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<OL> <LI> To characterize quality attributes and develop systems to measure quality of cereals, oilseeds, and bioprocess coproducts<LI> To develop methods to maintain quality, capture value, and preserve food safety at key points in the harvest to end product value chain<LI> To quantify and disseminate the impact of market-chain technologies on providing high value, food-safe, and bio-secure grains for global markets and bioprocess industries. </OL>NC-213 will continue to have a significant impact on improving the efficiency of the U.S. grain industry and capturing value along the cereals, oilseeds and coproducts supply chains. Although NC-213 is not specifically focused on biofuels, the influence of biofuels on the U.S. grain industry cannot be overlooked. Therefore, NC-213 will address quality issues for food, feed, fuels, coproducts, and the emerging bioproducts industries. NC-213 will continue to have strong ties with industry. This multi-state project will use industry input and collaboration to ensure relevance and to aid in developing initiatives that can obtain extramural funding.
Non-Technical Summary: Fusarium Head Scab is a serious disease of wheat, causing considerable economic losses due to both outright loss of the crop to the fungus, and to mycotoxin production in grain pre- and postharvest. The fungus Fusarium graminearum is incapable of spreading in wheat without the mycotoxin deoxynivalenol (DON); strains that lack DON are effectively harmless. Resistant varieties of wheat do not respond to DON - or F. graminearum - but may be susceptible to other pathogens, or lack optimal qualities for food, flour, etc. The exact plant genes responsible for DON response or resistance are not known. This research addresses the dialog between fungus and plant during the early stages of infection: what signal from the plant turns on DON production What genes in the plant respond to DON to let the fungus infect or, in resistant plants, to keep it from spreading By examining gene expression at the time of initial infection and the 72 hours afterwards, in wheat varieties that differ only in Fusarium resistance, we can identify the wheat genes that respond directly to the fungal toxin. These individual genes can then be evaluated one by one for their role in disease development, and may be used in very specific, targeted breeding programs to introduce one or a few disease-resistance genes into plants possessing other desirable characteristics. Whole-genome microarrays, a technique showing the expression of every gene in a genome for a given gene or set of conditions, will be used to identify wheat genes of interest. It is necessary to use a whole-genome technique, as it is not known what wheat genes are involved. The presence of the fungus, and toxin gene expression, will be monitored by qRT-PCR, a technique that allows quantification of the expression of individual genes where they are known. Toxin production will be confirmed by the use of a commercial kit for DON detection and quantification. <P> Approach: First phases of multistate project: Research will be performed in the greenhouses on the University of Nebraska-Lincoln East Campus. Near-isogenic lines of hard red winter wheat cv. Wesley, differing in the presence or absence of the head scab resistance QTL FHB1 will be inoculated with the sequenced strain of Fusarium graminearum, PH-1 (NRRL 31084). This strain produces the trichothecene mycotoxins DON and 15ADON, which act as virulence factors in wheat. Additionally, a TRI5 knockout strain of F. graminearum PH-1, incapable of producing trichothecenes, will be used as a control. Kernels adjacent to the inoculation point will be harvested from 3-9 days post-inoculation, the wheat and fungal RNA extracted, and mycotoxin levels quantified using ELISA. Quantitative reverse-transcript PCR (qRT-PCR) will be used to assess 1) the presence or absence of the fungus (using fungal housekeeping genes GAPDH and EF1A) and 2) the transcript abundance of genes in the trichothecene biosynthesis pathway (e.g. TRI5 and TRI8); this data will be correlated with the condition of the harvested wheat kernels and the measured levels of DON. The same RNA will be hybridized to the Affymetrix Wheat GeneChip to provide a transcriptional profile for the wheat. The cross-talk between host plant and fungus will be examined at fine scale (with sampling at 1-3 hour intervals beginning 12 hours before DON gene expression is first detected, and continuing until 24 hours after the DON mycotoxin is first detected - likely 5-7 days post-inoculation) to answer the following questions: What signal(s) from the plant initiate DON gene expression and DON synthesis in the fungus What plant genes allow DON to act as a virulence factor in the absence of FHB1, but not in its presence The use of the TRI5 knockout strain of PH-1 will enable us to differentiate between general plant response to pathogen attack, and specific response to the trichothecene mycotoxin; the resistant wheat plants will independently address the same issue. Affymetrix data will be analyzed using the limma module in Bioconductor, which provides statistical significance for comparisons between treatments in microarray data. All experiments will be replicated in triplicate. The exact times of harvest for data collection will be contingent upon initial mycotoxin and mycotoxin transcript data, collected daily from 3-9 days post-inoculation. Affymetrix GeneChips will not be used until the second phase investigating the cross-talk between plant and fungus with sampling at 1-3 hour intervals. Depending on the information obtained from the wheat GeneChips, qRT-PCR may eventually be used to query a subset of relevant wheat genes. Future stages of the project include collaboration with the wheat breeders to investigate the specific roles of individual wheat genes in the response of wheat to DON, with the possibility for field trials of promising plants, which would also be evaluated for grain quality.