Two new Aspergillus flavus reference genomes reveal a large insertion potentially contributing to isolate stress tolerance and aflatoxin production

Authors: FOUNTAIN, JAKE - University Of Georgia; CLEVENGER, JOSH - Mars, Inc; Nadon, Brian; YOUNGBLOOD, RAMEY - Mississippi State University; Chang, Perng Kuang; STARR, DAKOTA - University Of Georgia; Wang, Hongliang; WIGGINS, RAEGAN - University Of Georgia; KEMERAIT, ROBERT - University Of Georgia; Bhatnagar, Deepak; OZIAS-AKINS, PEGGY - University Of Georgia; VARSHNEY, RAJEEV - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; Scheffler, Brian; Vaughn, Justin; Guo, Baozhu

Submitted to: Genes, Genomes, and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/3/2020
Publication Date: 8/18/2020
Citation: Fountain, J.C., Clevenger, J.P., Nadon, B.D., Youngblood, R.C., Chang, P., Starr, D., Wang, H., Wiggins, R., Kemerait, R.C., Bhatnagar, D., Ozias-Akins, P., Varshney, R.K., Scheffler, B.E., Vaughn, J.N., Guo, B. 2020. Two new Aspergillus flavus reference genomes reveal a large insertion potentially contributing to isolate stress tolerance and aflatoxin production. Genes, Genomes, and Genomics. 10(9). https://doi.org/10.1534/g3.120.401405.
DOI: https://doi.org/10.1534/g3.120.401405

Interpretive Summary: The currently available genomes of Aspergillus flavus have been invaluable for and have enabled genomics-assisted experiments including characterization of genes involved in a number of primary and secondary metabolic pathways. Still, an understanding of A. flavus phenotypic diversity has been hindered by the lack of suitable and diverse references. Therefore, to address these concerns, and to investigate the structure and evolutionary history of this pathogen, here we present two chromosome-level, reference-quality genomes for the A. flavus isolates AF13 and NRRL3357. These isolates were chosen based on two biologically-driven questions: (1) what are the causes of variation in A. flavus isolates’ aflatoxin production; and (2) why do these isolates exhibit contrasting responses to reactive oxygen species (ROS), reactive compounds associated with drought stress which exacerbate aflatoxin production by A. flavus. These genomes were sequenced using both Illumina and PacBio sequencing technologies and scaffolds were bridged using optical mapping to produce full chromosomes. We confirmed the presence of a unique 310Kb insertion in AF13 containing 58 genes, identified a novel bZIP transcription factor, which may contribute to stress tolerance in A. flavus under drought stress conditions, and also show that the A. flavus section Flavi is polyphyletic. These reference genomes represent a valuable asset for use by the Aspergillus research community for the biology of these organisms, particularly for stress biology related to oxidative stress and aflatoxin production.

Technical Abstract: Efforts in genome sequencing in the Aspergillus genus have led to the development of quality reference genomes for several important species including A. nidulans, A. fumigatus, and A. oryzae. However, less progress has been made for A. flavus. As part of the effort of the USDA-ARS Annual Aflatoxin Workshop Fungal Genome Project, the isolate NRRL3357 was sequenced and resulted in a scaffold-level genome released in 2005. Our goal has been biologically driven, focusing on two areas: isolate variation in aflatoxin production and drought stress exacerbating aflatoxin production by A. flavus. Therefore, we developed two chromosome-level genomes for two isolates: AF13, a MAT1-2, highly stress tolerant, and highly aflatoxigenic isolate; and NRRL3357, a MAT1-1, less stress tolerant, and moderate aflatoxin producer in comparison to AF13. Here, we report these two reference-grade assemblies for these isolates through a combination of PacBio long-read sequencing and optical mapping, and coupled them with comparative, functional, and phylogenetic analyses. This analysis resulted in the identification of 153 and 45 unique genes in AF13 and NRRL3357, respectively. We also confirmed the presence of a unique 310Kb insertion in AF13 containing 58 genes. Analysis of this insertion revealed the presence of a bZIP transcription factor, named atfC, which may contribute to isolate pathogenicity and stress tolerance. Phylogenomic analyses comparing these and other available assemblies also suggest that A. flavus is polyphyletic, and that AF13 may represent a new sub-species, here named Aspergillus flavus subsp. valens.