title: Host-induced gene silencing of wheat leaf rust fungus Puccinia triticina pathogenicity genes mediated by the Barley stripe mosaic virus.
abstract: Rust fungi are devastating plant pathogens and several Puccinia species have a large economic impact on wheat production worldwide. Disease protection, mostly offered by introgressed host-resistance genes, is often race-specific and rapidly overcome by newly-emerging virulent strains. Extensive new genomic resources have identified vital pathogenicity genes but their study is hampered because of the biotrophic life styles of rust fungi. In cereals, Barley stripe mosaic virus (BSMV)-induced RNAi has emerged as a useful tool to study loss-of-function phenotypes of candidate genes. Expression of pathogen-derived gene fragments in this system can be used to obtain in planta-generated silencing of corresponding genes inside biotrophic pathogens, a technique termed host-induced gene silencing (HIGS). Here we test the effectiveness of BSMV-mediated HIGS in the wheat leaf rust fungus Puccinia triticina (Pt) by targeting three predicted pathogenicity genes, a MAPK, a cyclophilin, and a calcineurin regulatory subunit. Inoculation of BSMV RNAi constructs generated fungal gene-specific siRNA molecules in systemic leaves of wheat plant. Subsequent Pt inoculation resulted in a suppressed disease phenotype and a reduction in endogenous transcript levels of the targeted fungal genes indicating translocation of siRNA molecules from host to fungal cells. Efficiency of this host-generated trans-specific RNAi was enhanced by using BSMV silencing vectors defective in coat protein coupled with introducing fungal gene sequences simultaneously in sense and antisense orientation. The disease suppression indicated the likely involvement of these fungal genes in pathogenicity. This study demonstrates that BSMV-mediated in planta-generated RNAi is an effective strategy for functional genomics in rust fungi.
authors: Agriculture and Agri-Food Canada, Pacific Agri-Food Research Center, PO Box 5000, 4200 Hwy 97, Summerland, BC, V0H 1Z0, Canada.
authors: Vinay
authors: V
authors: Panwar
authors: Brent
authors: B
authors: McCallum
authors: Guus
authors: G
authors: Bakkeren
mesh_terms: D001487:Basidiomycota / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016679:Genome, Viral; D054884:Host-Pathogen Interactions; D018613:Microscopy, Confocal; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D009029:Mosaic Viruses / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D010935:Plant Diseases / Q000235:genetics / Q000382:microbiology* / Q000821:virology; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000382:microbiology / Q000821:virology; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014908:Triticum / Q000235:genetics / Q000378:metabolism / Q000382:microbiology* / Q000821:virology; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D005656:Fungal Proteins; D034741:RNA, Small Interfering; D012367:RNA, Viral; D037521:Virulence Factors; D019950:Mitogen-Activated Protein Kinase 1