Dataset Information

A comparative genomics study of 23 Aspergillus species from section Flavi.

ABSTRACT: Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high genome diversity, especially in sub-telomeric regions. We predict abundant CAZymes (598 per species) and prolific secondary metabolite gene clusters (73 per species) in section Flavi. However, the observed phenotypes (growth characteristics, polysaccharide degradation) do not necessarily correlate with inferences made from the predicted CAZyme content. Our work, including genomic analyses, phenotypic assays, and identification of secondary metabolites, highlights the genetic and metabolic diversity within section Flavi.

SUBMITTER: Kjærbolling I

PROVIDER: S-EPMC7046712 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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A comparative genomics study of 23 Aspergillus species from section Flavi.

Kjærbølling Inge I Vesth Tammi T Frisvad Jens C JC Nybo Jane L JL Theobald Sebastian S Kildgaard Sara S Petersen Thomas Isbrandt TI Kuo Alan A Sato Atsushi A Lyhne Ellen K EK Kogle Martin E ME Wiebenga Ad A Kun Roland S RS Lubbers Ronnie J M RJM Mäkelä Miia R MR Barry Kerrie K Chovatia Mansi M Clum Alicia A Daum Chris C Haridas Sajeet S He Guifen G LaButti Kurt K Lipzen Anna A Mondo Stephen S Pangilinan Jasmyn J Riley Robert R Salamov Asaf A Simmons Blake A BA Magnuson Jon K JK Henrissat Bernard B Mortensen Uffe H UH Larsen Thomas O TO de Vries Ronald P RP Grigoriev Igor V IV Machida Masayuki M Baker Scott E SE Andersen Mikael R MR

Nature communications 20200227 1

Section Flavi encompasses both harmful and beneficial Aspergillus species, such as Aspergillus oryzae, used in food fermentation and enzyme production, and Aspergillus flavus, food spoiler and mycotoxin producer. Here, we sequence 19 genomes spanning section Flavi and compare 31 fungal genomes including 23 Flavi species. We reassess their phylogenetic relationships and show that the closest relative of A. oryzae is not A. flavus, but A. minisclerotigenes or A. aflatoxiformans and identify high g ...[more]

PMID: 32107379

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Project description:For Aspergillus flavus, a pathogen of considerable economic and health concern, successful gene knockout work for more than a decade has relied nearly exclusively on using nonhomologous end-joining pathway (NHEJ)-deficient recipients via forced double-crossover recombination of homologous sequences. In this study, a simple CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) genome editing system that gave extremely high (>95%) gene-targeting frequencies in A. flavus was developed. It contained a shortened Aspergillus nidulans AMA1 autonomously replicating sequence that maintained good transformation frequencies and Aspergillus oryzae ptrA as the selection marker for pyrithiamine resistance. Expression of the codon-optimized cas9 gene was driven by the A. nidulans gpdA promoter and trpC terminator. Expression of single guide RNA (sgRNA) cassettes was controlled by the A. flavus U6 promoter and terminator. The high transformation and gene-targeting frequencies of this system made generation of A. flavus gene knockouts with or without phenotypic changes effortless. Additionally, multiple-gene knockouts of A. flavus conidial pigment genes (olgA/copT/wA or olgA/yA/wA) were quickly generated by a sequential approach. Cotransforming sgRNA vectors targeting A. flavus kojA, yA, and wA gave 52%, 40%, and 8% of single-, double-, and triple-gene knockouts, respectively. The system was readily applicable to other section Flavi aspergilli (A. parasiticus, A. oryzae, A. sojae, A. nomius, A. bombycis, and A. pseudotamarii) with comparable transformation and gene-targeting efficiencies. Moreover, it gave satisfactory gene-targeting efficiencies (>90%) in A. nidulans (section Nidulantes), A. fumigatus (section Fumigati), A. terreus (section Terrei), and A. niger (section Nigri). It likely will have a broad application in aspergilli. IMPORTANCE CRISPR/Cas9 genome editing systems have been developed for many aspergilli. Reported gene-targeting efficiencies vary greatly and are dependent on delivery methods, repair mechanisms of induced double-stranded breaks, selection markers, and genetic backgrounds of transformation recipient strains. They are also mostly strain specific or species specific. This developed system is highly efficient and allows knocking out multiple genes in A. flavus efficiently either by sequential transformation or by cotransformation of individual sgRNA vectors if desired. It is readily applicable to section Flavi species and aspergilli in other sections ("section" is a taxonomic rank between genus and species). This cross-Aspergillus section system is for wild-type isolates and does not require homologous donor DNAs to be added, NHEJ-deficient strains to be created, or forced recycling of knockout recipients to be performed for multiple-gene targeting. Hence, it simplifies and expedites the gene-targeting process significantly.

Project description:Fungal species within Aspergillus section Flavi contaminate food and feed with aflatoxins. These toxic fungal metabolites compromise human and animal health and disrupt trade. Genotypically and phenotypically diverse species co-infect crops, but temporal and spatial variation in frequencies of different lineages suggests that environmental factors such as temperature may influence structure of aflatoxin-producing fungal communities. Furthermore, though most species within Aspergillus section Flavi produce sclerotia, divergent sclerotial morphologies (small or S-type sclerotia vs. large or L-type sclerotia) and differences in types and quantities of aflatoxins produced suggest lineages are adapted to different life strategies. Temperature is a key parameter influencing pre- and post-harvest aflatoxin contamination of crops. We tested the hypothesis that species of aflatoxin-producing fungi that differ in sclerotial morphology will vary in competitive ability and that outcomes of competition and aflatoxin production will be modulated by temperature. Paired competition experiments between highly aflatoxigenic S-type species (A. aflatoxiformans and Lethal Aflatoxicosis Fungus) and L-type species (A. flavus L morphotype and A. parasiticus) were conducted on maize kernels at 25 and 30°C. Proportions of each isolate growing within and sporulating on kernels were measured using quantitative pyrosequencing. At 30°C, S-type fungi were more effective at host colonization compared to L-type isolates. Total aflatoxins and the proportion of B vs. G aflatoxins were greater at 30°C compared to 25°C. Sporulation by L-type isolates was reduced during competition with S-type fungi at 30°C, while relative quantities of conidia produced by S-type species either increased or did not change during competition. Results indicate that both species interactions and temperature can shape population structure of Aspergillus section Flavi, with warmer temperatures favoring growth and dispersal of highly toxigenic species with S-type sclerotia.

Project description:Microfungal isolates were routinely identified depending on both macro and micro morphological characteristics, sometimes, some fungal isolates appeared to be similar and such cases caused severe confusion for mycologists during the preliminary identification. During our previous studies dealing with isolation of fungi for some biotechnological applications; two mystifying species Aspergillus flavus and Aspergillus oryzae showed similar cultural and macroscopic features. Therefore, the current study aimed to easily distinguish between these two species depending on simple approaches which are routinely followed by a large segment of researchers. Investigation of the macroscopic features was performed to check the fungal growth on four different media (PDA, MEA, YES, and CYA) followed by microscopic examination using an ordinary light microscope, and scanning electron microscope SEM. Also, screening of secondary metabolites for both strains was preliminarily identified to find out the difference between their metabolic profiles. Finally, ITS rDNA was involved to clarify the molecular differences along their partial sequence. Conclusively, the BLAST strategy confirmed the similarity of ITS rDNA segments of both fungal strains that supported our hypothesis. The color of the fungal growth is a very critical factor whereas it is extensively influenced by the type of cultivation media. Accordingly, the YES medium was an inspiring tool assisting in prompt differentiation during the culture investigation step whereas A. oryzae and A. flavus appeared significant mustard yellow and olive green respectively. During the microscopic examination, the CYA medium also had a robust effect on the formation of the conidial chain whereas the knit long chain was observed in A. oryzae while the conidia appeared scattered and not in a chain in the case of A. flavus. Likewise, both two strains possessed different metabolic profiles where A. oryzae is not an Afla toxin producer, unlike A. flavus.

Dataset Information

A comparative genomics study of 23 Aspergillus species from section Flavi.

Publications

A comparative genomics study of 23 Aspergillus species from section Flavi.

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