Infectious in vivo transcripts of a plum pox potyvirus full-length cDNA clone containing the cauliflower mosaic virus 35S RNA promoter

Abstract

A full-length cDNA clone of an aphid non-transmissible isolate of plum pox potyvirus (PPV) was rendered biologically active when placed under the control of the cauliflower mosaic virus 35S RNA promoter and the nopaline synthase polyadenylation signal. The cDNA was constructed so that the exact 5' end of the PPV RNA was present at the transcription initiation site.Inoculation of plasmid DNA onto Nicotiana benthamiana led to systemic infection, whereas local lesions were produced in Chenopodium amaranticolor and C.quinoa, typical of an infection with PPV. Examination of infected plants revealed PPV-specific virus particles as well as viral RNA, the coat protein and the nonstructural large nuclear inclusion protein (NIb).Potyviruses belong to the largest group of plant viruses and cause serious diseases in a number of economically important crop plants. Members of the potyvirus group are flexuous rods 720 to 900 nm in length (Hollings & Brunt, 1981) and encapsidate a single positive-sense RNA molecule of about 10 kb, attached to a 5'-linked protein (VPg) and including a poly(A) tract at the 3' end. The RNA is translated into a large polyprotein which is subsequently processed by at least two virus-encoded proteases. Upon proteolytic cleavage this polyprotein yields the viral coat protein (CP) and the non-structural proteins helper component-protease, cylindrical inclusion protein and two nuclear inclusion proteins (NIa and NIb). In addition, at least two other gene products which have not yet been identified are predicted from the nucleotide sequence of all potyviruses sequenced (Allison et al., 1986;Balint et al., 1990;Domier et al., 1986;Lain et al., 1989;Maiss et al., 1989;Robaglia et al., 1989).The in vitro synthesis of biologically active RNAs from full-length cDNA clones has been reported for two potyviruses (Domier et al., 1989;Riechmann et al., 1990). Genetic manipulation of full-length cDNA together with the subsequent transcription of infectious RNA provide a powerful tool for the examination of the molecular biology of RNA viruses. Moreover, it has been shown that infectious RNA can be transcribed in vivo by unknown promoters from plasmids containing cDNA copies of RNA 3 of alfalfa mosaic virus (Dore & Pinck, 1988), tobacco necrosis satellite RNA (van Emmelo et al., 1987) and potato spindle tuber viroid (Crees et al., 1983). Recently, Mori et al. (1991) have described the infectivity of plasmids containing the 35S RNA promoter of cauliflower mosaic virus (CaMV) upstream of fulllength cDNAs of all three brome mosaic virus (BMV) RNAs. Also, Commandeur et al. (1991) have demonstrated the infectivity of clones containing the 35S RNA promoter and cDNAs of RNA 3 and RNA 4 of beet necrotic yellow vein virus when complemented with a virus isolate which contains only RNA 1 and RNA 2. In this paper, the construction of a plasmid containing a full-length cDNA of an aphid non-transmissible isolate of plum pox potyvirus (PPV-NAT;Maiss et al., 1989) linked to the 35S RNA promoter of CaMV is described. PPV host plan...

No recombination detected in artificial potyvirus mixed infections and between potyvirus derived transgenes and heterologous challenging potyviruses

“…The infectious full-length clone p35PPV-NAT of the non-aphid transmissible strain of PPV (Maiss et al, 1992) as well as wildtype virus isolates of TVMV (PV-0253, DSMZ, Germany), PVA (SCRI collection), PVY-O (SCRI collection), PPV-SK68 (Palkovics et al, 1993) and PVY-H (Thole et al, 1993) were used in the respective experiments.…”

“…A plasmid with a PPV-NAT/TVMV chimeric sequence was prepared by digesting a TVMV full-length clone (pXBS; Domier et al, 1986) with BamHI/EcoRI and cloning a 1748 bp fragment into pTrueBlue (Slilaty and Lebel, 1998) giving pTB_10T. After digestion of p35PPV-NAT (Maiss et al, 1992) with EcoRI/SacI, a 836 bp fragment was fused to the TVMV sequence of pTB_10T (pTB_10TP).…”

Cauliflower Mosaic Virus 35S Promoter-Controlled DNA Copies of Cowpea Mosaic Virus RNAs are Infectious on Plants

“…The constructs used in this study do not contain a polyadenylation signal downstream of the viral sequence, in contrast to the 35S-controlled clones constructed by Mori et al (1991), Commandeur et al (1991, MacFarlane et al (1992), Weber et al (1992 and Maiss et al (1992). Instead, the clones used in this study are linearized downstream of the viral sequence, thus preventing the synthesis of transcripts longer than the genome.…”

“…Recently, the construction of directly infectious cDNA clones of brome mosaic virus RNAs 1, 2 and 3 (Mori et al, 1991), beet necrotic yellow vein virus RNAs 3 and 4 (Commandeur et al, 1991), pea early browning virus RNAs 1 and 2 (MacFarlane et al, 1992), tomato mosaic virus RNA (Weber et al, 1992) and plum pox virus RNA (Maiss et al, 1992) have been reported. These constructs make use of the cauliflower mosaic virus (CaMV) 35S promoter sequence linked to the 5' ends of the viral cDNAs to generate infectious transcripts in the plant.…”

Biologically active transcripts from cloned cDNA of genomic grapevine fanleaf nepovirus RNAs

“…genomic cDNAs of several picorna-like plant viruses have been reported: CPMV (Vos et al, 1988), tobacco vein mottling virus (TVMV, Domier et al, 1989) and plum pox potyvirus (PPV, Riechmann et al, 1990;Maiss et al, 1992), but no infectious transcripts corresponding to genomic RNA of nepoviruses have so far been described. Here, we report the construction of clones containing the full-length cDNA of GFLV (isolate F13) RNA1 and -2 under the control of bacteriophage T3 or T7 RNA polymerase promoters.…”