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James M. Crosslin

-dependent RNA polymerase ( Sudarshana and Berger, 1998 ). One ORF near the 3′ end of RNA 1 called the 16 kDa protein ORF codes for a factor reportedly involved in nematode transmissibility ( Boccara et al., 1986 ) or possibly in virus gene regulation ( Liu et al

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Jean-Michel Hily, Michel Ravelonandro, Vern Damsteegt, Carole Bassett, Cesar Petri, Zongrang Liu, and Ralph Scorza

, but it also serves as a component of adaptive protection against mobile genetic elements, such as transposons and viruses ( Voinnet, 2005 ). A key component of the pathway is known as small interfering RNA (siRNA) ( Hamilton and Baulcombe, 1999

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P.B. McGarvey, M.S. Montasser, and J.M. Kaper

Transgenic tomato plants (Lycopersicon esculentum Mill.) expressing cucumber mosaic virus (CMV) satellite RNA fused to a gene for β-glucuronidase were produced using Agrobacterium-mediated transformation. The R1 progeny of self-crossed R0 plants were challenge-inoculated with virion or RNA preparations of CMV or tomato aspermy virus (TAV). The transgenic plants challenged with CMV-1 showed mild disease symptoms in the first 2 weeks postchallenge followed by a decrease in symptoms, resulting in little difference between the transgenic and uninfected control group by the fourth week. Enzyme-linked immunosorbent assay results showed about a 10-fold decrease in virus accumulation in the transgenic plants compared to controls. Tolerance was evident only in plants that contained the recombinant insert and produced mature unit-length satellite RNA after CMV infection. Plants challenged with TAV showed no significant tolerance to virus-induced symptoms.

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Jean-Michel Hily, Ralph Scorza*, and Michel Ravelonandro

We have shown that high-level resistance to plum pox virus (PPV) in transgenic plum clone C5 is based on post-transcriptional gene silencing (PTGS), otherwise termed RNA silencing (Scorza et al. Transgenic Res. 10:201-209, 2001). In order to more fully characterize RNA silencing in woody perennial crops, we investigated the production of short interfering RNA (siRNA) in transgenic plum clones C3 and C5, both of which harbor the capsid protein (CP) gene of PPV. We used as a control, plum PT-23, a clone only transformed with the two marker genes, NPTII and GUS. We show in the current report that C5 constitutively produces two classes of siRNA, the short (21-22 nucleotides) and long (≈27 nucleotides) species in the absence of PPV inoculation. Transgenic susceptible clone C3 and the control clone PT-23, when healthy, produce no siRNA. Upon infection, these clones produce only the short siRNA (21-22 nt). This siRNA production suggests that plum trees naturally respond to virus infection by initiating PTGS or PTGS-like mechanisms. This study also suggests that high-level virus resistance in woody perennials may require the production of both the short and long size classes of siRNA, as are produced by the resistant C5 plum clone.

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C.A. Powell, A. Hadidi, and J.M. Halbrendt

The ability of 32P-labeled transcribed cRNA probes to detect tomato ringspot virus (TmRSV) RNA in nucleic acid extracts from roots, bark, and leaves of nectarine (Prunus persica [L.] Batsch) trees with the Prunus stem-pitting disease was assessed and compared with detection of TmRSV antigen by enzyme-linked immunosorbent assay (ELISA) in the same tissues. Neither TmRSV-specific nucleic acid nor antigen was detected in nectarine leaf tissue. ELISA detected TmRSV antigen in root extracts from 71% of the diseased trees, while dot hybridization detected virus-specific nucleic acid in 18% of the same samples. However, ELISA detected TmRSV antigen in only 47% of bark extracts; whereas TmRSV-specific nucleic acid was detected in 100% of the bark extracts from samples collected at or near the soil line. When nucleic acid extracts from bark were prepared from various locations on diseased trees and tested for TmRSV-specific nucleic acid by dot hybridization, there was an almost perfect correlation between the presence of stem-pitting symptoms and the detection of TmRSV nucleic acid. Detection of TmRSV RNA from the bark tissue of rootstock suckers from TmRSV-infected `Delicious'/MM.lO6 apple (Malus × domestica Borkh.) trees was unsuccessful using dot hybridization. The viral RNA, however, was usually detected in either leaf or root tissue of these same trees.

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Shin Je Kim, Kyung-Hee Paek, and Byung-Dong Kim

CMV 117N satellite RNA. We thank J.R. Liu for his critical reading of this manuscript.. Sang-Jik Lee's technical assistance is greatly appreciated. This work was supported in part by a grant (G70300) to K.-H. Pack in aid for scientific research from

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J.R. Fisher and S.G.P. Nameth

Cucumber mosaic virus (CMV) was isolated from the perennial ornamental mint, Ajuga reptans L. `Royalty', using melon aphids (Aphis gossypii Glover). The isolate and its associated satellite RNA (satRNA) were biologically and chemically characterized. The satRNA was cloned and sequenced and is 338 nucleotides long and does not induce lethal necrosis on `Rutgers' tomato (Lycopersicon esculentum Mill.) or severe chlorosis on tobacco (Nicotiana L. spp.). The virus is ≈28 to 30 nm in diameter and reacts to CMV serological subgroup I antibodies. The virus is able to infect `Black Beauty' squash (Cucurbita pepo L.), cucumber (Cucumis sativus L.), and `Howden' pumpkin (Cucurbita pepo) but is not able to infect green bean (Phaseolus vulgaris L.) or cowpea [Vigna unguiculata (L.) Walp. ssp. unguiculata]. The virus is able to efficiently replicate its satRNA in tobacco and `Black Beauty' squash but replication is less efficient in cucumber, based on accumulation of double-stranded satRNA.

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J.R. Fisher and S.T. Nameth

Ajuga reptans L. is an herbaceous ornamental mint grown in borders or as a groundcover, and is commonly propagated vegetatively and by seed. Three hundred and fifty-six A. reptans samples were obtained from growers in Washington, Michigan, Iowa, and Ohio, and screened for alfalfa mosaic virus (AMV), tobacco streak ilarvirus (TSV), cucumber mosaic cucumovirus (CMV), tomato aspermy cucumovirus (TAV), tomato spotted wilt tospovirus (TSWV), impatiens necrotic spot tospovirus (INSV), tobacco mosaic tobamovirus (TMV), potato virus × potexvirus (PVX), and 80 potyviruses, using direct antibody sandwich (DAS) and indirect enzyme-linked immunosorbent assay (ELISA). Viral-associated double-stranded ribonucleic acid (dsRNA) analysis was used to detect an apparent satellite (sat) RNA, and northern hybridization using a digoxigenin (DIG) labeled (S) CARNA-5 cDNA probe was used to confirm the identity of the apparent satRNA. No incidences of TAV, TMV, TSWV, INSV, PVX, or potyviruses were detected. CMV was detected in 11%, AMV in 22.2%, TSV in 3.7%, and mixed infections of CMV and AMV in 1.1% of the samples. SatRNA was detected in 36 A. reptans `Royalty', two `Rainbow', and two `Burgundy Glow' samples by dsRNA analysis, and confirmed by hybridization in 29 `Royalty' and one `Burgundy Glow' samples. Sixteen A. reptans `Royalty' seedlings grown from seed harvested from CMV-infected plants were tested by ELISA for CMV, AMV, and TSV. All were positive for CMV, and two were positive for a mixed infection of CMV and AMV. SatRNA was detected in all 16 seedlings by RT-PCR.

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Ena Akamatsu, Takanori Kai, Hideaki Hirabaru, Chizuko Yukizaki, Miho Sakai, Hirofumi Uto, Hirohito Tsubouchi, and Hisato Kunitake

Blueberry (Vaccinium sp.) fruits contain high concentrations of polyphenols such as anthocyanin. It is well known that polyphenols have antioxidant activity, so it is likely that the fruit has a possible preventative effect against several diseases like cancer. However, only a few reports so far have studied the human health benefits of the leaves. In this study, the antioxidant activity and antiviral effects of blueberry leaves were investigated. The leaves of three groups of blueberry, northern highbush blueberry (NHB), southern highbush blueberry (SHB), and rabbiteye blueberry (REB), were examined. These leaves were harvested in July and extracted with 80% ethanol. Samples were analyzed for antioxidant activity (DPPH radical scavenging activity) and antiviral activity against hepatitis C virus using the replicon cell assay (Lomann et al., 1999). The antioxidant activity showed significant variability between cultivars and species, with REB having about two times the activity of NHB and SHB. Antiviral activity was observed in the extracts of the leaves and the fruit, and the activity of the leaves was higher than that of the fruit. Among the cultivars and species evaluated, the antiviral activity of REB was higher than that of NHB and SHB. In addition, we discovered a positive correlation (r=0.68) between the antioxidant activity and the antiviral activity, using the leaves of hybrid seedlings of REB. Therefore, it is possible to speculate that the antiviral activity bears some relation to the antioxidant activity.

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Zongrang Liu, Ralph Scorza, Jean-Michel Hily, Simon W. Scott, and Delano James

engineering of multivirus resistance in any crop. The mechanisms of virus resistance through PDR have been found to be either protein-mediated, usually resulting in moderate resistance to a broad range of isolates, or RNA-mediated resulting in extreme