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.