Double-stranded (ds) RNAs and imperfect hairpin RNAs of endogenous genes trigger post-transcriptional gene silencing (PTGS) and are cleaved by a Dicer-like nuclease into small interfering RNAs (siRNAs) and microRNs (miRNAs), respectively. Such small RNAs (siRNAs and miRNAs) then guide an RNA-induced silencing complex (RISC) for sequence-specific RNA degradation. While PTGS serves as an antiviral defense in plants, many plant viruses encode suppressors as a counter defense. Here we demonstrate that the PTGS suppressor (2b) of a severe strain (CM95R) of cucumber mosaic virus (CMV) can bind to in vitro synthesized siRNAs and even to long dsRNAs to a lesser extent. However, the 2b suppressor weakly bound to a miRNA (miR171) duplex in contrast to another small RNA-binding suppressor, p19 of tombusvirus that can effectively bind miRNAs. Because the 2b suppressor of an attenuated strain of CMV (CM95), which differs in a single amino acid from the 2b of CM95R, could barely bind siRNAs, we hypothesized that the weak suppressor activity of the attenuated strain resulted from a loss of the siRNA-binding property of 2b via a single amino acid change. Here we consider that 2b interferes with the PTGS pathway by directly binding siRNAs (or long dsRNA).

Apple chlorotic leaf spot virus (ACLSV) is the type species of the genus Trichovirus and its single-stranded, plus-sense RNA genome encodes a 216 kDa protein (P216) involved in replication, a 50 kDa movement protein (P50) and a 21 kDa coat protein (CP). In this study, it was investigated whether these proteins might have RNA silencing-suppressor activities by Agrobacterium-mediated transient assay in the green fluorescent protein-expressing Nicotiana benthamiana line 16c. The results indicated that none of these proteins could suppress local silencing in infiltrated leaves. However, systemic silencing in upper leaves induced by both single-and double-stranded RNA could be suppressed by P50, but not by a frame-shift mutant of P50, P216 or CP. Moreover, when P50 was expressed separately from where silencing signals were generated in a leaf, systemic silencing in upper leaves was inhibited. Collectively, our data indicate that P50 acts as a suppressor of systemic silencing without interfering with local silencing, probably by inhibiting the movement of silencing signals. INTRODUCTIONRNA silencing is a sequence-specific RNA-degradation mechanism conserved in a wide variety of eukaryotic organisms and has been termed post-transcriptional gene silencing in plants, quelling in Neurospora crassa and RNA interference (RNAi) in Caenorhabditis elegans and Drosophila melanogaster (Cogoni, 2001;Hannon, 2002;Zamore, 2002). The pathway is triggered initially by doublestranded RNAs, which are processed into small interfering RNAs (siRNAs) of 21-25 nt by an RNase III-like enzyme called Dicer (Hamilton & Baulcombe, 1999). These siRNAs are incorporated into a protein complex called RNAinduced silencing complex (RISC) and guide the RISC to degrade target RNAs that have sequences identical to those of the siRNAs (Hammond et al., 2000).In plants, RNA silencing functions as an immune system against viruses and transposons ( Vance & Vaucheret, 2001;Baulcombe, 2004;Ding et al., 2004;Voinnet, 2005a;Wang & Metzlaff, 2005). During virus infection, long doublestranded (ds) RNAs generated from the replication intermediates of virus RNA trigger RNA silencing. When RNA silencing is induced at one site, silencing signals then move both from cell to cell and long distance (Palauqui et al., 1997;Voinnet & Baulcombe, 1997;Guo & Ding, 2002;Himber et al., 2003) and trigger systemic silencing of target RNA in distant tissues of plants. If the silencing signals induced by virus replication spread in advance of virus movement, sequence-specific virus resistance may be established in whole plants and the virus cannot infect systemically. Although the exact nature of silencing signals remains to be elucidated, RNA is probably a key component to confer sequence specificity in RNA silencing (Mlotshwa et al., 2002;Voinnet, 2005b).To counteract RNA silencing, viruses have evolved RNAsilencing suppressors. Over 30 viral suppressors have been identified among plant, animal and insect viruses. However, these suppressors have no obvious sequence similarity to e...

No abstract

Tobacco plants (Nicotiana tabacum cv. Xanthi-nc) inoculated with a necrotic strain of Potato virus Y (PVY, T01 isolate) developed necrotic symptoms in some systemically infected leaves, but not in younger leaves. However, PVY expressed distinct symptoms not only in the older leaves, but also in the younger leaves, of plants that had been doubly inoculated with PVY and with Cucumber mosaic virus (CMV, strain Pepo). A tissue blot immunoassay of tissues from various positions of the stem detected PVY weakly in each stem, but not in the shoot apex, of singly infected plants, whereas PVY was detected at high levels in almost all sections of doubly infected plants. CMV was also detected at high levels in sections of singly and doubly infected plants. Immunohistochemistry of stem tissues showed that in singly infected plants, PVY was confined to external phloem cells and was not detected in internal phloem cells. However, in doubly infected plants, PVY was distributed uniformly throughout whole tissues, including the external phloem, xylem parenchyma and internal phloem cells. In plants that were doubly infected with PVY and Pepo Delta 2b, a modified CMV that cannot translate the 2b protein, the spread of PVY was restricted as in singly infected plants. These results suggested that the plant host has a counterdefence mechanism that restricts systemic spread of PVY T01, and that the 2b protein of CMV strain Pepo negates this restriction.

To cross-protect cucumber plants from Zucchini yellow mosaic virus (ZYMV), we used cold treatment to obtain an attenuated isolate of ZYMV, designated ZYMV-2002. ZYMV-2002 was obtained from a virulent ZYMV isolate after repeated low temperature treatment at 12.5 to 15°C followed by five cycles of single-plant transfer. The isolate produced very mild or no symptoms on cucurbit plants. In addition, inoculated cucumber plants had very similar fruit productivity to healthy control plants under field conditions. During field experiments in 2002 and 2003, when other viruses were also present, protected plants significantly suppressed infection with ZYMV, progression of disease severity, and reduction of fruit yield and quality. These results demonstrate that ZYMV-2002 is a potentially useful attenuated ZYMV isolate for reducing the impact of ZYMV.