Site-directed mutagenesis of a plant viral satellite RNA changes its phenotype from ameliorative to necrogenic.

Abstract

Comparison of the nucleotide sequences of cucumber mosaic virus (CMV) satellite RNAs, which induce necrosis on tomatoes, reveals a highly conserved region within their 3' halves. The sequence of WL1 satellite (WLl-sat) RNA, which attenuates CMV symptoms on tomatoes, differs from all necrogenic satellite RNAs at three nucleotide positions within this conserved region. These nucleotides were progressively mutated to determine what sequence is required for the induction ofnecrosis in tomatoes. Infectious transcripts from a cDNA clone of WLl-sat RNA, and its mutated derivatives, were assayed on tomato. Three of the four mutant satellite RNAs, in which two of the three nucleotides corresponded to those present in necrogenic satellite RNAs, and the rest of the molecule corresponded to WLl-sat RNA, attenuated CMV symptoms on tomatoes, and were phenotypically identical to the parental WLl-sat RNA.' One other mutant satellite RNA, in which all three mutated nucleotides corresponded to the sequence of necrogenic satellite RNAs and the rest of the molecule corresponded to WL1-sat RNA, induced a lethal necrosis on tomatoes. Necrosis was also observed when the same nmutant satellite RNA was associated with a second CMV strain as helper virus. These results indicate that a single nucleotide change at any of the three nucleotides examined distinguishes necrogenic from nonnecrogenic satellite RNAs. This necrosis was similar to that induced by a naturally necrogenic satellite RNA. The various mutations did not modify the effect of WL1-sat RNA on the symptoms induced by CMV on tobacco or squash. (3,7,8), induce chlorosis (7-9), induce necrosis (10), or induce combinations of these effects (11). In this report, we examine the molecular basis for the ability of CMV satellite RNAs to induce lethal necrosis on tomatoes.Earlier, Kurath and Palukaitis (11) conducted recombination experiments with an ameliorative satellite RNA, WL1-sat RNA (7,8), and a necrogenic satellite RNA, D-sat RNA (10), to delimit the sequences involved in the induction of necrosis in tomatoes. Infectious satellite RNAs were transcribed in vitro from full-length cDNA clones of WL1-sat RNA and D-sat RNA that were recombined at their common Nhe I site, located 4185 nucleotides from their respective 5' termini. These experiments indicated that the necrogenic domain is located within the 3'-terminal 150 nucleotides of D-sat RNA and presumably also in other necrogenic satellite RNAs (11). Results of others obtained by recombination of cDNA clones of the necrogenic Y-sat RNA and the ameliorative Ra-sat RNA reinforced this conclusion (12).Sequence comparisons also have provided information concerning the potential location of the necrogenic domain (13). In the 3'-terminal 150 nucleotides, within which the necrosis domain is located, necrogenic satellite RNA sequences are essentially conserved, apart from two regions of heterogeneity between nucleotides 231 and 234t and 337 and 340. Ameliorative satellite RNAs do not show such conservation within the 3' half (13...

Widely separated sequence elements within cucumber mosaic virus satellites contribute to their ability to induce lethal tomato necrosis

“…The conclusion is reinforced by the site-directed mutation experiment that regenerated some of the D-CARNA 5 sequence context in the mutant transcript DY-mt, and in which much (but not all) of the necrogenicity of D-CARNA 5 was restored ( Table 2). The latter result can also simply be seen as a widening of the boundaries of the 3'nDetm, also discussed by others (Devic et al, 1990;Sleat & Palukaitis, 1990).…”

“…With these variants it became apparent that a larger degree of sequence conservation existed among those which caused tomato necrosis than among those which did not . This finding and further work from different laboratories (Jacquemond & Lauquin, 1988;Masuta & Takanami, 1989;Devic et al, 1990;Sleat & Palukaitis, 1990) led to the localization of a tomato necrosis-determining conserved sequence element in the 3' half of CARNA 5 between positions 284 and 295 [sequence numbering is as in .…”

Cucumber Mosaic Virus D Satellite RNA-Induced Programmed Cell Death in Tomato

“…The molecular and cellular mechanisms of the disease, which can cause a catastrophic reduction in tomato production, remain unknown, although the sequences in D-satRNA responsible for the lethal necrosis have been determined (Sleat and Palukaitis, 1990;Sleat et al, 1994). By using in vitro and in vivo analyses of its secondary structure, researchers have correlated a helix and a tetraloop region near the 3 Ј end of the satRNA with the necrotic syndrome (Bernal and García-Arenal, 1997;Rodríguez-Alvarado and Roossinck, 1997).…”

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction

“…The cloning strategy and technical protocols used for site-specific mutagenesis are also outlined elsewhere (Kunkel et a/., 1987;Sleat and Palukaitis, 1990b). Mutant satellite cDNAs were cloned in the in-vitro transcription vector, pBS(+) (Strategene).…”