Putative core transcription factors affecting virulence in Aspergillus flavus during infection of maize

Authors: Gilbert, Matthew; Mack, Brian; Lebar, Matthew; Chang, Perng Kuang; Gross, Stephanie; Sweany, Rebecca; Cary, Jeffrey; Rajasekaran, Kanniah - Rajah

Submitted to: The Journal of Fungi
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2022
Publication Date: 1/14/2023
Citation: Gilbert, M.K., Mack, B.M., Lebar, M.D., Chang, P.-K., Gross, S.R., Sweany, R.R., Cary, J.W., Rajasekaran, K. 2023. Putative core transcription factors affecting virulence in Aspergillus flavus during infection of maize. The Journal of Fungi. 9(1):118. https://doi.org/10.3390/jof9010118.
DOI: https://doi.org/10.3390/jof9010118

Interpretive Summary: Aspergillus flavus is a fungal plant pathogen that is responsible for millions of dollars in crop losses annually and has negative health impacts on crop consumers globally. Some strains of this fungus have the potential to produce the potent carcinogen aflatoxin and other toxic secondary metabolites, which can often accumulate to high levels in the crop during fungal infection. In order to develop strategies to mitigate this impact on crop losses and human health, it's important to understand the biological processes of the fungus that allow it to invade host crops and produce these toxins. In this study we first look at gene expression data to understand which fungal genes are likely important for giving the fungus the ability to colonize plants. After selecting candidate genes, we then generated knockout strains whereby we remove the gene of interest from the fungus, and analyze how this affects the fungus ability to grow, produce reproductive bodies, produce toxins, and survive within stressful environments. This research identified three important genes that impact one or more of these processes. Importantly, we discovered that one of these genes is also responsible for the its ability to produce the toxic metabolite cyclopiazonic acid (CPA), which in addition to having health impacts, evidence suggests it allows the fungus to overcome defenses mounted by the plant. In summary, this data provides key pieces of information toward building our knowledge on basic fungal physiology and to better develop approaches towards mitigating the impact of A. flavus infection of crops.

Technical Abstract: Aspergillus flavus is an opportunistic pathogen responsible for millions of dollars in crop losses annually and negative health impacts on crop consumers globally. A. flavus strains have the potential to produce aflatoxin and other toxic secondary metabolites, which often increase during plant colonization. To mitigate the impacts of this international issue, we employ a range of strategies to directly impact fungal physiology, growth and development, thus requiring knowledge on the underlying molecular mechanisms driving these processes. Here we utilize RNA-sequencing data obtained from in situ assays, whereby Z. mays kernels are inoculated with A. flavus strains, is used to select transcription factors putatively driving virulence-related gene networks. We demonstrate through growth, sporulation, oxidative stress-response and aflatoxin/CPA analysis that three A. flavus strains with knockout mutations for the putative transcription factors AFLA_089270, AFLA_112760, and AFLA_031450 demonstrate characteristics such as reduced growth capacity and decreased aflatoxin/CPA accumulation in kernels consistent with decrease fungal pathogenicity. Furthermore, AFLA_089270, also known as HacA, eliminates CPA production and impacts the fungus’ capacity to respond to highly oxidative conditions, indicating an impact on plant colonization. Taken together this data provides a sound foundation for elucidating the downstream molecular pathways potentially contributing to fungal virulence.