Preliminary work for full -lenght CDNA clones construction of Italian Soil-borne cereal mosaic virus isolate

A mosaic disease of winter wheat was first described in the USA in 1919 and later was world wide associated to the Furovirus Soil-borne wheat mosaic virus (SBWMV), transmitted by the soil-borne plasmodiophorid Polymyxa graminis. Subsequent sequence analyises divided American, European and Chinese isolates into different species within the Furovirus genus: wheat mosaic disease is generally caused by the species SBWMV in the United States, Brazil and Canada, Soil-borne cereal mosaic virus (SBCMV ) in Europe and Chinese wheat mosaic virus (CWMV) in Asia. In Italy, SBCMV was first reported in 1960 on cultivars of common wheat (Triticum aestivum) grown in the Po valley, and subsequently also on durum wheat (Triticum durum) in the central and southern Italian regions. SBCMV has been shown to cause grain yield reductions of about 50-70% on the most susceptible varieties in Italy. The use of resistant varieties, due to the persistent nature of the virus within the soil, is the only practical and economical viable mean of control. The SBWMV, SBCMV and CWMV genomes consist of two plus-sense strands of RNA. RNA1 (about 7 kb) contains three open reading frames which encode for the RNA polymerase, the methyltransferase-helicase and the cell-to-cell movement proteins, The RNA2 (about 3.7 kb) encodes for the coat protein, for protein involved on lasmodiophorid transmission of the virus, pathogenicity determinant, suppressor of RNA silencing and for 25 kDa protein with unknown function. An Italian isolate of SBCMV has been derived from infected wheat plants collected in a field near Bologna in 2007. Instability of a viral cDNA insert in a bacterial plasmid vector has been previously reported with a number of plus-sense RNA viruses in the process of construction of full-length cDNA clones. Several approaches have been examined as a solution for this problem with the aim to produce cDNA clones of the complete SBCMV genome. Strategies based on the use of several plasmid vectors with different markers and high- or low-copy numbers, cDNA insertion in both orientations in the plasmid, insertion of multiple introns into a full-length cDNA, use of E. coli and Bacillus subtilis strains with different phenotypes have been employed in attempting the construction of the full-length SBCMV RNA-1 cDNA clone. Two clones, containing the 5’-terminal 3.9-kb region and the 3’-terminal 3.1-kb region, have been produced and a full-length form can be reconstituted from them. Plasmid containing SBCMV RNA-2 cDNA has been obtained cloning full-length PCR product. Biological activity of the clones has been tested by rub-inoculation on Chenopodium quinoa and T. durum plants of the in vitro transcripts produced from plasmids.