Construction of an infectious full-length cDNA clone of Sunflower chlorotic mottle virus

BEJERMAN, NICOLÁS; GIOLITTI, FABIÁN; DE BREUIL SOLEDAD; LENARDON, SERGIO

Congreso; 18th International Sunflower Conference; 2012

Sunflower chlorotic mottle virus (SuCMoV), which belongs to the genus Potyvirus, is the most prevalent sunflower virus present in Argentina. Two biologically different strains of SuCMoV have been described in Argentina: the common strain (C) and the chlorotic ringspot strain (CRS). Construction of full-length cDNAs of an RNA virus genome, from which infectious RNA can be transcribed in vivo is a crucial step not only towards mapping the genetic determinants involved in symptoms induction, but also to study the virus-sunflower interactions. Therefore, the objective of this work was to construct a full-length cDNA clone of the SuCMoV-C genome and test its infectivity. Three overlapping fragments which covered the complete genomic RNA of SuCMoV were amplified and assembled into a plasmid which contains a Cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and 20 adenine residues were present at the 3  end of the SuCMoV cDNA clone, which was named p35SuCMoV. Mechanical inoculation on Nicotiana occidentalis seedlings with 5µg of p35SuCMoV DNA at a concentration of 100 ng/µl in DEPC water resulted in 20% infection. The chlorotic mosaic symptoms induced by p35SuCMoV in N. occidentalis were identical to those caused by the wild-type SuCMoV on this host. However, a week delay in the establishment of infection was observed in those N. occidentalis infected with p35SuCMoV. Potyvirus like particles were observed in crude sap preparation of p35SuCMoV infected plants under electron microscopy. To further confirm that the symptoms observed in the p35SuCMoV-infected plants were induced by p35SuCMoV, we also carried out DAS-ELISA and RT-PCR analysis. All inoculated plants were analyzed for the presence of coat protein by DAS-ELISA using SuCMoV-C policlonal antisera. DAS-ELISA gave positive results only in those N. occidentalis showing chlorotic mosaic symptoms. Furthermore, the 5´ and 3´ end of the SuCMoV genome was detected using RT-PCR with sequence-specific primers. The 1495 nt and 1070 nt fragments, respectively, were amplified from p35SuCMoV-infected plants but not from a healthy plant. Finally, when the sap from p35SuCMoV-N. occidentalis infected plants was mechanically inoculated to sunflower seedlings, 100% plants developed chlorotic mottle symptoms, which were indistinguishable from that induced by the wild-type SuCMoV. No delay in the establishment of infection was observed. This indicates that the progeny virus from p35SuCMoV DNA inoculated plants is not phenotypically different from the original virus. These results demonstrate that the p35SuCMoV cDNA clone is infectious. Thus, this is the first report of the construction of a biologically active, full-length cDNA copy of the SuCMoV RNA genome. The infectious intact cDNA copy of the SuCMoV-C genome will be used to map the viral sequences involved in the virus symptoms induction by construction of hybrid viruses between SuCMoV-C and -CRS strains.