Plant Physiology Preview
Published on July 22, 2005; 10.1104/pp.105.062810
Received March 14, 2005
Returned for revision May 6, 2005
Accepted May 24, 2005
Virus-Induced Gene Silencing-Based Functional Characterization of Genes Associated with Powdery Mildew Resistance in Barley
Ingo Hein , Maria Barciszewska-Pacak , Katarina Hrubikova , Sandie Williamson , Malene Dinesen , Ida E. Soenderby , Suresh Sundar , Artur Jarmolowski , Ken Shirasu , and Christophe Lacomme *
Programme of Genome Dynamics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom
Programme of Cell-to-Cell Communication, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom; Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, 60-371 Poznan, Poland
Programme of Cell-to-Cell Communication, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom
Danish Institute of Agricultural Sciences, Biotechnology Group, DK-1871 Frederiksberg C, Copenhagen, Denmark
Denmark Plant Biochemistry Laboratory, Department of Plant Biology, The Royal Veterinary and Agricultural University, DK-1871 Frederiksberg C, Copenhagen, Denmark
Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, 60-371 Poznan, Poland
The Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom
* Corresponding author; email: clacom{at}scri.sari.ac.uk.
We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley.
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