Reduced intracellular content of methotrexate in an isolated MTX-resistant cell line of Nicotiana plumbaginifolia.

In plant cells methotrexate (MTX) may exert its toxic effect through several mechanisms, including inhibition of its target protein dihydrofolate reductase. Resistance based on a mechanism operating before MTX binds to proteins should confer protection to plant cells. A methotrexate-resistant cell line of Nicotiana plumbaginifolia was isolated by a stepwise selection procedure. This cell line survived in the presence of 10 µM MTX which is 50-100 fold higher than the lethal dose for the wild type cells. Neither alteration in kinetic characteristics of dihydrofolate reductase, nor elevated binding capacity of (3)H-MTX to target protein(s), were observed. However, in comparison with wild type cells, markedly lower amounts of intracellular (3)H-MTX were found after the selected cell line was incubated with (3)H-MTX, indicating that either reduced uptake or enhanced efflux of MTX is the major reason for MTX-resistance in this cell line.

Enhanced tobacco mosaic virus production and suppressed synthesis of a virus inhibitor in protoplasts exposed to antibiotics.

Actinomycin D and chloramphenicol, when added up to 24 hr after inoculation, markedly increased tobacco mosaic virus (TMV) replication in protoplasts of Samsun NN, a cultivar in which the infection in the intact plant is localized. No increase was observed when TMV-infected protoplasts of Samsun, a systemic-responding cultivar, were incubated in the presence of these antimetabolites. Concomitant with the increase in virus replication in protoplasts of Samsun NN, production of the inhibitor of virus replication (IVR) (G. Loebenstein and A. Gera, Virology, 114, 132-139, 1981) from these protoplasts was suppressed almost completely. These results strengthen the suggestion that IVR is associated with the localizing mechanism by suppressing virus replication.

Effect of 2,4-dichlorophenoxyacetic acid on multiplication of tobacco mosaic virus in protoplasts from local-lesion and systemic-responding tobaccos.

Tobacco mosaic virus (TMV) multiplication was markedly enhanced in protoplasts from Samsun NN and Xanthi-nc, two local-lesion-responding cultivars, by 2,4-dichlorophenoxyacetic acid (2,4-D) added to the incubation medium after inoculation of the protoplasts. In Samsun, a cultivar which reacts systemically to TMV infection, 2,4-D reduced virus multiplication. These findings may explain the discrepancy observed between the low virus multiplication in intact tobaccos with the N gene and the high titers reached in protoplasts from these plants (in the presence of 2,4-D), which were similar to those found in protoplasts from a systemic cultivar.