Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus 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 A. fumigatus 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 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 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 A. fumigatus 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 in vitro 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 erg11A (cyp51A), as well as multi-drug transporters, erg25, 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 A. fumigatus or other heterothallic pathogens, especially for virulence associated traits.

Large-scale transcriptional response to hypoxia in Aspergillus fumigatus observed using RNAseq identifies a novel hypoxia regulated ncRNA.

We utilized RNAseq analysis of the Aspergillus fumigatus response to early hypoxic condition exposure. The results show that more than 89% of the A. fumigatus genome is expressed under normoxic and hypoxic conditions. Replicate samples were highly reproducible; however, comparisons between normoxia and hypoxia revealed that >23 and 35% of genes were differentially expressed after 30 and 120 min of hypoxia exposure, respectively. Consistent with our previous report detailing transcriptomic and proteomic responses at later time points, the results here show major repression of ribosomal function and induction of ergosterol biosynthesis, as well as activation of alternate respiratory mechanisms at the later time point. RNAseq data were used to define 32 hypoxia-specific genes, which were not expressed under normoxic conditions. Transcripts of a C6 transcription factor and a histidine kinase-response regulator were found only in hypoxia. In addition, several genes involved in the phosphoenylpyruvate and D-glyceraldehyde-3-phosphate metabolism were only expressed in hypoxia. Interestingly, a 216-bp ncRNA Afu-182 in the 3' region of insA (AFUB_064770) was significantly repressed under hypoxia with a 40-fold reduction in expression. A detailed analysis of Afu-182 showed similarity with several genes in the genome, many of which were also repressed in hypoxia. The results from this study show that hypoxia induces very early and widely drastic genome-wide responses in A. fumigatus that include expression of protein-coding and ncRNA genes. The role of these ncRNA genes in regulating the fungal hypoxia response is an exciting future research direction.

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.

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.

Mobile elements and mitochondrial genome expansion in the soil fungus and potato pathogen Rhizoctonia solani AG-3.

The soil fungus Rhizoctonia solani is an economically important pathogen of agricultural and forestry crops. Here, we present the complete sequence and analysis of the mitochondrial genome of R. solani, field isolate Rhs1AP. The genome (235 849 bp) is the largest mitochondrial genome of a filamentous fungus sequenced to date and exhibits a rich accumulation of introns, novel repeat sequences, homing endonuclease genes, and hypothetical genes. Stable secondary structures exhibited by repeat sequences suggest that they comprise functional, possibly catalytic RNA elements. RNA-Seq expression profiling confirmed that the majority of homing endonuclease genes and hypothetical genes are transcriptionally active. Comparative analysis suggests that the mitochondrial genome of R. solani is an example of a dynamic history of expansion in filamentous fungi.