Mycovirus-encoded suppressors of RNA silencing: Possible allies or enemies in the use of RNAi to control fungal disease in crops

Abstract

Plants, fungi, and many other eukaryotes have evolved an RNA interference (RNAi) mechanism that is key for regulating gene expression and the control of pathogens. RNAi inhibits gene expression, in a sequence-specific manner, by recognizing and deploying cognate double-stranded RNA (dsRNA) either from endogenous sources (e.g. pre-micro RNAs) or exogenous origin (e.g. viruses, dsRNA, or small interfering RNAs, siRNAs). Recent studies have demonstrated that fungal pathogens can transfer siRNAs into plant cells to suppress host immunity and aid infection, in a mechanism termed cross-kingdom RNAi. New technologies, based on RNAi are being developed for crop protection against insect pests, viruses, and more recently against fungal pathogens. One example, is host-induced gene silencing (HIGS), which is a mechanism whereby transgenic plants are modified to produce siRNAs or dsRNAs targeting key transcripts of plants, or their pathogens or pests. An alternative gene regulation strategy that also co-opts the silencing machinery is spray-induced gene silencing (SIGS), in which dsRNAs or single-stranded RNAs (ssRNAs) are applied to target genes within a pathogen or pest. Fungi also use their RNA silencing machinery against mycoviruses (fungal viruses) and mycoviruses can deploy virus-encoded suppressors of RNAi (myco-VSRs) as a counter-defence. We propose that myco-VSRs may impact new dsRNA-based management methods, resulting in unintended outcomes, including suppression of management by HIGS or SIGS. Despite a large diversity of mycoviruses being discovered using high throughput sequencing, their biology is poorly understood. In particular, the prevalence of mycoviruses and the cellular effect of their encoded VSRs are under-appreciated when considering the deployment of HIGS and SIGS strategies. This review focuses on mycoviruses, their VSR activities in fungi, and the implications for control of pathogenic fungi using RNAi.

Keywords: HIGS; RNA interference; SIGS; dsRNA; mycovirus; suppressor of silencing.

Figure 1

Various scenarios depicting the impact of fungal control using spray-induced gene silencing (SIGS) targeting fungal silencing machinery and mycovirus infection. These scenarios are particularly relevant for those fungi that produce dsRNA to target the host defence response. (A) Fungus produces either double stranded RNA (dsRNAs) and/or small interfering RNAs (siRNAs) to suppress host plant immunity or regulate virulence genes (effectors). (B) The dsRNAs and/or siRNAs from SIGS are taken up by a fungus and processed by RNAIII enzyme, DICER. Then, Argonaute (AGO) and RNA-induced silencing complex (RISC) include one siRNA strand that guides silencing of DCL1/2 and AGO1/2 messenger mRNA (mRNA). Alternatively, siRNAs prime RNA-dependent RNA polymerase (RdRP) to create more dsRNA that amplifies the RNAi machinery to target more DCL1/2 and AGO1/2 mRNAs. In scenario B the RNAi activity in the cell will be reduced resulting in few siRNA required for pathogenesis. If the fungus relies on siRNAs as key secreted virulence factors, then fungal fitness and pathogenicity will be reduced. (C) Fungus is infected by mycovirus with no or weak virus-encoded suppressors of RNAi (VSRs) resulting in mycovirus replication that is limited by active fungal host RNAi (dependent on specific mycovirus and fungal host interaction). (D) This scenario depicts the combinations of A and B, where the fungus has a mycovirus and SIGS targets either DICER or AGO resulting in reduced antiviral activity, increased mycovirus replication and reducing fungal virulence compared with scenario (C) virulence. (E) Mycovirus produces VSRs to deactivate the fungal RNAi machinery (DICER, AGO, RdRP or other RNAi targets/activities), resulting in mycovirus accumulation and reduction in fungal fitness. (F) Depicts a scenario of a fungus with SIGS (targeting DICER and AGO) and a mycovirus encoding strong VSR activity; the outcome is unknown, and probably dependent on a balance between mycovirus, VSR activity and strength, and the efficiency of the dsRNA to achieve silencing. (G) Depicts a fungus with SIGS applied to target β-tubulin (a protein not involved in RNAi). Fungal death results from the inhibition of tubulin polymerization. (H) depicts a scenario that combines E and G whereby the fungal cell is infected with a mycovirus with strong VSR activity and tubulin polymerization is inhibited via RNAi. Fitness of the fungus in this scenario will depend on the balance between SIGS-induced RNAi efficacy and VSR deactivation of the RNAi machinery similar to scenario (F). The illustration was created with BioRender.