease when grown in insect cuticle medium but not in plant cell wall medium, while the plant pathogen V. albo-atrum showed a diminished regulatory component in the production of this enzyme. The opportunistic pathogens (V. fungicola and V. coccosporum) and the saprobic species (V. rexianum) were less specific in the production and regulation of several proteases as well as cellulases and xylanases. A dendrogram based on cluster analysis compiled from fungal API-ZYM profiles showed commonalties in a broad array of extracellular enzymes within a host-pathogen group (i.e. insect or plant pathogen). The opportunistic pathogens were dispersed throughout the dendrogram, suggestive of the diversity in type and expression of extracellular enzymes.Key words: extracellular enzymes, pathogenic fungi.

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Deacetylation of Fungal Exopolysaccharide Mediates Adhesion and Biofilm Formation

mBio ◽

10.1128/mbio.00252-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 39

Author(s):

Mark J. Lee ◽

Alexander M. Geller ◽

Natalie C. Bamford ◽

Hong Liu ◽

Fabrice N. Gravelat ◽

...

Keyword(s):

Biofilm Formation ◽

Fungal Pathogens ◽

Molecular Mechanisms ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Bacterial Biofilm ◽

Invasive Infection ◽

Biosynthetic Gene ◽

Adhesive Properties

ABSTRACTThe moldAspergillus fumigatuscauses invasive infection in immunocompromised patients. Recently, galactosaminogalactan (GAG), an exopolysaccharide composed of galactose andN-acetylgalactosamine (GalNAc), was identified as a virulence factor required for biofilm formation. The molecular mechanisms underlying GAG biosynthesis and GAG-mediated biofilm formation were unknown. We identified a cluster of five coregulated genes that were dysregulated in GAG-deficient mutants and whose gene products share functional similarity with proteins that mediate the synthesis of the bacterial biofilm exopolysaccharide poly-(β1-6)-N-acetyl-d-glucosamine (PNAG). Bioinformatic analyses suggested that the GAG cluster geneagd3encodes a protein containing a deacetylase domain. Because deacetylation ofN-acetylglucosamine residues is critical for the function of PNAG, we investigated the role of GAG deacetylation in fungal biofilm formation. Agd3 was found to mediate deacetylation of GalNAc residues within GAG and render the polysaccharide polycationic. As with PNAG, deacetylation is required for the adherence of GAG to hyphae and for biofilm formation. Growth of the Δagd3mutant in the presence of culture supernatants of the GAG-deficient Δuge3mutant rescued the biofilm defect of the Δagd3mutant and restored the adhesive properties of GAG, suggesting that deacetylation is an extracellular process. The GAG biosynthetic gene cluster is present in the genomes of members of thePezizomycotinasubphylum of theAscomycotaincluding a number of plant-pathogenic fungi and a single basidiomycete species,Trichosporon asahii, likely a result of recent horizontal gene transfer. The current study demonstrates that the production of cationic, deacetylated exopolysaccharides is a strategy used by both fungi and bacteria for biofilm formation.IMPORTANCEThis study sheds light on the biosynthetic pathways governing the synthesis of galactosaminogalactan (GAG), which plays a key role inA. fumigatusvirulence and biofilm formation. We find that bacteria and fungi use similar strategies to synthesize adhesive biofilm exopolysaccharides. The presence of orthologs of the GAG biosynthetic gene clusters in multiple fungi suggests that this exopolysaccharide may also be important in the virulence of other fungal pathogens. Further, these studies establish a molecular mechanism of adhesion in which GAG interacts via charge-charge interactions to bind to both fungal hyphae and other substrates. Finally, the importance of deacetylation in the synthesis of functional GAG and the extracellular localization of this process suggest that inhibition of deacetylation may be an attractive target for the development of novel antifungal therapies.

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Comparative Phylogenomic Analysis of Cytochrome P450 Monooxygenases From Fusarium Species

10.21203/rs.3.rs-1097665/v1 ◽

2021 ◽

Author(s):

Wadzani Palnam Dauda ◽

Elkanah Glen ◽

Peter Abraham ◽

Charles Oluwaseun Adetunji ◽

Daji Morumda ◽

...

Keyword(s):

Cytochrome P450 ◽

Gene Cluster ◽

Polyketide Synthase ◽

Fusarium Species ◽

Pathogenic Fungi ◽

Plant Diseases ◽

Biological Research ◽

Cytochrome P450 Monooxygenases ◽

Phylogenomic Analysis ◽

P450 Monooxygenases

Abstract Cytochrome P450s (P450s) are a unique multifamily class of enzymes that possess the capability to exhibit catalytic versatility in several biochemical reactions which entails metabolite biosynthesis, primary and secondary metabolism. Fusarium spp. is an important microorganism with many members known to produce secondary metabolites that cause plant diseases and mycotoxicoses in animals and humans. In this present study, from the initially screened 4,579 proteins, we elucidated the nature of abundance, evolutionary relationships, classification and cellular location of 320 cytochrome P450 from 17 phytopathogenic members of Fusarium species. The total CYPs protein sequences were phylogenetically grouped into seventeen (17) clades. Eighty-six (86) CYPs families and forty-eight (48) clans were identified. Twenty-seven (27) families were each found in only one species. The CYPs were found to be majorly localized in the endoplasmic reticulum. The non-ribosomal peptide synthetase-like (NRPS-like) gene cluster was the predominant secondary metabolic-related gene cluster across all the seventeen selected Fusarium species except in F. cucurbiticola and F. solani, where PolyKetide Synthase (PKS) was the most prevalent. The presence of numerous families and clans as observed in in this study shows the expansions of the CYPs families across Fusarium species, this CYPs family and clan expansion is often associated with the evolvement of several fungal traits that include their pathogenicity adaptation to survive on an extensive range of toxic substrates. Identification of P450 proteins in these pathogenic fungi provides fundamental information for further basic and applied biological research into the physiological and toxigenic roles of P450s in Fusarium species.

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