Drought stress and aflatoxin contamination: transcriptional responses of Aspergillus flavus to oxidative stress are related to stress tolerance and aflatoxin production capability

Oilseed crops such as maize and peanut are staple food crops which are vital for global food security. The contamination of these crops with carcinogenic aflatoxins during infection by Aspergillus flavus under drought stress conditions is a serious threat to the safety of these commodities. In order to better understand the role of aflatoxin production in the biology of this pathogen under environmental stress, a collaborative transcriptome project was undertaken to examine the transcriptional responses of toxigenic and atoxigenic isolates of A. flavus to oxidative stress. Selected isolates were cultured in aflatoxin production-conducive and non-conducive media amended with varying levels of H2O2. Isolates which possessed greater tolerance to H2O2 stress and aflatoxin production capability exhibited fewer differentially expressed genes (DEGs) than those which possessed less tolerance and lower aflatoxin production. Primary metabolic mechanisms were also stimulated in response to stress along with antioxidant enzyme-encoding genes. Genes related to fungal development such as aminobenzoate degradation genes and conidiation regulators were also differentially expressed in response to stress. Secondary metabolite biosynthetic processes also formed a large component of the isolate responses to stress including those for aflatoxin, aflatrem, and kojic acid. Co-expression analyses also showed that aflatoxin biosynthetic gene expression along with antioxidant genes were highly correlated with toxigenic isolate biomass under variable stresses. These results along with others in the literature suggest that the production of these secondary metabolites may provide supplemental oxidative stress alleviation. Additional data validation using proteomics, metabolomics and whole genome resequencing (WGRS) approaches will also be discussed

Responses of Aspergillus flavus to Oxidative Stress Are Related to Fungal Development Regulator, Antioxidant Enzyme, and Secondary Metabolite Biosynthetic Gene Expression

The infection of maize and peanut with Aspergillus flavus and subsequent contamination with aflatoxin pose a threat to global food safety and human health, and is exacerbated by drought stress. Drought stress-responding compounds such as reactive oxygen species (ROS) are associated with fungal stress responsive signaling and secondary metabolite production, and can stimulate the production of aflatoxin by A. flavus in vitro. These secondary metabolites have been shown to possess diverse functions in soil-borne fungi including antibiosis, competitive inhibition of other microbes, and abiotic stress alleviation. Previously, we observed that isolates of A. flavus showed differences in oxidative stress tolerance which correlated with their aflatoxin production capabilities. In order to better understand these isolate-specific oxidative stress responses, we examined the transcriptional responses of field isolates of A. flavus with varying levels of aflatoxin production (NRRL3357, AF13, and Tox4) to H2O2-induced oxidative stress using an RNA sequencing approach. These isolates were cultured in an aflatoxin-production conducive medium amended with various levels of H2O2. Whole transcriptomes were sequenced using an Illumina HiSeq platform with an average of 40.43 million filtered paired-end reads generated for each sample. The obtained transcriptomes were then used for differential expression, gene ontology, pathway, and co-expression analyses. Isolates which produced higher levels of aflatoxin tended to exhibit fewer differentially expressed genes than isolates with lower levels of production. Genes found to be differentially expressed in response to increasing oxidative stress included antioxidant enzymes, primary metabolism components, antibiosis-related genes, and secondary metabolite biosynthetic components specifically for aflatoxin, aflatrem, and kojic acid. The expression of fungal development-related genes including aminobenzoate degradation genes and conidiation regulators were found to be regulated in response to increasing stress. Aflatoxin biosynthetic genes and antioxidant enzyme genes were also found to be co-expressed and highly correlated with fungal biomass under stress. This suggests that these secondary metabolites may be produced as part of coordinated oxidative stress responses in A. flavus along with antioxidant enzyme gene expression and developmental regulation

Oxidative stress and carbon metabolism influence Aspergillus flavus transcriptome composition and secondary metabolite production

Contamination of crops with aflatoxin is a serious global threat to food safety. Aflatoxin production by Aspergillus flavus is exacerbated by drought stress in the field and by oxidative stress in vitro. We examined transcriptomes of three toxigenic and three atoxigenic isolates of A. flavus in aflatoxin conducive and non-conducive media with varying levels of H2O2 to investigate the relationship of secondary metabolite production, carbon source, and oxidative stress. We found that toxigenic and atoxigenic isolates employ distinct mechanisms to remediate oxidative damage, and that carbon source affected the isolates’ expression profiles. Iron metabolism, monooxygenases, and secondary metabolism appeared to participate in isolate oxidative responses. The results suggest that aflatoxin and aflatrem biosynthesis may remediate oxidative stress by consuming excess oxygen and that kojic acid production may limit iron-mediated, non-enzymatic generation of reactive oxygen species. Together, secondary metabolite production may enhance A. flavus stress tolerance, and may be reduced by enhancing host plant tissue antioxidant capacity though genetic improvement by breeding selection

Comparative transcriptome analysis of Aspergillus flavus isolates under different oxidative stresses and culture media

Aspergillus flavus and aflatoxin contamination in the field are known to be influenced by numerous stress factors, particularly drought and heat stress. However, the purpose of aflatoxin production is unknown. Here, we report transcriptome analyses comprised of 282.6 Gb of sequencing data describing 11,144 of 13,487 (82.6%) annotated A. flavus genes, which provides the gene expression comparisons among different A. flavus isolates and between two culture media, aflatoxin conducive (sucrose) and non-conducive (peptone). We identified genes that are differentially expressed (DEGs) in response to oxidative stress in media with H2O2. Isolates tolerating greater levels of oxidative stress exhibited fewer DEGs in comparison to those with less tolerance (r = -0.6). We found that proteolytic genes were more expressed in the non-conducive medium, while carbohydrate catabolic and glucose transporter genes (e.g. MFS transporters) were more expressed in the conducive medium. Among the observed DEGs, components of polyketide (aflatoxin) and isoprenoid (aflatrem) secondary metabolite production along with kojic acid biosynthesis and monooxygenase genes were prevalent. To our knowledge, this is the first study that explores the molecular responses of different A. flavus isolates to H2O2-induced stress in different culture media. Together, these results demonstrate a potential key role for secondary metabolite production in A. flavus oxidative stress responses

Insights on host-pathogen interaction between groundnut (Arachis hypogaea) and Aspergillus flavus

Aflatoxin contamination, caused by fungal pathogen Aspergillus flavus, is the major quality and health problem delimiting the trade and consumption of groundnut (Arachis hypogaea L.) worldwide. Three types of aflatoxin resistance mechanisms namely, resistance to in-vitro seed colonization (IVSC), pre-harvest aflatoxin contamination (PAC) and aflatoxin production (AP) have been reported in groundnut. Transcriptome sequencing approach was used to study the differentially expressed genes that differ in-vitro seed colonization (IVSC) in resistant (J 11) and susceptible (JL 24) genotypes. A total of 1,344 million raw reads with an average of 84 million reads per sample were generated from 16 libraries from four different stages of fungal infection. A total of 737.75 and 770.83 million reads were mapped on the progenitor genomes- A subgenome (A. duranensis) and B subgenome (A. ipaensis) of cultivated groundnut (A. hypogaea), respectively. In groundnut, defense related genes like senescence associated proteins, resveratrol synthase, seed linoleate 9s-lipoxygenases (9s-LOX), pathogenesis related proteins, peroxidases, glutathione- S-transferases, chalcone synthase, defensin and chitinases were differentially expressed. In A. flavus, the genes involved in growth and development of fungus, aflatoxin biosynthesis, binding and transporter proteins were found to be induced in compatible interaction. In addition to IVSC resistance, we have also carried out transcriptome sequencing for PAC and AP resistance. In summary, this study will provide greater insights on the resistance mechanisms and discovery of candidate genes for all the three mechanisms that can further be used as expression markers in genomics-enabled aflatoxin resistance breeding

Insights on host-pathogen interaction between groundnut (Arachis hypogaea) and Aspergillus flavus

Aflatoxin contamination, caused by fungal pathogen Aspergillus flavus, is the major quality and health problem delimiting the trade and consumption of groundnut (Arachis hypogaea L.) worldwide. Three types of aflatoxin resistance mechanisms namely, resistance to in-vitro seed colonization (IVSC), pre-harvest aflatoxin contamination (PAC) and aflatoxin production (AP) have been reported in groundnut. Transcriptome sequencing approach was used to study the differentially expressed genes that differ in-vitro seed colonization (IVSC) in resistant (J 11) and susceptible (JL 24) genotypes. A total of 1,344 million raw reads with an average of 84 million reads per sample were generated from 16 libraries from four different stages of fungal infection. A total of 737.75 and 770.83 million reads were mapped on the progenitor genomes- A subgenome (A. duranensis) and B subgenome (A. ipaensis) of cultivated groundnut (A. hypogaea), respectively. In groundnut, defense related genes like senescence associated proteins, resveratrol synthase, seed linoleate 9s-lipoxygenases (9s-LOX), pathogenesis related proteins, peroxidases, glutathione- S-transferases, chalcone synthase, defensin and chitinases were differentially expressed. In A. flavus, the genes involved in growth and development of fungus, aflatoxin biosynthesis, binding and transporter proteins were found to be induced in compatible interaction. In addition to IVSC resistance, we have also carried out transcriptome sequencing for PAC and AP resistance. In summary, this study will provide greater insights on the resistance mechanisms and discovery of candidate genes for all the three mechanisms that can further be used as expression markers in genomics-enabled aflatoxin resistance breeding

Drought stress and aflatoxin contamination: transcriptional responses of Aspergillus flavus to oxidative stress are related to stress tolerance and aflatoxin production capability

Oilseed crops such as maize and peanut are staple food crops which are vital for global food security. The contamination of these crops with carcinogenic aflatoxins during infection by Aspergillus flavus under drought stress conditions is a serious threat to the safety of these commodities. In order to better understand the role of aflatoxin production in the biology of this pathogen under environmental stress, a collaborative transcriptome project was undertaken to examine the transcriptional responses of toxigenic and atoxigenic isolates of A. flavus to oxidative stress. Selected isolates were cultured in aflatoxin production-conducive and non-conducive media amended with varying levels of H2O2. Isolates which possessed greater tolerance to H2O2 stress and aflatoxin production capability exhibited fewer differentially expressed genes (DEGs) than those which possessed less tolerance and lower aflatoxin production. Primary metabolic mechanisms were also stimulated in response to stress along with antioxidant enzyme-encoding genes. Genes related to fungal development such as aminobenzoate degradation genes and conidiation regulators were also differentially expressed in response to stress. Secondary metabolite biosynthetic processes also formed a large component of the isolate responses to stress including those for aflatoxin, aflatrem, and kojic acid. Co-expression analyses also showed that aflatoxin biosynthetic gene expression along with antioxidant genes were highly correlated with toxigenic isolate biomass under variable stresses. These results along with others in the literature suggest that the production of these secondary metabolites may provide supplemental oxidative stress alleviation. Additional data validation using proteomics, metabolomics and whole genome resequencing (WGRS) approaches will also be discussed