Symptomatology and Movement of a Cucumber Necrosis Virus Mutant Lacking the Coat Protein Protruding Domain

Abstract

A cucumber necrosis virus (CNV) mutant which lacks the coding sequence for the coat protein protruding domain, PD(-), was constructed by site-directed mutagenesis of an infectious CNV cDNA clone, pK2/M5 (wild-type, 4701 nt). Transcripts of PD(-) were infectious on Nicotiana clevelandii; however, local lesions produced were significantly smaller than those on the corresponding leaves of plants inoculated with wild-type transcript. In addition, systemic symptoms took 8 to 12 days longer to develop than in wild-type-inoculated plants. The distinctive PD(-) phenotype was lost when N. clevelandii was inoculated with sap from systemically infected leaves of PD(-) transcript-inoculated plants and was replaced by symptoms that were the same as those with wild-type infections. High-molecular-weight RNA from mutant- and wild-type-infected plants was extracted and analyzed by Northern blotting. Full-length PD(-) RNA could be detected only rarely in RNA preparations from transcript-inoculated leaves; a further deleted, stable RNA species of approximately 3800 nucleotides was found in preparations from systemically infected leaves of PD(-) transcript- and sap-inoculated plants. CNV coat protein could not be detected by ELISA or ISEM in PD(-)infected leaf material. The ca. 3.8-kb RNA, when cloned and sequenced, was found to have lost all but 74 of the 1140 nucleotides of the CNV coat protein open reading frame. Transcripts from this coat proteinless CNV cDNA clone produced wild-type symptoms on N. clevelandii. It would appear that CNV is able to replicate and move systemically, in both transcript-inoculated and sap-inoculated N. clevelandii, in the absence of a functional coat protein. Additionally, mechanical transmission of this virus occurs in the absence of the coat protein; however, such transmission is less efficient when compared with wild-type infections.