Xianbing Wang

RNAi-mediated viral immunity requires amplification of virus-derived siRNAs in Arabidopsis thaliana

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5′-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3′ half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.

The 21-Nucleotide, but Not 22-Nucleotide, Viral Secondary Small Interfering RNAs Direct Potent Antiviral Defense by Two Cooperative Argonautes in Arabidopsis thaliana

This work identifies cooperative action of ARGONAUTE1 and ARGONAUTE2 in virus resistance conferred by 21-nucleotide virus-derived small interfering RNAs (siRNAs). It also reveals that 22-nucleotide viral siRNAs do not guide efficient antiviral defense, demonstrating a qualitative difference between 21- and 22-nucleotide classes of siRNAs in RNA silencing. Arabidopsis thaliana defense against distinct positive-strand RNA viruses requires production of virus-derived secondary small interfering RNAs (siRNAs) by multiple RNA-dependent RNA polymerases. However, little is known about the biogenesis pathway and effector mechanism of viral secondary siRNAs. Here, we describe a mutant of Cucumber mosaic virus (CMV-Δ2b) that is silenced predominantly by the RNA-DEPENDENT RNA POLYMERASE6 (RDR6)-dependent viral secondary siRNA pathway. We show that production of the viral secondary siRNAs targeting CMV-Δ2b requires SUPPRESSOR OF GENE SILENCING3 and DICER-LIKE4 (DCL4) in addition to RDR6. Examination of 25 single, double, and triple mutants impaired in nine ARGONAUTE (AGO) genes combined with coimmunoprecipitation and deep sequencing identifies an essential function for AGO1 and AGO2 in defense against CMV-Δ2b, which act downstream the biogenesis of viral secondary siRNAs in a nonredundant and cooperative manner. Our findings also illustrate that dicing of the viral RNA precursors of primary and secondary siRNA is insufficient to confer virus resistance. Notably, although DCL2 is able to produce abundant viral secondary siRNAs in the absence of DCL4, the resultant 22-nucleotide viral siRNAs alone do not guide efficient silencing of CMV-Δ2b. Possible mechanisms for the observed qualitative difference in RNA silencing between 21- and 22-nucleotide secondary siRNAs are discussed.

Oomycete pathogens encode RNA silencing suppressors

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

Viral Suppressors of RNA-Based Viral Immunity: Host Targets

RNA-based viral immunity (RVI) operates in fungi, plants, and invertebrates to specifically destroy viral RNAs using the cellular RNA interference machinery. Discovery of diverse viral proteins as suppressors of RNA silencing provides strong validation for the effectiveness of RVI. Here we review recent studies that have revealed new mechanistic insights into plant and insect viral suppressors of RVI or suggested a role for RNA silencing suppression during mammalian viral infection.

Virus infection triggers widespread silencing of host genes by a distinct class of endogenous siRNAs in Arabidopsis

Significance RNAi-mediated antiviral immunity directs specific virus resistance by virus-derived siRNAs in contrast to broad-spectrum resistance triggered in innate immunity by host pattern recognition receptors. Here we show that induction of antiviral RNAi in Arabidopsis is associated with production of a genetically distinct class of virus-activated siRNAs (vasiRNAs) by RNA-dependent RNA polymerase-1 to target hundreds of host genes for RNA silencing by Argonaute-2. Production of vasiRNAs is induced by viruses from two different supergroups of RNA virus families, targeted for inhibition by Cucumber mosaic virus, and correlated with virus resistance independently of viral siRNAs. We propose that antiviral RNAi activates broad-spectrum antiviral activity via widespread silencing of host genes directed by vasiRNAs in addition to specific antiviral defense by viral siRNAs. Antiviral immunity controlled by RNA interference (RNAi) in plants and animals is thought to specifically target only viral RNAs by the virus-derived small interfering RNAs (siRNAs). Here we show that activation of antiviral RNAi in Arabidopsis plants is accompanied by the production of an abundant class of endogenous siRNAs mapped to the exon regions of more than 1,000 host genes and rRNA. These virus-activated siRNAs (vasiRNAs) are predominantly 21 nucleotides long with an approximately equal ratio of sense and antisense strands. Genetically, vasiRNAs are distinct from the known plant endogenous siRNAs characterized to date and instead resemble viral siRNAs by requiring Dicer-like 4 and RNA-dependent RNA polymerase 1 (RDR1) for biogenesis. However, loss of EXORIBONUCLEASE4/THYLENE-INSENSITIVE5 enhances vasiRNA biogenesis and virus resistance without altering the biogenesis of viral siRNAs. We show that vasiRNAs are active in directing widespread silencing of the target host genes and that Argonaute-2 binds to and is essential for the silencing activity of vasiRNAs. Production of vasiRNAs is readily detectable in Arabidopsis after infection by viruses from two distinct supergroups of plant RNA virus families and is targeted for inhibition by the silencing suppressor protein 2b of Cucumber mosaic virus. These findings reveal RDR1 production of Arabidopsis endogenous siRNAs and identify production of vasiRNAs to direct widespread silencing of host genes as a conserved response of plants to infection by diverse viruses. A possible function for vasiRNAs to confer broad-spectrum antiviral activity distinct to the virus-specific antiviral RNAi by viral siRNAs is discussed.

Two virus-encoded RNA silencing suppressors, P14 ofBeet necrotic yellow vein virus and S6 ofRice black streak dwarf virus

Functional analysis for gene silencing suppressor of P14 geneof Beet necrotic yellow vein virus and S6 gene ofRice black streak dwarf virus was carried out by agro-infiltration with recombinant vectors ofPotato virus X. The phenotype observation of green fluorescent protein (GFP) expression and Northern blot showed that the gene silencing ofgfp transgenicNicotiana benthamiana induced by homologous sequence was strongly suppressed by the immixture infiltration of either the P14 or the S6. In the suppressed plants, thegfp mRNA accumulation was higher than that in the non-suppressed controls and the symptoms caused by PVX infection became more severe, especially thegfp DNA methylation of plant genome was significantly inhabited when co-infiltrated with RBSDV S6 gene. These results suggested that these two virus genes were potentially to encode for proteins as RNA silencing suppressors.

Oral administration of plant-based rotavirus VP6 induces antigen-specific IgAs, IgGs and passive protection in mice

Plant viruses show significant potential as expression vectors for producing foreign proteins in plants. In this study, codon-optimized VP6 gene (sVP6) of human rotavirus was engineered as a replacement to the coat protein (CP) open reading frame of Beet black scorch virus (BBSV). In vitro-generated RNA transcripts corresponding to the engineered virus were infectious when inoculated onto the leaves of Chenopodium amaranticolor. Molecular analysis revealed that sVP6 was efficiently expressed and accounted for 0.25% of the total soluble protein (TSP) in plant leaves on 7 dpi. On average, a high level 1.54 microg of sVP6 was expressed in each gram of infected leaves. Oral immunization of female BALB/c mice with the plant-based sVP6 protein induced high titers of anti-VP6 mucosal IgA and serum IgG. 60% of suckling pups born from immunized dams were protected against the virulent rotavirus challenge and those infected pups developed less severe diarrhea. These results suggested that it is feasible to induce lactogenic immunity against an enteric pathogen through oral vaccination, by using the antigen produced in a new BBSV-based plant protein expression system.

Morphogenesis of Endoplasmic Reticulum Membrane-Invaginated Vesicles during Beet Black Scorch Virus Infection: Role of Auxiliary Replication Protein and New Implications of Three-Dimensional Architecture

ABSTRACT All well-characterized positive-strand RNA viruses[(+)RNA viruses] induce the formation of host membrane-bound viral replication complexes (VRCs), yet the underlying mechanism and machinery for VRC formation remain elusive. We report here the biogenesis and topology of the Beet black scorch virus (BBSV) replication complex. Distinct cytopathological changes typical of endoplasmic reticulum (ER) aggregation and vesiculation were observed in BBSV-infected Nicotiana benthamiana cells. Immunogold labeling of the auxiliary replication protein p23 and double-stranded RNA (dsRNA) revealed that the ER-derived membranous spherules provide the site for BBSV replication. Further studies indicated that p23 plays a crucial role in mediating the ER rearrangement. Three-dimensional electron tomographic analysis revealed the formation of multiple ER-originated vesicle packets. Each vesicle packet enclosed a few to hundreds of independent spherules that were invaginations of the ER membranes into the lumen. Strikingly, these vesicle packets were connected to each other via tubules, a rearrangement event that is rare among other virus-induced membrane reorganizations. Fibrillar contents within the spherules were also reconstructed by electron tomography, which showed diverse structures. Our results provide the first, to our knowledge, three-dimensional ultrastructural analysis of membrane-bound VRCs of a plant (+)RNA virus and should help to achieve a better mechanistic understanding of the organization and microenvironment of plant (+)RNA virus replication complexes. IMPORTANCE Assembly of virus replication complexes for all known positive-strand RNA viruses depends on the extensive remodeling of host intracellular membranes. Beet black scorch virus, a necrovirus in the family Tombusviridae, invaginates the endoplasmic reticulum (ER) membranes to form spherules in infected cells. Double-stranded RNAs, the viral replication intermediate, and the viral auxiliary replication protein p23 are all localized within such viral spherules, indicating that these are the sites for generating progeny viral RNAs. Furthermore, the BBSV p23 protein could to some extent reorganize the ER when transiently expressed in N. benthamiana. Electron tomographic analysis resolves the three-dimensional (3D) architecture of such spherules, which are connected to the cytoplasm via a neck-like structure. Strikingly, different numbers of spherules are enclosed in ER-originated vesicle packets that are connected to each other via tubule-like structures. Our results have significant implications for further understanding the mechanisms underlying the replication of positive-strand RNA viruses.

Deep sequencing-based transcriptome profiling reveals comprehensive insights into the responses of Nicotiana benthamiana to beet necrotic yellow vein virus infections containing or lacking RNA4.

Background Beet necrotic yellow vein virus (BNYVV), encodes either four or five plus-sense single stranded RNAs and is the causal agent of sugar beet rhizomania disease, which is widely distributed in most regions of the world. BNYVV can also infect Nicotiana benthamiana systemically, and causes severe curling and stunting symptoms in the presence of RNA4 or mild symptoms in the absence of RNA4. Results Confocal laser scanning microscopy (CLSM) analyses showed that the RNA4-encoded p31 protein fused to the red fluorescent protein (RFP) accumulated mainly in the nuclei of N. benthamiana epidermal cells. This suggested that severe RNA4-induced symptoms might result from p31-dependent modifications of the transcriptome. Therefore, we used next-generation sequencing technologies to analyze the transcriptome profile of N. benthamiana in response to infection with different isolates of BNYVV. Comparisons of the transcriptomes of mock, BN3 (RNAs 1+2+3), and BN34 (RNAs 1+2+3+4) infected plants identified 3,016 differentially expressed transcripts, which provided a list of candidate genes that potentially are elicited in response to virus infection. Our data indicate that modifications in the expression of genes involved in RNA silencing, ubiquitin-proteasome pathway, cellulose synthesis, and metabolism of the plant hormone gibberellin may contribute to the severe symptoms induced by RNA4 from BNYVV. Conclusions These results expand our understanding of the genetic architecture of N. benthamiana as well as provide valuable clues to identify genes potentially involved in resistance to BNYVV infection. Our global survey of gene expression changes in infected plants reveals new insights into the complicated molecular mechanisms underlying symptom development, and aids research into new strategies to protect crops against viruses.

Identification of two RNA silencing suppressors from banana bunchy top virus.

In order to suppress RNA silencing, many plant and some animal viruses encode RNA silencing suppressors to achieve infection. In this study, we report that B3 and B4, encoded by DNA3 and DNA4 of banana bunchy top virus (BBTV), exhibit RNA silencing suppression activity. B3 and B4 were able to increase the transient expression of green fluorescent protein (GFP) and dramatically enhanced the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana. B4 was able to reverse established gene silencing on an inoculated leaf or on an upper leaf. B3, however, was only active during infection of an inoculated leaf. Furthermore, B4, but not B3, was able to enhance GFP expression in the transgenic N. benthamiana line 16c. In conclusion, B3 and B4 are the RNA silencing suppressors of BBTV, and they may act at different steps in the RNA silencing pathways.