Proteom analysis of Aspergillus flavus isolate-specific responses to oxidative stress in relationship to aflatoxin production capability

Authors: FOUNTAIN, JAKE - University Of Georgia; KOH, JIN - University Of Florida; YANHG, LIMING - University Of Georgia; BAJAJ, PRASAD - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; PANDEY, MANISH - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; NAYAK, SPURTHI - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; CHEN, WEI - Fujian Agricultural & Forestry University; ZHUANG, WEIJIAN - Fujian Agricultural & Forestry University; CHEN, ZHI-YUAN - Louisana State University; KEMERAIT, ROBERT - University Of Georgia; CHEN, SIXUE - University Of Florida; VARSHNEY, RAJEEV - International Crops Research Institute For Semi-Arid Tropics (ICRISAT) - India; Guo, Baozhu

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/3/2018
Publication Date: 2/21/2018
Citation: Fountain, J.C., Koh, J., Yanhg, L., Bajaj, P., Pandey, M.K., Nayak, S.N., Chen, W., Zhuang, W., Chen, Z., Kemerait, R.C., Chen, S., Varshney, R.K., Guo, B. 2018. Proteom analysis of Aspergillus flavus isolate-specific responses to oxidative stress in relationship to aflatoxin production capability. Scientific Reports. 8:3430.

Interpretive Summary: Aspergillus flavus is a facultative plant pathogen, which is capable of infecting corn and peanut, resulting in significant economic losses and a serious health issue to human and animal due to the production of carcinogenic aflatoxins. Outbreaks of aflatoxin contamination typically occur in regions prone to drought stress, which has been shown to stimulate the production of reactive oxygen species (ROS) in plant tissues. Recent studies have suggested that these ROS and their reactive byproducts may influence the production of aflatoxin by A. flavus. We observed that isolates, which produced higher levels of aflatoxin and possessed greater tolerance to oxidative stress, exhibited less differential gene expression compared to less tolerant, atoxigenic isolates. To examine the oxidative stress responses of A. flavus at the protein/enzymatic level, we examined the proteomic responses of select field isolates of A. flavus to oxidative stress. The selected isolates exhibited distinct responses to oxidative stress which provide insights into potential targets for enhancing host resistance and biological control. Greater numbers of differentially expressed proteins were detected in isolates with less oxidative stress tolerance. Correlative analysis between this proteome data and the transcriptome data showed a weak correlation (r = 0.1114) indicating a possible post-transcriptional regulation. Highly toxigenic isolates exhibited greater expression of lytic enzymes and sclerotial developmental proteins while less toxigenic isolates mainly displayed regulation of antioxidant and primary metabolic pathways. The environmental stress tolerance mechanisms employed by these isolates provide direction for the enhancement of host resistance through the manipulation of host antioxidant capacity and lytic enzyme inhibitor activity using biomarker selection in breeding programs and through novel biotechnologies such as genome editing in crops.

Technical Abstract: Aspergillus flavus is a facultative pathogen of crops such as maize and peanut which produces carcinogenic aflatoxins during infection, particularly in drought stressed host plants. Reactive oxygen species (ROS) have been shown to both accumulate in host plant tissues during drought and to stimulate the production of aflatoxin by A. flavus both in vitro and in vivo. In order to understand the role of aflatoxin production in oxidative stress responses in A. flavus, we previously examined the transcriptomes of field isolates of A. flavus. To validate this and to examine these responses at the protein level, here we performed iTRAQ (Isobaric Tags for Relative and Absolute Quantification) proteomics for three isolates: AF13 (+++), NRRL3357 (+), and K54A (-) in aflatoxin conducive medium amended with varying levels of H2O2. Proteomic analysis showed that 1173 proteins were detected in at least two replicates. Of these greater numbers of differentially expressed proteins were detected in isolates with less oxidative stress tolerance. Correlative analysis between this proteome data and the transcriptome data for the same conditions showed a weak correlation (r = 0.1114) indicative of possible post-transcriptional regulation. Biochemical pathways including carbon metabolism, glutathione metabolism, oxidative stress regulation, and secondary metabolite biosynthesis comprised the most commonly differentially expressed mechanisms observed. Highly toxigenic isolates exhibited greater expression of lytic enzymes and sclerotial developmental proteins while less toxigenic isolates mainly displayed regulation of antioxidant and primary metabolic pathways. The environmental stress tolerance mechanisms employed by these isolates provide direction for the enhancement of host resistance through the manipulation of host antioxidant capacity and lytic enzyme inhibitor activity using biomarker selection in breeding programs and through novel biotechnologies such as genome editing in crops.