Brief Summary of Minutes of Annual Meeting

See attached "Copy of Minutes" file for NC1183's 2014 annual report.

Accomplishments: Investigators enhanced knowledge and practical ability regarding bioinformatic analyses of fungal genome sequencing and assembly and advanced in transition from proteomics to metabolomics via hands-on experience and interacting with respective colleagues during the American Society for Mass Spectrometry meeting. A research associate received training in operation of liquid chromatography - mass spectrometry instrumentation for analysis of small molecules and the software-based analysis of raw mass spectrometry data. We developed new bioenergy curricula with content related to mycotoxins. We have been providing trainings in molecular lab techniques and data acquisition and analysis to MSU graduate students, undergraduate students, and also high school students from the community to provide opportunities that students can be exposed to up to date molecular biology techniques in the research area. We also hosted international visiting scholars. We maintained outreach services to the community by hosting agricultural and food/feed safety experiences within our laboratory. Individuals from the community (74) participated in the following activities: “What killed my bees and contaminated my honey?” activity as a forensic experience for Bug Camp; “An Agricultural Crime Case-Focusing on Mycotoxin Analysis” activity as a forensic/feed safety experience for Winston Academy; Facility tours were provided to 4-H Participants during Summer 2014 highlighting food and feed safety. We also offered a demonstration of the practical application of analytical testing for petroleum products for environmental and mechanical engineers (50 individuals). Approximately a dozen PhD students in Toxicology and other disciplines graduated; at least that many other graduate students were mentored. Many undergraduate students were engaged in these studies as well. Professional Master's students have benefited from new curricula developed related to this project. Results have been presented at professional meetings in chemistry, plant pathology and veterinary medicine. Presentations were made at the NCC annual meetings at Purdue University, and at Iowa State University, The Genetics Society of America, Gordon Research Conference, Food Research Institute (FRI) annual meetings, the ASMS Conference on Mass Spectrometry and Allied Topics, June 2014 and at the American Society of Brewing Chemists, and informally at the annual US Wheat and Barley Scab Forum. Scientists received a report at the MSA meetings hosted by MSU; results were shared with companies. Graduate students working on these projects attended and presented material at national research conferences. Students provided extended abstracts and they were awarded ACS National Travel Grants. A number of papers were published. Obj. 1: Surveys for mycotoxins in grain storage elevator (KS) was conducted across the project. Numerous corn samples were collected and tested for the presence of aflatoxin, fumonisin, and deoxynivalenol (DON). Few samples were contaminated with fumonisin. The fumonisin levels in were very low (0.01 ppm to 7.71 ppm). Typically the samples showed a negative result for aflatoxin contamination. DON level was also below the allowable limit and ranged from 0.00 to 0.01 ppm. Generally in years lacking weather extremes, the mycotoxin analysis results were negative, also showed proper harvesting and storage of the grains. As part of this work the formation of Deoxynivalenol-3-glucoside (DON3G) during malting was investigated. DON3G is a bound, or conjugated, form of deoxynivalenol (DON). There is concern over the presence of DON3G in commercial samples of barley and malt, as it is not measured by routine analytical methods for DON. It has been proposed that DON3G may be broken down or transformed, to free DON during digestion or in food processing, and if this is the case, DON3G and other conjugated forms could make a significant contribution to the tolerable daily intake (TDI), and should be measured. DON-3-G was observed to increase by an average of 48-fold during malting. Levels ranged from the limit of detection to 65.84 mg/kg on the malt. In all cases, the levels of DON3G, greatly exceeded those of DON. The formation of DON3G during malting is attributed to the presence of UDP-glucosyl-transferase enzymes in germinating barley. A preservative/antioxidant blend mitigated DON toxicity in swine in terms of improved average daily gain. DON at 5 ppm decreased liver selenium compared with controls in swine fed for 120 d. various mycotoxin binders were tested in rodents and livestock, with generally protective results. A system was developed to study the toxicity of DON that uses Caenorhabditis elegans. The lifespan and egg-laying capacity of DON-treated nematode worms is significantly reduced compared to controls. This assay system will allow us to study mechanisms of toxicity of DON and other mycotoxins, and to test potential mitigation strategies, in a simple animal model. We performed RNA-Seq using the C. elegans system in order to identify genes that are potential targets for DON and which may also be involved in detoxification mechanisms. Genes that are up-regulated by DON include a number involved in detoxification and innate immunity, while down-regulated genes are involved in metabolism and development. The significance of these genes can now be assessed through the use of RNAi suppression or overexpression in transgenic worms. Obj. 2: Research focused on the development of new protocols for production biofuels and chemicals. Genetically modified lines of maize containing newer Bacillus thuringensis (BT) genes were assessed for content of fumonisins (FB) and susceptibility to insect damage (IA). A new BT gene was very effective in reducing FB contents compared with the older BT versions. Ethanol was made from corn containing up to 8 ppm FB, which did not adversely affect ethanol yield. Spiking ethanol fermentation with even higher levels of FB also did not affect ethanol production. Dried distillers grains had about 3 fold enrichment of FB. Batches showing lesser increase of FB are planned to be investigated further. The role of mycotoxins as virulence-enhancing factors in plant disease was studied in seedling maize, soybeans, and wheat. We have identified 4 compounds that affect the production of aflatoxin by Aspergillus and deoxynivalenol by Fusarium graminearum. We have examined their effect in culture using pure synthesized compounds. This project accomplished the first study describing a QuEChERS method for the quantitative determination of AFM1 in raw milk using HPLC-MS. The effectiveness of the binders to separate AFM1 from the milk using QuEChERS as an extraction method was possible as QuEChERS produced 3 layers separating the AFM1 in the organic layer, the middle layer contained the binder from the sample (and AFM1 if the binder was efficient), and the lower aqueous layer and the excess salts. Obj. 3: The genome sequence data for Stenocarpella maydis was refined with a more complete assembly. This work was performed in close collaboration with other members of the multi-state working group (IN, KY). This work produced a genome assembly that is comparable to published de novo genome sequences in other filamentous fungal pathogens. Work on FB production by black Aspergillus spp. (IA) showed that a good portion of these isolates produce FB2 in the laboratory, but low levels compared with F. verticillioides or F. proliferatum. Drier regions had greater black Aspergillus in maize, which co-occurred with A. flavus. The interactions between fungal species and mycotoxigenesis are planned to be further studied. We developed some novel markers and used them to analyze a population of Fusarium graminearum from Kentucky (Bec et al., 2014). We learned that most of the isolates belonged to the dominant chemotype, but that genetically they showed signs of being an isolated divergent population, suggesting there is not a lot of mixing of isolates from outside of Kentucky. We found two species that had not been previously described on symptomatic wheat heads, but these did not cause symptoms. We postulate that these colonize the tissues killed by the scab fungus. This is significant because these other species produce different types of mycotoxins. We have developed Brachypodium distachyon as a model system for studying infection by F. graminearum and the effects of DON. Detached leaves of B. distachyon can be infected with GFP-labeled wild type F. graminearum and tri5-mutant F. graminearum strains, and are also sensitive to the DON application. We have now developed B. distachyon and barley tissue culture and transformation protocols using immature and mature seeds, and have started a program to exploit CRISPR/Cas gene editing technology to engineer FHB resistance in these plants. The model plant Arabidopsis thaliana is being used to test efficacy of CRISPR/Cas gene editing to improve FHB resistance. A number of genes required for FHB susceptibility and DON detoxification are currently being targeted using this technology. A number of transgenic barley plants expressing genes involved in resistance and susceptibility have now been introduced and are currently being verified for gene expression. Functional testing against FHB will proceed once gene expression levels are known. This project’s research can potentially be used to analyze large qRT-PCR datasets in combination with the corresponding maize DNA marker data and maize phenotypic data. This is essential to the identification of aflatoxin resistance DNA markers for incorporation of maize resistance into elite commercial maize lines. A number of candidate genes differing between resistant and susceptible lines were identified. Newly found antifungal agents may have a wide array of applications – from crop protection, to wood decay prevention, to use in household cleaning products. In regards to major goals, the initial (year 1) research provided critical outcome in the form of a set of statistically significant and tentatively identified corn metabolites that are differentially abundant in fungus-resistant genotype versus fungus-susceptible genotype. We established a mechanism of bZIP regulation of fungal secondary metabolites (SMs) through RsmA, a recently discovered YAP-like bZIP protein. RsmA greatly increases SM production by binding to two sites in the Aspergillus nidulans AflR promoter region, a C6 transcription factor known for activating production of the carcinogenic and anti-predation SM, sterigmatocystin. Deletion of aflR in an overexpression rsmA (OE:rsmA) background not only eliminates sterigmatocystin production but also significantly reduces asperthecin synthesis. Furthermore, the fungivore, Folsomia candida, exhibited a distinct preference for feeding on wild type rather than an OE:rsmA strain. RsmA may thus have a critical function in mediating direct chemical resistance against predation. Taken together, these results suggest RsmA represents a bZIP pathway hardwired for defensive SM production. We also used laeA mutants as tools to elucidate virulence attributes in Aspergillus flavus. Microarray expression profiles of ?laeA and over-expression laeA (OE::laeA) were compared to wild type A. flavus. Strikingly, several nitrogen metabolism genes were oppositely mis-regulated in the ?laeA and OE::laeA mutants. One of the nitrogen regulatory genes, the bZIP encoding meaB, was up-regulated in ?laeA. Significantly, over-expression of meaB (OE::meaB) phenocopied the decreased virulence attributes of a ?laeA phenotype including decreased colonization of host seed, reduced lipase activity and loss of aflatoxin B1 production in seed. However, a double knock-down of laeA and meaB (KD::laeA,meaB) demonstrated that KD::laeA,meaB closely resembled ?laeA rather than wild type or ?meaB in growth, aflatoxin biosynthesis and sclerotia production thus suggesting that meaB does not contribute to the ?laeA phenotype. MeaB and LaeA appear to be part of regulatory networks that allow them to have both shared and distinct roles in fungal biology. The velvet genes in A. flavus are an ideal target for control strategies, as disruption of these genes can reduce the fungus ability to spread and produce toxin. We found a new member of velvet family, VelD, which only found in A. flavus and A. oryzae. We have generated vosA, velB, velC, and velD deletion mutants in A. flavus. The deletion of velB caused severely impaired (number, size and morphology) conidiation and the lack of sclerotia production. Moreover, the velB deletion mutant no longer produced AFB1. The deletion of vosA causes earlier conidiation and shows 2 fold more conidia number in 4 day culture. Besides, the vosA deletion mutant produces significantly less AFB1 comparing to WT. velB and vosA deletion mutant conidia contain only ~30% of trehalose compared to wild type spores, suggesting that both may be required for the spore viability in A. flavus. Deletion mutants of the osaA gene homologues in A. flavus show aberrations in development and aflatoxin biogenesis. For that reason, we conclude that OsaA is a key regulatory factor that participates in controlling the process of development and mycotoxin biosynthesis in Aspergillus species. We also did the first study to elucidate WetA function on mycotoxin production in A. flavus. The loss of wetA leads to reduced conidia viability and conidia autolysis in 3 days after inoculation. The wetA null mutant showed a reduced growth rate. Deletion of wetA also decreased fungal stress tolerance. The Velvet proteins, OsaA, and WetA are involved in either sporogenesis and/or mycotoxin production. Understanding the regulatory mechanisms of these proteins, we have more confidence to control both fungal dissemination and mycotoxin production in fields. We have identified several global regulators of mycotoxin production in A. nidulans, such as LaeB that regulates sterigmatocystin production. LaeA regulates all mycotoxins in all fungi. We have recently found that LaeA regulates production of spore toxins through the transcription factor known as BrlA. We found that LaeA regulates brlA expression via chromatin remodeling of the brlA promoter. A human immunotoxin in A. fumigatus, endocrocin, was found to be regulated by LaeA through BrlA. We proposed that G protein coupled receptors (GPCR) are the likely receptors involved in quorum sensing; we have published the deletion of 14 of 16 GPCRs (the 2 others were already known). We are now testing these mutants to see if they are impaired in density development, which will further advance understanding of mycotoxin regulation.

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Tucson, Arizona