Virulence-attenuating hypoviruses of the species Cryphonectria hypovirus 1 (CHV1) encode a papain-like protease, p29, that shares similarities with the potyvirus-encoded suppressor of RNA silencing HC-Pro. We now report that hypovirus CHV1-EP713-encoded p29 can suppress RNA silencing in the natural host, the chestnut blight fungus Cryphonectria parasitica. Hairpin RNA-triggered silencing was suppressed in C. parasitica strains expressing p29, and transformation of a transgenic green fluorescent protein (GFP)-silenced strain with p29 resulted in an increased number of transformants with elevated GFP expression levels. The CHV1-EP713 p29 protein was also shown to suppress both virus-induced and agroinfiltration-induced RNA silencing and systemic spread of silencing in GFP-expressing transgenic Nicotiana benthamiana line 16c plants. The demonstration that a mycovirus encodes a suppressor of RNA silencing provides circumstantial evidence that RNA silencing in fungi may serve as an antiviral defense mechanism. The observation that a phylogenetically conserved protein of related plant and fungal viruses functions as a suppressor of RNA silencing in both fungi and plants indicates a level of conservation of the mechanisms underlying RNA silencing in these two groups of organisms.RNA-mediated, sequence-specific silencing of gene expression, termed RNA silencing, has been reported for plants (33,48), fungi (39), and animals (16) and variously referred to as posttranscriptional gene silencing, quelling, and RNA interference, respectively. A common feature of RNA silencing is the processing of structured or double-stranded RNA into small interfering RNAs (siRNAs) of 21 to 25 nucleotides by members of the RNase III family of double-stranded RNA-specific endonucleases (Dicers). These siRNAs are then incorporated into an RNA-induced silencing complex that guides sequencespecific degradation of homologous RNA (reviewed in reference 56).RNA silencing plays a key antiviral defense role in plants (reviewed in reference 52) and has been demonstrated to influence virus replication in animal cells (31). Viruses, in turn, produce proteins capable of suppressing host cell RNA silencing (reviewed in reference 46). RNA silencing is part of a larger set of regulatory pathways involving small RNAs that include microRNA (miRNA)-mediated developmental regulation in plants and animals (reviewed in reference 20). Recent findings (29) indicate that some viral suppressors of RNA silencing also contribute to virus-induced disease symptoms by interfering with miRNA-controlled developmental pathways.Although RNA silencing in fungi is generally portrayed as having originated as an ancient antiviral defense mechanism, there is currently no experimental evidence to support this assumption. Our understanding of RNA silencing in fungi comes primarily from studies with the model filamentous fungus Neurospora crassa (4, 6, 11). However, no experimental virus system is available for this organism. In this regard, the chestnut blight fungus, Cryphonectria ...
The nuclear localized C2 protein of the monopartite begomovirus Tomato yellow leaf curl virus-China (TYLCV-C) contributes to viral pathogenicity. Here, we have investigated TYLCV-C C2 protein domains that play a role in the phenotype. Alignment of the C2 protein with 67 homologues from monopartite and bipartite begomoviruses revealed that a putative zinc-finger motif C36-X1-C38-X7-C46-X6-H53-X4-H58C59 and four potential phosphorylation sites (T52, S61, Y68, and S74) are highly conserved. When expressed from a Potato virus X (PVX) vector, TYLCV-C C2 protein mutants C2-T52M, C2-H58S, C2-C59S, C2-S61R, and C2-S74D, like the wild-type C2 protein, induced local necrotic ringspots and systemic necrosis in Nicotiana benthamiana plants. Mutants C2-H53P and C2-Y68D produced irregular necrotic lesions on inoculated leaves that were distinct from the wild-type phenotype. In contrast, mutants C2-C36R, C2-C38N, and C2-C46I induced chlorosis and mosaic symptoms rather than necrosis. We demonstrate that TYLCV-C C2, like its counterpart in the bipartite begomovirus African cassava mosaic virus, mediates suppression of posttranscriptional gene silencing (PTGS). Moreover, the individual mutations C36R, C38N, and C46I abolished the ability of C2 protein to suppress PTGS. These results suggest that the three cysteine residues within the putative zinc-finger motif are essential for C2 protein to induce necrosis and to act as a suppressor of PTGS.
The nucleus-localized C2 protein of Tomato yellow leaf curl virus-China (TYLCV-C) is an active suppressor of posttranscriptional gene silencing (PTGS). Consistently, infection with TYLCV-C resulted in PTGS arrest in plants. The C2 protein possesses a functional, arginine-rich nuclear localization signal within the basic amino acid-rich region 17 KVQHRIAKKTTRRRR 31 . When expressed from potato virus X, C2-RRRR 31 DVGG (in which the four consecutive arginine residues 28 RRRR 31 were replaced with DVGG) that had been tagged with a green fluorescent protein (GFP) failed to transport GFP into nuclei and was dysfunctional in inducing necrosis and suppressing PTGS in plants. Amino acid substitution mutants C2-K 17 D-GFP, C2-HR 21 DV-GFP, and C2-KK 25 DI-GFP localized to nuclei and produced necrosis, but only C2-K 17 D-GFP suppressed PTGS. The N-terminal portions C2 1-31 and C2 17-31 fused in frame to GFP were capable of targeting GFP to nuclei, but neither caused necrosis nor affected PTGS. Our data establish that nuclear localization is likely required for C2 protein to function in C2-mediated induction of necrosis and suppression of PTGS, which may follow diverse pathways in plants. Possible mechanisms of how the C2 protein involves these biological functions are discussed.Posttranscriptional gene silencing (PTGS), RNA interference, and gene quelling represent a conserved cellular defense system for controlling foreign gene expression across the plant, animal, and fungal kingdoms (2,6,8,17,32,42,45). These silencing systems involve double-stranded RNA (dsRNA) from which a 21-to 26-nucleotide (nt) short interfering RNA (siRNA) is derived by the action of an RNase III-like dicer, and they share a common molecular mechanism in which a target RNA is degraded in an RNA homology-dependent manner by an RNA-induced silencing multisubunit RNase complex under the guidance of siRNA (2,3,(15)(16)(17)32). In plants, PTGS defends the host against virus infection, down-regulates transgene expression, and may also participate in the control of development (42,45). To counterattack, plant viruses have evolved the ability to encode proteins (i.e., PTGS suppressors) capable of suppressing PTGS by targeting various stages of the PTGS process, including initiation, propagation, and maintenance (6,29,30,42,45). For example, the potyvirus protein HC-Pro affects PTGS maintenance by interfering with a step coincident with, or upstream of, the production of siRNAs (25,27). HC-Pro interacts with a calmodulin-related protein that can suppress PTGS in plants (1). However, the p25 cell-to-cell movement protein of Potato virus X (PVX) and the 2b protein of Cucumber mosaic virus (CMV) preclude the spread of silencing signals (13, 43). On the other hand, the viral PTGS suppressors are often found to be pathogenicity determinants, and their PTGS suppression activity is associated with pathogenicity determination (44). It is worth noting that certain mutants of the CMV 2b protein have been reported to be functional in pathogenesis but dysfunctional in...
A Turnip crinkle virus (TCV)-based system was devised to discriminate cell-to-cell and systemic long-distance spread of RNA silencing in plants. Modified TCV-GFP⌬CP, constructed by replacing the coat protein (CP) gene with the green fluorescent protein (GFP) gene, replicated in single epidermal cells but failed to move from cell to cell in Nicotiana benthamiana. Mechanical inoculation of TCV-GFP⌬CP induced effective RNA silencing in single epidermal cells which spread from cell to cell to form silenced foci on inoculated leaves, but no long-distance systemic spread of RNA silencing occurred. Agroinfiltration of TCV-GFP⌬CP was, however, able to induce both local and systemic RNA silencing. TCV coinfection arrested TCV-GFP⌬CP-mediated local induction of RNA silencing. Possible mechanisms involved in cell-to-cell and long-distance spread of RNA silencing are discussed.RNA silencing, including gene quelling, RNA interference, and posttranscriptional gene silencing are sequence-specific RNA degradation mechanisms that operate in fungi, animals, and plants (6,7,30). RNA silencing is triggered by doublestranded RNA and requires a conserved set of gene products (1,13,14). The double-stranded RNA is processed into small interfering RNAs (siRNAs) of 21 to 25 nucleotides (nt), and these siRNAs become associated with an RNA-induced silencing complex that degrades specific target RNA sequences (3,11,12). RNA silencing plays a natural role in protecting fungi, plants, and animals against viral infection. To withstand the RNA-silencing defense, viruses across kingdoms have evolved diverse mechanisms either to avoid or actively suppress RNA silencing (22,35,40).One intriguing feature of RNA silencing is that it is not cell autonomous. In plants, RNA silencing can be induced locally and then spread to distal parts (16,(36)(37)(38). While RNA-silencing induction and RNA degradation have been elucidated in detail, much less is known about how RNA silencing moves from cell to cell and spreads systemically in plants. No mobile silencing signal has been characterized, although the sequence specificity of RNA silencing implies that nucleic acids, possibly siRNAs, may be a component of such an RNA-silencing signal (26). We have used Turnip crinkle virus (TCV) to explore the requirements for cell-to-cell and long-distance spread of RNA silencing in plants.TCV, a member of the Carmovirus genus, has a positive single-strand genomic RNA (4,053 nt), packaged in icosahedral capsids, which contains five major open reading frames (5). The p28 and p88 proteins are translated from genomic RNA, by readthrough of the p28 terminator, and are involved in viral RNA replication. Two overlapping proteins, p8 and p9, are expressed from subgenomic RNA1 and are required for cell-to-cell movement and systemic spread of the virus (10, 23).The 3Ј-proximal open reading frame encodes the 38,000-molecular-weight coat protein (CP), which also plays an essential role in cell-to-cell movement of TCV in Nicotiana benthamiana (8) and acts as an effective suppressor ...
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