Characterization of silencing suppressor 2b of cucumber mosaic virus based on examination of its small RNA-binding abilities.

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 50 kDa movement protein acts as a suppressor of systemic silencing without interfering with local silencing in Nicotiana benthamiana.

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.

Effectiveness of an Attenuated Zucchini yellow mosaic virus Isolate for Cross-Protecting Cucumber.

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.

A New Technique to Select Mild Strains of Cucumber mosaic virus.

An efficient technique to select a good attenuated virus to control Cucumber mosaic virus (CMV) disease was developed. Preliminary screenings were conducted to assess the virulence of virus recovered from dark-green islands and yellow tissues of mosaic leaves of Nicotiana rustica after co-inoculation with an attenuated mutant P2bR46C of CMV and its original severe isolate Pepo. All single-lesion isolates (SLIs) obtained from dark-green islands had the attenuated P2bR46C phenotype, but the SLIs from yellow tissue had either the virulent Pepo or the P2bR46C phenotype. When Pepo-infected N. rustica and tomato plants were grown at 15 or 36°C for 30 days, 17 of 288 SLIs obtained from the treated leaves elicited mosaic and dark-green spots without malformation. Dark-green tissue from each plant infected with 1 of these 17 SLIs then was used to inoculate one plant of N. rustica. All 17 plants had either very mild mosaic or no visible symptoms. One of these potential mild strains, 36R37, had an amino acid substitution on the 2b gene encoding the 2b protein. Isolate 36R37 also was highly cross-protective, and its symptom attenuation was stable for three serial host passages. After cold or heat treatment, the dark-green tissue proved to be a good source for isolating mild strains of the virus.

Cucumber mosaic virus 2b protein compensates for restricted systemic spread of Potato virus Y in doubly infected tobacco.

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.

Cucumber mosaic virus Establishes Systemic Infection at Increased Temperature Following Viral Entrance Into the Phloem Pathway of Tetragonia expansa.

ABSTRACT A potential regulatory site for Cucumber mosaic virus (CMV, pepo strain) movement necessary to establish systemic infection was identified through immunological and hybridization studies on Tetragonia expansa, which was systemically infected by CMV at 36 degrees C but not at 24 degrees C. In inoculated leaves, cell-to-cell movement of CMV was enhanced at 36 degrees C compared with that observed at 24 degrees C. CMV was distributed in the phloem cells of minor veins as well as epidermal and mesophyll cells at both 36 and 24 degrees C. CMV was detected in the petioles of inoculated leaves, stems, and petioles of uninoculated upper leaves at 36 degrees C, whereas CMV was detected only in the petioles of inoculated leaves and in stems at 24 degrees C. CMV moved into the phloem and was transported to the stem within 24 h postinoculation (hpi) at 36 degrees C. However, it did not accumulate in the petioles of the upper leaves until 36 hpi. In petioles of inoculated leaves at 24 degrees C, CMV was detected in the external phloem but not in the internal phloem. From these results, we conclude that systemic infection is established after viral entrance into the phloem pathway in T. expansa at 36 degrees C.