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Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

ABSTRACT: In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1?g). Using analytical tools, including elemental analysis, ultraviolet/infrared absorption spectrophometry and MALDI-TOF mass spectrometry analysis, we characterized both pigment content in mycelia and secreted to the culture media as 1,8-dihydroxynaphthalene (DHN)-melanin type compound. This is sole melanin-type in M. fijiensis. Isolated melanins irradiated with a Nd:YAG laser at 532 nm produced monomol light emission at 1270 nm, confirming generation of O2 (1?g), a highly reactive oxygen specie (ROS) that causes cellular death by reacting with all cellular macromolecules. Intermediary polyketides accumulated in culture media by using tricyclazole and pyroquilon (two inhibitors of DHN-melanin synthesis) were identified by ESI-HPLC-MS/MS. Additionally, irradiation at 532 nm of that mixture of compounds and whole melanized mycelium also generated O2 (1?g). A pigmented-strain generated more O2 (1?g) than a strain with low melanin content. Banana leaves of cultivar Cavendish, naturally infected with different stages of black Sigatoka disease, were collected from field. Direct staining of the naturally infected leaf tissues showed the presence of melanin that was positively correlated to the disease stage. We also found hydrogen peroxide (H2O2) but we cannot distinguish the source. Our results suggest that O2 (1?g) photogenerated by DHN-melanin may be involved in the destructive effects of Mycosphaerella fijiensis on banana leaf tissues. Further studies are needed to fully evaluate contributions of melanin-mediated ROS to microbial pathogenesis.

SUBMITTER: Beltran-Garcia MJ

PROVIDER: S-EPMC3960117 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

Beltrán-García Miguel J MJ Prado Fernanda M FM Oliveira Marilene S MS Ortiz-Mendoza David D Scalfo Alexsandra C AC Pessoa Adalberto A Medeiros Marisa H G MH White James F JF Di Mascio Paolo P

PloS one 20140319 3

In pathogenic fungi, melanin contributes to virulence, allowing tissue invasion and inactivation of the plant defence system, but has never been implicated as a factor for host cell death, or as a light-activated phytotoxin. Our research shows that melanin synthesized by the fungal banana pathogen Mycosphaerella fijiensis acts as a virulence factor through the photogeneration of singlet molecular oxygen O2 (1Δg). Using analytical tools, including elemental analysis, ultraviolet/infrared absorpti ...[more]

PMID: 24646830

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Project description:Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Project description:The haploid fungus Pseudocercospora fijiensis causes black Sigatoka in banana and is chiefly controlled by extensive fungicide applications, threatening occupational health and the environment. The 14α-Demethylase Inhibitors (DMIs) are important disease control fungicides, but they lose sensitivity in a rather gradual fashion, suggesting an underlying polygenic genetic mechanism. In spite of this, evidence found thus far suggests that P. fijiensis cyp51 gene mutations are the main responsible factor for sensitivity loss in the field. To better understand the mechanisms involved in DMI resistance, in this study we constructed a genetic map using DArTseq markers on two F1 populations generated by crossing two different DMI resistant strains with a sensitive strain. Analysis of the inheritance of DMI resistance in the F1 populations revealed two major and discrete DMI-sensitivity groups. This is an indicative of a single major responsible gene. Using the DMI-sensitivity scorings of both F1 populations and the generation of genetic linkage maps, the sensitivity causal factor was located in a single genetic region. Full agreement was found for genetic markers in either population, underlining the robustness of the approach. The two maps indicated a similar genetic region where the Pfcyp51 gene is found. Sequence analyses of the Pfcyp51 gene of the F1 populations also revealed a matching bimodal distribution with the DMI resistant. Amino acid substitutions in P. fijiensis CYP51 enzyme of the resistant progeny were previously correlated with the loss of DMI sensitivity. In addition, the resistant progeny inherited a Pfcyp51 gene promoter insertion, composed of a repeat element with a palindromic core, also previously correlated with increased gene expression. This genetic approach confirms that Pfcyp51 is the single explanatory gene for reduced sensitivity to DMI fungicides in the analysed P. fijiensis strains. Our study is the first genetic analysis to map the underlying genetic factors for reduced DMI efficacy.

Project description:Bananas are an important staple food crop in tropical and subtropical regions in Asia, sub-Saharan Africa, and Central and South America. The plant is affected by numerous diseases, with the fungal leaf disease black Sigatoka, caused by Mycosphaerella fijiensis Morelet [anamorph: Pseudocercospora fijiensis (Morelet) Deighton], considered one of the most economically important phytosanitary problem. Although the development of resistant cultivars is recognized as most effective method for long term control of the disease, the majority of today's cultivars are susceptible. In order to gain insights into this pathosystem, this first systematic literature review on the topic is presented. Utilizing six databases (PubMed Central, Web of Science, Google Academic, Springer, CAPES and Scopus Journals) searches were performed using pre-established inclusion and exclusion criteria. From a total of 3,070 published studies examined, 24 were relevant with regard to the Musa-P. fijiensis pathosystem. Relevant papers highlighted that resistant and susceptible cultivars clearly respond differently to infection by this pathogen. M. acuminata wild diploids such as Calcutta 4 and other diploid cultivars can harbor sources of resistance genes, serving as parentals for the generation of improved diploids and subsequent gene introgression in new cultivars. From the sequenced reference genome of Musa acuminata, although the function of many genes in the genome still require validation, on the basis of transcriptome, proteome and biochemical data, numerous candidate genes and molecules have been identified for further evaluation through genetic transformation and gene editing approaches. Genes identified in the resistance response have included those associated with jasmonic acid and ethylene signaling, transcription factors, phenylpropanoid pathways, antioxidants and pathogenesis-related proteins. Papers in this study also revealed gene-derived markers in Musa applicable for downstream application in marker assisted selection. The information gathered in this review furthers understanding of the immune response in Musa to the pathogen P. fijiensis and is relevant for genetic improvement programs for bananas and plantains for control of black Sigatoka.

Project description:Mycosphaerella fijiensis, causal agent of black Sigatoka disease of banana, is a Dothideomycete fungus closely related to fungi that produce polyketides important for plant pathogenicity. We utilized the M. fijiensis genome sequence to predict PKS genes and their gene clusters and make bioinformatics predictions about the types of compounds produced by these clusters. Eight PKS gene clusters were identified in the M. fijiensis genome, placing M. fijiensis into the 23rd percentile for the number of PKS genes compared to other Dothideomycetes. Analysis of the PKS domains identified three of the PKS enzymes as non-reducing and two as highly reducing. Gene clusters contained types of genes frequently found in PKS clusters including genes encoding transporters, oxidoreductases, methyltransferases, and non-ribosomal peptide synthases. Phylogenetic analysis identified a putative PKS cluster encoding melanin biosynthesis. None of the other clusters were closely aligned with genes encoding known polyketides, however three of the PKS genes fell into clades with clusters encoding alternapyrone, fumonisin, and solanapyrone produced by Alternaria and Fusarium species. A search for homologs among available genomic sequences from 103 Dothideomycetes identified close homologs (>80% similarity) for six of the PKS sequences. One of the PKS sequences was not similar (< 60% similarity) to sequences in any of the 103 genomes, suggesting that it encodes a unique compound. Comparison of the M. fijiensis PKS sequences with those of two other banana pathogens, M. musicola and M. eumusae, showed that these two species have close homologs to five of the M. fijiensis PKS sequences, but three others were not found in either species. RT-PCR and RNA-Seq analysis showed that the melanin PKS cluster was down-regulated in infected banana as compared to growth in culture. Three other clusters, however were strongly upregulated during disease development in banana, suggesting that they may encode polyketides important in pathogenicity.

Project description:Meiosis in the haploid plant-pathogenic fungus Mycosphaerella graminicola results in eight ascospores due to a mitotic division following the two meiotic divisions. The transient diploid phase allows for recombination among homologous chromosomes. However, some chromosomes of M. graminicola lack homologs and do not pair during meiosis. Because these chromosomes are not present universally in the genome of the organism they can be considered to be dispensable. To analyze the meiotic transmission of unequal chromosome numbers, two segregating populations were generated by crossing genetically unrelated parent isolates originating from Algeria and The Netherlands that had pathogenicity towards durum or bread wheat, respectively. Detailed genetic analyses of these progenies using high-density mapping (1793 DArT, 258 AFLP and 25 SSR markers) and graphical genotyping revealed that M. graminicola has up to eight dispensable chromosomes, the highest number reported in filamentous fungi. These chromosomes vary from 0.39 to 0.77 Mb in size, and represent up to 38% of the chromosomal complement. Chromosome numbers among progeny isolates varied widely, with some progeny missing up to three chromosomes, while other strains were disomic for one or more chromosomes. Between 15-20% of the progeny isolates lacked one or more chromosomes that were present in both parents. The two high-density maps showed no recombination of dispensable chromosomes and hence, their meiotic processing may require distributive disjunction, a phenomenon that is rarely observed in fungi. The maps also enabled the identification of individual twin isolates from a single ascus that shared the same missing or doubled chromosomes indicating that the chromosomal polymorphisms were mitotically stable and originated from nondisjunction during the second division and, less frequently, during the first division of fungal meiosis. High genome plasticity could be among the strategies enabling this versatile pathogen to quickly overcome adverse biotic and abiotic conditions in wheat fields.

Project description:Mycosphaerella fijiensis, causative agent of the black Sigatoka disease of banana, is considered the most economically damaging banana disease. Despite its importance, the genetics of pathogenicity are poorly understood. Previous studies have characterized polyketide pathways with possible roles in pathogenicity. To identify additional candidate pathogenicity genes, we compared the transcriptome of this fungus during the necrotrophic phase of infection with that during saprophytic growth in medium.Transcriptome analysis was conducted, and the functions of differentially expressed genes were predicted by identifying conserved domains, Gene Ontology (GO) annotation and GO enrichment analysis, Carbohydrate-Active EnZymes (CAZy) annotation, and identification of genes encoding effector-like proteins. The analysis showed that genes commonly involved in secondary metabolism have higher expression in infected leaf tissue, including genes encoding cytochrome P450s, short-chain dehydrogenases, and oxidoreductases in the 2-oxoglutarate and Fe(II)-dependent oxygenase superfamily. Other pathogenicity-related genes with higher expression in infected leaf tissue include genes encoding salicylate hydroxylase-like proteins, hydrophobic surface binding proteins, CFEM domain-containing proteins, and genes encoding secreted cysteine-rich proteins characteristic of effectors. More genes encoding amino acid transporters, oligopeptide transporters, peptidases, proteases, proteinases, sugar transporters, and proteins containing Domain of Unknown Function (DUF) 3328 had higher expression in infected leaf tissue, while more genes encoding inhibitors of peptidases and proteinases had higher expression in medium. Sixteen gene clusters with higher expression in leaf tissue were identified including clusters for the synthesis of a non-ribosomal peptide. A cluster encoding a novel fusicoccane was also identified. Two putative dispensable scaffolds were identified with a large proportion of genes with higher expression in infected leaf tissue, suggesting that they may play a role in pathogenicity. For two other scaffolds, no transcripts were detected in either condition, and PCR assays support the hypothesis that at least one of these scaffolds corresponds to a dispensable chromosome that is not required for survival or pathogenicity.Our study revealed major changes in the transcriptome of Mycosphaerella fijiensis, when associating with its host compared to during saprophytic growth in medium. This analysis identified putative pathogenicity genes and also provides support for the existence of dispensable chromosomes in this fungus.

Dataset Information

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

Publications

Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas.

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