Plants and fungi produce exosomes that contain sRNAs and proteins that play a role in modulating plant-fungal interactions. However, it is not well understood if barley uses exosomes to transfer extracellular proteins and sRNAs to F. graminearum and how those exosomes may impact fungal growth. The primary goal of this research is to isolate exosomes from mock inoculated and F. g. infected barley seedlings and characterize the contents by high-throughput analysis of exosomal proteins and sRNAs. Global analysis of exosome cargo will provide a catalog of candidate genes that can be quickly tested for their role in pathogenicity. This work will provide novel insights into how barley controls Fusarium infection and identify new proteins and sRNAs that can be used to improve resistance to Fusarium head blight (FHB).  Apoplastic barley exosomes (50-100 nM vesicles) are isolated using ultracentrifugation and density gradient purification from barley leaf tissue. We identified an Arabidopsis TET8 antibody, a marker for exosomes, that binds a barley TET8-orthologous protein isolated from our barley exosome total protein preparations. In addition, proteomic analysis (LC-MS/MS by spectral count) revealed enrichment of specific exosome proteins, including the syntaxin, PENETRATION 1 (PEN1) in the purified apoplastic preparations compared to the raw exosome preparations (not purified via density gradient methods). Recent research from Arabidopsis suggests that exosomes inhibit fungal penetration. Our current work is exploring which fractions of the barley exosome preparations reduce fungal spread when tested directly on barley leaves.  Additionally, we are exploring the use of tangential flow filtration (TFF) along with polyethylene glycol (PEG) precipitation to isolate the highest quality exosomes from the apoplast.  This method has advantages over the ultracentrifugation methods which may impact both the exosome content cataloging and fungal bioassays.

ACKNOWLEDGEMENT AND DISCLAIMER

 This material is based upon work supported by the U.S. Department of Agriculture, under Agreement No. 59-0206-0-148 (McLaughlin and Tumer).  We thank Hailing Jin (University of California, Riverside) for the TET8 antibodies gift.  We also thank Peter Lobel and his team at the Biological Mass Spectrometry Facility of Robert Wood Johnson Medical School and Rutgers.  This is a cooperative project with the U.S. Wheat & Barley Scab Initiative. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture.