Draft genome sequence of the plant-pathogenic soil fungus Rhizoctonia solani anastomosis group 3 strain Rhs1AP

The soil fungus Rhizoctonia solani is a pathogen of agricultural crops. Here, we report on the 51,705,945 bp draft consensus genome sequence of R. solani strain Rhs1AP. A comprehensive understanding of the heterokaryotic genome complexity and organization of R. solani may provide insight into the plant disease ecology and adaptive behavior of the fungus

Sequencing of mitochondrial genomes of nine Aspergillus and Penicillium species identifies mobile introns and accessory genes as main sources of genome size variability

BACKGROUND: The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. RESULTS: Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25–36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. CONCLUSIONS: The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing

Sequencing of mitochondrial genomes of nine <it>Aspergillus</it> and <it>Penicillium</it> species identifies mobile introns and accessory genes as main sources of genome size variability

Abstract Background The genera Aspergillus and Penicillium include some of the most beneficial as well as the most harmful fungal species such as the penicillin-producer Penicillium chrysogenum and the human pathogen Aspergillus fumigatus, respectively. Their mitochondrial genomic sequences may hold vital clues into the mechanisms of their evolution, population genetics, and biology, yet only a handful of these genomes have been fully sequenced and annotated. Results Here we report the complete sequence and annotation of the mitochondrial genomes of six Aspergillus and three Penicillium species: A. fumigatus, A. clavatus, A. oryzae, A. flavus, Neosartorya fischeri (A. fischerianus), A. terreus, P. chrysogenum, P. marneffei, and Talaromyces stipitatus (P. stipitatum). The accompanying comparative analysis of these and related publicly available mitochondrial genomes reveals wide variation in size (25–36 Kb) among these closely related fungi. The sources of genome expansion include group I introns and accessory genes encoding putative homing endonucleases, DNA and RNA polymerases (presumed to be of plasmid origin) and hypothetical proteins. The two smallest sequenced genomes (A. terreus and P. chrysogenum) do not contain introns in protein-coding genes, whereas the largest genome (T. stipitatus), contains a total of eleven introns. All of the sequenced genomes have a group I intron in the large ribosomal subunit RNA gene, suggesting that this intron is fixed in these species. Subsequent analysis of several A. fumigatus strains showed low intraspecies variation. This study also includes a phylogenetic analysis based on 14 concatenated core mitochondrial proteins. The phylogenetic tree has a different topology from published multilocus trees, highlighting the challenges still facing the Aspergillus systematics. Conclusions The study expands the genomic resources available to fungal biologists by providing mitochondrial genomes with consistent annotations for future genetic, evolutionary and population studies. Despite the conservation of the core genes, the mitochondrial genomes of Aspergillus and Penicillium species examined here exhibit significant amount of interspecies variation. Most of this variation can be attributed to accessory genes and mobile introns, presumably acquired by horizontal gene transfer of mitochondrial plasmids and intron homing.</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

Total number of SNP of sequenced AFB62 or AFB62F9 intermediate (bold) and high level resistant isolates in itraconazole, posaconazole, or voriconazole after in vitro selection.

<p>Number of mutations in relevant genes <i>erg11A</i>, <i>erg25A</i>, <i>hmg1</i>, <i>ssc70</i>, <i>ganA</i>, and the ABC transporter (AFUA_92.m00226) are presented. Strains in <b>bold</b> were selected under intermediate drug concentrations, strain in <i>italics</i> were direct derivatives of strains in bold selected at high drug concentrations. Numbers in parentheses refer to the number of individual SNPs identified in each gene. Details on the specific mutations are found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004834#ppat.1004834.s006" target="_blank">S4 Table</a>. Itra—itraconazole, Posa—posaconazole, Vori—voriconazole.</p><p>Total number of SNP of sequenced AFB62 or AFB62F9 intermediate (bold) and high level resistant isolates in itraconazole, posaconazole, or voriconazole after in vitro selection.</p

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

Virulence studies in murine and larvae models.

<p>Mice with chronic granulomatous disease (CGD) (A), hydrocortisone treated BALB/c mice (B) and <i>Galleria mellonella</i> larvae (C) were inoculated with <i>Aspergillus fumigatus MAT1-1</i> (AFB62) or <i>MAT1-2</i> (AFB62F9) strains. CGD (5 mice per group) and BALB/c (10 mice per group) mice received 30 μl of 3.33x10⁵ and 3.33x10⁷ conidia/ml, respectively, via pharyngeal aspiration. Larvae (15 larvae per group) were injected with 5 μl of 2x10⁷ conidia/ml. Survival data was analyzed using log rank test.</p

Lungs of mice with chronic granulomatous disease (CGD) or hydrocortisone-treated BALB/c mice inoculated with <i>Aspergillus fumigatus</i> isogenic strains, AFB62 and AFB62F9.

<p>(A-D) Lungs of CGD mice. (E-H) Lungs of hydrocortisone-treated BALB/c mice. (A, B, E and F) Lungs of mice inoculated with AFB62. (C, D, G and H) Lungs of mice inoculated with AFB62F9. Numerous granulomas (A and C) with extensive hyphal growth (B and D) are seen in the lungs of CGD mice at nine days post fungal inoculation. Extensive hyphal growth (arrows in F and H) is seen in the bronchial tree in lungs of hydrocortisone-treated BALB/c mice at seven days post fungal inoculation. CGD and hydrocortisone-treated BALB/c mice were inoculated with 30 μl of 3.33x10⁵ and 3.33x10⁷ conidia/ml, respectively, via pharyngeal aspiration. Sections were stained with hematoxylin and eosin (A, C, E and G) and Gomori methenamine silver (B, D, F and H).</p

Azole resistance in spontaneous and engineered strains.

<p>MIC determined by broth dilution method.</p><p>Azole resistance in spontaneous and engineered strains.</p

Azole resistance experimental design.

<p>(A) Sensitive AFB62 or AFB62F9 cells were incubated on solid media containing sub-MIC concentrations of each of itraconazole, voriconazole, or posaconazole. Resulting spores were plated on selective media with at least 1X MIC of each antifungal to obtain fewer than 100 colonies. Three resistant colonies (purple) from each selective plate were subsequently and independently plated on highly selective plates containing 10 – 50X MIC of each antifungal. Three highly resistant strains (purple) were selected for sequencing and for sexual crosses. (B) Each of the highly resistant isolates from (A) was crossed with the sensitive isogenic strain of the opposite mating type. Ascospores from selected crosses were grown on media with no drug, 1X MIC and 10X MIC of the drug and their DNA sequenced to identify resistance allele frequencies. Mutant alleles are depicted in purple and yellow reference alleles in green. Cells filled with a single color represent an allele frequency of 1, cells with two colors represent an equal proportion of mutant and reference alleles. The purple mutation shows the expected frequencies for an allele associated with resistance, while the yellow mutation shows an expected frequency for alleles not associated with resistance.</p