The nucleotide sequence of a satellite RNA associated with a strawberry isolate (H) of strawberry latent ringspot nepovirus (SLRSV) was determined from cDNA copies and the 5' end sequence was deduced from directly sequenced virion RNA. At the 3' end a poly(A) sequence was identified. A long open reading frame encoding a polypeptide of 331 amino acids (Mr 36488) was determined. Sequence comparisons showed that SLRSV satellite RNA has no extensive homology with other sequences in the GenEmbl and Swiss-Prot databases.
The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The Nterminal amino acid sequences ofvirion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97 % identical to that of SLRSV-H. Except in the 3' and 5' terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.
The coding sequences in RNA2 for the coat proteins (CP) of strawberry latent ringspot virus (SLRSV) were modified and amplified using polymerase chain amplification reactions (PCR) to facilitate their expression in Agrobacterium tumefaciens-transformed Nicotiana tabacum Xanthi-nc. The coding sequences for the smaller capsid protein (S, 29kDa) and that for the theoretical precursor of L and S (P, 73kDa) had ATG 'initiation' codon sequences added at the 5'-proximal Ser/Gly (S/G) cleavage site in the unmodified sequence. The sequence coding for the larger of the two proteins of mature SLRSV capsids (L, 44kDa) had an ATG codon added at its 5' S/G site and a TAG 'stop' codon sequence added at the 3~-proximal S/G site. The P, L and S proteins were expressed in planta to a maximum concentration of 0.01% of total extractable proteins but did not assemble into virus-like particles. When challenged by mechanical inoculation with virus particles or viral RNA, and compared with control plants, tobacco plants (primary transgenic clones or S 1 and $2, kanamycin-resistant seedlings) expressing the virus capsid subunits separately, or their precursor, decreased the accumulation of SLRSV particles in inoculated leaves and fewer plants became invaded systemically. In experiments in which the roots of seedlings were exposed to SLRSV-carrying vector nematodes (Xiphinema diversicaudatum), SLRSV was detected in the roots of non-transformed control tobacco plants (6/20) and in transgenic tobacco expressing the L protein (7/40), but not in any of 25 tobacco plants expressing the S protein or in 35 expressing the P protein. This is the second example of CP-mediated resistance to virus inoculation by nematode vectors.
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