The discovery of sRNA uptake by fungal pathogens of barley and delivery of sRNAs and potentially proteins and other small molecules into fungal pathogens by exosomes provides a strong rationale to understand the contents of barley exosomes and the impact on Fusarium graminearum.  Recent work in Arabidopsis has shown that sRNAs generated by the plant accumulate at infection sites and are taken up by fungal cells, host sRNAs that function to silence fungal genes that are critical for pathogenicity.  Previously in our laboratory, we showed, using confocal microscopy, that infection of the barley variety Conlon with Ph1 spores induces multivesicular bodies (MVBs) accumulation. To determine if sRNAs and proteins are packaged into exosomes and delivered into F.g. during infection of barley, we isolated exosomes from barley apoplast fluid. Transgenic barley (Golden Promise) plants that accumulate the Arabidopsis (AtLTP4.4-GFP) and wheat (TaLTP3-GFP) nsLTP proteins in the apoplast were used in the experiment.  We followed the Rutter and Innes exosome protocol to isolate and quantify plant extracellular vesicles.  Proteins isolated from exosomes purified using a discontinuous iodixanol density gradient (OptiPrep) were run on a SDS-PAGE gel and analyzed by Western blotting using Arabidopsis TET8 antibodies.  We found that the isolated barley exosome proteins contain an orthologous TET8 protein that will cross react with the Arabidopsis TET8 antibodies, indicating a promising marker for barley exosomes.  Proteomic analysis via spectral scanning was performed between exosomes purified only by ultracentrifugation and exosomes purified by the OptiPrep gradient.  An approximate 20% reduction in Rubisco was observed in the post-OptiPrep samples indicating enhanced purification.  Gene Ontology (GO) term enrichment was identified for vesicle mediated transport, endomembrane system, GTPase activity, and several other classes of proteins.  Specific proteins of potential interest identified in the enriched exosome fraction (http://microvesicles.org/) include small heat shock proteins, annexins, GAPDH enzymes, and GSH enzymes.

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 sending us TET8 antibodies.  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.