Showing papers by "Shou-Wei Ding published in 2022"

Mouse circulating extracellular vesicles contain virus‐derived siRNAs active in antiviral immunity

Abstract: Induction and suppression of antiviral RNA interference (RNAi) has been observed in mammals during infection with at least seven distinct RNA viruses, including some that are pathogenic in humans. However, while the cell‐autonomous immune response mediated by antiviral RNAi is gradually being recognized, little is known about systemic antiviral RNAi in mammals. Furthermore, extracellular vesicles (EVs) also function in viral signal spreading and host immunity. Here, we show that upon antiviral RNAi activation, virus‐derived small‐interfering RNAs (vsiRNAs) from Nodamura virus (NoV), Sindbis virus (SINV), and Zika virus (ZIKV) enter the murine bloodstream via EVs for systemic circulation. vsiRNAs in the EVs are biologically active, since they confer RNA–RNA homology‐dependent antiviral activity in both cultured cells and infant mice. Moreover, we demonstrate that vaccination with a live‐attenuated virus, rendered deficient in RNAi suppression, induces production of stably maintained vsiRNAs and confers protective immunity against virus infection in mice. This suggests that vaccination with live‐attenuated VSR (viral suppressor of RNAi)‐deficient mutant viruses could be a new strategy to induce immunity.

Identification of positive and negative regulators of antiviral RNA interference in Arabidopsis thaliana

Abstract: Virus-host coevolution often drives virus immune escape. However, it remains unknown whether natural variations of plant virus resistance are enriched in genes of RNA interference (RNAi) pathway known to confer essential antiviral defense in plants. Here, we report two genome-wide association study screens to interrogate natural variation among wild-collected Arabidopsis thaliana accessions in quantitative resistance to the endemic cucumber mosaic virus (CMV). We demonstrate that the highest-ranked gene significantly associated with resistance from both screens acts to regulate antiviral RNAi in ecotype Columbia-0. One gene, corresponding to Reduced Dormancy 5 (RDO5), enhances resistance by promoting amplification of the virus-derived small interfering RNAs (vsiRNAs). Interestingly, the second gene, designated Antiviral RNAi Regulator 1 (VIR1), dampens antiviral RNAi so its genetic inactivation by CRISPR/Cas9 editing enhances both vsiRNA production and CMV resistance. Our findings identify positive and negative regulators of the antiviral RNAi defense that may play important roles in virus-host coevolution.

Current rice production is highly vulnerable to insect-borne viral diseases

Abstract: Rice is one of the most important food crops and feeds more than half of the world’s population [1].Arthropod-borne rice viruses have caused devastating epidemics in Asian countries and are amajor threat to food security [2]. However, little is known about the vulnerability of rice crops to viral pathogens [3,4], especially southern rice black-streaked dwarf virus (SRBSDV), rice black-streaked dwarf virus (RBSDV) and rice gall dwarf virus (RGDV), known to induce annual outbreaks by insect transmission in some localities in Asia. To address this question, we first investigated the susceptibility of 136 conventional and hybrid rice varieties widely cultivated or approved for release in China to SRBSDV, which has been circulating in Asian countries since its first report in 2008 [2]. We monitored symptom development and asymptomatic infection in seedlings after inoculation with viruliferous white-backed plant hoppers (Sogatella furcifera, Horváth) in a greenhouse. We found that most of the varieties examinedwere highly susceptible to SRBSDV and developed the characteristic disease symptoms at 45 days postinoculation (Fig. 1A and Supplementary Table S1). The results from greenhouse inoculation with viruliferous insect vectors predict widespread vulnerability of rice cultivars to SRBSDV in rice fields. To test this hypothesis, we planted seedlings of 528 varieties in 3 consecutive years under open field conditions at locations of Nanning and Guilin, Guangxi Province that have recorded a multiyear SRBSDV outbreak from plant hopper transmission. We found that ≥25% of the seedlings from 80%–93% of the examined hybrid or conventional indica

Transgene silencing, RNA interference, and the antiviral defense mechanism directed by small interfering RNAs.

Abstract: One very important discovery in plant pathology over recent decades is the natural antiviral defense mechanism mediated by RNA interference (RNAi). In antiviral RNAi, virus infection triggers Dicer processing of virus-specific double-stranded RNA into small interfering RNAs (siRNAs). Frequently further amplified by host enzyme and cofactors, these virus-derived siRNAs direct specific virus clearance in an Argonaute protein-containing effector complex. The siRNAs derived from viruses and viroids accumulate to very high levels during infection. Because they overlap extensively in nucleotide sequence this allows for deep sequencing and bioinformatics assembly of total small RNAs for rapid discovery and identification of viruses and viroids. Antiviral RNAi acts as the primary defense mechanism against both RNA and DNA viruses in plants, yet viruses still successfully infect plants. They do so because all currently recognized plant viruses combat the RNAi response by encoding at least one protein as viral suppressor of RNAi (VSR) required for infection even though plant viruses have small genome sizes with a limited coding capacity. This review article will recapitulate the key findings that have revealed the genetic pathway for the biogenesis and antiviral activity of viral siRNAs and the specific role of VSRs in infection by antiviral RNAi suppression. Moreover, I also discuss how early pioneering studies on transgene silencing, RNAi, and virus-plant/virus-virus interactions paved the road to the discovery of antiviral RNAi.

Discovery of aphid-transmitted Rice tiller inhibition virus from native plants through metagenomic sequencing

Abstract: A major threat to rice production is the disease epidemics caused by insect-borne viruses that emerge and re-emerge with undefined origins. It is well known that some human viruses have zoonotic origins from wild animals. However, it remains unknown whether native plants host new endemic viruses with spillover potential to rice (Oryza sativa) as emerging pathogens. Here, we discovered rice tiller inhibition virus (RTIV), a novel RNA virus species, from colonies of Asian wild rice (O. rufipogon) in a genetic reserve by metagenomic sequencing. We identified the specific aphid vector to transmit RTIV and found RTIV would cause low-tillering disease in rice cultivar after transmission. We further demonstrated that an infectious molecular clone of RTIV initiated systemic infection and causes low-tillering disease in an elite rice variety after Agrobacterium-mediated inoculation or stable plant transformation, and RTIV can also be transmitted from transgenic rice plant through its aphid vector to cause disease. Finally, global transcriptome analysis indicated that RTIV may disturb defense and tillering pathway to cause low tillering disease in rice cultivar. Thus, our results show that new rice viral pathogens can emerge from native habitats, and RTIV, a rare aphid-transmitted rice viral pathogen from native wild rice, can threaten the production of rice cultivar after spillover.

Culture-Independent Discovery of Viroids by Deep Sequencing and Computational Algorithms.

Abstract: Viroids are single-stranded circular RNA molecules that cause diseases in plants and do not encode any protein. Classical approaches for the identification of new viroids are challenging for many plant pathology laboratories as viroid cDNA synthesis and sequencing require purification and enrichment of the naked viroid RNA by two-dimensional gel electrophoresis. Conventional metagenomic approaches are not effective for viroid discovery because the total number of known viroids is small, and distinct viroids share limited nucleotide sequence similarity. In this chapter, we describe a homology-independent approach for the identification of both known and new viroids in disease samples. It is known that viroid infection of plants triggers production of overlapping viroid-derived small interfering RNAs (siRNAs) targeting the entire genome with high densities and that replication of viroids occurs via a rolling-circle mechanism to yield head-to-tail multiple-repeat replicative intermediates. Our approach involves deep sequencing of either long or small RNAs in a disease sample followed by viroid identification with a unique computational algorithm, progressive filtering of overlapping small RNAs (PFOR). Among the sequenced total small RNAs, PFOR retains viroid-derived siRNAs for viroid genome assembly by progressively eliminating nonoverlapping small RNAs and those that overlap but cannot be assembled into a direct repeat RNA, a unique feature of viroid RNA replication. In contrast, long RNAs sequenced after depletion of ribosomal RNAs are cut into 21-nucleotide virtual overlapping small RNAs with the algorithm SLS (splitting longer read into shorter fragments) before PFOR. We show that new viroids or viroids from the two known families are readily identified and their full-length sequences recovered by PFOR from long or small RNAs sequenced directly from infected plants. We propose that our approach can be used for viroid discovery in both plants and potentially animals since PFOR identifies viroids by searching for circular RNAs or a unique replication intermediate of the viroid genome in a sequence homology-independent manner.