Viral Silencing Suppressors Research Articles

Dose-dependent long-distance movement of microRNA399 duplex regulates phosphate homeostasis in Arabidopsis.

MicroRNA399 (miR399), a phosphate (Pi) starvation-induced long-distance signal, is first produced in shoots and moves to roots to suppress PHO2 encoding a ubiquitin conjugase, leading to enhanced Pi uptake and root-to-shoot translocation. However, the molecular mechanism underlying miR399 long-distance movement remains elusive. Hypocotyl grafting with various Arabidopsis mutants or transgenic lines expressing artificial miR399f was employed. The movement of miR399 across graft junction and the rootstock PHO2 transcript and scion Pi levels were analyzed to elucidate the potential factors involved. Our results showed that miR399f precursors are cell-autonomous and mature miR399f movement is independent of its biogenesis, sequence context, and length (21 or 22 nucleotides). Expressing viral silencing suppressor P19 in the root stele or blocking unloading in the root phloem pore pericycle (PPP) antagonized its silencing effect, suggesting that the miR399f/miR399f* duplex is a mobile entity unloaded through PPP. Notably, the scion miR399f level positively correlates with its amount translocated to rootstocks, implying dose-dependent movement. This study uncovers the molecular basis underlying the miR399-mediated long-distance silencing in coordinating shoot Pi demand with Pi acquisition and translocation activities in the roots.

A selective autophagy receptor VISP1 induces symptom recovery by targeting viral silencing suppressors

Selective autophagy is a double-edged sword in antiviral immunity and regulated by various autophagy receptors. However, it remains unclear how to balance the opposite roles by one autophagy receptor. We previously identified a virus-induced small peptide called VISP1 as a selective autophagy receptor that facilitates virus infections by targeting components of antiviral RNA silencing. However, we show here that VISP1 can also inhibit virus infections by mediating autophagic degradation of viral suppressors of RNA silencing (VSRs). VISP1 targets the cucumber mosaic virus (CMV) 2b protein for degradation and attenuates its suppression activity on RNA silencing. Knockout and overexpression of VISP1 exhibit compromised and enhanced resistance against late infection of CMV, respectively. Consequently, VISP1 induces symptom recovery from CMV infection by triggering 2b turnover. VISP1 also targets the C2/AC2 VSRs of two geminiviruses and enhances antiviral immunity. Together, VISP1 induces symptom recovery from severe infections of plant viruses through controlling VSR accumulation.

Role of Plant Virus Movement Proteins in Suppression of Host RNAi Defense.

One of the systems of plant defense against viral infection is RNA silencing, or RNA interference (RNAi), in which small RNAs derived from viral genomic RNAs and/or mRNAs serve as guides to target an Argonaute nuclease (AGO) to virus-specific RNAs. Complementary base pairing between the small interfering RNA incorporated into the AGO-based protein complex and viral RNA results in the target cleavage or translational repression. As a counter-defensive strategy, viruses have evolved to acquire viral silencing suppressors (VSRs) to inhibit the host plant RNAi pathway. Plant virus VSR proteins use multiple mechanisms to inhibit silencing. VSRs are often multifunctional proteins that perform additional functions in the virus infection cycle, particularly, cell-to-cell movement, genome encapsidation, or replication. This paper summarizes the available data on the proteins with dual VSR/movement protein activity used by plant viruses of nine orders to override the protective silencing response and reviews the different molecular mechanisms employed by these proteins to suppress RNAi.

The interaction between the lemon ribosomal protein ClRPS9-2 and citrus yellow vein clearing virus coat protein affects viral infection and gene silencing suppressor activity.

Citrus yellow vein clearing virus (CYVCV) is an emerging virus that causes serious economic damage to the lemon industry worldwide. The coat protein (CP) of CYVCV is a strong RNA silencing suppressor and is associated with the severity of symptoms in citrus, yet the interaction between CP and host factors remains unknown. In this study, the 40S ribosomal subunit protein S9-2 (ClRPS9-2) was identified as a CP-binding partner using the yeast two-hybrid system from a lemon (cv. Eureka) cDNA library, and the interaction between CP and ClRPS9-2 was demonstrated by in vivo methods. The results suggest that the N-terminal 8-108 amino acid sequence of ClRPS9-2 is crucial for its interaction with CP and may be associated with the nuclear localization of ClRPS9-2. The accumulation and silencing suppressor activity of CP were reduced by transient expression of ClRPS9-2 in Nicotiana benthamiana. Reverse transcription-quantitative PCR analysis showed that the content of CYVCV in ClRPS9-2 transgenic Eureka lemon plants was approximately 50% of that in CYVCV-infected wild-type plants 1 month after inoculation, and mild yellowing and vein clearing symptoms were observed in the transgenic plants. These findings demonstrate that ClRPS9-2 plays a role in host defensive reactions, and the enhanced resistance of transgenic plants to CYVCV may be associated with the up-regulation of salicylic acid-related and R genes.

Grapevine red blotch virus C2 and V2 are suppressors of post-transcriptional gene silencing

Grapevine red blotch virus (GRBV) is the causative agent of grapevine red blotch disease (GRBD) which is one of the major threats faced by grapevine industry in the United States. Since its initial identification in 2011, the disease has rapidly spread in the major US grape-growing regions of the Pacific Northwest, causing major economic impacts. Geminiviruses, the largest family of plant viruses, can induce and be targeted by host post-transcriptional gene-silencing (PTGS) anti-viral mechanisms. As a counter-defense mechanism, viruses have evolved viral silencing suppressor proteins to combat PTGS mechanisms and establish a successful infection in host plants. Here we provide characterization of two ORFs of GRBV, C2 and V2 as viral silencing suppressors. In Nicotiana benthamiana line 16c GFP marker plants, synergism or additive effects of C2 and V2 suppressors was observed at the mRNA level when they are expressed together transiently. Additionally, we showed there is no evidence by yeast two-hybrid of self-interaction (dimerization) of C2 or V2 proteins, and no evidence of physical interaction between these two suppressors.

Special issue: Relationship between insect vectors and plant pathogens

Special issue: Relationship between insect vectors and plant pathogens

In vivo transient expression of a viral silencing suppressor, NSs, derived from tomato spotted wilt virus decreases insect RNAi efficiencies.

Tomato spotted wilt virus is a single-stranded RNA virus and causes a serious plant disease. Its horizontal transmission depends on some thrips species including Frankliniella occidentalis. Its genome encodes a nonstructural protein, nonstructural (NSs), which acts as a silencing suppressor and plays a crucial role in the pathogenicity by defending antiviral immunity using RNA interference (RNAi) in plant hosts. However, its physiological function as a silencing suppressor was not well clarified in insect vectors. This study assessed any change of RNAi efficiencies in two other insect systems by NSs expression. To this end, the gene was cloned into a eukaryotic expression vector and transiently expressed in two different insect species via in vivo transient expression (IVTE). After feeding the recombinant construct to non-viruliferous F. occidentalis, NSs expression was observed for over 2 days in the thrips. Under this expression of NSs, thrips were rescued from a treatment of a toxic double stranded RNA specific to v-ATPase. Interestingly, the thrips treated with IVTE significantly suppressed the expression of RNAi machinery genes such as SID and Dicer-2. The recombinant vector expressing NSs was injected to a non-vector insect, Spodoptera exigua, larvae. The larvae expressing NSs by the IVTE were highly susceptible to an infection of a RNA virus called iflavirus. These suggest that NSs acts as a silencing suppressor in insects and would be used for a synergist for RNA pathogens to control insect pests.

Analysis of the RNA-Dependent RNA Polymerase 1 (RDR1) Gene Family in Melon.

RNA-dependent RNA polymerase 1 (RDR1) plays a crucial defense role against plant viruses by secondary amplification of viral double-stranded RNA in the gene-silencing pathway. In this study, it was found that melon (Cucumis melo) encodes four RDR1 genes (CmRDR1a, b, c1 and c2) similar to the CsRDR1 gene family of cucumber (C. sativus). However, in contrast to cucumber, melon harbors a truncated CmRDR1b gene. In healthy plants, CmRDR1a was expressed, whereas the expression of CmRDR1c1/c2 was not detected. CmRDR1a expression level increased 20-fold upon cucumber mosaic virus (CMV) infection and was not increased in melon plants infected with zucchini yellow mosaic virus (ZYMV), cucumber vein yellowing virus (CVYV) and cucumber green mottle mosaic virus (CGMMV). The expression of CmRDR1c1/c2 genes was induced differentially by infection with viruses from different families: high levels of ~340-, 172- and 115-fold increases were induced by CMV, CVYV and CGMMV, respectively, and relatively low-level increases by potyvirus infection (4- to 6-fold). CMV mutants lacking the viral silencing suppressor 2b protein did not cause increased CmRDR1c/c2 expression; knockout of CmRDR1c1/c2 by CRISPR/Cas9 increased susceptibility to CMV but not to ZYMV. Therefore, it is suggested that the sensitivity of melon to viruses from different families is a result of the loss of function of CmRDR1b.

Promoting virus-induced gene silencing of pepper genes by a heterologous viral silencing suppressor.

Pepper is an important vegetable cultivated worldwide. Pepper fruits accumulate unique metabolites, capsanthin and capsaicin, which are important raw materials for natural pigment and medicine. Pepper plants are rich in genetic diversity and are attractive subjects for fruit developmental studies. The release of pepper reference genomes provided key genetic information for dissecting function of pepper genes underlying various interesting phenotypes (Kim et al., 2014; Qin et al., 2014). However, due to the difficulty in transformation, dissecting genetic mechanisms in pepper remained technically challenging.

Impact of viral silencing suppressors on plant viral synergism: a global agro-economic concern.

Plant viruses are known for their devastating impact on global agriculture. These intracellular biotrophic pathogens can infect a wide variety of plant hosts all over the world. The synergistic association of plant viruses makes the situation more alarming. It usually promotes the replication, movement, and transmission of either or both the coexisting synergistic viral partners. Although plants elicit a robust antiviral immune reaction, including gene silencing, to limit these infamous invaders, viruses counter it by encoding viral suppressors of RNA silencing (VSRs). Growing evidence also suggests that VSRs play a driving role in mediating the plant viral synergism. This review briefly discusses the evil impacts of mixed infections, especially synergism, and then comprehensively describes the emerging roles of VSRs in mediating the synergistic association of plant viruses. KEY POINTS: • Synergistic associations of plant viruses have devastating impacts on global agriculture. • Viral suppressors of RNA silencing (VSRs) play key roles in driving plant viral synergism.

Cucumber Ribosomal Protein CsRPS21 Interacts With P22 Protein of Cucurbit Chlorotic Yellows Virus.

Cucurbit chlorotic yellows virus (CCYV) is a cucurbit-infecting crinivirus. RNA silencing can be initiated as a plant defense against viruses. Viruses encode various RNA silencing suppressors to counteract antiviral silencing. P22 protein encoded by RNA1 of CCYV is a silencing suppressor, but its mechanism of action remains unclear. In this study, the cucumber ribosomal-like protein CsRPS21 was found to interact with P22 protein in vitro and in vivo. A conserved CsRPS21 domain was indispensable for its nuclear localization and interaction with P22. Transient expression of CsRPS21 in Nicotiana benthamiana leaves interfered with P22 accumulation and inhibited P22 silencing suppressor activity. CsRPS21 expression in N. benthamiana protoplasts inhibited CCYV accumulation. Increasing numbers of ribosomal proteins are being found to be involved in viral infections of plants. We identified a P22-interacting ribosomal protein, CsRPS21, and uncovered its role in early viral replication and silencing suppressor activity. Our study increases knowledge of the function of ribosomal proteins during viral infection.

Identification and Characterization of Plant-Interacting Targets of Tomato Spotted Wilt Virus Silencing Suppressor.

Tomato spotted wilt virus (TSWV; species Tomato spotted wilt orthotospovirus) is an economically important plant virus that infects multiple horticultural crops on a global scale. TSWV encodes a non-structural protein NSs that acts as a suppressor of host RNA silencing machinery during infection. Despite extensive structural and functional analyses having been carried out on TSWV NSs, its protein-interacting targets in host plants are still largely unknown. Here, we systemically investigated NSs-interacting proteins in Nicotiana benthamiana via affinity purification and mass spectrometry (AP-MS) analysis. Forty-three TSWV NSs-interacting candidates were identified in N. benthamiana. Gene Ontology (GO) and protein–protein interaction (PPI) network analyses were carried out on their closest homologs in tobacco (Nicotiana tabacum), tomatoes (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). The results showed that NSs preferentially interacts with plant defense-related proteins such as calmodulin (CaM), importin, carbonic anhydrase and two heat shock proteins (HSPs): HSP70 and HSP90. As two major nodes in the PPI network, CaM and importin subunit α were selected for the further verification of their interactions with NSs via yeast two-hybrid (Y2H) screening. Our work suggests that the downstream signaling, transportation and/or metabolic pathways of host-NSs-interacting proteins may play critical roles in NSs-facilitated TSWV infection.

Development of a Virus-Induced Gene Silencing System for Dioecious Coccinia grandis.

Coccinia grandis is an interesting model system to understand dioecy in Cucurbitaceae family. Recent transcriptomics and proteomics studies carried out to understand the sex expression in C. grandis have resulted in identification of many candidate sex-biased genes. In absence of an efficient genetic transformation protocol for C. grandis, virus-induced gene silencing (VIGS) would be a powerful tool to enable gene functional analysis. In current study, we explored the apple latent spherical virus (ALSV) for gene knockdown in C. grandis. The viral infection was achieved through mechanical inoculation of ALSV-infected Chenopodium quinoa leaf extract onto the cotyledons of C. grandis. ALSV-VIGS mediated knockdown of CgPDS gene was successfully achieved in C. grandis by mechanical inoculation method resulting in characteristic photobleaching. Subsequently, we developed agroinfiltration compatible vectors for direct infection of C. grandis and shortened the time-frame by skipping viral propagation in C. quinoa. Typical yellow-leaf phenotype was observed in C. grandis plants agroinfiltrated with ALSV-CgSU constructs, indicating robust silencing of CgSU gene. In addition, we improved the infection efficiency of ALSV by co-infiltration of P19 viral silencing suppressor. These results suggest that ALSV-VIGS is suitable for characterization of gene function in dioecious C. grandis and it can help us understand the mechanism of sex expression.

Viral silencing suppressors and cellular proteins partner with plant RRP6-like exoribonucleases.

RNA silencing and RNA decay are functionally interlaced, regulate gene expression and play a pivotal role in antiviral responses. As a counter-defensive strategy, many plant and mammalian viruses encode suppressors which interfere with both mechanisms. However, the protein interactions that connect these pathways remain elusive. Previous work reported that RNA silencing suppressors from different potyviruses, together with translation initiation factors EIF(iso)4E, interacted with the C-terminal region of the tobacco exoribonuclease RRP6-like 2, a component of the RNA decay exosome complex. Here, we investigate whether other viral silencing suppressors and cellular proteins might also bind RRP6-like exoribonucleases. A candidate search approach based on yeast two-hybrid protein interaction assays showed that three other unrelated viral suppressors, two from plant viruses and one from a mammalian virus, bound the C-terminus of the tobacco RRP6-like 2, the full-length of the Arabidopsis RRP6L1 protein and its C-terminal region. In addition, RRP6-like proteins were found to interact with members of the cellular double-stranded RNA-binding protein (DRB) family involved in RNA silencing. The C-terminal regions of RRP6L proteins are engaged in homotypic and heterotypic interactions and were predicted to be disordered. Collectively, these results suggest a protein interaction network that connects components of RNA decay and RNA silencing that is targeted by viral silencing suppressors.

Protein expression and gene editing in monocots using foxtail mosaic virus vectors.

Plant viruses can be engineered to carry sequences that direct silencing of target host genes, expression of heterologous proteins, or editing of host genes. A set of foxtail mosaic virus (FoMV) vectors was developed that can be used for transient gene expression and single guide RNA delivery for Cas9‐mediated gene editing in maize, Setaria viridis, and Nicotiana benthamiana. This was accomplished by duplicating the FoMV capsid protein subgenomic promoter, abolishing the unnecessary open reading frame 5A, and inserting a cloning site containing unique restriction endonuclease cleavage sites immediately after the duplicated promoter. The modified FoMV vectors transiently expressed green fluorescent protein (GFP) and bialaphos resistance (BAR) protein in leaves of systemically infected maize seedlings. GFP was detected in epidermal and mesophyll cells by epifluorescence microscopy, and expression was confirmed by Western blot analyses. Plants infected with FoMV carrying the bar gene were temporarily protected from a glufosinate herbicide, and expression was confirmed using a rapid antibody‐based BAR strip test. Expression of these proteins was stabilized by nucleotide substitutions in the sequence of the duplicated promoter region. Single guide RNAs expressed from the duplicated promoter mediated edits in the N. benthamiana Phytoene desaturase gene, the S. viridis Carbonic anhydrase 2 gene, and the maize HKT1 gene encoding a potassium transporter. The efficiency of editing was enhanced in the presence of synergistic viruses and a viral silencing suppressor. This work expands the utility of FoMV for virus‐induced gene silencing (VIGS), virus‐mediated overexpression (VOX), and virus‐enabled gene editing (VEdGE) in monocots.

Characterization of cucumber mosaic virus infecting coleus (Plectranthus barbatus) in Karnataka.

Plectranthus barbatus also known by the synonym Coleus forskohlii it is called as forskohlii and Indian coleus. It is a tropical perennial herb belongs to the family Lamiaceae widely cultivated in India used as traditional medicinal crop. Its tuberous roots produce forskolin, an extract useful for pharmaceutical preparations and research in cell biology. The incidence of mosaic with dark and light green patches, mottling, leaf distortion and reduction growth was noticed in commercial cultivation of coleus. For identification of the virus, the infected leaf sample extract was mechanically inoculated to different hosts such as chilli, tobacco, tomato, cucumber, cowpea and Chenopodium amaranticolor. Host range studies revealed that the virus showed severe mosaic symptoms on Nicotiana spp. and Cucumis spp. The virus produced systemic and local lesion symptoms in a different host. The Leaf dip preparation of virus infected leaf extract was observed under an electron microscope showed the presence of isometric particles of 28nm in size. The healthy and infected samples were tested using DAC-ELISA against antibodies of CMV, GBNV and TSV the infected samples showed strong positive reaction with 1.85 optical density to CMV antibodies indicated the presence of CMV. For molecular identification, total RNA was isolated and used for RT-PCR amplification using CMV specific primers. RT-PCR resulted in the positive amplification in virus infected samples but not from a healthy control. The complete genome of CMV RNA-1 consists of 3360 nucleotides (nt) encoding replicase gene of 807 amino acids (aa). The CMV RNA-2 was 2983 nt in length containing 2a (859 aa) encoding RNA dependent RNA polymerase protein and 2b encoding viral silencing suppressor (112 aa), while RNA-3 encoding 3a movement protein (280 aa) and coat protein (219 aa) was 2223 nt in length. Phylogenetic analyses of nucleotide sequences of coleus CMV isolate is closely related to subgroup IB than to subgroup IA or II with other CMV isolates. In recombination analysis, the recombination event occurs between the subgroups of I, II as well as IA and IB in RNA 1, RNA2 and RNA3 of coleus isolate with other CMV isolates. To best of our knowledge, this is the first report of CMV infection in coleus.

Differential gene expression and physiological changes during acute or persistent plant virus interactions may contribute to viral symptom differences.

Viruses have different strategies for infecting their hosts. Fast and acute infections result in the development of severe symptoms and may cause the death of the plant. By contrast, in a persistent interaction, the virus can survive within its host for a long time, inducing only mild symptoms. In this study, we investigated the gene expression changes induced in CymRSV-, crTMV-, and TCV-infected Nicotiana benthamiana and in PVX- and TMV-U1-infected Solanum lycopersicum plants after the systemic spread of the virus by two different high-throughput methods: microarray hybridization or RNA sequencing. Using these techniques, we were able to clearly differentiate between acute and persistent infections. We validated the gene expression changes of selected genes by Northern blot hybridization or by qRT-PCR. We show that, in contrast to persistent infections, the drastic shut-off of housekeeping genes, downregulation of photosynthesis-related transcripts and induction of stress genes are specific outcomes with acute infections. We also show that these changes are not a consequence of host necrosis or the presence of a viral silencing suppressor. Thermal imaging data and chlorophyll fluorescence measurements correlated very well with the molecular changes. We believe that the molecular and physiological changes detected during acute infections mostly contribute to virus symptom development. The observed characteristic physiological changes associated with economically more dangerous acute infections could serve as a basis for the elaboration of remote monitoring systems suitable for detecting developing virus infections in crops. Moreover, as molecular and physiological changes are characteristics of different types of virus lifestyles, this knowledge can support risk assessments of recently described novel viruses.

Interaction between Brassica yellows virus silencing suppressor P0 and plant SKP1 facilitates stability of P0 invivo against degradation by proteasome and autophagy pathways.

Summary P0 protein of some polerovirus members can target ARGONAUTE1 (AGO1) to suppress RNA silencing. Although P0 harbors an F‐box‐like motif reported to be essential for interaction with S phase kinase‐associated protein 1 (SKP1) and RNA silencing suppression, it is the autophagy pathway that was shown to contribute to AGO1 degradation. Therefore, the role of P0–SKP1 interaction in silencing suppression remains unclear.We conducted global mutagenesis and comparative functional analysis of P0 encoded by Brassica yellows virus (BrYV) (P0Br).We found that several residues within P0Br are required for local and systemic silencing suppression activities. Remarkably, the F‐box‐like motif mutant of P0Br, which failed to interact with SKP1, is destabilized in vivo. Both the 26S proteasome system and autophagy pathway play a role in destabilization of the mutant protein. Furthermore, silencing of a Nicotiana benthamiana SKP1 ortholog leads to the destabilization of P0Br. Genetic analyses indicated that the P0Br–SKP1 interaction is not directly required for silencing suppression activity of P0Br, but it facilitates stability of P0Br to ensure efficient RNA silencing suppression. Consistent with these findings, efficient systemic infection of BrYV requires P0Br.Our results reveal a novel strategy used by BrYV for facilitating viral suppressors of RNA silencing stability against degradation by plant cells.

An atypical RNA silencing suppression strategy provides a snapshot of the evolution of sweet potato-infecting potyviruses

Plant viruses usually encode proteins with RNA silencing suppression (RSS) activity to counteract plant defenses. In Potyvirus, the largest genus in the family Potyviridae, this role is taken over by the multifunctional HCPro, also involved in aphid transmission, polyprotein processing and virion formation. Recently, the large P1 of Sweet potato feathery mottle virus (SPFMV) was characterized finding an extra ORF produced after polymerase slippage, which originates the product P1N-PISPO. Transient expression assays showed that SPFMV P1 and P1N-PISPO presented RSS activity, while HCPro did not. In this work, we analyze possible differences between HCPro of SPFMV and other potyviruses, testing HCPro RSS activity in a transient expression assay, and using a Plum pox virus-based system to test the ability of SPFMV P1N-PISPO and HCPro to serve as RNA silencing suppressors in the context of a viral infection. Our results indicate that not only P1 and P1N-PISPO, but also HCPro display RSS activity when expressed in a suitable context, stressing the importance of the selected experimental system for testing anti-silencing capacity of proteins. The presence of multiple viral silencing suppressors in SPFMV adds complexity to an already intricate RSS system, and provides insight into the hypothetical evolution of sweet potato-infecting potyvirids.

Cotton Leaf Curl Multan virus C4 protein suppresses both transcriptional and post-transcriptional gene silencing by interacting with SAM synthetase.

Gene silencing is a natural antiviral defense mechanism in plants. For effective infection, plant viruses encode viral silencing suppressors to counter this plant antiviral response. The geminivirus-encoded C4 protein has been identified as a gene silencing suppressor, but the underlying mechanism of action has not been characterized. Here, we report that Cotton Leaf Curl Multan virus (CLCuMuV) C4 protein interacts with S-adenosyl methionine synthetase (SAMS), a core enzyme in the methyl cycle, and inhibits SAMS enzymatic activity. By contrast, an R13A mutation in C4 abolished its capacity to interact with SAMS and to suppress SAMS enzymatic activity. Overexpression of wild-type C4, but not mutant C4R13A, suppresses both transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). Plants infected with CLCuMuV carrying C4R13A show decreased levels of symptoms and viral DNA accumulation associated with enhanced viral DNA methylation. Furthermore, silencing of NbSAMS2 reduces both TGS and PTGS, but enhanced plant susceptibility to two geminiviruses CLCuMuV and Tomato yellow leaf curl China virus. These data suggest that CLCuMuV C4 suppresses both TGS and PTGS by inhibiting SAMS activity to enhance CLCuMuV infection in plants.