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Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNAdependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positivestrand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130-and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the nonmembrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.The virus particles of positive-strand RNA [(ϩ)RNA] viruses contain single-stranded, messenger-sense genomic RNA, and these viruses replicate via complementary, negative-strand RNA [(Ϫ)RNA] templates. Most plant viruses and many animal viruses are (ϩ)RNA viruses. After infection, the genomes of these viruses are translated to produce viral proteins that function in viral RNA replication. The replication of eukaryotic (ϩ)RNA viruses takes place in replication complexes that are formed on the intracellular membranes of infected cells and that contain the viral RNA replication proteins and endogenous (Ϫ)RNA templates. The replication complexes synthesize viral (ϩ)RNAs from ribonucleoside triphosphates (rNTPs) and release them into the cytoplasm (4, 24).The genus Tobamovirus, which belongs to the alphavirus-like superfamily of (ϩ)RNA viruses, includes Tobacco mosaic virus and Tomato mosaic virus (ToMV) and many related viruses. The genome of a tobamovirus is a 6.4-kilobase RNA that encodes a nonstructural protein with an approximate molecular mass of 130 kDa (130,000-molecular-weight [130K] protein), its read-through product of 180 kDa (180K prote...

Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.

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