Identification of an intermediate for 1,8-dihydroxynaphthalene-melanin synthesis in a race-2 isolate of Fulvia fulva (syn. Cladosporium fulvum).
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SUBMITTER: Medina R
PROVIDER: S-EPMC6299106 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:1,8-Dihydroxynaphthalene (1,8-DHN) is a key intermediate in the biosynthesis of DHN melanin, which is specific to fungi. In this study, we characterized the enzymatic properties of the gene products of an operon consisting of soceCHS1, bdsA, and bdsB from the Gram-negative bacterium Sorangium cellulosum Heterologous expression of soceCHS1, bdsA, and bdsB in Streptomyces coelicolor caused secretion of a dark-brown pigment into the broth. High-performance liquid chromatography (HPLC) analysis of the broth revealed that the recombinant strain produced 1,8-DHN, indicating that the operon encoded a novel enzymatic system for the synthesis of 1,8-DHN. Simultaneous incubation of the recombinant SoceCHS1, BdsA, and BdsB with malonyl-coenzyme A (malonyl-CoA) and NADPH resulted in the synthesis of 1,8-DHN. SoceCHS1, a type III polyketide synthase (PKS), catalyzed the synthesis of 1,3,6,8-tetrahydroxynaphthalene (T4HN) in vitro T4HN was in turn converted to 1,8-DHN by successive steps of reduction and dehydration, which were catalyzed by BdsA and BdsB. BdsA, which is a member of the aldo-keto reductase (AKR) superfamily, catalyzed the reduction of T4HN and 1,3,8-tetrahydroxynaphthalene (T3HN) to scytalone and vermelone, respectively. The stereoselectivity of T4HN reduction by BdsA occurred on the si-face to give (R)-scytalone with more than 99% optical purity. BdsB, a SnoaL2-like protein, catalyzed the dehydration of scytalone and vermelone to T3HN and 1,8-DHN, respectively. The fungal pathway for the synthesis of 1,8-DHN is composed of a type I PKS, naphthol reductases of the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (SD). These findings demonstrated 1,8-DHN synthesis by novel enzymes of bacterial origin.IMPORTANCE Although the DHN biosynthetic pathway was thought to be specific to fungi, we discovered novel DHN synthesis enzymes of bacterial origin. The biosynthesis of bacterial DHN utilized a type III PKS for polyketide synthesis, an AKR superfamily for reduction, and a SnoaL2-like NTF2 superfamily for dehydration, whereas the biosynthesis of fungal DHN utilized a type I PKS, SDR superfamily enzyme, and SD-like NTF2 superfamily. Surprisingly, the enzyme systems comprising the pathway were significantly different from each other, suggesting independent, parallel evolution leading to the same biosynthesis. DHN melanin plays roles in host invasion and adaptation to stress in pathogenic fungi and is therefore important to study. However, it is unclear whether DHN biosynthesis occurs in bacteria. Importantly, we did find that bacterial DHN biosynthetic enzymes were conserved among pathogenic bacteria.
| S-EPMC5930340 | biostudies-literature
Project description:Dothistroma septosporum (Ds) and Fulvia fulva (Ff; previously called Cladosporium fulvum) are two closely related Dothideomycete fungal species that cause Dothistroma needle blight in pine and leaf mold in tomato, respectively. During host colonization, these pathogens secrete virulence factors termed effectors to promote infection. In the presence of corresponding host immune receptors, however, these effectors activate plant defenses, including a localized cell death response that halts pathogen growth. We identified two apoplastic effector protein families, Ecp20 and Ecp32, which are conserved between the two pathogens. The Ecp20 family has four paralogues in both species, while the Ecp32 family has four paralogues in D. septosporum and five in F. fulva. Both families have members that are highly expressed during host infection. Members of the Ecp20 family have predicted structural similarity to proteins with a β-barrel fold, including the Alt a 1 allergen from Alternaria alternata, while members of the Ecp32 family have predicted structural similarity to proteins with a β-trefoil fold, such as trypsin inhibitors and lectins. Using Agrobacterium tumefaciens-mediated transient transformation assays, each family member was assessed for its ability to trigger cell death in leaves of the non-host species Nicotiana benthamiana and N. tabacum. Using this approach, FfEcp20-2, DsEcp20-3, and FfEcp20-3 from the Ecp20 family, and all members from the Ecp32 family, except for the Ds/FfEcp32-4 pair, triggered cell death in both species. This cell death was dependent on secretion of the effectors to the apoplast. In line with recognition by an extracellular immune receptor, cell death triggered by Ds/FfEcp20-3 and FfEcp32-3 was compromised in N. benthamiana silenced for BAK1 or SOBIR1, which encode extracellular co-receptors involved in transducing defense response signals following apoplastic effector recognition. We then investigated whether DsEcp20-3 and DsEcp20-4 triggered cell death in the host species Pinus radiata by directly infiltrating purified protein into pine needles. Strikingly, as in the non-host species, DsEcp20-3 triggered cell death, while DsEcp20-4 did not. Collectively, our study describes two new candidate effector families with cell death-eliciting activity from D. septosporum and F. fulva and provides evidence that members of these families are recognized by plant immune receptors.
| S-EPMC9493481 | biostudies-literature
Project description:Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.
| S-EPMC6312243 | biostudies-literature
Project description:Aspergillus fumigatus produces heavily melanized infectious conidia. The conidial melanin is associated with fungal virulence and resistance to various environmental stresses. This 1,8-dihydroxynaphthalene (DHN) melanin is synthesized by enzymes encoded in a gene cluster in A. fumigatus, including two laccases, Abr1 and Abr2. Although this gene cluster is not conserved in all aspergilli, laccases are critical for melanization in all species examined. Here we show that the expression of A. fumigatus laccases Abr1/2 is upregulated upon hyphal competency and drastically increased during conidiation. The Abr1 protein is localized at the surface of stalks and conidiophores, but not in young hyphae, consistent with the gene expression pattern and its predicted role. The induction of Abr1/2 upon hyphal competency is controlled by BrlA, the master regulator of conidiophore development, and is responsive to the copper level in the medium. We identified a developmentally regulated putative copper transporter, CtpA, and found that CtpA is critical for conidial melanization under copper-limiting conditions. Accordingly, disruption of CtpA enhanced the induction of abr1 and abr2, a response similar to that induced by copper starvation. Furthermore, nonpigmented ctpA? conidia elicited much stronger immune responses from the infected invertebrate host Galleria mellonella than the pigmented ctpA? or wild-type conidia. Such enhancement in eliciting Galleria immune responses was independent of the ctpA? conidial viability, as previously observed for the DHN melanin mutants. Taken together, our findings indicate that both copper homeostasis and developmental regulators control melanin biosynthesis, which affects conidial surface properties that shape the interaction between this pathogen and its host.
| S-EPMC3889567 | biostudies-literature
Project description:Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mould of tomato. Analysis of its genome suggested a high potential for production of secondary metabolites (SM), which might be harmful to plants and animals. Here, we have analysed in detail the predicted SM gene clusters of C. fulvum employing phylogenetic and comparative genomic approaches. Expression of the SM core genes was measured by RT-qrtPCR and produced SMs were determined by LC-MS and NMR analyses. The genome of C. fulvum contains six gene clusters that are conserved in other fungal species, which have undergone rearrangements and gene losses associated with the presence of transposable elements. Although being a biotroph, C. fulvum has the potential to produce elsinochrome and cercosporin toxins. However, the corresponding core genes are not expressed during infection of tomato. Only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue. In vitro SM profiling detected only one major compound that was identified as cladofulvin. PKS6 is likely involved in the production of this pigment because it is the only core gene significantly expressed under these conditions. Cladofulvin does not cause necrosis on Solanaceae plants and does not show any antimicrobial activity. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies on C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.
| S-EPMC3895014 | biostudies-literature
Project description:Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species. IMPORTANCE Opportunistic pathogens like Aspergillus fumigatus have strategies to protect themselves against reactive oxygen species like hydrogen peroxides and superoxides that are produced by immune cells. DHN-melanin is the green pigment on conidia of Aspergillus fumigatus and more than 2 decades ago was reported to protect conidia against hydrogen peroxide. Here, we correct this misinterpretation by showing that DHN-melanin actually is not involved in protection of conidia against hydrogen peroxide. We show that UV mutagenesis that was previously used to select a pksP mutant generated many more genome-wide mutations. We discovered that a mutation in the mycelial catalase gene cat1 could explain the observed phenotype of increased hydrogen peroxide sensitivity. Our work shows that UV mutagenesis is not the preferred methodology to be used for generating mutants. It requires genome sequencing with single-nucleotide polymorphism analysis as well as additional validations to discard unwanted and confirm correct phenotypes.
| S-EPMC8730813 | biostudies-literature
Project description:Cf-12 is an effective gene for resisting tomato leaf mold disease caused by Cladosporium fulvum (C. fulvum). Unlike many other Cf genes such as Cf-2, Cf-4, Cf-5, and Cf-9, no physiological races of C. fulvum that are virulent to Cf-12 carrying plant lines have been identified. In order to better understand the molecular mechanism of Cf-12 gene resistance response, RNA-Seq was used to analyze the transcriptome changes at three different stages of C. fulvum infection (0, 4, and 8 days post infection [dpi]). A total of 9100 differentially expressed genes (DEGs) between 4 and 0 dpi, 8643 DEGs between 8 and 0 dpi and 2547 DEGs between 8 and 4 dpi were identified. In addition, we found that 736 DEGs shared among the above three groups, suggesting the presence of a common core of DEGs in response to C. fulvum infection. These DEGs were significantly enriched in defense-signaling pathways such as the calcium dependent protein kinases pathway and the jasmonic acid signaling pathway. Additionally, we found that many transcription factor genes were among the DEGs, indicating that transcription factors play an important role in C. fulvum defense response. Our study provides new insight on the molecular mechanism of Cf resistance to C. fulvum, especially the unique features of Cf-12 in responding to C. fulvum infection.
| S-EPMC5212946 | biostudies-literature
Project description:Heterologous expression of the fungal pathogen Cladosporium fulvum Avr2 in Arabidopsis plants. Experiment Overall Design: Samples used for microarray analyses were replicated three times in independent experiments and each replication consisted of 10 Avr2-expressing Arabidopsis plants and 10 Col-0 plants grown for four weeks under standard greenhouse conditions
2008-10-26 | E-GEOD-10801 | biostudies-arrayexpress
Project description:BackgroundDuring tomato cultivation, tomato leaf mould is a common disease caused by Cladosporium fulvum (C. fulvum). By encoding Cf proteins, which can recognize corresponding AVR proteins produced by C. fulvum, Cf genes provide resistance to C. fulvum, and the resistance response patterns mediated by different Cf genes are not identical. Plants carrying the Cf-19 gene show effective resistance to C. fulvum in the field and can be used as new resistant materials in breeding. In this study, to identify key regulatory genes related to resistance and to understand the resistance response process in tomato plants carrying Cf-19, RNA sequencing (RNA-seq) was used to analyse the differences between the response of resistant plants (CGN18423, carrying the Cf-19 gene) and susceptible plants (Moneymaker (MM), carrying the Cf-0 gene) at 0, 7 and 20 days after inoculation (dai).ResultsA total of 418 differentially expressed genes (DEGs) were identified specifically in the CGN18423 response process. Gene Ontology (GO) analysis revealed that GO terms including "plasma membrane (GO_Component)", "histidine decarboxylase activity (GO_Function)", and "carboxylic acid metabolic process (GO_Process)", as well as other 10 GO terms, were significantly enriched. The "plant hormone signal transduction" pathway, which was unique to CGN18423 in the 0-7 dai comparison, was identified. Moreover, ten key regulatory points were screened from the "plant hormone signal transduction" pathway and the "plant pathogen interaction" pathway. Hormone content measurements revealed that the salicylic acid (SA) contents increased and peaked at 7 dai, after which the contents deceased and reached minimum values in both CGN18423 and MM plants at 20 dai. The jasmonic acid (JA) content increased to a very high level at 7 dai but then decreased to nearly the initial level at 20 dai in CGN18423, while it continued to increase slightly during the whole process from 0 to 20 dai in MM.ConclusionsThe initial responses are very different between the resistant and susceptible plants. The "plant hormone signal transduction" pathway is important for the formation of Cf-19-mediated immunity. In addition, both JA and SA play roles in regulating the Cf-19-dependent resistance response.
| S-EPMC6923989 | biostudies-literature
Project description:Entomopathogenic fungi have been used for biocontrol of insect pests for many decades. However, the efficacy of such fungi in field trials is often inconsistent, mainly due to environmental stresses, such as UV radiation, temperature extremes, and desiccation. To circumvent these hurdles, metabolic engineering of dihydroxynaphthalene (DHN) melanin biosynthetic genes (polyketide synthase, scytalone dehydratase, and 1,3,8-trihydroxynaphthalene reductase genes) cloned from Alternaria alternata were transformed into the amelanotic entomopathogenic fungus Metarhizium anisopliae via Agrobacterium-mediated transformation. Melanin expression in the transformant of M. anisopliae was verified by spectrophotometric methods, liquid chromatography/mass spectrometry (LC/MS), and confocal microscopy. The transformant, especially under stresses, showed notably enhanced antistress capacity and virulence, in terms of germination and survival rate, infectivity, and reduced median time to death (LT50) in killing diamondback moth (Plutella xylostella) larvae compared with the wild type. The possible mechanisms in enhancing the stress tolerance and virulence, and the significance and potential for engineering melanin biosynthesis genes in other biocontrol agents and crops to improve antistress fitness are discussed.
| S-EPMC3127726 | biostudies-literature