Construction and evaluation of infectious cDNA clones and analysis of packaging determinants of soybean dwarf virus

Soybean dwarf virus (SbDV) is a phloem-limited, aphid-transmitted luteovirus that causes dwarfing of soybean plants. In the present study, infectious clones of two dwarfing isolates of SbDV were constructed, and methods for Agrobacterium-mediated inoculation of plants were standardized and used to introduce them into fava bean, Nicotiana benthamiana, ‘Puget’ pea and red clover plants. SbDV virions purified from N. benthamiana leaves infiltrated with either of these cDNA clones encapsidated genomic RNA (gRNA), but small subgenomic RNA (SsgRNA) was encapsidated by only one isolate and hence its packaging was isolate specific. Site-specific, deletion and domain-exchange mutagenesis indicated that, open reading frames (ORFs) 1, 2, 4, and 5 and interacting stem-loop structures in the 5’ and 3’ untranslated regions (UTRs) were required for replication of SbDV gRNA and that sequences in the 3’ UTR were involved in isolate-specific encapsidation of SbDV SsgRNA. Preferential trans-encapsidation of non-replicating mutant gRNAs and large subgenomic RNA (LsgRNA) by coat protein provided by replicating wild-type virus suggested that encapsidation of SbDV gRNA is replication independent, and that two interacting signals located in ORFs 1 and 5 differentially regulate encapsidation of SbDV gRNA and LsgRNA. The present study also showed that soybean aphids (Aphis glycines) can vector SbDV, that the readthrough protein (RTP), a minor structural protein involved in specificity of aphid transmission, is much more diverse than the major coat protein among Midwestern SbDV isolates, and that no mutations occurred in the RTP as a result of aphid transmission of SbDV from red clover into soybean plants.