Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi

Mehrabi, Rahim; Gohari, Amir Mirzadi; Silva, Gilvan Ferreira da; Steinberg, Gero; Kema, G.H.J.; Wit, P.J.G.M. de

doi:10.1016/j.fgb.2015.03.016

Cited by 16 publications

(11 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Correct integration of the hph gene, which confers hygromycin resistance [49], and kmt1 or kmt6 deletion were verified by PCR and Southern analyses (Fig S1). We generated a double deletion mutant by deleting the kmt1 gene in a kmt6 deletion mutant background by using resistance to nourseothricin conferred by the nat gene [50] as an additional selection marker. We isolated several independent transformants, including eight Δkmt1 , six Δkmt6 and ten Δkmt1 Δkmt6 double mutants (from here on abbreviated Δk1/k6 ).…”

Section: Resultsmentioning

confidence: 99%

Destabilization of chromosome structure by histone H3 lysine 27 methylation

Moeller

Schotanus

Soyer

et al. 2018

Preprint

View full text Add to dashboard Cite

27Chromosome and genome stability are important for normal cell function as instability often 28 correlates with disease and dysfunction of DNA repair mechanisms. Many organisms maintain 29 supernumerary or accessory chromosomes that deviate from standard chromosomes. The 30 pathogenic fungus Zymoseptoria tritici has as many as eight accessory chromosomes, which are 31 highly unstable during meiosis and mitosis, transcriptionally repressed, show enrichment of 32 repetitive elements, and enrichment with heterochromatic histone methylation marks, e.g., 33trimethylation of H3 lysine 9 or lysine 27 (H3K9me3, H3K27me3). To elucidate the role of 34 heterochromatin on genome stability in Z. tritici, we deleted the genes encoding the 35 methyltransferases responsible for H3K9me3 and H3K27me3, kmt1 and kmt6, respectively, and 36 generated a double mutant. We combined experimental evolution and genomic analyses to 37 determine the impact of these deletions on chromosome and genome stability, both in vitro and 38 in planta. We used whole genome sequencing, ChIP-seq, and RNA-seq to compare changes in 39 genome and chromatin structure, and differences in gene expression between mutant and 40 wildtype strains. Analyses of genome and ChIP-seq data in H3K9me3-deficient strains revealed 41 dramatic chromatin reorganization, where H3K27me3 is mostly relocalized into regions that are 42 enriched with H3K9me3 in wild type. Many genome rearrangements and formation of new 43 chromosomes were found in the absence of H3K9me3, accompanied by activation of transposable 44 elements. In stark contrast, loss of H3K27me3 actually increased the stability of accessory 45 chromosomes under normal growth conditions in vitro, even without large scale changes in gene 46 activity. We conclude that H3K9me3 is important for the maintenance of genome stability 47 because it disallows H3K27me3 in these regions. In this system, H3K27me3 reduces the overall 48 stability of accessory chromosomes, generating a "metastable" state for these quasi-essential 49 regions of the genome. 50 3 Author Summary 51 Genome and chromosome stability are essential to maintain normal cell function and viability. 52However, differences in genome and chromosome structure are frequently found in organisms 53 that undergo rapid adaptation to changing environmental conditions, and in humans are often 54 found in cancer cells. We study genome instability in a fungal pathogen that exhibits a high degree 55 of genetic diversity. Regions that show extraordinary diversity in this pathogen are the 56 transposon-rich accessory chromosomes, which contain few genes that are of unknown benefit 57 to the organism but maintained in the population and thus considered "quasi essential". 58Accessory chromosomes in all fungi studied so far are enriched with markers for 59 heterochromatin, namely trimethylation of H3 lysine 9 and 27 (H3K9me3, H3K27me3). We show 60 that loss of these heterochromatin marks has strong but opposing effects on genome stability. 61While loss of the transposon-associate...

show abstract

Section: Resultsmentioning

confidence: 99%

Destabilization of chromosome structure by histone H3 lysine 27 methylation

Moeller

Schotanus

Soyer

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Using the same methods, A . tumefaciens AGL1 was transformed with plasmid pRM254 , which contains GFP and geneticin-resistance genes [66] to yield AT-pRM254 strain. The plasmid was introduced into the OE .…”

Section: Methodsmentioning

confidence: 99%

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum

et al. 2018

View full text Add to dashboard Cite

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.

show abstract

“…Using the same methods, A. tumefaciens AGL1 was transformed with plasmid pRM254, which contains GFP and geneticin resistance genes (Mehrabi et al ., ), to yield AT‐pRM254. The plasmid was introduced into the C. fulvum OE.claE transformant as described above.…”

Section: Methodsmentioning

confidence: 99%

Down‐regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato

Griffiths¹,

Mesarich²,

Overdijk³

et al. 2017

Molecular Plant Pathology

View full text Add to dashboard Cite

Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during the colonization of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato in a similar manner to the wild-type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which, in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low-temperature stress. Overall, this study demonstrates that the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.

show abstract

Flexible gateway constructs for functional analyses of genes in plant pathogenic fungi

Cited by 16 publications

References 22 publications

Destabilization of chromosome structure by histone H3 lysine 27 methylation

Destabilization of chromosome structure by histone H3 lysine 27 methylation

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum

Down‐regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato

Contact Info

Product

Resources

About