Triticum mosaic poacevirus enlists P1 rather than HC-Pro to suppress RNA silencing-mediated host defense

Triticum mosaic virus (TriMV) is the type species of the newly established Poacevirus genus in the family Potyviridae. In this study, we demonstrate that in contrast to the helper component-proteinase (HC-Pro) of Potyvirus species, the P1 proteins of TriMV and Sugarcane streak mosaic poacevirus function in suppression of RNA silencing (SRS). TriMV P1 effectively suppressed silencing induced by single- or double-stranded RNAs (ss/ds RNAs), and disrupted the systemic spread of silencing signals at a step after silencing signal production. Interestingly, contrary to enhanced SRS activity of potyviral HC-Pro by co-expression with P1, the presence of TriMV HC-Pro reduced SRS activity of TriMV P1. Furthermore, TriMV P1 suppressed systemic silencing triggered by dsRNA more efficiently than the HC-Pro of Turnip mosaic potyvirus. Furthermore, TriMV P1 enhanced the pathogenicity of a heterologous virus. Our results established poaceviral P1 as a potent RNA silencing suppressor that probably employs a novel mechanism to suppress RNA silencing-based antiviral defense.     

The multifunctional plant viral suppressor of gene silencing P19 interacts with itself and an RNA binding host protein

Tomato bushy stunt virus (TBSV) is an RNA plant virus encoding a protein of approximately 19 kDa (P19) that is involved in various activities important for pathogenicity, including virus transport and suppression of gene silencing. In this study, we provide evidence in vivo and in vitro that P19 specifically interacts with itself to predominantly form dimers, and with a novel host protein, Hin19. Hin19 has a high degree of similarity with a class of RNA-binding proteins of which many are involved in RNA processing. The binding of P19 to itself and to Hin19 both depend on a structurally important central region of P19 that was previously shown critical for its biological function in plants. Our findings provide evidence for a model in which virus spread through suppression of defense-related gene silencing involves the formation of a complex that includes P19 dimers and a newly identified host RNA-binding protein.     

V2 protein encoded by Tomato yellow leaf curl China virus is an RNA silencing suppressor

The V2 protein of Tomato yellow leaf curl China virus (TYLCCNV) was identified as an RNA silencing suppressor by Agrobacterium-mediated co-infiltration. The V2 protein could inhibit local RNA silencing, systemic RNA silencing of the green fluorescent protein (GFP) gene and the spread of a systemic GFP RNA silencing signal. However, the V2 could not interfere with the cell-to-cell spread of RNA silencing. Subcellular localization assay indicated that the V2 protein was distributed in the cytoplasm of Nicotiana benthamiana cells, and accumulated in irregular cytoplasmic bodies. The V2 bound 21 nt and 24 nt small interfering RNA (siRNA) duplexes and 24 nt single-stranded (ss)-siRNA but not 21 nt ss-siRNA in electrophoresis mobility shift assays. Expression of the V2 protein via the Potato virus X (PVX) vectors heterogenous system induced severe symptoms in N. benthamiana. In a yeast two-hybrid system, TYLCCNV V2 could interact with itself, but not with SlSGS3, which is known to been involved in RNA silencing pathway and to interact with a closely related Tomato yellow leaf curl virus (TYLCV) V2. These results indicate that TYLCCNV V2 is an RNA silencing suppressor, possibly through sequestering siRNA molecules.     

Importance of coat protein and RNA silencing in satellite RNA/virus interactions

RNA silencing is a major defense mechanism plants use to fight an invading virus. The silencing suppressor of Turnip crinkle virus (TCV) is the viral coat protein (CP), which obstructs the DCL2/DCL4 silencing pathway. TCV is associated with a virulent satellite RNA (satC) that represses the accumulation of TCV genomic RNA and whose accumulation is repressed by the TCV CP. To investigate if reduced TCV accumulation due to satC involves RNA silencing and/or the suppressor activity of the CP, TCV was altered to contain a mutation reported to target CP silencing suppressor activity (Deleris et al., Science 313, 68, 2006). However, the mutation did not cause an exclusive defect in silencing suppression, but rather produced a generally non-functional protein. We demonstrate that a functional CP, but not DCL2/DCL4, is required for satC-mediated repression of TCV. In addition, enhancement of satC accumulation in the absence of a functional CP requires DCL2/DCL4.     

The Red clover necrotic mosaic virus RNA-2 encoded movement protein is a second suppressor of RNA silencing

The replication complex of Red clover necrotic mosaic virus (RCNMV) has been shown to possess silencing suppression activity. Here a newly developed viral-based assay for the identification of silencing suppression activity was used to provide evidence for a second, mechanistically distinct method of silencing suppression provided for by the RCNMV movement protein (MP). This new assay relies on Turnip crinkle virus with its capsid protein replaced with green fluorescent protein to act as a reporter (TCV-sGFP). In the presence of a protein with silencing suppression activity TCV-sGFP readily moves from cell-to-cell, but in the absence of such a protein TCV-sGFP is confined to small foci of infection. This TCV-sGFP assay was used to identify MP as a suppressor of RNA silencing, to delimit essential amino acids for this activity and uncouple silencing and movement functions.     

The CUG-initiated larger form coat protein of Chinese wheat mosaic virus binds to the cysteine-rich RNA silencing suppressor

Some viruses use alternative translation initiation at non-AUG codons as a strategy to produce multiple proteins during gene expression. Here we show that, using this strategy, Chinese wheat mosaic virus (CWMV; Furovirus) expresses a larger form of coat protein (N-ext/CP) in infected plants. Site-directed mutagenesis and transient expression analysis confirmed that CWMV N-ext/CP is initiated at an upstream in-frame CUG codon at nucleotide position 207–209 of RNA 2, which adds a 39 amino acid (aa) N-terminal extension to the major CP. Interestingly, in planta and in vitro analyses indicated that CWMV N-ext/CP but not CP interacts with the CWMV cysteine-rich protein (CRP), an RNA silencing suppressor. We further determined that the N-terminal 39 aa extension, particularly the 10 aa region immediately upstream of the major CP coding region is responsible for the interaction of N-ext/CP with CRP. In an Agrobacterium co-infiltration assay, co-expression with N-ext/CP did not affect CRP silencing suppression activity. Thus the alternative translation initiation at a CUG codon provides the CWMV N-ext/CP with the ability to bind to the viral silencing suppressor.     

Overexpression of a host factor TOM1 inhibits tomato mosaic virus propagation and suppression of RNA silencing

A plant integral membrane protein TOM1 is involved in the multiplication of Tomato mosaic virus (ToMV). TOM1 interacts with ToMV replication proteins and has been suggested to tether the replication proteins to the membranes where the viral RNA synthesis takes place. We have previously demonstrated that inactivation of TOM1 results in reduced ToMV multiplication. In the present study, we show that overexpression of TOM1 in tobacco also inhibits ToMV propagation. TOM1 overexpression led to a decreased accumulation of the soluble form of the replication proteins and interfered with the ability of the replication protein to suppress RNA silencing. The reduced accumulation of the soluble replication proteins was also observed in a silencing suppressor-defective ToMV mutant. Based on these results, we propose that RNA silencing suppression is executed by the soluble form of the replication proteins and that efficient ToMV multiplication requires balanced accumulation of the soluble and membrane-bound replication proteins.     

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant's RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0^PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0^PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0^PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.     

Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus

Rice stripe virus (RSV) is a single-stranded (ss) RNA virus belonging to the genus Tenuivirus. RSV is present in many East Asian countries and causes severe diseases in rice fields, especially in China. In this study, we analyzed six proteins encoded by the virus for their abilities to suppress RNA silencing in plant using a green fluorescent protein (GFP)-based transient expression assay. Our results indicate that NS3 encoded by RSV RNA3, but not other five RSV encoded proteins, can strongly suppress local GFP silencing in agroinfiltrated Nicotiana benthamiana leaves. NS3 can reverse the GFP silencing, it can also prevent long distance spread of silencing signals which have been reported to be necessary for inducing systemic silencing in host plants. The NS3 protein can significantly reduce the levels of small interfering RNAs (siRNAs) in silencing cells, and was found to bind 21-nucleotide ss-siRNA, siRNA duplex and long ssRNA but not long double-stranded (ds)-RNA. Both N and C terminal of the NS3 protein are critical for silencing suppression, and mutation of the putative nuclear localization signal decreases its local silencing suppression efficiency and blocks its systemic silencing suppression. The NS3-GFP fusion protein and NS3 were shown to accumulate predominantly in nuclei of onion, tobacco and rice cells through transient expression assay or immunocytochemistry and electron microscopy. In addition, transgenic rice and tobacco plants expressing the NS3 did not show any apparent alteration in plant growth and morphology, although NS3 was proven to be a pathogenicity determinant in the PVX heterogenous system. Taken together, our results demonstrate that RSV NS3 is a suppressor of RNA silencing in planta, possibly through sequestering siRNA molecules generated in cells that are undergoing gene silencing.     

Synergism of a DNA and an RNA virus: enhanced tissue infiltration of the begomovirus Abutilon mosaic virus (AbMV) mediated by Cucumber mosaic virus (CMV)

Replication of the begomovirus Abutilon mosaic virus (AbMV) is restricted to phloem nuclei, generating moderate levels of virus DNA. Co-infection with Cucumber mosaic virus (CMV) evidently increased AbMV titers in Nicotiana benthamiana, tobacco, and tomato, resulting in synergistic symptom enhancement. In situ hybridization revealed that in double-infected leaves an increased number of nuclei contained elevated amounts of AbMV. Additionally, the begomoviral phloem-limitation was broken. Whereas CMV 3a movement protein-expressing tobacco plants did not exert any similar influence, the presence of CMV 2b silencing suppressor protein lead to enhanced AbMV titers and numbers of infected vascular cells. The findings prove that AbMV can replicate in nonvascular cells and represent the first report on a true synergism of an RNA/ssDNA virus combination in plants, in which CMV 2b protein plays a role. They indicate considerable consequences of mixed infections between begomo- and cucumoviruses on virus epidemiology and agriculture.     

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: formation of dense fluorescent aggregates for sensitive virus tracking

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering a cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic peptides cleaved GFP from HC-Pro but expressed GFP inefficiently. WSMV-GFP vectors with homologous NIa-Pro heptapeptide cleavage sites did not release GFP from HC-Pro, but efficiently expressed GFP as dense fluorescent aggregates. However, insertion of one or two spacer amino acids on either side of NIb/CP heptapeptide cleavage site or deletion in HC-Pro cistron improved processing by NIa-Pro. WSMV-GFP vectors were remarkably stable in wheat for seven serial passages and for 120 days postinoculation. Mite transmission efficiencies of WSMV-GFP vectors correlated with the amount of free GFP produced. WSMV-GFP vectors infected the same range of cereal hosts as wild-type virus, and GFP fluorescence was detected in most wheat tissues.     

Enhanced resistance and neutralization of defense responses by suppressors of RNA silencing

The effects of transgenic expression of the potato virus Y (PVY) HCPro silencing suppressor in tobacco were examined on infection by several viruses. Infection by tobacco mosaic virus (TMV) was reduced at 25 °C, but not at 33 °C. By contrast, systemic infection at 33 °C by the TMV expressing green fluorescent protein was promoted by the HCPro. Infection by tobacco rattle virus (TRV) was restricted to local necrotic lesions by the PVY HCPro. However, this resistance was neutralized by expression of the cucumber mosaic virus (CMV) 2b protein from TRV. By contrast, infection by either wild-type CMV or CMV with a deletion of the 2b gene was not affected. Similarly, infection by cauliflower mosaic virus, red clover necrotic mosaic virus (both limited to infection of the inoculated leaves of tobacco) or tomato bushy stunt virus (systemically infecting tobacco) was not altered by the expression of PVY HCPro. Therefore, it appeared that the PVY HCPro was able to induce defense responses at 25 °C, but not at 33 °C, where it actually neutralized a pre-existing defense response. Moreover, the CMV 2b protein was able to neutralize a defense response activated by HCPro in combination with TRV.     

Interactions between p27 and p88 replicase proteins of Red clover necrotic mosaic virus play an essential role in viral RNA replication and suppression of RNA silencing via the 480-kDa viral replicase complex assembly

Red clover necrotic mosaic virus (RCNMV), a positive-sense RNA virus with a bipartite genome, encodes p27 and p88 replicase proteins that are required for viral RNA replication and suppression of RNA silencing. In this study, we indentified domains in p27 and p88 responsible for their protein-protein interactions using in vitro pull-down assays with the purified recombinant proteins. Coimmunoprecipitation analysis in combination with blue-native polyacrylamide gel electrophoresis using mutated p27 proteins showed that both p27-p27 and p27-p88 interactions are essential for the formation of the 480-kDa complex, which has RCNMV-specific RNA-dependent RNA polymerase activity. Furthermore, we found a good correlation between the accumulated levels of the 480-kDa complex and replication levels and the suppression of RNA silencing activity. Our results indicate that interactions between RCNMV replicase proteins play an essential role in viral RNA replication and in suppressing RNA silencing via the 480-kDa replicase complex assembly.     

Allele dependent silencing of COL1A2 using small interfering RNAs

Osteogenesis imperfecta (OI) is generally caused by a dominant mutation in Collagen I, encoded by the genes COL1A1 and COL1A2. To date there is no satisfactory therapy for OI, but inactivation of the mutant allele through small interfering RNAs (siRNA) is a promising approach, as siRNAs targeting each allele of a polymorphism could be used for allele-specific silencing irrespective of the location of the actual mutations. In this study we examined the allele dependent effects of several tiled siRNAs targeting a region surrounding an exonic COL1A2 T/C polymorphism (rs1800222) in heterozygous primary human bone cells. Relative abundances of COL1A2 alleles were determined by cDNA sequencing and overall COL1A2 abundance was analyzed by quantitative PCR. One of the siRNAs decreased overall COL1A2 abundance by 71% of which 75% was due to silencing of the targeted T-allele. In conclusion, allele-preferential silencing of Collagen type I genes may be a future therapeutic approach for OI.     

Characterization and genetic diversity of sugarcane streak mosaic virus causing mosaic in sugarcane

Sixty-three sugarcane leaf samples were collected from fifty-eight sugarcane varieties, evolved from eleven major sugarcane growing states in India, Australia, South Africa and USA. In RT-PCR, using gene specific primers for sugarcane streak mosaic virus (SCSMV)-CP, 58 of 63 sugarcane samples were found positive to the virus infection and rest of the five samples were negative. Partial CP gene sequences of 42 SCSMV isolates including an isolate from aphid colony (Melanaphis indosacchari) infested on sugarcane variety from this study were characterized after cloning and sequencing for selective isolates represented by at least one isolate from each location. The new sequences identified in the study were named as SCSMV-CB isolates. Fifty two sequences including the 10 database sequences (complete CP cds) deposited earlier from this institute were compared with each other as well as GenBank database sequences of Potyviridae members viz., Rymovirus, Potyvirus, Ipomovirus, Tritimovirus and eight sequences of SCSMV reported from elsewhere. Among the SCSMV-CB isolates sequenced in the study, 85.7–100% (nucleotide) and 89.9–100% (amino acid) sequence identities were observed and with the other data base sequences of SCSMV, the respective identities were 82.2–97.5 and 89.7–98.6%. Grouping of the isolates by the maximum likelihood with molecular clock model, distributed 60 SCSMV sequences including the eight database sequences deposited by other SCSMV working groups from India and USA in 16 different phylogenetic groups. Although the isolates of SCSMV were relatively close to Ipomovirus and Tritimovirus, they were sandwiched between Rymovirus and Ipomovirus. The sequence comparison and phylogenetic studies revealed that the relatedness of SCSMV with the potyviral related genera was comparatively low to consider it as a member of earlier described potyviral genera, hence the genus “Susmovirus” (sugarcane streak mosaic virus) has been proposed, with SCSMV as the sole species to be included. The 52 SCSMV-CB isolates from this institute were distributed in 14 phylogenetic groups and the grouping pattern revealed that the virus isolates could not be grouped based on geographical origin of the host varieties or longevity of the host variety.