A full-length Beet black scorch virus (BBSV) cDNA clone (pUBF52) was constructed by RT-PCR. The clone contains an upstream T7 RNA polymerase promoter designed for in vitro transcription of infectious RNAs from the linearized plasmid that faithfully represent the viral cDNA. Leaves of Chenopodium amaranticolor inoculated with in vitro transcripts developed the same symptoms and disease phenotype as the wild type virus. The presence of BBSV RNA and coat protein in the leaves was confirmed respectively by Northern blotting and Western blotting. Comparisons of specific immunoreactions between the expression product of the BBSV p24 gene in E. coli and antiserum against purified BBSV virions demonstrated that the p24 gene encodes the coat protein. Based on the sequence of the pUBF52 cDNA, a frame-shift mutant and two deletion mutants were generated. One of the deletions encompasses the entire CP ORF and the other truncates 174 amino acids from the central region of the protein. Transcripts derived from the frame-shift CP mutant, which terminates the CP after the first 23 amino acids, elicited the same symptom phenotype and levels of RNA accumulation as the wild type virus, but the leaves infected with the CP deletion mutants exhibited greatly reduced RNA accumulation. In addition, leaves inoculated with in vitro transcripts of the mutant in which the entire CP gene was deleted had lower local lesions than wild type virus transcripts. Two expression vectors, pBGFP and pBGUS, were constructed by fusing the GFP and GUS genes to the 23 N-terminal amino acids of the CP gene, respectively. Leaves infected with in vitro transcripts of pBGFP and pBGUS exhibited expression of GFP and GUS proteins as assessed by laser confocal microscopy and histochemical staining, respectively. The high levels of expression of the GFP and GUS proteins provide tools that can be used for studies of replication and movement of the virus, and indicate that BBSV has considerable biotechnology potential as a plant virus expression vehicle.