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.

Gene silencing and gene expression in phytopathogenic fungi using a plant virus vector

“…Thus, VIGS is a powerful reverse genetics tool originally developed for plants, but new protocols and viral vectors have expanded its utility also for functional genomics in fungi. For example, the barley stripe mosaic virus (BSMV)-VIGS system has been used successfully for RNAi of specific pathogenicity genes in Puccinia triticina by siRNA generated in planta through infection of the BSMV vector expressing dsRNAs from P. triticina genes (9). Besides the complexity of some models that include transformation and handling of transgenic plants, the stability of these dsRNA transcripts also has been questioned in fungi (10) and in viral vectors (11).…”

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals

“…Some of these are discussed in greater detail. Based on recent studies, HIGS of pathogen‐specific deemed 'essential for life' genes could be an efficient strategy to control FHB, as well as other fungal diseases and pests 37, 45, 46. Broad spectrum control of multiple pathogens using a single approach is highly desirable.…”

Biotech Potatoes in the 21st Century: 20 Years Since the First Biotech Potato

“…Host induced gene silencing (HIGS) is a relatively new approach for controlling plant pathogens that relies on RNA interference to target the expression of essential pathogen genes. This strategy has been used to target a wide range of pathogen types including insects (reviews by Baum et al 2007;Huvenne and Smagghe 2010), nematodes (reviewed by Huang et al 2006;Yadav et al 2006;Fairbairn et al 2007;Sindhu et al 2009), fungi (Nowara et al 2010;Tinoco et al 2010;Yin et al 2011;Zhang et al 2012;Koch et al 2013;Panwar et al 2013;Pliego et al 2013;Ghag et al 2014), parasitic weeds (Tomilov et al 2008), and oomycetes (Govindarajulu et al 2014;VegaArreguin et al 2014;Jahan et al 2015). Pathogen effectors essential for virulence or Bhousekeeping^genes necessary for normal pathogen growth are typically the targets for HIGS.…”