Export and Tolerance of Toxins Important for Plant Disease
Cereal Disease Laboratory
Project Number: 5062-22000-024-02
General Cooperative Agreement
Start Date: Dec 15, 2013
End Date: Nov 14, 2016
Many fungal phytopathogens produce toxins critical for determining the outcome of plant disease interactions. While much progress has been made in understanding the enzymatic pathways mediating toxin biosynthesis, little is known about how toxins are exported from fungal cells and delivered to the host. Additionally, how toxigenic fungi escape the inhibitory effects of those compounds is largely unknown. Cellular processes that allow self-tolerance to toxins and toxin export may be critical for fungal virulence or pathogenicity. Recently we have discovered that a transmembrane transporter protein involved in toxin tolerance and enzymes involved in toxin biosynthesis are sequestered in novel cellular organelles. Our goal is to examine the role of multidrug transporters and toxin sequestration in trichothecene tolerance as well as to determine whether vesicular transport and exocytosis facilitate export of toxins. Proposed studies will focus on trichothecenes produced by the phytopathogenic fungus Fusarium graminearum. The objectives of this proposal are to: (1) Identify secretory or trafficking pathways essential for trichothecene export or vesicular sequestration by immuno-localization of trichothecenes and by blocking transit pathways by chemical inhibition or directed mutagenesis.(2) Identify additional co-regulated multidrug resistance transporters essential for maximum tolerance to trichothecenes. Functional genomic methodologies will be used to delete and over-express all known Fusarium multidrug resistance proteins responding to induction of trichothecene biosynthesis.(3) Develop transgenic wheat lines with increased trichothecene tolerance achieved by expression of Fusarium proteins conferring resistance. This transgenic approach may represent a novel strategy by which wheat may escape the effects of pathogen-produced toxins.
1) Secretory pathways utilized for trichothecene export will be identified by a combination of tagging native trafficking proteins with fluorescent proteins and directed mutagenesis. Trafficking and sequestration of trichothecenes will be directly measured using fluorescence-lifetime imaging microscopy, infrared chemical imaging and physical organellar fractionation. 2) Functional genomic methodologies will be used to delete and over-express all known Fusarium multidrug resistance proteins responding to induction of trichothecene biosynthesis in order to determine their relative contribution to overcoming toxicity. 3) With input from the cooperator, genes responsible to trichothecene tolerance as determined in Fusarium will be expressed in transgenic wheat to determine if they function similarly.