Genetic analysis of cowpea mosaic virus mutants by supplementation and reassortment tests

During an infection with cowpea mosaic virus (CPMV) both virion assembly and formation of tubules associated with plasmodesmata are required for cell-to-cell movement. These functions are encoded by the M-RNA of CPMV. To study the mechanism of CPMV movement, mutant N123 was used in complementation studies with sunn-hemp mosaic virus (SHMV), a legume-infecting tobamovirus. Previous studies have shown that N123 fails to spread in cowpea plants because of mutation(s) in its M-RNA. However, the mutant was efficiently replicated in cowpea protoplasts, in which virions were formed and tubular transport structures were induced. After high-dose inoculation of cowpeas with N123, only a few infected protoplasts could be isolated, indicating that cell-to-cell transport of N123 was greatly impaired, if not completely abolished. Upon coinoculation with SHMV, mutant N123 infected cowpea plants systemically and accumulated to levels which were comparable to those of wild-type CPMV. In contrast, separate B-RNA of CPMV and a CPMV deletion mutant lacking the tubule-inducing function, were complemented by SHMV to only low levels. It is concluded that SHMV-facilitated spread of CPMV in the non-virion tobamovirus mode is inefficient and that spread of mutant N123 is probably in the CPMV mode, SHMV providing an as yet unidentified helper function.

Section: Infectivity Of N123 In Cowpea Plantssupporting

confidence: 53%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Defective cell-to-cell movement of cowpea mosaic virus mutant N123 is efficiently complemented by sunn-hemp mosaic virus

Taliansky

Jager²,

Wellink³

et al. 1993

“…B and M components were separated in a linear 15 to 30% sucrose gradient by zonal centrifugation (Beckman Ti 15 rotor, 16 h, 23000 rev/min at 10 °C). This procedure was repeated twice to give an M component free of B, and a B component contaminated with less than 0.2% of M, as determined by the local lesion infectivity test (De Jager, 1976). Protoplast isolation and inoculation.…”

Section: Methodsmentioning

confidence: 99%

Limits to the Independence of Bottom Component RNA of Cowpea Mosaic Virus

Rezelman

Franssen

Goldbach

et al. 1982

“…The RF was assayed for infectivity in a local lesion host and a systemic host. In neither host was undenatured RF infectious, although infectivity was restored upon denaturation.Cowpea mosaic virus (CPMV) is a virus of legumes whose genome consists of two separately encapsidated, positive-sense RNA molecules, both of which are required for infection (van Kammen, 1967;de Jager, 1976). Both RNA molecules have recently been sequenced, the larger, B RNA having 5889 nucleotides, the smaller, M RNA having 3481 nucleotides (Lomonossoff & Shanks, 1983 ;van Wezenbeek et al, 1983).…”

mentioning

confidence: 99%

Double-stranded, Replicative Form RNA Molecules of Cowpea Mosaic Virus Are Not Infectious

Shanks

Lomonossoff

Evans

1985

SUMMARYVirus-specific double-stranded replicative form (RF) RNA was isolated from cowpeas infected with cowpea mosaic virus. The RF was assayed for infectivity in a local lesion host and a systemic host. In neither host was undenatured RF infectious, although infectivity was restored upon denaturation.Cowpea mosaic virus (CPMV) is a virus of legumes whose genome consists of two separately encapsidated, positive-sense RNA molecules, both of which are required for infection (van Kammen, 1967;de Jager, 1976). Both RNA molecules have recently been sequenced, the larger, B RNA having 5889 nucleotides, the smaller, M RNA having 3481 nucleotides (Lomonossoff & Shanks, 1983 ;van Wezenbeek et al., 1983). Each RNA has a small protein (VPg) covalently attached to its 5" terminus (Stanley et al., 1978: Daubert et al., 1978 and is polyadenylated at the 3' end (El Manna & Bruening, 1973). Translation in vitro and protoplast studies have shown that each RNA is initially translated into a large polypeptide which is subsequently cleaved by virusspecific proteases to functional virus proteins (Davies et al., 1977;Pelham, 1979;: Rottier et al., 1980Goldbach et al., 1981).Replication of CPMV RNA in infected plants takes place in a membrane-associated replication complex containing a 110K polypeptide encoded by the B RNA and two hostspecified polypeptides (Dorssers et al., 1984: Franssen et al., 1984. As intermediates in the replication process, partially double-stranded RNA molecules, termed replicative intermediates (RI), and completely double-stranded RNA molecules, termed replicative forms (RF), are produced (van Griensven & van Kammen, 1969: van Griensven et al., 1973. In this short communication we describe the isolation of RF molecules from CPMV-infected cowpeas and assays of their infectivity.The method of purification of the two RF forms of CPMV, RF-M and RF-B, involving DNase I treatment and CF-11 and Sepharose column chromatography, is essentially a standard method for preparing double-stranded RNA and has been widely used in the preparation of plant virus RFs (Zelcer et al., 1981: Jackson et al., 1971 including those from CPMV (Dorssers et al., 1984). Primary leaves of Vigna unguiculata var. 'Blackeye Early Ramshorn' were harvested 5 or 6 days after inoculation with CPMV strain SB. The mid-ribs were excised and the leaf tissue was frozen in liquid nitrogen and the RNA extracted and treated with DNase I as described by Zelcer et al. (1981). After the elimination of the DNase I by phenol extraction, the RNA was precipitated with ethanol and redissolved in water. An equal volume of 4 M-LiC1 was added and the solution was kept at -20 °C overnight. After centrifugation, the supernatant containing double-stranded RNA and tRNA was precipitated with ethanol. Partially double-stranded RNA (RI) could be recovered from the LiCl-insoluble precipitate.The LiCl-soluble RNA was dissolved in 100 mM-NaCI, 50 mM-Tris-HC1 pH 7.4, 1 mM-EDTA (STE) and ethanol was added to a final concentration of 17.5 % (v/v). The solution was loaded on to a...