Tritimovirus P1 functions as a suppressor of RNA silencing and an enhancer of disease symptoms

Wheat streak mosaic virus (WSMV) is an eriophyid mite-transmitted virus of the genus Tritimovirus, family Potyviridae. Complete deletion of helper component-proteinase (HC-Pro) has no effect on WSMV virulence or disease synergism, suggesting that a different viral protein suppresses RNA silencing. RNA silencing suppression assays using Nicotiana benthamiana 16C plants expressing GFP were conducted with each WSMV protein; only P1 suppressed RNA silencing. Accumulation of GFP siRNAs was markedly reduced in leaves infiltrated with WSMV P1 at both 3 and 6 days post infiltration relative to WSMV HC-Pro and the empty vector control. On the other hand, helper component-proteinase (HC-Pro) of two species in the mite-transmitted genus Rymovirus, family Potyviridae was demonstrated to be a suppressor of RNA silencing. Symptom enhancement assays were conducted by inoculating Potato virus X (PVX) onto transgenic N. benthamiana. Symptoms produced by PVX were more severe on transgenic plants expressing WSMV P1 or potyvirus HC-Pro compared to transgenic plants expressing GFP or WSMV HC-Pro.     

Inhibition of long-distance movement of RNA silencing signals in Nicotiana benthamiana by Apple chlorotic leaf spot virus 50 kDa movement protein

Apple chlorotic leaf spot virus 50 kDa movement protein (P50) acts as a suppressor of systemic silencing in Nicotiana benthamiana. Here, we investigate the mode of action of P50 suppressor. An agroinfiltration assay in GFP-expressing N. benthamiana line16c (GFP-plant) showed that P50 could not prevent the short-distance spread of silencing. In grafting experiments, the systemic silencing was inhibited in GFP-plants (scion) grafted on P50-expressing N. benthamiana (P50-plant; rootstock) when GFP silencing was induced in rootstock. In double-grafted plants, GFP-plant (scion)/P50-plant (interstock)/GFP-plant (rootstock), the systemic silencing in scion was inhibited when GFP silencing was induced in rootstock. Analysis of P50 deletion mutants indicated that the N-terminal region (amino acids 1-284) is important for its suppressor activity. In gel mobility shift assay, P50 lacks binding ability with siRNAs. These results indicated that P50 has a unique suppressor activity that specifically inhibits the long-distance movement of silencing signals.     

The Citrus leaf blotch virus movement protein acts as silencing suppressor

To counteract plant antiviral defense based on RNA silencing, many viruses express proteins that inhibit this mechanism at different levels. The genome of Citrus leaf blotch virus (CLBV) encodes a 227-kDa protein involved in replication, a 40-kDa movement protein (MP), and a 41-kDa coat protein (CP). To determine if any of these proteins might have RNA silencing suppressor activities, we have used Agrobacterium-mediated transient assays in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c. Only CLBV MP was able to suppress intracellular GFP silencing induced by expression of either single- or double-stranded (ds) GFP RNA, but not cell-to-cell or long distance spread of the silencing signal. The MP suppressor activity was weak compared to other characterized viral suppressor proteins. Overall our data indicate that MP acts as a suppressor of local silencing probably by interfering in the silencing pathway downstream of the steps of dsRNA and small RNAs generation.     

p2 <Emphasis Type="Italic">of Rice grassy stunt virus</Emphasis> (RGSV) and p6 and p9 of <Emphasis Type="Italic">Rice ragged stunt virus</Emphasis> (RRSV) isolates from Vietnam exert suppressor activity on the RNA silencing pathway

In Vietnam, the two main viruses that cause disease in rice are the Rice grassy stunt virus (RGSV) and the Rice ragged stunt virus (RRSV). Outbreaks of these two viruses have dramatically decreased rice production in Vietnam. Because natural resistance genes are unknown, an RNAi strategy may be an alternative method to develop resistance to RGSV and RRSV. However, this strategy will be efficient only if putative silencing suppressors encoded by the two viruses are neutralized. To identify these suppressors, we used the classical green fluorescent protein (GFP) agroinfiltration method in Nicotiana benthamiana. Then, we investigated the effects of viral candidate proteins on GFP expression and GFP siRNA accumulation and their interference with the short- or long-range signal of silencing. RGSV genes s2gp1, s5gp2, and s6gp1 and RRSV genes s5gp1, s6gp1, s9gp1, and s10gp1 were selected for viral silencing suppressor investigation according to their small molecular weight, the presence of cysteines, or the presence of a GW motif in related protein products. We confirmed that protein p6 of RRSV displays mild silencing suppressor activity and affects long-range silencing by delaying the systemic silencing signal. In addition, we identified two new silencing suppressors that displayed mild activity: p2 of RGSV and p9 of RRSV.     

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.     

RNA viruses and their silencing suppressors boost Abutilon mosaic virus, but not the Old World Tomato yellow leaf curl Sardinia virus

Mixed viral infections can induce different changes in symptom development, genome accumulation and tissue tropism. These issues were investigated for two phloem-limited begomoviruses, Abutilon mosaic virus (AbMV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in Nicotiana benthamiana plants doubly infected by either the potyvirus Cowpea aphid-borne mosaic virus (CABMV) or the tombusvirus Artichoke mottled crinkle virus (AMCV). Both RNA viruses induced an increase of the amount of AbMV, led to its occasional egress from the phloem and induced symptom aggravation, while the amount and tissue tropism of TYLCSV were almost unaffected. In transgenic plants expressing the silencing suppressors of CABMV (HC-Pro) or AMCV (P19), AbMV was supported to a much lesser extent than in the mixed infections, with the effect of CABMV HC-Pro being superior to that of AMCV P19. Neither of the silencing suppressors influenced TYLCSV accumulation. These results demonstrate that begomoviruses differentially respond to the invasion of other viruses and to silencing suppression.     

Isolation and molecular characterization of a distinct begomovirus and its associated betasatellite infecting Hedyotis uncinella (Hook. et Arn.) in Vietnam

A begomovirus isolate VN1 associated with symptomatic Hedyotis uncinella Hook. et Arn. from Vietnam was characterized. The virus, which we provisionally name H. uncinella yellow mosaic virus (HUYMV), has a monopartite genome of 2,749 nucleotides (nts). Pairwise comparisons of DNA-A sequences showed that HUYMV had a maximum nt sequence identity with Soybean crinkle leaf virus (SbLCV) and Premna leaf curl virus (PLCuV) at 82.1 and 81.9 %, respectively, which are less than the 89 % identity in the complete genome, which has been used as the threshold value for demarcation of species in the genus Begomovirus, the family Geminiviridae. One recombination event was detected for HUYMV, which involves an unknown begomovirus as the major parent and Tomato leaf curl Philippines virus (ToLCPV) as the minor parent, with nt 2163 and nt 2452 as the beginning and ending breakpoints, respectively. A betasatellite was found to be associated with HUYMV. The betasatellite showed the highest nt sequence identity (70 %) with Tomato leaf curl Philippine betasatellite—[Philippines:Laguna2:2006]. The name H. uncinella yellow mosaic betasatellite [Vietnam: Binh Dinh: 2013] was proposed for the betasatellite.     

HC-Pro,a potyvirus RNA silencing suppressor,cancels cycling of <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants

The mixed infection of Cucumber mosaic virus (CMV) and a potyvirus has been known to increase CMV titer in Nicotiana benthamiana plants, resulting in synergistic viral symptoms. We found that among three potyviruses—Potato virus Y (PVY), Turnip mosaic virus (TuMV), and Clover yellow vein virus (C1YVV)—synergistic effects on CMV (or a recombinant CMV vector) titers were most efficiently induced by a co-infection with PVY in N. benthamiana plants. In addition, the helper component-proteinase (HC-Pro) gene of PVY expressed by transgenic plants, which is a viral RNA silencing suppressor, was sufficient to cancel the cycling pattern of CMV titer, resulting in increased levels of overall CMV accumulation. Surprisingly, we found that the levels of CMV and the foreign protein expressed from the CMV vector were much higher in the HC-Pro-transgenic plants than the levels detected in the plants mixed-infected with CMV and PVY. The mechanism for canceling the cyclic infection of CMV by the HC-Pro protein alone is discussed in view of the interaction between RNA silencing and HC-Pro, as well as the possible involvement of the 3a protein.     

Molecular dissection of Flock house virus protein B2 reveals that electrostatic interactions between N-terminal domains of B2 monomers are critical for dimerization

Flock house virus (FHV) encodes a suppressor protein B2 to overcome antiviral RNA silencing during infection. Biochemical analyses have shown that a homodimer of B2 binds to double-stranded RNA to inhibit dicer-mediated cleavage of dsRNA and incorporation of small interfering RNAs into the RNA-induced silencing complex. In this study, using FHV-Nicotiana benthamiana system, we identified that the charged amino acids at the N-terminus of B2 are critical for dimerization. Interestingly, B2 mutants defective in dimerization exhibited enhanced silencing suppressor activity, Furthermore, we found that the C-terminal charged amino acids are dispensable for B2 dimerization and viral RNA silencing suppression but are critical for transgene silencing suppression. Additional yeast two hybrid assays revealed that dimerization of B2 is not essential for interacting with the RNA silencing machinery. Taken together, our data provide evidence that both monomeric and dimeric B2 proteins function in different modes to suppress RNA silencing.     

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.     

Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors

The coat protein of satellite panicum mosaic virus (SPCP) is known to effectively protect its cognate RNA from deleterious events, and here, we tested its stabilizing potential for heterologous virus-based gene vectors in planta. In support of this, a Potato virus X (PVX) vector carrying the SPMV capsid protein (PVX-SPCP) gene was stable for at least three serial systemic passages through Nicotiana benthamiana. To test the effect of SPCP in trans, PVX-SPCP was co-inoculated onto N. benthamiana together with a Tomato bushy stunt virus (TBSV) vector carrying a green fluorescent protein (GFP) gene that normally does not support systemic GFP expression. In contrast, co-inoculation of TBSV-GFP plus PVX-SPCP resulted in GFP accumulation and concomitant green fluorescent spots in upper, non-inoculated leaves in a temperature-responsive manner. These results suggest that the multifaceted SPMV CP has intriguing effects on virus-host interactions that surface in heterologous systems.     

Endoplasmic reticulum targeting of the Red clover necrotic mosaic virus movement protein is associated with the replication of viral RNA1 but not that of RNA2

Red clover necrotic mosaic virus (RCNMV) is a positive-strand RNA virus with a bipartite genome. The movement protein (MP) encoded by RNA2 is essential for viral movement. To obtain further insights into the viral movement mechanism, subcellular localizations of RCNMV MP fused with green fluorescent protein (MP:GFP) were examined in Nicotiana benthamiana epidermal cells and protoplasts. The MP:GFP expressed from the recombinant virus first appeared in the cell wall and subsequently was observed on the cortical endoplasmic reticulum (ER) as punctate spots. In contrast, the MP:GFP expressed transiently in the absence of other viral components was localized exclusively in the cell wall. Transient expression of the MP:GFP with a variety of RCNMV components revealed that the ER localization of the MP:GFP was associated with RNA1 replication, or its negative-strand RNA synthesis, but not those of RNA2 or replicase proteins per se. A model of RCNMV cell-to-cell movement is discussed.     

Molecular characterization of a distinct begomovirus infecting tobacco in Yunnan, China

Summary.  Virus isolate Y3V, obtained from tobacco showing leaf curl symptoms in Yunnan, China, had particles with the size and morphology typical of geminiviruses. In reactions with a set of monoclonal antibodies raised against begomoviruses, Y3V was readily differentiated from two previously studied Chinese Begomovirus isolates. The complete nucleotide sequence of a DNA-A-like molecule of Y3V was determined; it comprises 2744 nucleotides and has a typical Begomovirus genome organization. When compared with the DNA-A sequences of other begomoviruses, the total DNA-A of Y3V was most closely related to that of Ageratum yellow vein virus (AYVV) (85% sequence identity), but the Y3V intergenic region differed greatly from those of the other sequences (maximum 70% identity). In contrast, the deduced coat protein of Y3V is most like that of Tomato yellow leaf curl Thailand virus-[1] (TYLCTHV-[1]) (92% amino acid sequence identity). The molecular data show that the Yunnan isolate of Tobacco leaf curl virus is a distinct Begomovirus species, for which the name Tobacco leaf curl Yunnan virus (TLCYnV) is proposed. Received February 27, 2001/Accepted May 29, 2001     

A betasatellite-dependent begomovirus infects ornamental rose: characterization of begomovirus infecting rose in Pakistan

The Begomovirus genus of the family Geminiviridae comprises the largest group of geminiviruses. The list of begomoviruses is continuously increasing as a result of improvement in the methods for identification. Ornamental rose plants (Rosa chinensis) with highly stunted growth and leaf curling were found in Faisalabad, Pakistan. Plants were analyzed for begomovirus infection, through rolling circle amplification and PCR methods. Based on complete genome sequence homologies with other begomoviruses, a new begomovirus species infecting the rose plants was discovered. In this paper, we propose a new species name, Rose leaf curl virus (RoLCuV), for the virus. RoLCuV showed close identity (83 %) with Tomato leaf curl Pakistan virus, while associated betasatellite showed 96 % identity with Digera arvensis yellow vein betasatellite (DiAYVB), justifying a new isolate for the betasatellite. Recombination analysis of newly identified begomovirus revealed it as a recombinant of tomato leaf curl Pakistan virus from its coat protein region. The infectious molecules for virus/satellite were prepared and inoculated through Agrobacterium tumefaciens to N. benthamiana plants. RoLCuV alone was unable to induce any level of symptoms on N. benthamiana plants, but co-inoculation with cognate betasatellite produced infection symptoms. Further investigation to understand the trans-replication ability of betasatellites revealed their flexibility to interact with Rose leaf curl virus.     

Characterization of an RNA silencing suppressor encoded by maize yellow dwarf virus-RMV2

Maize yellow dwarf virus-RMV2 (MYDV-RMV2) causes dwarfing and yellowing symptoms on leaves in field-grown maize plants in Anhui province in China. Herein, we evaluated the RNA silencing suppressor (RSS) activity of the P0 protein from MYDV-RMV2 by co-infiltration assays using wild-type and GFP-transgenic Nicotiana benthamiana (line 16C). The P0 of MYDV-RMV2 exhibited RSS activity and inhibited RNA silencing both locally and systemically. MYDV-RMV2 P0 acts as an F-box-like motif, and mutations to Ala at positions 67, 68, and 81 in the F-box-like motif (67LPxx81P) abolished the RSS activity of P0. However, MYDV-RMV2 P0 failed to interact with AGO1 from Arabidopsis thaliana. Expressing P0 induced developmental defects. P0 was targeted to both the nuclei and cytoplasm of plant cells. These findings expand our knowledge of the role of polerovirus P0 proteins in RNA silencing.