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

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-14-0261-fi ◽

2015 ◽

Vol 28 (3) ◽

pp. 232-248 ◽

Cited By ~ 109

Author(s):

Ana-Rosa Ballester ◽

Marina Marcet-Houben ◽

Elena Levin ◽

Noa Sela ◽

Cristina Selma-Lázaro ◽

...

Keyword(s):

Secondary Metabolites ◽

Secondary Metabolism ◽

Plant Pathogens ◽

Citrus Fruit ◽

Genomic Analysis ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Penicillium Expansum ◽

Pome Fruit ◽

Penicillium Species

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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Pseudomonas chlororaphis Produces Two Distinct R-Tailocins That Contribute to Bacterial Competition in Biofilms and on Roots

Applied and Environmental Microbiology ◽

10.1128/aem.00706-17 ◽

2017 ◽

Vol 83 (15) ◽

Cited By ~ 17

Author(s):

Robert J. Dorosky ◽

Jun Myoung Yu ◽

Leland S. Pierson ◽

Elizabeth A. Pierson

Keyword(s):

Gene Cluster ◽

Gene Clusters ◽

Associated Bacteria ◽

Content Type ◽

Bacterial Competition ◽

Transmission Electron ◽

Lysis Cassette ◽

First Time ◽

Insight Into

ABSTRACT R-type tailocins are high-molecular-weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it contains the structural components to produce two R-tailocins of different ancestral origins. Two distinct R-tailocin populations differing in length were observed in UV-induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectra of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin-sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette but differing in their killing spectra. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere. IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface-attached biofilms and on roots.

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RubisCO Gene Clusters Found in a Metagenome Microarray from Acid Mine Drainage

Applied and Environmental Microbiology ◽

10.1128/aem.03400-12 ◽

2013 ◽

Vol 79 (6) ◽

pp. 2019-2026 ◽

Cited By ~ 17

Author(s):

Xue Guo ◽

Huaqun Yin ◽

Jing Cong ◽

Zhimin Dai ◽

Yili Liang ◽

...

Keyword(s):

Acid Mine Drainage ◽

Gene Cluster ◽

Molecular Phylogenetics ◽

Mine Drainage ◽

Calvin Cycle ◽

Gene Clusters ◽

Rrna Gene ◽

Bisphosphate Carboxylase ◽

Acid Mine ◽

Horizontal Transfers

ABSTRACTThe enzyme responsible for carbon dioxide fixation in the Calvin cycle, ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), is always detected as a phylogenetic marker to analyze the distribution and activity of autotrophic bacteria. However, such an approach provides no indication as to the significance of genomic content and organization. Horizontal transfers of RubisCO genes occurring in eubacteria and plastids may seriously affect the credibility of this approach. Here, we presented a new method to analyze the diversity and genomic content of RubisCO genes in acid mine drainage (AMD). A metagenome microarray containing 7,776 large-insertion fosmids was constructed to quickly screen genome fragments containing RubisCO form I large-subunit genes (cbbL). Forty-sixcbbL-containing fosmids were detected, and six fosmids were fully sequenced. To evaluate the reliability of the metagenome microarray and understand the microbial community in AMD, the diversities ofcbbLand the 16S rRNA gene were analyzed. Fosmid sequences revealed that the form I RubisCO gene cluster could be subdivided into form IA and IB RubisCO gene clusters in AMD, because of significant divergences in molecular phylogenetics and conservative genomic organization. Interestingly, the form I RubisCO gene cluster coexisted with the form II RubisCO gene cluster in one fosmid genomic fragment. Phylogenetic analyses revealed that horizontal transfers of RubisCO genes may occur widely in AMD, which makes the evolutionary history of RubisCO difficult to reconcile with organismal phylogeny.

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The Genomic Sequence of Pseudomonas fluorescens Pf-5: Insights Into Biological Control

Phytopathology ◽

10.1094/phyto-97-2-0233 ◽

2007 ◽

Vol 97 (2) ◽

pp. 233-238 ◽

Cited By ~ 80

Author(s):

Joyce E. Loper ◽

Donald Y. Kobayashi ◽

Ian T. Paulsen

Keyword(s):

Biological Control ◽

Pseudomonas Fluorescens ◽

Secondary Metabolite ◽

Plant Pathogens ◽

Genomic Sequence ◽

Biological Control Agent ◽

Seedling Emergence ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Membrane Receptors

The complete sequence of the 7.07 Mb genome of the biological control agent Pseudomonas fluorescens Pf-5 is now available, providing a new opportunity to advance knowledge of biological control through genomics. P. fluorescens Pf-5 is a rhizosphere bacterium that suppresses seedling emergence diseases and produces a spectrum of antibiotics toxic to plant-pathogenic fungi and oomycetes. In addition to six known secondary metabolites produced by Pf-5, three novel secondary metabolite biosynthesis gene clusters identified in the genome could also contribute to biological control. The genomic sequence provides numerous clues as to mechanisms used by the bacterium to survive in the spermosphere and rhizosphere. These features include broad catabolic and transport capabilities for utilizing seed and root exudates, an expanded collection of efflux systems for defense against environmental stress and microbial competition, and the presence of 45 outer membrane receptors that should allow for the uptake of iron from a wide array of siderophores produced by soil microorganisms. As expected for a bacterium with a large genome that lives in a rapidly changing environment, Pf-5 has an extensive collection of regulatory genes, only some of which have been characterized for their roles in regulation of secondary metabolite production or biological control. Consistent with its commensal lifestyle, Pf-5 appears to lack a number of virulence and pathogenicity factors found in plant pathogens.

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Complex Evolutionary Origins of Specialized Metabolite Gene Cluster Diversity among the Plant Pathogenic Fungi of the Fusarium graminearum Species Complex

Genome Biology and Evolution ◽

10.1093/gbe/evz225 ◽

2019 ◽

Vol 11 (11) ◽

pp. 3106-3122

Author(s):

Sabina Moser Tralamazza ◽

Liliana Oliveira Rocha ◽

Ursula Oggenfuss ◽

Benedito Corrêa ◽

Daniel Croll

Keyword(s):

Gene Cluster ◽

Fusarium Graminearum ◽

Species Complex ◽

Plant Pathogens ◽

De Novo ◽

Fusarium Species ◽

Gene Clusters ◽

Death Process ◽

Fusarium Graminearum Species Complex ◽

Fungal Genomes

Abstract Fungal 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 the Fusarium graminearum species complex (FGSC) composed of plant pathogens producing potent mycotoxins and causing Fusarium head blight on cereals. We de novo assembled genomes of previously uncharacterized FGSC members (two strains of F. austroamericanum, F. cortaderiae, and F. meridionale). Our analyses of 8 species of the FGSC in addition to 15 other Fusarium species 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 the Fusarium genus. 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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