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Title:   Author  item
                                            NEWMISTER, SEAN - University Of Wisconsinitem
                                            Dahleen, Lynnitem
                                            McCormick, Susanitem
                                            RAYMENT, IVAN - University Of Wisconsin
  Submitted to: National Fusarium Head Blight Forum
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 11/18/2010
                                    Publication  Date: 12/7/2010
                                    Citation: Newmister, S.A., Dahleen, L.S., Mccormick, S.P., Rayment, I. 2010. Development and testing of improved enzymes for transgenic control of FHB. National Fusarium Head Blight Forum Proceedings. pg. 27.Interpretive Summary:
                                Technical Abstract: The primary goal of the present study is to develop improved enzymes for the inactivation of trichothecene mycotoxins associated with Fusarium head blight and test their efficacy in barley. Trichothecene mycotoxins such as DON play a prominent role in the establishment of FHB and have been implicated in pathogen virulence. A primary agent for the inactivation of trichothecene mycotoxins is the trichothecene 3-O-acetylase (TRI101) enzyme. TRI101 catalyzes the acetylation of the 3-OH on the trichothecene toxin resulting in a 100-fold decrease in toxicity. Therefore efforts to use TRI101 from Fusarium sporotrichioides as a transgenic resistance factor have been implemented in wheat (Triticum aestivum), barley (Hordeum vulgare), and rice (Oryza sativa). These transgenic cereals have shown moderate resistance to FHB in greenhouse tests, but have shown little success in field trials. In vitro kinetic analysis of the TRI101 enzymes from Fusarium sporotrichioides (FsTRI101) and Fusarium graminearum (FgTRI101) reveal that FgTRI101 has 100-fold greater efficiency (kcat/KM) for the acetylation of DON. It is proposed that this significant kinetic difference accounts for the poor performance of transgenic cereals in field trials. Consequently the present work is focused on optimization of the kinetically superior FgTRI101 for expression in barley. The 3-dimensional structure of FgTRI101 was used to engineer several point mutations to improve the stability and solubility of the enzyme in its transgenic host. Strategies such as entropic stabilization, consensus mutagenesis, and surface charge introduction were employed to create an optimized FgTRI101. An increase of 4.7 'C in enzyme melting temperature and a catalytic efficiency comparable to the wild type FgTRI101 were observed for the optimized enzyme. Both the wild type and optimized FgTRI101 have been inserted into plasmid pBract214 and have been utilized in Agrobacterium-mediated transformation of barley to create transgenic strains. Transformed plants have been obtained and will be analyzed for resistance to FHB once homozygous lines are identified. Additionally, an antibody-based purification protocol has been established for TRI101 expressed in transgenic barley. This protocol has been used to isolate FsTRI101 from barley and has shown that the transgenic enzyme has retained enzymatic activity although western blots indicate that the enzyme has been post-translationally modified. Future studies will examine the nature of this modification and also characterize the optimized and wild type FgTRI101 enzymes from transgenic barley. These studies will establish a connection between the in vitro and in vivo studies of TRI101.


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