RNA 2 of Red Clover Necrotic Mosaic Virus Determines Lesion Morphology and Systemic Invasion in Cowpea

Abstract

SUMMARYRed clover necrotic mosaic virus (RCNMV) has two RNA species of mol. wt. about 1.5 x 106 (RNA 1) and 0.5 × 106 (RNA 2). An English strain (H) and a Czechoslovakian strain (TpM-34)of RCNMV could be distinguished serologically, by solid-phase RNA hybridization analysis (Northern blotting) and by the symptoms they induced in cowpea. Studies of pseudorecombinants, formed following inoculation of plants with heterologous combinations of the RNA species of each strain, showed that RNA 2 determines the morphology of lesions induced by the isolates in cowpea and their ability to invade the plants systemically.Red clover necrotic mosaic virus (RCNMV) belongs to the dianthovirus group (Matthews, 1982), members of which are characterized by isometric particles 27 to 35 nm in diameter, a genome composed of two single-stranded RNA molecules, RNA 1 (mol. wt. 1.35 x 106 to 1.55 X 106) and RNA 2 (mol. wt. 0.5 × 106 to 0.6 x 106), both of which are required for infectivity, and a single capsid polypeptide species, mol. wt. 38 000 to 41000, encoded by RNA 1 (Hollings & Stone, 1977;Dodds et al., 1977; Gould et al., 1981 ;Okuno et al., 1983;Morris-Krsinich et al., 1983). The properties of pseudorecombinants formed between RCNMV and two other dianthoviruses, sweet clover necrotic mosaic virus and clover primary leaf necrosis virus, indicated that symptom type in several plant species was determined by RNA 1 or by interaction between RNA 1 and RNA 2 (Okuno et al., 1983). We now describe two properties of RCNMV which are determined primarily by RNA 2.English isolate H and Czechoslovakian isolate TpM-34 of RCNMV (Musil, 1969;Hollings & Stone, 1977;Musil & Gallo, 1982) (kindly provided by Drs A. A. Brunt and O. M. Stone) were each subjected to three cycles of single lesion isolation in Chenopodium quinoa to obtain strains as homogeneous as possible. The viruses were propagated in Phaseolus vulgaris (French bean) cv.'The Prince' (Hollings & Stone, 1977) and extracted and purified by differential and sucrose density gradient centrifugations (Gould et al., 1981). Double immunodiffusion analysis of virus particles in 1 ~o agarose gels was as described by Buck et al. (1981). Rabbit antisera to the two virus strains were kindly provided by Dr A. A. Brunt. Capsid polypeptide species were analysed by SDS-PAGE (Laemmli, 1970). RNA was isolated from virus particles by phenol/SDS extraction (Okuno et al., 1983) and the two species were separated by two cycles of centrifugation in sucrose/formamide gradients. Virus RNA in buffered 85 ~ formamide (85 vol. of deionized formamide mixed with 15 vol. 10 mM-Tris-HC1, 1 mM-EDTA, pH 7-5) was heatdenatured (60 °C, 3 min), then cooled to 20 °C and layered onto a 5 ml linear gradient of 2 to 10~ (w/v) sucrose in buffered 85~o formamide. Gradients were centrifuged in a Beckman SW50.1 rotor at 40000 r.p.m, for 17 h at 20°C and then 0.1 ml fractions were collected from the bottom of the tubes. RNA in each fraction was recovered by precipitation with 2.5 vol. ethanol and analysed, after glyoxalation, by el...

A comparative study of the RNA-2 nucleotide sequences of two sweet clover necrotic mosaic virus strains

“…It has been demonstrated that physicochemical and serological features of progeny virus are determined by the donor RNA-1 (Pappu & Hiruki, 1989;Okuno et al, 1983). Similar work on red clover necrotic mosaic virus (RCNMV) Osman et al, 1986Osman et al, , 1991aPaje-Manalo & Lommel, 1989), another member of the dianthovirus group, has demonstrated that RNA-2 is responsible for cell-to-cell virus movement and that it plays a role in the development of disease symptoms in infected plants. To gain further understanding of the function of SCNMV RNA-2, we have determined the primary structures of RNA-2s of SCNMV-38 and -59.…”

Nucleotide Sequence of RNA 2 of a Czechoslovakian Isolate of Red Clover Necrotic Mosaic Virus

“…When RNAs of RCNMV-TpM-34 and RCNMV-Aus were separated (Osman et al, 1986;Osman & Buck, 1987) and inoculated in homologous or heterologous mixtures to cowpea leaves, lesions were formed in numbers similar to those induced by inocula of the parent isolates. Hence these two isolates can form pseudorecombinants in both directions.…”

“…Hence these two isolates can form pseudorecombinants in both directions. Previous studies have shown that isolates of RCNMV and other dianthoviruses can form pseudorecombinants either bilaterally (Okuno et al, 1983;Osman et al, 1986) or unilaterally (Rao & Hiruki, 1987). The ability to form viable pseudorecombinants probably depends, amongst other things, on the ability of the viral RNA replicase to recognize sequences at the 5' and 3' ends of the heterologous RNA.…”

“…Both RNA 1 and RNA 2 are required for systemic infection of plants (Gould et al, 1981 ;Okuno et al, 1983;Osman et al, 1986;Paje-Manalo & Lommel, 1989), but RNA 1 can replicate and give rise to virus particles in protoplasts in the absence of RNA 2 (Osman & Buck, 1987;PajeManalo & Lommel, 1989), implying a role for RNA 2 in cell-to-cell movement of the virus. RNA 2 encodes a protein of Mr 34K to 35K (Morris-Krsinich et al, 1983;Lommel et al, 1988), which may be analogous to the movement proteins of other plant viruses (reviewed by Hull, 1989).…”

“…RCNMV-TpM-34 was propagated, virus particles were extracted and purified, and viral RNA was isolated as described previously (Osman et al, 1986). Doublestranded cDNA was synthesized (Gubler & Hoffman, 1983) either using unfractionated viral RNA as a template with random oligonueleotide primers (Taylor et (Ahlquist et al, 1981) viral RNA, using TloG as a primer.…”