Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley.
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.
Hein
Ingo
I
Programme of Genome Dynamics, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, United Kingdom.
Barciszewska-Pacak
Maria
M
Hrubikova
Katarina
K
Williamson
Sandie
S
Dinesen
Malene
M
Soenderby
Ida E
IE
Sundar
Suresh
S
Jarmolowski
Artur
A
Shirasu
Ken
K
Lacomme
Christophe
C
D002352:Carrier Proteins / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D017343:Genes, Plant; D005822:Genetic Vectors; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D001467:Hordeum / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics* / Q000382:microbiology; D018515:Plant Leaves / Q000382:microbiology / Q000502:physiology; D010940:Plant Proteins / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D010942:Plant Viruses / Q000235:genetics* / Q000502:physiology; D030821:Plants, Genetically Modified
D002352:Carrier Proteins; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010940:Plant Proteins; C499582:Rar1 protein, Hordeum vulgare