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

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

A Coordinated Effort to Manage Soybean Rust in North America: A Success Story in Soybean Disease Monitoring

Existing crop monitoring programs determine the incidence and distribution of plant diseases and pathogens and assess the damage caused within a crop production region. These programs have traditionally used observed or predicted disease and pathogen data and environmental information to prescribe management practices that minimize crop loss (3,69). Monitoring programs are especially important for crops with broad geographic distribution or for diseases that can cause rapid and great economic losses. Successful monitoring programs have been developed for several plant diseases, including downy mildew of cucurbits, Fusarium head blight of wheat, potato late blight, and rusts of cereal crops (13,36,51,80)

Aspergillus flavus functional genomics: Toward enhancing host resistance to aflatoxin contamination under drought using biotechnology

The contamination of maize and peanut with aflatoxin during Aspergillus flavus infection is exacerbated by drought stress. This is correlated with the accumulation of reactive oxygen species (ROS) in host tissues. These ROS also stimulate the production of aflatoxin by A. flavus, which is postulated to provide fringe antioxidant benefits. In order to investigate the functional causes for isolate-to-isolate variation in oxidative stress responses and to characterize components pertinent to host resistance, 10 field isolates of A. flavus and A. parasiticus were used for whole genome re-sequencing (WGRS). Sequencing reads were aligned to the NRRL3357 reference genome with an average of 86.6X coverage for each isolate. Variant calling between the re-sequenced isolates and the reference genome found that toxigenic isolates exhibited fewer non-synonymous single nucleotide polymorphisms (SNPs) than atoxigenic isolates with averages of 22,601 and 34,294, respectively. Also, greater numbers of non-synonymous SNPs were observed than synonymous SNPs suggesting possible niche specialization in progress proportional to observed stress tolerance. Gene family variant enrichment analysis and variant influences on gene expression are under investigation. Understanding the factors influencing A. flavus stress responses and aflatoxin production will allow for targeted enhancement of host resistance through breeding, and the application of novel biotechnologies

Phenotypic assessments of peanut nested association mapping (NAM) populations

Nested association mapping (NAM) is a valuable innovation and multi-parental mapping population strategy in peanut genetics which increases the power to map quantitative trait loci and assists in extending the gene pool of elite peanut lines. In the peanut research community, two structured mapping populations were developed using a 2 × 8 (common by unique) factorial nested association mapping design, each with eight founders and a reference line. Here, we demonstrate its usefulness by assessing the phenotypic diversity of two assembled NAM populations (2 × 4). The common parents are Tifrunner and Florida-07 while the four unique parents are N08082oilct, C76-16, NC3033 and SPT06-06. We initially screened the phenotypic characteristics of the RILs including morphological and disease resistance traits. We found that leaf length and width, plant size, main stem height, and leaf spot resistance segregated within the assembled population and exhibited normal distributions. We also calculated the variance and heritability of each trait, and found that plant size had the lowest narrow sense heritability (0.06) while disease resistance had the highest (0.67) in the Tifrunner NAM population. In the Florida-07 population, main stem height had the lowest (0.27) and leaf width had the highest (0.73). Phenotyping of pod and kernel traits is underway along with further genotyping by sequencing. The NAM concept will promote the evaluation of the genetic diversity present in peanut gene pool

Proteomic Profile of Aspergillus flavus responses to oxidative stress

Aflatoxin production by A. flavus and related species of fungi is regulated in concert with other secondary metabolites, developmental processes, and stress-responsive enzymes in response to environmental stress. Specifically, oxidative stress has been shown to be a pre-requisite and stimulator of aflatoxin production. This is of particular interest given the observation that drought stress results in compromised host resistance to aflatoxin contamination, and that drought stress results in the accumulation of ROS in host plant tissues. The objectives of this study were to use proteomics to provide insights into the pathogen responses to H2O2-derived oxidative stress, and to identify potential biomarkers for fungal infection and targets for host resistance breeding. Three isolates, AF13, NRRL3357, and K54A with high, moderate, and no aflatoxin production, and cultured in medium supplemented with varying levels of H2O2, were examined using an iTRAQ approach. Overall, 1,173 proteins were identified of which 238 were found to be differentially expressed (DEPs). Observed DEPs encompassed metabolic pathways including antioxidants, carbohydrates, pathogenicity, and secondary metabolism. Increased lytic enzyme, secondary metabolite, and developmental pathway expression in AF13 was correlated with increased tolerance to oxidative stress, likely assisting in host plant infection and microbial competition. Elevated expression of energy and cellular component production in NRRL3357 and K54A implies a greater focus on oxidative damage remediation. These trends provide insight into important mechanisms relevant to host plant interactions under drought stress allowing for more targeted efforts in host resistance research

Clonality and geographic structure of host-specialized populations of Corynespora cassiicola causing emerging target spot epidemics in the southeastern United States.

Corynespora cassiicola is a destructive plant-pathogenic fungus causing widespread target spot epidemics, including outbreaks on cotton, soybean, and tomato in the southeastern United States. Previous studies revealed that populations from the three hosts are genetically distinct and host specialized. Although variation in aggressiveness to cotton and tomato were observed, no genetic diversity was detected within populations sampled from each of these hosts. We aimed to gain a better understanding of the emerging target spot epidemics by developing microsatellite markers for C. cassiicola to assess genetic variation, population structure, and to infer modes of reproduction and mechanisms of dispersal. Two hundred sixty-five isolates from cotton, soybean, tomato, and other host plants were genotyped with 13 microsatellite markers. Genotypic diversity revealed genetic variation within each of the populations collected from different hosts, with the population from cotton dominated by clonal genotypes and showing the least genetic diversity. In addition, C. cassiicola populations on different host species were genetically distinct and structured based on host species. No association between genetic and geographic distances was identified in the tomato populations, and the association in cotton populations was low. However, significant regional geographic structure was detected in the soybean populations of C. cassiicola. These results further support previous findings of introduced host specialized isolates or the evolution of more aggressive strains on each host. The lack of geographic structure suggests that the clones on cotton and tomato spread rapidly, or similar founder populations were established by human-mediated dispersal, and that dispersal is not limited. However, regional geographic structure of populations on soybean suggests limited dispersal among more established populations of C. cassiicola, or genetic differences in founder populations that colonized different geographic areas

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