ORF C4 OF TOMATO LEAF CURL GEMINIVIRUS IS A DETERMINANT OF SYMPTOM SEVERITY

A tomato leaf curl virus (TLCV) mutant has been constructed in vitro t hat contains T-to-C replacements at nucleotides 2457 and 2463 within t he C4 open reading frame (ORF). The mutations destroy the two possible initiator AUG codons for the C4 ORF without disrupting the coding cap acity of the C1 ORF which entirely overlaps the C4 ORF. Agroinoculatio n of the C4 mutant TLCV into three alternative experimental hosts for the virus (Datura stramonium, Lycopersicon esculentum, and Nicotiana t abacum) gives rise to infections which show dramatically reduced sympt oms when compared to a wild-type infection, while retaining wild-type levels of all viral DNA species. In most cases the mutations were stab ly inherited by progeny virus. However, a single tomato plant inoculat ed with the mutant developed phenotypically wild-type symptoms and was subsequently shown to contain progeny virus in which the mutation at position 2457 had reverted to wild-type sequence, indicating that this AUG may be the site of initiation of translation of the C4 product in the wild-type virus. The results suggest that the C4 ORF encodes a po lypeptide which is not required by TLCV to replicate or to spread thro ugh the host plant, but is involved in symptom development. (C) 1994 A cademic Press, Inc.