Abstract
Host-induced gene silencing (HIGS) based on trans-kingdom RNA interference (RNAi) has been successfully exploited to engineer host resistance to pests and pathogens, including fungi and oomycetes. However, revealing the mechanisms underlying trans-kingdom RNAi between hosts and pathogens lags behind applications. The effectiveness and durability of trans-kingdom silencing of pathogenic genes are uncharacterized. In this study, using our transgenic 35S-VdH1i cotton plants in which dsVdH1-derived small RNAs (siVdH1) accumulated, small RNA sequencing analysis revealed that siVdH1s exclusively occur within the double-stranded (ds)VdH1 region, and no transitive siRNAs were produced beyond this region in recovered hyphae of Verticillium dahliae (V. dahliae). Accordingly, we found that VdH1 silencing was reduced over time in recovered hyphae cultured in vitro, inferring that once the fungus got rid of the 35S-VdH1i cotton plants would gradually regain their pathogenicity. To explore whether continually exporting dsRNAs/siRNAs from transgenic plants into recipient fungal cells guaranteed the effectiveness and stability of HIGS, we created GFP/RFP double-labeled V. dahliae and transgenic Arabidopsis expressing dsGFP (35S-GFPi plants). Confocal images visually demonstrate the efficient silencing of GFP in V. dahliae that colonized host vascular tissues. Taken together, our results demonstrate that HIGS effectively triggers long-lasting trans-kingdom RNAi during plant vasculature V. dahliae interactions, despite no amplification or transitivity of RNAi being noted in this soil-borne fungal pathogen.
Highlights
RNA interference (RNAi, or RNA silencing) is universal in eukaryotes, including animals, plants, and fungi
Small RNAs isolated from VdWT−1st and VdVdH1i−1st colonies as well as 35S-VdH1i transgenic cotton plants were sequenced. siRNAs mapped to VdH1 were included in our analysis
The newly developed Host-induced gene silencing (HIGS) is based on a sequence-specific RNAi mechanism that is friendly to the environment
Summary
RNA interference (RNAi, or RNA silencing) is universal in eukaryotes, including animals, plants, and fungi. Compared with RNAi in animals and plants, the mechanisms of fungal RNAi are diverse based on studies in the model fungus, Neurospora crassa [1–3]. The existence of RNAi in plant phytopathogenic fungi is largely unknown [4–6]. RNAi in fungi is significantly diversified, and the numbers of RNA silencing proteins differ considerably among fungal species [24]. This diversification may lead to reduced efficacy of RNAi in some fungal species, such as Ustilago maydis, in which the entire RNA silencing machinery appears to have been lost [25]
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