New Beet soil-borne mosaic virus RNA-4 form isolation from infected sugar beet roots.

Beet soil-borne mosaic virus (BSBMV) is a member of Benyvirus genus together with Beet necrotic yellow vein virus (BNYVV)necrotic yellow vein virus (BNYVV), both vectored by Polymyxa betae. BSBMV is widely distributed only in the United States and, up to date, it has not been reported in others countries. It was first identified in Texas as a sugar beet virus morphologically similar but serologically distinct to BNYVV. Subsequent sequence analysis of BSBMV RNAs showed similar genomic organization to that of BNYVV but sufficient molecular differences to distinct BSMBV and BNYVV in two different species. The described BSBMV genome consists of four RNAs capped at the 5’end and 3’ polyadenylated. RNA-1, and 2 are, respectively, 6,683 and 4,615 nucleotides in length and contain putative ORFs similar to which identified on BNYVV RNA-1 and 2. The 1,720 nt BSBMV RNA-3 has a 29 kDa ORF that share 23% amino acid sequence identity with the 25 kDa ORF of BNYVV RNA3. A 1,203 nt long BSBMV RNA-4 form (GenBank accession number: NC_003508) has been so far described with a single putative ORF with a predicted mass (13 kDa) considerably smaller than the BNYVV 31 kDa RNA-4 product. During our research program, pointed to the studies of the molecular interactions between sugar beets and Benyviruses, unexpected 1,731 nucleotides long form of BSBMV RNA-4 has been detected from sugar beet roots grown on BSBMV infected soil kindly supplied by Marc Richard-Molard (ITB, Paris). The new BSBMV RNA-4 form has been molecularly and functionally characterised. Sequence analysis revealed high identity (~100%) with NC_003508 sequence between nucleotides 1-608 and 1,138-1,731, however the new form shows 529 additionally nucleotides between positions 608-1,138. Two putative ORFs has been identified, the first one encode a protein with predicted mass of 32 kDa (p32) and the second one express an expected product of 13 kDa. Recombination experiments demonstrated BNYVV viral machinery ability to replicate and to encapsidate BSBMV RNA-4 in planta and the capability of BSBMV RNA4 to promote BNYVV RNA-1, 2 and 3 transmission through the vector P. betae in Beta vulgaris plants. Start codons and self-activation domains of the BSBMV RNA4-encoded proteins were also identified by mutagenesis and yeast one-hybrid approaches respectively. Finally the expression of p32 by BNYVV RNA-3 based replicon in the BNYVV RNA-1 and 2 context allow to associate p32 to the appearance of local necrotic lesions