RESUMO
The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.
Assuntos
Família Multigênica , Micotoxinas , Policetídeo Sintases , Stachybotrys , Stachybotrys/genética , Stachybotrys/metabolismo , Família Multigênica/genética , Policetídeo Sintases/genética , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Vias Biossintéticas/genética , Engenharia Genética/métodos , Metabolismo Secundário/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMO
Claviceps purpurea is a plant pathogenic fungus which is still highly relevant in modern agriculture as it infects grasses such as rye and wheat. The disease caused by the consumption of contaminated grain or flour has been known since the Middle Ages and is termed ergotism. The main cause for the toxicity of this fungus is attributed to the ergot alkaloids. Apart from these alkaloids and the ergochromes known as ergot pigments, the secondary metabolism of C. purpurea is not well investigated. This study demonstrated the function of the polyketide synthase PKS7 in C. purpurea by determining the effect of its overexpression on metabolite profiles. For the first time, the depsides lecanoric acid, ethyl lecanorate, gerfelin, and C10-deoxy gerfelin were discovered as secondary metabolites of C. purpurea. Additionally, to estimate the contribution of isolated secondary metabolites to the toxic effects of C. purpurea, lecanoric acid, ethyl lecanorate, and orsellinic acid were tested on HepG2 and CCF-STTG1 cell lines. This study provides the first report on the function of C. purpurea PKS7 responsible for the production of depsides, among which lecanoric acid and ethyl lecanorate were identified as main secondary metabolites.
Assuntos
Claviceps/genética , Alcaloides de Claviceps/biossíntese , Policetídeo Sintases/genética , Salicilatos/metabolismo , Claviceps/metabolismo , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Alcaloides de Claviceps/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Triticum/microbiologiaRESUMO
It has long been known that hormones affect the interaction of a phytopathogen with its host plant. The pathogen can cause changes in plant hormone homeostasis directly by affecting biosynthesis or metabolism in the plant or by synthesizing and secreting the hormone itself. We previously demonstrated that pathogenic fungi of the Fusarium species complex are able to produce three major types of hormones: auxins, cytokinins, and gibberellins. In this work, we explore changes in the levels of these hormones in maize and mango plant tissues infected with Fusarium. The ability to produce individual phytohormones varies significantly across Fusarium species and such differences likely impact host specificity inducing the unique responses noted in planta during infection. For example, the production of gibberellins by F. fujikuroi leads to elongated rice stalks and the suppression of gibberellin biosynthesis in plant tissue. Although all Fusarium species are able to synthesize auxin, sometimes by multiple pathways, the ratio of its free form and conjugates in infected tissue is affected more than the total amount produced. The recently characterized unique pathway for cytokinin de novo synthesis in Fusarium appears silenced or non-functional in all studied species during plant infection. Despite this, a large increase in cytokinin levels was detected in F. mangiferae infected plants, caused likely by the up-regulation of plant genes responsible for their biosynthesis. Thus, the accumulation of active cytokinins may contribute to mango malformation of the reproductive organs upon infection of mango trees. Together, our findings provide insight into the complex role fungal and plant derived hormones play in the fungal-plant interactions.
RESUMO
The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones. Recent genome sequencing revealed the genetic capacity for the biosynthesis of 46 additional secondary metabolites besides the industrially produced GAs. Among them are the pigments bikaverin and fusarubins, as well as mycotoxins, such as fumonisins, fusarin C, beauvericin, and fusaric acid. However, half of the potential secondary metabolite gene clusters are silent. In recent years, it has been shown that the fungal specific velvet complex is involved in global regulation of secondary metabolism in several filamentous fungi. We have previously shown that deletion of the three components of the F. fujikuroi velvet complex, vel1, vel2, and lae1, almost totally abolished biosynthesis of GAs, fumonisins and fusarin C. Here, we present a deeper insight into the genome-wide regulatory impact of Lae1 on secondary metabolism. Over-expression of lae1 resulted in de-repression of GA biosynthetic genes under otherwise repressing high nitrogen conditions demonstrating that the nitrogen repression is overcome. In addition, over-expression of one of five tested histone acetyltransferase genes, HAT1, was capable of returning GA gene expression and GA production to the GA-deficient Δlae1 mutant. Deletion and over-expression of HAT1 in the wild type resulted in downregulation and upregulation of GA gene expression, respectively, indicating that HAT1 together with Lae1 plays an essential role in the regulation of GA biosynthesis.
Assuntos
Proteínas Fúngicas/genética , Fusarium/genética , Regulação Fúngica da Expressão Gênica , Histona Acetiltransferases/genética , Metabolismo Secundário/genética , Proteínas Fúngicas/metabolismo , Fusarium/enzimologia , Deleção de Genes , Giberelinas/metabolismo , Histona Acetiltransferases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Família Multigênica , Micotoxinas/biossíntese , Oryza/microbiologia , Doenças das Plantas/microbiologia , Ativação TranscricionalRESUMO
Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles.
Assuntos
Fusarium/genética , Fusarium/patogenicidade , Oryza/microbiologia , Doenças das Plantas/microbiologia , Proteínas Fúngicas/biossíntese , Fusariose/genética , Fusarium/metabolismo , Genes Fúngicos/genética , Filogenia , VirulênciaRESUMO
GATA-type transcription factors (TFs) such as the nitrogen regulators AreA and AreB, or the light-responsive TFs WC-1 and WC-2, play global roles in fungal growth and development. The conserved GATA TF NsdD is known as an activator of sexual development and key repressor of conidiation in Aspergillus nidulans, and as light-regulated repressor of macroconidia formation in Botrytis cinerea. In the present study, we functionally characterized the NsdD ortholog in Fusarium fujikuroi, named Csm1. Deletion of this gene resulted in elevated microconidia formation in the wild-type (WT) and restoration of conidiation in the non-sporulating velvet mutant Δvel1 demonstrating that Csm1 also plays a role as repressor of conidiation in F. fujikuroi. Furthermore, biosynthesis of the PKS-derived red pigments, bikaverin and fusarubins, is de-regulated under otherwise repressing conditions. Cross-species complementation of the Δcsm1 mutant with the B. cinerea ortholog LTF1 led to full restoration of WT-like growth, conidiation and pigment formation. In contrast, the F. fujikuroi CSM1 rescued only the defects in growth, the tolerance to H2O2 and virulence, but did not restore the light-dependent differentiation when expressed in the B. cinerea Δltf1 mutant. Microarray analysis comparing the expression profiles of the F. fujikuroi WT and the Δcsm1 mutant under different nitrogen conditions revealed a strong impact of this GATA TF on 19 of the 47 gene clusters in the genome of F. fujikuroi. One of the up-regulated silent gene clusters is the one containing the sesquiterpene cyclase-encoding key gene STC1. Heterologous expression of STC1 in Escherichia coli enabled us to identify the product as the volatile bioactive compound (-)-germacrene D.
RESUMO
The 2H-pyran-2-one gibepyrone A and its oxidized derivatives gibepyrones B-F have been isolated from the rice pathogenic fungus Fusarium fujikuroi already more than 20 years ago. However, these products have not been linked to the respective biosynthetic genes, and therefore, their biosynthesis has not yet been analyzed on a molecular level. Feeding experiments with isotopically labeled precursors clearly supported a polyketide origin for the formal monoterpenoid gibepyrone A, whereas the terpenoid pathway could be excluded. Targeted gene deletion verified that the F. fujikuroi polyketide synthase PKS13, designated Gpy1, is responsible for gibepyrone A biosynthesis. Next to Gpy1, the ATP-binding cassette transporter Gpy2 is encoded by the gibepyrone gene cluster. Gpy2 was shown to have only a minor impact on the actual efflux of gibepyrone A out of the cell. Instead, we obtained evidence that Gpy2 is involved in gene regulation as it represses GPY1 gene expression. Thus, GPY1 was up-regulated and gibepyrone A production was enhanced both extra- and intracellularly in Δgpy2 mutants. Furthermore, expression of GPY genes is strictly repressed by members of the fungus-specific velvet complex, Vel1, Vel2, and Lae1, whereas Sge1, a major regulator of secondary metabolism in F. fujikuroi, affects gibepyrone biosynthesis in a positive manner. The gibepyrone A derivatives gibepyrones B and D were shown to be produced by cluster-independent P450 monooxygenases, probably to protect the fungus from the toxic product. In contrast, the formation of gibepyrones E and F from gibepyrone A is a spontaneous process and independent of enzymatic activity.
Assuntos
Proteínas Fúngicas/genética , Fusarium/genética , Oryza/genética , Doenças das Plantas/genética , Policetídeo Sintases/metabolismo , Pironas/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/crescimento & desenvolvimento , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Família Multigênica , Oryza/metabolismo , Oryza/microbiologia , Doenças das Plantas/microbiologiaRESUMO
In this study, we compared the secondary metabolite profile of Fusarium fujikuroi and the histone deacetylase mutant ΔHDA1. We identified a novel peak in ΔHDA1, which was identified as beauvericin (BEA). Going in line with a 1000-fold increased BEA production, the respective non-ribosomal peptide synthetase (NRPS)-encoding gene (BEA1), as well as two adjacent genes (BEA2-BEA3), were significantly up-regulated in ΔHDA1 compared to the wild type. A special role was revealed for the ABC transporter Bea3: deletion of the encoding gene resulted in significant up-regulation of BEA1 and BEA2 and drastically elevated product yields. Furthermore, mutation of a conserved sequence motif in the promoter of BEA1 released BEA repression and resulted in elevated product levels. Candidate transcription factors (TFs) that could bind to this motif are the cluster-specific TF Bea4 as well as a homolog of the global mammalian Kruppel-like TF Yin Yang 1 (Yy1), both acting as repressors of BEA biosynthesis. In addition to Hda1, BEA biosynthesis is repressed by the activity of the H3K27 methyltransferase Kmt6. Consistently, Western blot analyses revealed a genome-wide enrichment of H3K27 acetylation (H3K27ac) in the ΔHDA1 and KMT6 knock-down mutants. Subsequent chromatin immunoprecipitation (ChIP) experiments showed elevated H3K27ac modification levels at the BEA cluster.
Assuntos
Depsipeptídeos/biossíntese , Proteínas Fúngicas/metabolismo , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Metiltransferases/metabolismo , Acetilação , Proteínas Fúngicas/genética , Fusarium/enzimologia , Fusarium/genética , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Metiltransferases/genética , Família Multigênica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Species of the Fusarium fujikuroi species complex (FFC) cause a wide spectrum of often devastating diseases on diverse agricultural crops, including coffee, fig, mango, maize, rice, and sugarcane. Although species within the FFC are difficult to distinguish by morphology, and their genes often share 90% sequence similarity, they can differ in host plant specificity and life style. FFC species can also produce structurally diverse secondary metabolites (SMs), including the mycotoxins fumonisins, fusarins, fusaric acid, and beauvericin, and the phytohormones gibberellins, auxins, and cytokinins. The spectrum of SMs produced can differ among closely related species, suggesting that SMs might be determinants of host specificity. To date, genomes of only a limited number of FFC species have been sequenced. Here, we provide draft genome sequences of three more members of the FFC: a single isolate of F. mangiferae, the cause of mango malformation, and two isolates of F. proliferatum, one a pathogen of maize and the other an orchid endophyte. We compared these genomes to publicly available genome sequences of three other FFC species. The comparisons revealed species-specific and isolate-specific differences in the composition and expression (in vitro and in planta) of genes involved in SM production including those for phytohormome biosynthesis. Such differences have the potential to impact host specificity and, as in the case of F. proliferatum, the pathogenic versus endophytic life style.
Assuntos
Fusarium/genética , Genoma Fúngico , Especificidade de Hospedeiro/genética , Polimorfismo Genético , Evolução Molecular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/isolamento & purificação , Fusarium/patogenicidade , Mangifera/microbiologia , Metaboloma , Orchidaceae/microbiologia , Zea mays/microbiologiaRESUMO
Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety.
Assuntos
Transporte Biológico/genética , Vias Biossintéticas/genética , Ácido Fusárico/biossíntese , Fusarium/enzimologia , Fusarium/genética , Ácido Fusárico/análogos & derivados , Ácido Fusárico/genética , Fusarium/metabolismo , Dados de Sequência Molecular , Família Multigênica/genética , Oryza/genética , Policetídeo Sintases/genética , Fatores de Transcrição/genéticaRESUMO
Fusarium fujikuroi is a member of the Gibberella fujikuroi species complex and well known for the production of gibberellins and mycotoxins including fusarins and fusaric acid. A recent genome sequencing study revealed that the fungus has the genetic potential to produce many more secondary metabolites than have been reported. This paper describes the structure elucidation of the products of the cryptic and silent PKS19 gene cluster that were recently identified (fujikurins A-D). We present the complete NMR data for the structure elucidation of the main compound fujikurin D, which shows tautomeric 1,3-diketo elements. The different tautomeric structures could be confirmed using quantum chemical calculations. Additionally, the structures of the minor compounds fujikurins A-C were elucidated by high-resolution mass spectrometric fragmentation experiments. It emerged that fujikurin A was identical to the bioactive compound CR377 of the taxonomically unclassified Fusarium strain CR377, while fujikurins B-D have not been reported from other fungi.
Assuntos
Fusarium/química , Fusarium/genética , Giberelinas/metabolismo , Micotoxinas/biossíntese , Sequência de Bases , Ácido Fusárico/biossíntese , Fusarium/metabolismo , Estrutura Molecular , Família Multigênica , Micotoxinas/metabolismo , Pironas/química , Tricotecenos/químicaRESUMO
The fungus F. fujikuroi is well known for its production of gibberellins causing the 'bakanae' disease of rice. Besides these plant hormones, it is able to produce other secondary metabolites (SMs), such as pigments and mycotoxins. Genome sequencing revealed altogether 45 potential SM gene clusters, most of which are cryptic and silent. In this study we characterize a new non-ribosomal peptide synthetase (NRPS) gene cluster that is responsible for the production of the cyclic tetrapeptide apicidin F (APF). This new SM has structural similarities to the known histone deacetylase inhibitor apicidin. To gain insight into the biosynthetic pathway, most of the 11 cluster genes were deleted, and the mutants were analyzed by HPLC-DAD and HPLC-HRMS for their ability to produce APF or new derivatives. Structure elucidation was carried out be HPLC-HRMS and NMR analysis. We identified two new derivatives of APF named apicidin J and K. Furthermore, we studied the regulation of APF biosynthesis and showed that the cluster genes are expressed under conditions of high nitrogen and acidic pH in a manner dependent on the nitrogen regulator AreB, and the pH regulator PacC. In addition, over-expression of the atypical pathway-specific transcription factor (TF)-encoding gene APF2 led to elevated expression of the cluster genes under inducing and even repressing conditions and to significantly increased product yields. Bioinformatic analyses allowed the identification of a putative Apf2 DNA-binding ("Api-box") motif in the promoters of the APF genes. Point mutations in this sequence motif caused a drastic decrease of APF production indicating that this motif is essential for activating the cluster genes. Finally, we provide a model of the APF biosynthetic pathway based on chemical identification of derivatives in the cultures of deletion mutants.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Fusarium/metabolismo , Família Multigênica , Peptídeos Cíclicos/química , Peptídeos Cíclicos/genética , Clonagem Molecular , Proteínas Fúngicas/metabolismo , Fusarium/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Concentração de Íons de Hidrogênio , Nitrogênio/metabolismo , Peptídeos Cíclicos/metabolismo , Mutação Puntual , Metabolismo SecundárioRESUMO
The "bakanae" fungus Fusarium fujikuroi is a common pathogen of rice and produces a variety of mycotoxins, pigments, and phytohormones. Fusaric acid is one of the oldest known secondary metabolites produced by F. fujikuroi and some other Fusarium species. Investigation of its biosynthesis and regulation is of great interest due to its occurrence in cereal-based food and feed. This study describes the identification and characterization of the fusaric acid gene cluster in F. fujikuroi consisting of the PKS-encoding core gene and four co-regulated genes, FUB1-FUB5. Besides fusaric acid, F. fujikuroi produces two fusaric acid-like derivatives: fusarinolic acid and 9,10-dehydrofusaric acid. We provide evidence that these derivatives are not intermediates of the fusaric acid biosynthetic pathway, and that their formation is catalyzed by genes outside of the fusaric acid gene cluster. Target gene deletions of all five cluster genes revealed that not all of them are involved in fusaric acid biosynthesis. We suggest that only two genes, FUB1 and FUB4, are necessary for the biosynthesis. Expression of the FUB genes and production of fusaric acid and the two derivatives are favored under high nitrogen. We show that nitrogen-dependent expression of fusaric acid genes is positively regulated by the nitrogen-responsive GATA transcription factor AreB, and that pH-dependent regulation is mediated by the transcription factor PacC. In addition, fusaric acid production is regulated by two members of the fungal-specific velvet complex: Vel1 and Lae1. In planta expression studies show a higher expression in the favorite host plant rice compared to maize.
Assuntos
Proteínas Fúngicas/metabolismo , Ácido Fusárico/metabolismo , Fusarium/genética , Família Multigênica/genética , Proteínas Fúngicas/genética , Fusarium/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
Apicidins are cyclic tetrapeptides with histone deacetylase inhibitory activity. Since their discovery in 1996 a multitude of studies concerning the activity against protozoa and certain cancer cell lines of natural and synthetic apicidin analogues have been published. Until now, the only published natural sources of apicidin are the fungus Fusarium pallidoroseum, later known as F. semitectum and two unspecified Fusarium strains. The biosynthetic origin of apicidins could be associated with a gene cluster, and a biosynthetic pathway has been proposed. Recently, our group was able to identify for the first time an apicidin-like gene cluster in F. fujikuroi that apparently does not lead to the production of any known apicidin analogue. By overexpressing the pathway-specific transcription factor we were able to identify a new apicidin-like compound. The present study provides the complete structure elucidation of the new compound, named apicidin F. Activity evaluation against Plasmodium falciparum showed good in vitro activity with an IC50 value of 0.67 µM.
Assuntos
Antimaláricos/isolamento & purificação , Antimaláricos/farmacologia , Fusarium/química , Inibidores de Histona Desacetilases/isolamento & purificação , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Peptídeos Cíclicos/isolamento & purificação , Peptídeos Cíclicos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Antimaláricos/química , Inibidores de Histona Desacetilases/química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Peptídeos Cíclicos/químicaRESUMO
In this work, the biosynthesis and regulation of the polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS)-derived mutagenic mycotoxin fusarin C was studied in the fungus Fusarium fujikuroi. The fusarin gene cluster consists of nine genes (fus1-fus9) that are coexpressed under high-nitrogen and acidic pH conditions. Chromatin immunoprecipitation revealed a correlation between high expression and enrichment of activating H3K9-acetylation marks under inducing conditions. We provide evidence that only four genes are sufficient for the biosynthesis. The combination of genetic engineering with nuclear magnetic resonance and mass-spectrometry-based structure elucidation allowed the discovery of the putative fusarin biosynthetic pathway. Surprisingly, we indicate that PKS/NRPS releases its product with an open ring structure, probably as an alcohol. Our data indicate that 2-pyrrolidone ring closure, oxidation at C-20, and, finally, methylation at C-20 are catalyzed by Fus2, Fus8, and Fus9, respectively.
Assuntos
Fusarium/enzimologia , Fusarium/genética , Peptídeo Sintases/metabolismo , Polienos/metabolismo , Policetídeo Sintases/metabolismo , Acetilação , Vias Biossintéticas , Fusarium/metabolismo , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Engenharia Genética , Família Multigênica , Peptídeo Sintases/genética , Polienos/química , Policetídeo Sintases/genéticaRESUMO
The fungus Fusarium fujikuroi causes "bakanae" disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of F. fujikuroi that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the F. fujikuroi genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus Fusarium. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to F. fujikuroi, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (PKS19) and another that includes a non-ribosomal peptide synthetase gene (NRPS31) are unique to F. fujikuroi. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered F. fujikuroi strains overexpressing cluster genes. In planta expression studies suggest a specific role for the PKS19-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of F. fujikuroi as a rice pathogen.
Assuntos
Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/metabolismo , Genoma Fúngico/fisiologia , Estudo de Associação Genômica Ampla , Oryza/microbiologia , Doenças das Plantas/microbiologiaRESUMO
The gold standard for quantitation of contaminants with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is the use of isotopically labeled standards. Herein, we report a new strategy for the synthesis of isotopically labeled 21-d3-fusarin C via a genetically modified Fusarium strain, followed by a one-step derivatization reaction. Fusarin C is a Fusarium mycotoxin, which is mutagenic after metabolic activation. Its occurrence has been demonstrated recently in corn-based samples, but up to now, little is known about the contamination of other grain samples. To collect further data, the quantitation method was enhanced by application of the 21-d3-fusarin C and the use of a QTRAP 5500 mass spectrometer. This new method has a limit of detection (LOD) of 1 µg/kg, a limit of quantitation (LOQ) of 4 µg/kg, and a recovery rate of 99%. A total of 21 corn samples and 13 grain samples were analyzed, with resulting fusarin C levels varying from not detectable to 24.7 µg/kg.
Assuntos
Fusarium/genética , Técnicas de Inativação de Genes/métodos , Marcação por Isótopo/métodos , Polienos/análise , Polienos/síntese química , Espectrometria de Massas em Tandem/normas , Ração Animal/análise , Cromatografia Líquida de Alta Pressão/métodos , Diazometano/química , Análise de Alimentos/métodos , Análise de Alimentos/normas , Fusarium/metabolismo , Engenharia Genética , Limite de Detecção , Polienos/metabolismo , Pirrolidinonas/metabolismo , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Zea mays/química , Zea mays/microbiologiaRESUMO
The heterothallic ascomycete Fusarium fujikuroi is a notorious rice pathogen causing super-elongation of plants due to the production of terpene-derived gibberellic acids (GAs) that function as natural plant hormones. Additionally, F. fujikuroi is able to produce a variety of polyketide- and non-ribosomal peptide-derived metabolites such as bikaverins, fusarubins and fusarins as well as metabolites from yet unidentified biosynthetic pathways, e.g. moniliformin. The key enzymes needed for their production belong to the family of polyketide synthases (PKSs) and non-ribosomal peptide synthases (NRPSs) that are generally known to be post-translationally modified by a Sfp-type 4'phosphopantetheinyl transferase (PPTase). In this study we provide evidence that the F. fujikuroi Sfp-type PPTase FfPpt1 is essentially involved in lysine biosynthesis and production of bikaverins, fusarubins and fusarins, but not moniliformin as shown by analytical methods. Concomitantly, targeted Ffppt1 deletion mutants reveal an enhancement of terpene-derived metabolites like GAs and volatile substances such as α-acorenol. Pathogenicity assays on rice roots using fluorescent labeled wild-type and Ffppt1 mutant strains indicate that lysine biosynthesis and iron acquisition but not PKS and NRPS metabolism is essential for establishment of primary infections of F. fujikuroi. Additionally, FfPpt1 is involved in conidiation and sexual mating recognition possibly by activating PKS- and/or NRPS-derived metabolites that could act as diffusible signals. Furthermore, the effect on iron acquisition of Ffppt1 mutants led us to identify a previously uncharacterized putative third reductive iron uptake system (FfFtr3/FfFet3) that is closely related to the FtrA/FetC system of A. fumigatus. Functional characterization provides evidence that both proteins are involved in iron acquisition and are liable to transcriptional repression of the homolog of the Aspergillus GATA-type transcription factor SreA under iron-replete conditions. Targeted deletion of the first Fusarium homolog of this GATA-type transcription factor-encoding gene, Ffsre1, strongly indicates its involvement in regulation of iron homeostasis and oxidative stress resistance.