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Molecular Plant Pathology

Independent Replication of Red Clover Necrotic Mosaic Virus RNA-1 in Electroporated Host and Nonhost Nicotiana Species Protoplasts. L. L. Paje-Manalo, Former graduate research assistant, Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan 66506, Present address: Department of Biochemistry, University of New Hampshire, Durham 03824; S. A. Lommel, assistant professor, Department of Plant Pathology, Throckmorton Hall, Kansas State University, Manhattan 66506, Present address: Department of Plant Pathology, Box 7616, North Carolina State University, Raleigh 27695. Phytopathology 79:457-461. Accepted for publication 10 November 1988. Copyright 1989 The American Phytopathological Society. DOI: 10.1094/Phyto-79-457.

The two nonhomologous genomic single-stranded RNA components (RNA-1 and -2) of red clover necrotic mosaic virus (RCNMV) failed to elicit systemic symptoms when mechanically inoculated individually to Nicotiana clevelandii, a systemic host. However, plants previously inoculated with RNA-1 and then complemented with RNA-2 48 and 72 hr later became diseased within 45 days. In contrast, when RNA-2 was used as the primary inoculum, infections were obtained only when follow-up inoculations with RNA-1 were performed within 24 hr. RCNMV virions and genomic RNA were inoculated by electroporation into protoplasts of N. clevelandii and the N. tabacum-derived cell line BY-2 (a nonsystemic host for RCNMV). Total RNA extracts prepared from RNA-1-inoculated protoplasts after 48 and 72 hr incubation hybridized exclusively to RNA-1 specific probes, whereas RNA-2 electroporated protoplast extracts hybridized to neither RNA-1 nor RNA-2 probes. Capsid protein was synthesized in RNA-1-electroporated protoplasts but was not detected in protoplasts inoculated with RNA-2 alone. The independent replication of RCNMV RNA-1 in isolated protoplasts and results of the in planta experiments support the hypothesis that the 35-kDa polypeptide encoded by RNA-2 is involved in cell-to-cell movement. Additionally, the fact that RCNMV can replicate in the initially inoculated cells of the N. tabacum-derived cell line, BY-2, but does not result in systemic infection on plants of N. tabacum suggests that the cell-to-cell movement gene may also act as a host range determinant gene.

Additional keywords: cell-to-cell movement, electroporation, protoplasts, RNA-1 replication, suspension cell culture.