Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production

Authors: MEDINA, ANGEL - Cranfield University; Gilbert, Matthew; Mack, Brian; OBRIAN, GREGORY - North Carolina State University; RODRIGUEZ, ALICIA - University Of Extremadura; Bhatnagar, Deepak; PAYNE, GARY - North Carolina State University; MAGAN, NARESH - University Of Extremadura

Submitted to: International Journal of Food Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/22/2017
Publication Date: 5/26/2017
Citation: Medina, A., Gilbert, M.K., Mack, B.M., OBrian, G.R., Rodriguez, A., Bhatnagar, D., Payne, G., Magan, N. 2017. Interactions between water activity and temperature on the Aspergillus flavus transcriptome and aflatoxin B1 production. International Journal of Food Microbiology. 256:36-44.

Interpretive Summary: Future climate conditions are predicted to have a highly significant impact on agricultural commodity production and the technologies behind food production. Among the various changing environmental conditions expected to be of impact, the availability of water and global temperatures are among the most studied. The goal of this study is to examine the effects of altered water availability and the effects of increased temperature on the pathogenic fungus, Aspergillus flavus (A. flavus). Maize corn is susceptible to infection by A. flavus, which may then proliferate under conducive environmental conditions. Several strains of A. flavus are capable of producing highly carcinogenic and toxigenic compounds, thus spoiling the food supply. The consumption of contaminated food supplies can lead to liver damage, stunted growth, and other deleterious ailments. In order to continue the development of remediation methods, it is important to understand the physiology and biochemistry of A. flavus when it interacts with maize kernels in different environmental conditions. To this end, we used RNA sequencing technology to determine which biological processes in the fungus are affected by sltered water availability and altered temperature. Importantly, this data was collected while the fungus was growing inside and interacting with maize kernels. This study showed that aflatoxin, one of the most potent carcinogens produced by the fungus, production on maize grain was higher when water was freely available (0.99 water activity, aw) compared to drier conditions (0.91 aw), and that higher amounts were produced at 37° than 30°C. Increasing temperature from 30° to 37°C at both 0.99 and 0.91 aw resulted in 12 gene categories being upregulated and 9 significantly downregulated. Decreasing aw at both 30 and 37°C resulted in 22 gene ontological categories being significantly upregulated and 25 downregulated. The biological processes affected by the changes in temperature and water include several metabolic processes, and we identified four key genes involved in the synthesis of aflatoxin that are up regulated due to both temperature and water induced stresses. This research will contribute to a better understanding of the role climate change will play in the pre-harvest contamination of maize by a pathogenic fungus.

Technical Abstract: The objectives of this study were to examine the effects of Aspergillus flavus colonization of maize kernels under different water activity (aw; 0.99 and 0.91) and temperature (30 and 37°C) conditions on (a) aflatoxin B1 (AFB1) production and (b) impacts on the transcriptome using RNAseq. This study showed that AFB1 production on maize grain was higher when water was freely available (0.99 aw) compared to drier conditions (0.91 aw), and that higher amounts were produced at 37 than 30°C. Comparisons of the various environmental conditions were made using gene ontology enrichment analysis of the RNA-seq results. Comparisons of the effect of changing temperature at 0.99 aw showed that there was differential expression of 224 and 481 genes of A. flavus with 381 genes in common. At 0.91 aw these were 4307 and 702 genes with 1527 genes in common at the two temperatures. Increasing temperature from 30° to 37°C at both 0.99 and 0.91 aw resulted in 12 gene categories being upregulated and 9 significantly downregulated. Decreasing aw at both 30 and 37°C resulted in 22 gene ontological categories being significantly upregulated and 25 downregulated. The effect on the secondary metabolite gene clusters for aflatoxins and cyclopiazonic acid (CPA) were also examined. Overall, regulation of secondary metabolite clusters showed that both aw and temperature had an effect on several clusters although 9 clusters were not significantly affected. Variations in aw led to more pronounced changes in gene regulation than temperature. Thus changes in aw affected Cluster 9 (production of a siderophore) and Cluster 44 (production of 6-hydroxy-7-methyl-3- nonylisoquinoline-5,8-dione). There were also an elevated number of genes which were co-regulated by both aw and temperature. An interaction effect was observed between temperature and aw for 4 of the 25 aflatoxin biosynthetic genes (aflR, aflS, aflB and aflT). The former two are regulatory genes for aflatoxin biosynthesis and transcription activators. For Cluster 55, responsible for CPA production there were marked changes in expression of all 5 biosynthetic genes in response to aw stress regardless of temperature. In contrast, changes on temperature only modified expression of two genes (AFLA_139460, AFLA_139480). These results are discussed in the context of changes in individual and interacting environmental stresses on the molecular functioning of A. flavus in relation to aflatoxin contamination of maize grain.