Gene Ontology categories of genes that are significantly enriched in strain AF70 and NRRL 3357.

<p>Gene Ontology categories of genes that are significantly enriched in strain AF70 and NRRL 3357.</p

Whole genome comparison of <i>Aspergillus flavus</i> L-morphotype strain NRRL 3357 (type) and S-morphotype strain AF70

<div><p><i>Aspergillus flavus</i> is a saprophytic fungus that infects corn, peanuts, tree nuts and other agriculturally important crops. Once the crop is infected the fungus has the potential to secrete one or more mycotoxins, the most carcinogenic of which is aflatoxin. Aflatoxin contaminated crops are deemed unfit for human or animal consumption, which results in both food and economic losses. Within <i>A</i>. <i>flavus</i>, two morphotypes exist: the S strains (small sclerotia) and L strains (large sclerotia). Significant morphological and physiological differences exist between the two morphotypes. For example, the S-morphotypes produces sclerotia that are smaller (< 400 μm), greater in quantity, and contain higher concentrations of aflatoxin than the L-morphotypes (>400 μm). The morphotypes also differ in pigmentation, pH homeostasis in culture and the number of spores produced. Here we report the first full genome sequence of an <i>A</i>. <i>flavus</i> S morphotype, strain AF70. We provide a comprehensive comparison of the <i>A</i>. <i>flavus</i> S-morphotype genome sequence with a previously sequenced genome of an L-morphotype strain (NRRL 3357), including an in-depth analysis of secondary metabolic clusters and the identification SNPs within their aflatoxin gene clusters.</p></div

Percentage of genes identified as being present in <i>A</i>. <i>flavus</i> strains AF70 and NRRL 3357 that are putatively involved in the production of the indicated toxin^*.

<p>Percentage of genes identified as being present in <i>A</i>. <i>flavus</i> strains AF70 and NRRL 3357 that are putatively involved in the production of the indicated toxin^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199169#t002fn001" target="_blank">*</a>}.</p

Secondary metabolic gene cluster backbone genes.

<p>The “backbone enzymes” of secondary metabolic gene clusters in <i>A</i>. <i>flavus</i> strains AF70 and NRRL 3357 (S- and L-morphotypes, respectively) were compared and clustered according to amino acid sequence similarity. The colored segments indicate domains identified by InterProScan. A) Polyketide synthases (PKSs), B) Polyketide synthase-nonribosomal peptide synthetase hybrids (PKS-NRPSs), C) nonribosomal peptide synthetases (NRPSs), and D) dimethylallyl tryptophan synthases (DMATs).</p

Genes in the aflatoxin biosynthetic cluster with SNP impacts classified as high, moderate, or low impact.

<p>Genes in the aflatoxin biosynthetic cluster with SNP impacts classified as high, moderate, or low impact.</p

Summary of SNPs present in the <i>A</i>. <i>flavus</i> NRRL 3357 genome when queried against the <i>A</i>. <i>flavus</i> AF70 genome.

<p>Summary of SNPs present in the <i>A</i>. <i>flavus</i> NRRL 3357 genome when queried against the <i>A</i>. <i>flavus</i> AF70 genome.</p

Schematic of the aflatoxin cluster as a comparison of polymorphisms that differentiate an <i>A</i>. <i>flavus</i> L-morphotype strain (NRRL 3357) from an <i>A</i>. <i>flavus</i> S-morphotype strain (AF70).

<p>The genes are shown as arrows and the intergenic regions are shown as boxes (A and B). Shading for each gene and intergenic region relates to the quantity of transition and transversion SNPs observed (legend). Any number noted above or below a gene or intergenic region for panel A represents the quantity of base pair deletions (bpd) found within the NRRL 3357 or AF70 cluster sequences, respectively. The boxed <i>aflF/aflU</i> regions in panel A are enlarged in panel B, for which the genes in this region (for both <i>A</i>. <i>flavus</i> morphotypes) are compared to the same (complete) genomic region in the SU-1 <i>A</i>. <i>parasiticus</i> strain. Areas noted with bpd indicate large-scale deletions observed.</p

Genome comparisons of <i>A</i>. <i>flavus</i> strain AF70 and NRRL 3357.

<p>A) Number of genes identified as unique or shared between strains AF70 and NRRL 3357 (S and L strains, respectively) indicate 94% of genes are shared (orthologous). B) Protein sequences were compared and used to illustrate the hierarchal relationship between strains AF70, NRRL 3357 and closely related species, and indicate that NRRL 3357 (“A_flavus_NRRL3357”) is more closely related to <i>A</i>. <i>oryzae</i> (“A_oryzae_RIB40”) than <i>A</i>. <i>flavus</i> AF70 (“A_flavus 70”). Values on main tree indicate bootstrap values. Values on inset indicate branch length.</p

Genetic Analysis Using an Isogenic Mating Pair of <i>Aspergillus fumigatus</i> Identifies Azole Resistance Genes and Lack of <i>MAT</i> Locus’s Role in Virulence

<div><p>Invasive aspergillosis (IA) due to <i>Aspergillus fumigatus</i> is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of <i>A</i>. <i>fumigatus</i> opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of <i>A</i>. <i>fumigatus</i> strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (<i>MAT1-1</i>) with a highly fertile (<i>MAT1-2</i>) progeny. Genome sequencing showed that the F₉<i>MAT1-2</i> progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in <i>A</i>. <i>fumigatus</i> was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional <i>in vitro</i> drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in <i>erg11A</i> (<i>cyp51A</i>), as well as multi-drug transporters, <i>erg25</i>, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in <i>A</i>. <i>fumigatus</i> or other heterothallic pathogens, especially for virulence associated traits.</p></div

Genomic comparison of AFB62F9 and AFB62.

<p>(A) Whole genome coverage plot using AFB62F9 reads mapped to the AFB62 genome showing percent identity (y-axis) per chromosome (x-axis). The red line and dots represents regions of the AFB62 genome covered by AFB62F9 reads at greater than 90% identity. (B) Close-up of mapped reads to the region surrounding the <i>MAT1-2</i> locus in AFB62. Numbers on top are coordinates on contig 677 (coordinates 1495670–1582078 on chromosome III). Top track in dark blue: average coverage of the genome was 48X with a maximum of 56 read coverage. The <i>MAT1-2</i> locus in AFB62F9 had no mapped reads so the coverage drops to 0X (~27 Kb in this figure). Second track: SNP density per 1,000 bp is depicted as a red histogram. Maximum SNP/Kb was 10. Note the sharp increase surrounding the <i>MAT1-1</i> locus and extending in the 3’ direction for an additional ~230Kb not depicted in this figure. Bottom track: annotated protein-coding genes in the AFB62 assembly. Direction of arrows depicts the coding strand.</p