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- Ianiri, Giuseppe, et al.
(author)
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Development of resources for the analysis of gene function in Pucciniomycotina red yeasts
- 2011
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In: Fungal Genetics and Biology. - : Elsevier BV. - 1087-1845 .- 1096-0937. ; 48:7, s. 685-695
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Journal article (peer-reviewed)abstract
- The Pucciniomycotina is an important subphylum of basidiomycete fungi but with limited tools to analyze gene functions. Transformation protocols were established for a Sporobolomyces species (strain IAM 13481), the first Pucciniomycotina species with a completed draft genome sequence, to enable assessment of gene function through phenotypic characterization of mutant strains. Transformation markers were the URA3 and URA5 genes that enable selection and counter-selection based on uracil auxotrophy and resistance to 5-fluoroorotic acid. The wild type copies of these genes were cloned into plasmids that were used for transformation of Sporobolomyces sp. by both biolistic and Agrobacterium-mediated approaches. These resources have been deposited to be available from the Fungal Genetics Stock Center. To show that these techniques could be used to elucidate gene functions, the LEU1 gene was targeted for specific homologous replacement, and also demonstrating that this gene is required for the biosynthesis of leucine in basidiomycete fungi. T-DNA insertional mutants were isolated and further characterized, revealing insertions in genes that encode the homologs of Chs7, Erg3, Kre6, Kexl, Pik1, Sad 1, Ssu1 and Tlg1. Phenotypic analysis of these mutants reveals both conserved and divergent functions compared with other fungi. Some of these strains exhibit reduced resistance to detergents, the antifungal agent fluconazole or sodium sulfite, or lower recovery from heat stress. While there are current experimental limitations for Sporobolomyces sp. such as the lack of Mendelian genetics for conventional mating, these findings demonstrate the facile nature of at least one Pucciniomycotina species for genetic manipulation and the potential to develop these organisms into new models for understanding gene function and evolution in the fungi.
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| 2. |
- Sankaranarayanan, Sundar Ram, et al.
(author)
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Loss of centromere function drives karyotype evolution in closely related Malassezia species
- 2020
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In: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 9
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Journal article (peer-reviewed)abstract
- Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3-5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome-chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation.
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| 3. |
- Wright, Sandra, A. I., 1962, et al.
(author)
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A rapid assay for patulin degradation by the basidiomycetous yeast Rhodotorula glutinis strain LS11
- 2008
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In: COST Action 924. Novel approaches for the control of postharvest diseases and disorders.. ; , s. 19-29
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Conference paper (other academic/artistic)abstract
- The mycotoxin patulin is produced by the green mould pathogen Penicillium expansum in rotting apples during postharvest storage. Strains of epiphytic yeasts and yeast-like organisms have the capacity to protect apples from infection by green mould. One of these, Rhodotorula glutinis strain LS11 can degrade patulin to two non-toxic products, of which one is desoxypatulinic acid. The aim of the work was to develop a rapid, high-throughput assay for patulin degradation in order to enable efficient screening of mutants and library clones to identify genes of LS11 that are involved in patulin degradation. Escherichia coli is highly sensitive to patulin (6-10 μg/ml) but is insensitive to desoxypatulinic acid. This information was utilized to test if a method for patulin detection that was based on inhibition zones in a lawn seeded with E. coli could replace the existing TLC plate method, which is cumbersome and time-consuming. Indeed, an assay for patulin based on the sensitivity of E. coli was developed and validated. In addition, a degradation assay using 96-well microtiter plates was made more efficient by cutting the assay time from ten to three days, by shaking the culture, reducing the assay volume and reducing the amount of patulin utilized. R. glutinis LS11 was mutagenized by random insertion of a hygromycin B resistance cassette, through Agrobacterium-mediated transformation. The putative transformants were tested for stability and assayed for patulin degradation using the method developed. Some of the mutants did not degrade patulin and all of these also had lost the ability to grow in the presence of this mycotoxin. The results show that the screening method developed was suited to detecting mutants, library clones or strains with altered patulin degradation profiles in liquid culture.
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