Viral RNA synthesis in tomato spotted wilt virus-infected Nicotiana rustica plants

Abstract

The synthesis of viral RNA species in tomato spotted wilt virus-infected Nicotiana rustica plants was followed in terms of time and relative abundance. Systemic symptoms were visible after 4 days postinoculation (p.i.), but viral (v) and viral-complementary (vc) strands of all three genomic RNA segments [large (L) RNA, medium (M) RNA and small (S) RNA] were detected from 2 days p.i. In addition, two subgenomic mRNAs, derived from S RNA, were detected. For the L RNA segment no subgenomic mRNAs were detected, suggesting that this segment is expressed via the synthesis of a genome-sized vc mRNA. A possible M-specific subgenomic mRNA was detected, showing a similar time course of appearance as the subgenomic mRNAs derived from the S RNA segment. Analysis of cytoplasmic RNA fractions revealed that both v and vc strands of all three genomic segments associate with the nucleocapsid protein into nucleocapsid structures, the vcRNA species being present in lower amounts. Intact, enveloped virus particles contained only the v strand of the L RNA segment and, surprisingly, both v and vc strands of the M and S RNA segment, though in different ratios.

Tospovirus : induction and suppression of RNA silencing

“…Tospoviral RNA genome segments are known to be tightly encapsidated with N protein and therefore not freely exposed to become targeted by RNA silencing, in contrast to their (sub)genomic mRNA molecules (Kormelink et al, 1992a). The latter is supported by the observation that TSWV is still able to replicate in protoplasts from TSWV NSm transgenic plants that confer RNA silencing-mediated resistance to TSWV (Prins et al, 1997).…”

“…1.2). The two ORFs in the ambisense segments are separated by an AU-rich noncoding intergenic region (IGR) that is predicted to fold into a stable hairpin structure (de Haan et al, 1990;Kormelink et al, 1992a). Both ORFs are transcribed, from opposite (v and vc) strands, into subgenomic mRNAs.…”

“…America as previously described (de Avila et al, 1990). Genomic RNA was purified using hot phenol extraction followed by ethanol precipitation (Kormelink et al, 1992a Southern blotting, siRNA purification and mapping on TSWV and TYRV S RNA Total RNA was purified from systemically infected N.benthamiana leafs using Trizol (Life Technologies). The S RNA segment was RT-PCR-amplified, using Superscript RT (Invitrogen), in 6-7 fragments of similar size and spanning the entire S RNA segment from TSWV and TYRV respectively.…”

Plant virus gene expression strategies

“…The first report of a plant virus using such a mechanism for transcription of the viral genome was described for TSWV by Kormelink et al (1992). Subgenomic mRNAs for the S RNA segment were partially purified from total RNA extracts of TSWV-infected Nicotiana rustica and analysed by primer extension analysis.…”

Characterization of a New Tomato Spotted Wilt Virus Isolates Found in Hippeastrum hybridum (Hort.) Plants in Poland

“…The M RNA encodes a 33.6-kDa viral movement protein (NSm) in the v sense and a 127.4-kDa precursor to G1 and G2 (envelope membrane glicoproteins) in the vc sense (Kormelink et al 1992a). The 8.9-kb L RNA has a completely negative sense and contains a single ORF in the vc sense encoding the 331-kDa putative RNA-dependent RNA polymerase (Kormelink et al 1992b).…”