RNA-directed RNA polymerases from healthy and from virus-infected cucumber.

Abstract

Much work has been done on the isolation, purification, and characterization of the RNA-directed RNA polymerase (EC 2.7.7.48) of cucumber mosaic virus (CMV)-infected cucumbers. Uninfected plants were reported to have no such enzyme, but we recently detected low levels of the activity in cucumber. Since tobacco and cowpea contain such an enzyme that is variably increased in amount by various virus (as well as viroid) infections, we assumed that this would also be the case upon CMV infection of cucumber. However, further purification and characterization of the RNA-directed RNA polymerases from healthy and from infected cucumber suggests that these are different enzymes. The presumed CMV replicase was obtained pure and consists of a major polypeptide of Mr 100,000 and minor components of Mr 110,000 and about 10,000. The Km is 5 pM ([3H]GTP) when tobacco mosaic virus RNA is used as template.The role of plant RNA-directed RNA polymerases (EC 2.7.7.48) in the replication ofRNA viruses remains uncertain. The RNA of cowpea mosaic virus, a virus resembling picornaviruses (1), appears to become replicated without participation of the cowpea RNA-directed RNA polymerase, although the amount of the enzyme is greatly increased by that virus infection (2-4). Several studies of viral replication complexes-particulate aggregates containing several proteins, membranous structures, and RNA-have given conflicting results in terms of containing the host enzyme and/or one or several viral gene products (5)(6)(7). In barley infected with brome mosaic virus (BMV), evidence for a virus-specific component in viral RNA replication was reported (5, 8). However, in tobacco no evidence was found for any role of a protein encoded by the infecting tobacco mosaic virus (TMV) in viral RNA replication (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20).The tobacco enzyme, as well as those of other plants, have molecular weights of 130,000-140,000. In contrast, the presumed viral RNA replicases have approximate molecular weights of 180,000 plus possibly 130,000 (TMV and turnip yellow mosaic virus) or 110,000 and possibly 70,000 [BMV and cucumber mosaic virus (CMV)]. Yet the molecular weight of the largest gene product of tobacco necrosis virus is only about 65,000. The need for both host and viral components for viral RNA replication is quite likely. This has been observed to be the case with the RNA bacteriophages and picornaviruses. Our present concern has been with cucumber, a plant reported to lack RNA-directed RNA polymerase, and the replication of CMV in this plant. We have found small amounts ofRNA-directed RNA polymerase in cucumber, partially characterized it, and compared it with the enzyme, produced in greater amount and obtained in pure form, from CMV-infected cucumber. It appears that the presumed CMV replicase has a molecular weight of about 100,000 and 110,000, whereas that of the cucumber enzyme is about 140,000. The earlier report that the Mr 100,000 enzyme differs in peptide pattern from the viral gene product corresponding ...

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

“…In several studies, elevated RdRP activity was found in virus-infected plants and has been speculated to be one of the host factors involved in virus replication (5,(8)(9)(10). In the present study, we have shown that RdRP activity is induced not only by virus infection but also by defense-inducing compounds (Fig.…”

“…In these assays, we detected synthesis of not only the high molecular weight TMV RNAs by TMV replicase but also smaller RNAs by an unknown RNA polymerase. Synthesis of smaller RNAs has been previously observed in similar assays and attributed to the activity of a plant RdRP (5,(8)(9)(10). Interestingly, in our assays, synthesis of these smaller RNAs was drastically increased in SA-treated tobacco plants (data not shown).…”

“…R NA-dependent RNA polymerase (RdRP) activities have been detected in a number of plants (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Recently, a tomato RdRP has been purified and its corresponding gene cloned (11)(12)(13).…”

Analysis of rice RNA-dependent RNA polymerase 1 (OsRDR1) in virus-mediated RNA silencing after particle bombardment

“…These results suggest that OsRDR1 is required at the RNA level after transcription for RNA silencing. Previous studies show that RDR1 is induced by SA treatment, virus and viroid infection (Dorssers et al 1984;Khan et al 1986;Wassenegger and Krczal 2006). The lossof-function in the RDR1 mutant was expressed as enhanced disease susceptibility to various viruses (Xie et al 2001;Yu et al 2003).…”

“…Recent findings indicate that RDR1, RDR2 and RDR6 act in different biological processes of RNA silencing (Dalmay et al 2000;Mourrain et al 2000;Xie et al 2004). RDR1 expression is induced by salicylic acid (SA), a defensesignaling hormone that accumulates during infection by many viruses and viroids (Dorssers et al 1984;Khan et al 1986;Xie et al 2001). More recent studies with Cucumber mosaic virus (CMV) demonstrate that the production of small interfering RNAs (siRNAs) is strongly dependent on RDR1 without interference by the virus-encoded 2b silencing suppressor protein called viral suppressors of RNA silencing, and RDR1-dependent secondary virusderived siRNAs (viRNAs) play a key role in non-cellautonomous antiviral silencing (Diaz-Pendon et al 2007).…”