Genome-wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis

Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) are the two most prevalent viruses infecting orchids and causing economic losses worldwide. Mixed infection of CymMV and ORSV could induce intensified symptoms as early at 10 days post-inoculation in inoculated Phalaenopsis amabilis, where CymMV pathogenesis was unilaterally enhanced by ORSV. To reveal the antiviral RNA silencing activity in orchids, we characterized the viral small-interfering RNAs (vsiRNAs) from CymMV and ORSV singly or synergistically infecting P. amabilis. We also temporally classified the inoculated leaf-tip tissues and noninoculated adjacent tissues as late and early stages of infection, respectively. Regardless of early or late stage with single or double infection, CymMV and ORSV vsiRNAs were predominant in 21- and 22-nt sizes, with excess positive polarity and under-represented 5ʹ-guanine. While CymMV vsiRNAs mainly derived from RNA-dependent RNA polymerase-coding regions, ORSV vsiRNAs encompassed the coat protein gene and 3ʹ-untranslated region, with a specific hotspot residing in the 3ʹ-terminal pseudoknot. With double infection, CymMV vsiRNAs increased more than 5-fold in number with increasing virus titres. Most vsiRNA features remained unchanged with double inoculation, but additional ORSV vsiRNA hotspot peaks were prominent. The potential vsiRNA-mediated regulation of the novel targets in double-infected tissues thereby provides a different view of CymMV and ORSV synergism. Hence, temporally profiled vsiRNAs from taxonomically distinct CymMV and ORSV illustrate active antiviral RNA silencing in their natural host, Phalaenopsis, during both early and late stages of infection. Our findings provide insights into offence–defence interactions among CymMV, ORSV and orchids.     

Differential Expression of Tomato Spotted Wilt Virus-Derived Viral Small RNAs in Infected Commercial and Experimental Host Plants

Background

Viral small RNAs (vsiRNAs) in the infected host can be generated from viral double-stranded RNA replicative intermediates, self-complementary regions of the viral genome or from the action of host RNA-dependent RNA polymerases on viral templates. The vsiRNA abundance and profile as well as the endogenous small RNA population can vary between different hosts infected by the same virus influencing viral pathogenicity and host response. There are no reports on the analysis of vsiRNAs of Tomato spotted wilt virus (TSWV), a segmented negative stranded RNA virus in the family Bunyaviridae, with two of its gene segments showing ambisense gene arrangement. The virus causes significant economic losses to numerous field and horticultural crops worldwide.

Principal Findings

Tomato spotted wilt virus (TSWV)-specific vsiRNAs were characterized by deep sequencing in virus-infected experimental host Nicotiana benthamiana and a commercial, susceptible host tomato. The total small (s) RNA reads in TSWV-infected tomato sample showed relatively equal distribution of 21, 22 and 24 nt, whereas N. benthamiana sample was dominated by 24 nt total sRNAs. The number of vsiRNA reads detected in tomato was many a magnitude (~350:1) higher than those found in N. benthamiana, however the profile of vsiRNAs in terms of relative abundance 21, 22 and 24 nt class size was similar in both the hosts. Maximum vsiRNA reads were obtained for the M RNA segment of TSWV while the largest L RNA segment had the least number of vsiRNAs in both tomato and N. benthamiana. Only the silencing suppressor, NSs, of TSWV recorded higher antisense vsiRNA with respect to the coding frame among all the genes of TSWV.

Significance

Details of the origin, distribution and abundance of TSWV vsiRNAs could be useful in designing efficient targets for exploiting RNA interference for virus resistance. It also has major implications toward our understanding of the differential processing of vsiRNAs in antiviral defense and viral pathogenicity.     

Deep sequencing of mycovirus‐derived small RNAs from Botrytis species

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer‐like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus‐derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double‐stranded RNA (dsRNA), (+)single‐stranded RNA (ssRNA) and (–)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5′ end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.     

Dynamics of Small RNA Profiles of Virus and Host Origin in Wheat Cultivars Synergistically Infected by Wheat Streak Mosaic Virus and Triticum Mosaic Virus: Virus Infection Caused a Drastic Shift in the Endogenous Small RNA Profile

Co-infection of wheat (Triticum aestivum L.) by Wheat streak mosaic virus (WSMV, a Tritimovirus) and Triticum mosaic virus (TriMV, a Poacevirus) of the family Potyviridae causes synergistic interaction. In this study, the effects of the synergistic interaction between WSMV and TriMV on endogenous and virus-derived small interfering RNAs (vsiRNAs) were examined in susceptible (‘Arapahoe’) and temperature-sensitive resistant (‘Mace’) wheat cultivars at 18°C and 27°C. Single and double infections in wheat caused a shift in the profile of endogenous small RNAs from 24 nt being the most predominant in healthy plants to 21 nt in infected wheat. Massive amounts of 21 and 22 nt vsiRNAs accumulated in singly and doubly infected Arapahoe at both temperatures and in Mace at 27°C but not 18°C. The plus- and minus-sense vsiRNAs were distributed throughout the genomic RNAs in Arapahoe at both temperature regimens and in Mace at 27°C, although some regions served as hot-spots, spawning an excessive number of vsiRNAs. The vsiRNA peaks were conserved among cultivars, suggesting that the Dicer-like enzymes in susceptible and resistant cultivars similarly accessed the genomic RNAs of WSMV or TriMV. Accumulation of large amounts of vsiRNAs in doubly infected plants suggests that the silencing suppressor proteins encoded by TriMV and WSMV do not prevent the formation of vsiRNAs; thus, the synergistic effect observed is independent from RNA-silencing mediated vsiRNA biogenesis. The high-resolution map of endogenous and vsiRNAs from WSMV- and/or TriMV-infected wheat cultivars may form a foundation for understanding the virus-host interactions, the effect of synergistic interactions on host defense, and virus resistance mechanisms in wheat.     

Efficient Dicer processing of virus-derived double-stranded RNAs and its modulation by RIG-I-like receptor LGP2

The interferon-regulated antiviral responses are essential for the induction of both innate and adaptive immunity in mammals. Production of virus-derived small-interfering RNAs (vsiRNAs) to restrict virus infection by RNA interference (RNAi) is a recently identified mammalian immune response to several RNA viruses, which cause important human diseases such as influenza and Zika virus. However, little is known about Dicer processing of viral double-stranded RNA replicative intermediates (dsRNA-vRIs) in mammalian somatic cells. Here we show that infected somatic cells produced more influenza vsiRNAs than cellular microRNAs when both were produced by human Dicer expressed de novo, indicating that dsRNA-vRIs are not poor Dicer substrates as previously proposed according to in vitro Dicer processing of synthetic long dsRNA. We report the first evidence both for canonical vsiRNA production during wild-type Nodamura virus infection and direct vsiRNA sequestration by its RNAi suppressor protein B2 in two strains of suckling mice. Moreover, Sindbis virus (SINV) accumulation in vivo was decreased by prior production of SINV-targeting vsiRNAs triggered by infection and increased by heterologous expression of B2 in cis from SINV genome, indicating an antiviral function for the induced RNAi response. These findings reveal that unlike artificial long dsRNA, dsRNA-vRIs made during authentic infection of mature somatic cells are efficiently processed by Dicer into vsiRNAs to direct antiviral RNAi. Interestingly, Dicer processing of dsRNA-vRIs into vsiRNAs was inhibited by LGP2 (laboratory of genetics and physiology 2), which was encoded by an interferon-stimulated gene (ISG) shown recently to inhibit Dicer processing of artificial long dsRNA in cell culture. Our work thus further suggests negative modulation of antiviral RNAi by a known ISG from the interferon response.     

利用小RNA深度测序技术鉴定半夏病毒病病原

RNA沉默是真核生物体内由病毒来源的干扰小RNA（virus derived small interfering RNA, vsiRNA）沉默复合物介导目标RNA特异降解的一种保守机制,通过对vsiRNA分析可进行植物病毒病原鉴定。本文利用小RNA深度测序技术对感病半夏叶片进行鉴定,结果发现,表现典型花叶症状的半夏叶片受到大豆花叶病毒（Soybean mosaic virus, SMV）、黄瓜花叶病毒（Cucumber mosaic virus, CMV）、芋花叶病毒（Dasheen mosaic virus, DsMV）、魔芋花叶病毒（Konjac mosaic virus, KoMV）、烟草花叶病毒（Tobacco mosaic virus, TMV）等多种病毒的复合侵染。为明确SMV山西半夏分离物（SMV-SXBX）的进化关系,进行SMV-SXBX全基因组克隆与分析,获得SMV-SXBX全长为9 735 nt,编码一个由3 105个氨基酸组成的多聚蛋白质。通过核苷酸与氨基酸序列比对发现,SMV-SXBX与半夏分离物P同源性最高,分别为91.1%和94.1%,且系统发育分析表明,SMV-SXBX与半夏SMV分离物P聚为一簇。同时,也对vsiRNA进行了系统分析,研究结果有望为半夏SMV的有效防治提供一定科学依据。     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

The ORF5 of Grapevine virus A is involved in symptoms expression in Nicotiana benthamiana plants

Grapevine virus A (GVA), a member of the genus Vitivirus which belongs to the family Flexiviridae, has a single‐stranded RNA genome of about 7.4 kb that comprises five open reading frames (ORFs). ORF5 encodes a small 10‐kDa protein (p10), which is believed to interact with nucleic acids and to suppress the plant's RNA‐ silencing response. We obtained molecular and biological data indicating that ORF5‐encoded product, specifically its N‐terminus, affects the appearance of symptoms in Nicotiana benthamiana plants. The ORF5‐encoded products of the severe GR5 and the mild GTR1‐1 isolates were found to affect RNA silencing similarly in mesophyll cells of N. benthamiana, despite being involved in different expressions of symptoms on this host.     

A Turnip crinkle virus Isolate Lacking the CP Counter‐Defence Protein Gene Providing Protection Against the Wild‐Type Strain is Associated with Highly Localized RNA Silencing

Cross‐protection has been used successfully and commercially to control a range of virus diseases for which the selection of suitable mild strains of plant viruses is necessary. Turnip crinkle virus (TCV) is highly pathogenic on Arabidopsis plants and its silencing suppressor‐defective mutant, TCVΔCP, can induce highly localized RNA silencing which is differs from that of other protective strains. We found that TCVΔCP provides some protection against wild‐type TCV but lacks complete protection, and the relative locations of the protective virus and challenge virus affect the degree of cross‐protection. However, similar cross‐protection afforded by TCVΔCP is not observed in Nicotiana benthamiana plants. As expected, TCVΔCP pre‐infected Arabidopsis plants fail to protect against infection with the unrelated Cucumber mosaic virus, strain Fhy. It appears that cross‐protection afforded by TCVΔCP requires that the challenge virus be very similar in sequence, which is a characteristic of RNA silencing. In order to investigate whether the protection is associated with the highly localized RNA silencing, mutant plants involved in key silencing pathway genes of RNA silencing machinery, including dcl2, dcl4 and triple dcl2/dcl3/dcl4 mutants were used. The results demonstrate that cross‐protection afforded by TCVΔCP is dependent on host RNA silencing, and both DCL2 and DCL4 play important roles in this process.     

Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing‐mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA‐dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus‐derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non‐transgenic N. benthamiana plants as well as transgene‐derived siRNAs (t‐siRNAs) derived from the immune‐transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t‐siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t‐siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t‐siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24‐nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24‐nucleotide t‐siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24‐nucleotide t‐siRNAs is associated with crinivirus immunity in transgenic plants.     

Structural and Functional Analysis of Viral siRNAs

A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5′-Phosphate-Dependent Exonuclease to study the 5′ end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5′ monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5′ end of short RNAs or after replacing any potential 5′ ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.     

Global Analyses of Small Interfering RNAs Derived from Bamboo mosaic virus and Its Associated Satellite RNAs in Different Plants

Background

Satellite RNAs (satRNAs), virus parasites, are exclusively associated with plant virus infection and have attracted much interest over the last 3 decades. Upon virus infection, virus-specific small interfering RNAs (vsiRNAs) are produced by dicer-like (DCL) endoribonucleases for anti-viral defense. The composition of vsiRNAs has been studied extensively; however, studies of satRNA-derived siRNAs (satsiRNAs) or siRNA profiles after satRNA co-infection are limited. Here, we report on the small RNA profiles associated with infection with Bamboo mosaic virus (BaMV) and its two satellite RNAs (satBaMVs) in Nicotiana benthamiana and Arabidopsis thaliana.

Methodology/Principal Findings

Leaves of N. benthamiana or A. thaliana inoculated with water, BaMV alone or co-inoculated with interfering or noninterfering satBaMV were collected for RNA extraction, then large-scale Solexa sequencing. Up to about 20% of total siRNAs as BaMV-specific siRNAs were accumulated in highly susceptible N. benthamiana leaves inoculated with BaMV alone or co-inoculated with noninterfering satBaMV; however, only about 0.1% of vsiRNAs were produced in plants co-infected with interfering satBaMV. The abundant region of siRNA distribution along BaMV and satBaMV genomes differed by host but not by co-infection with satBaMV. Most of the BaMV and satBaMV siRNAs were 21 or 22 nt, of both (+) and (−) polarities; however, a higher proportion of 22-nt BaMV and satBaMV siRNAs were generated in N. benthamiana than in A. thaliana. Furthermore, the proportion of non-viral 24-nt siRNAs was greatly increased in N. benthamiana after virus infection.

Conclusions/Significance

The overall composition of vsiRNAs and satsiRNAs in the infected plants reflect the combined action of virus, satRNA and different DCLs in host plants. Our findings suggest that the structure and/or sequence demands of various DCLs in different hosts may result in differential susceptibility to the same virus. DCL2 producing 24-nt siRNAs under biotic stresses may play a vital role in the antiviral mechanism in N. benthamiana.     

In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris

Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an “RNAi-of-RNAi” experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17–18) and also not of SBPV (n = 11–12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections.     

C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis

The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development.