|Luster, Douglas - Doug|
|Mcmahon, Michael - Mike|
|Edwards, Herbert - Western Illinois University|
|Boerma, Britney - Western Illinois University|
|Miller, Sally - The Ohio State University|
|Dorrance, Anne - The Ohio State University|
Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2012
Publication Date: 3/23/2012
Citation: Luster, D.G., Mcmahon, M.B., Edwards, H.H., Boerma, B.L., Miller, S.A., Dorrance, A.E. 2012. Novel phakopsora pachyrhizi extracellular proteins are ideal targets for immunological diagnostic assays. Applied and Environmental Microbiology. 78(11):3890-3895. Interpretive Summary: Asian soybean rust (ASR), continues to expand across the southeast and mid-south regions of the U.S., resulting in increased fungicide applications and associated cost inputs for producers. Our objectives in this research were to identify molecular targets for development of diagnostic assays, preferably expressed early in infected plants, before ASR symptoms appear, so that producers and extension agents can identify ASR infections and recommend fungicide treatments before the disease becomes widespread. We have identified and partially characterized a set of ASR proteins that are expressed in infected plants, and subsequently developed diagnostic reagents specific for the ASR molecular targets. These diagnostic reagents will prove applicable in early detection and diagnosis of ASR in the field, and to identify ASR spores captured in spore traps from U.S. sentinel surveillance plots.
Technical Abstract: Phakopsora pachyrhizi, the causal agent of Asian soybean rust (ASR), continues to expand across the southeast and mid-south regions of the U.S., resulting in increased fungicide applications for producers. Our objectives in this research were to identify ASR protein targets for development of immunodiagnostic assays, preferably expressed in planta and early in infection. We have identified and partially characterized a small gene family encoding extracellular proteins in the P. pachyrhizi urediniospore wall, termed PHEPs (for PHakopsora Extracellular Protein). Two high titer protein family members, PHEP 107 and PHEP 369, were selected as ideal immunodiagnostic targets after localization studies confirmed their extracellular nature, and western blot analysis detected PHEPs in plants as early as 3 DPI. Monoclonal antibodies (MAbs 2E8E5-1 and 3G6H7-3) generated against recombinant PHEP 369 were tested for sensitivity against the recombinant protein and extracts from ASR-infected plants, and for specificity against a set of common soybean pathogens (from cultures and infected soybeans). MAb 3G6H7-3 was highly specific for the target protein, detected as little as100 pg protein, and did not react with any of the soybean pathogens or urediniospores of related rust fungi. These antibodies will prove applicable in immunodiagnostic assays with infected soybeans and to identify ASR spores from sentinel surveillance plots.