A full-length cDNA clone of rice yellow mottle sobemovirus (RYMV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter sequence. Capped-RNA transcripts produced in vitro were infectious when mechanically inoculated onto rice plants (Oryza sativa L). Individual full-length clones varied in their degree of infectivity but all were less infectious than native viral RNA. A representative clone, designated RYMV-FL5, caused a disease phenotype identical to that produced by viral RNA except that symptoms were somewhat slower to appear than those induced by viral RNA. The infectivity of RYMV-FL5 was verified by ELISA, Western blot analysis, Northern blot hybridization, RT-PCR, and Southern blot hybridization. Frameshift and deletion mutations introduced into the coat protein cistron demonstrated that the coat protein was dispensable for RNA replication in rice protoplasts. However, the coat protein was required for full infectivity in rice plants, presumably by playing a role in phloem-mediated long-distance movement and possibly in cell-to-cell movement.

The genome of rice yeUow mottle virus (RYMV) is a single-stranded positive-sense RNA that is not polyadenylated, and has an M r of 1.4 x 106. We present here the 4550 nucleotide (nt) sequence of RYMV RNA, and its predicted genomic organization. The RYMV genomic

RNA transcribed from cloned satellite tobacco mosaic virus (STMV) cDNA replicated in Nicotiana benthamiana protoplasts when co-inoculated with tobacco mild green mosaic virus (TMGMV) genomic RNA, but degraded when inoculated alone. STMV genomic RNA extracted from wild-type virions replicated in protoplasts when co-inoculated with TMGMV, tobacco mosaic virus (TMV) or tomato mosaic virus (ToMV). Transcripts from clones of two STMV coat protein (CP) mutants accumulated to the same level as wild-type transcripts in protoplasts when co-inoculated with TMGMV, whereas a third mutant accumulated to detectable levels in some, but not all, experiments. These results confirm that STMV RNA requires helper virus for replication, and that the helper specificity exhibited by cloned STMV reflects a specific requirement for the TMGMV replicase. It also demonstrates that the low accumulation of STMV CP mutants observed previously in whole plants cannot be attributed to inefficient RNA replication.Satellite tobacco mosaic virus (STMV) shares, with other satellite viruses and satellite RNAs, a requirement for a helper virus, in this case one of several tobamoviruses, to establish infection. Based on studies in whole tobacco plants, the helper virus is believed to provide factors required for the replication of STMV (Valverde et al., 1986(Valverde et al., , 1987(Valverde et al., , 1991. Subclones of STMV cDNA, from which biologically active RNA can be transcribed, have been created (Mirkov et al., 1990 ;Kurath et al., 1993). Interestingly, these transcripts only accumulated in tobacco plants when co-inoculated with tobacco mild green mosaic virus (TMGMV ; a California isolate previously referred to as TMV-U5 was used). In contrast, STMV RNA purified from virions accumulated systemically in plants when coinoculated with any one of several tobamoviruses (Valverde et al., 1991). Deletion and frameshift mutations have been engineered into one of the original STMV cDNA clones. Approximately 80 % of the internal coat protein (CP) coding sequence can be removed without entirely abolishing the ability of these mutants to accumulate in whole plants ; however, these CP-deletion mutants did not accumulate in every plant that was co-inoculated with mutant STMV RNA and TMGMV RNA. Viability of these STMV CP mutants was affected by the conditions under which the plants were maintained, and when they were biologically active they accumulated to approximately 100-fold lower levels than the original clone (Routh et al., 1995).To initiate an analysis of the replication of wild-type and mutant STMV, Nicotiana benthamiana protoplasts were inoculated with combinations of various tobamoviruses and STMV RNAs. The objectives of this study were to verify that replication of STMV was dependent upon factors provided by the helper virus, to determine if the specificity for TMGMV helper exhibited by cloned STMV RNA occurred at the level of replication, and to analyse the replication efficiency of STMV CP mutants which accumulated poorly in whole tobacco plants. ...

Isolates of tobacco mild green mosaic tobamovirus (TMGMV) were obtained from 58 plants of Nicotiana glauca in southern California and placed in one of two groups (Small type and Large type) based on the size of the subgenomic RNA for the coat protein (CP). The CP sequence differed by no more than one amino acid for the two types, and the Small type was identical to that published for TMGMV. Thirty-six of the isolates had a double-stranded (ds)RNA profile that matched that of type TMGMV, and the nucleotide sequence of the 3h untranslated region (3hUTR) of six of these isolates was similar to the published sequence of TMGMV. Twenty-two isolates had a larger dsRNA for the CP subgenomic RNA. Six of these were sequenced and all had a repeat sequence of between 147 and 165 bases in the part of the 3hUTR that is involved in the formation of pseudoknots. These novel but common isolates are predicted to have six rather than three pseudoknots. Small types (three pseudoknots l type TMGMV) yielded twice as much virus after purification as Large types (six pseudoknots). The two groups of isolates could be distinguished in N. rustica (Large type, but not Small type gave a systemic infection), and N. clevelandii (Small type but not Large type induced systemic lethal necrosis). Almost all isolates of TMGMV used in this study were initially associated with satellite tobacco mosaic virus (STMV), and both types supported STMV experimentally.

The genome of satellite tobacco mosaic virus (STMV) adapted to tobacco mosaic virus (TMV), tomato mosaic virus or green tomato atypical mosaic virus consistently had two single base deletions at positions 1 and 61, corresponding to bases A and G, respectively, as compared to the type-strain genome which is naturally adapted to tobacco mild green mosaic virus (TMGMV). Transcript RNAs (STMV(TMV)) from clone pSTMV(TMV) which captured the deletions at positions 1 and 61 were infectious when co-inoculated to tobacco plants with either TMV or TMGMV at infection frequencies of > 90%. Two new STMV variants were created to investigate whether both deletions were essential for adaptation to TMV. These were STMV(TMGMV) deltaA1, which had the A at position 1 (A1) deleted, and STMV(TMGMV) deltaG61, which lacked G61. STMV(TMGMV) deltaA1 was infectious (75% frequency) in the presence of either TMV or TMGMV. Virion RNA of STMV(TMGMV) deltaA1 lost G61 after one infection cycle with TMV. This deletion did not occur in co-infections with TMGMV. STMV(TMGMV) deltaG61, like the clone STMV(TMGMV), was infectious (100% frequency) with TMGMV but TMV did not support this clone. When Nicotiana benthamiana protoplasts were transfected with STMV(TMGMV), STMV(TMGMV) deltaA1 or STMV(TMV), STMV replicated when TMGMV was the helper virus. STMV(TMV) and STMV(TMGMV) deltaA1 replicated in the presence of helper TMV, but STMV(TMGMV) did not, the same result as in whole plants. The deletion of A1 is thus essential for initial STMV adaptation to TMV and the eventual deletion of G61 is a predicted additional change.