Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

Abstract

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.

Nuclear Targeting in Plants

“…By histochemical analysis of GUS, it was shown that a sevenamino acid SV40 sequence can function as an NLS in transgenic tobacco plants (24), but a mutant SV40 NLS (PKTKRKV), in which an essential K was mutated to T, cannot. Nuclear localization was also observed when the 100-kD T7 RNA polymerase protein was fused to the SV40 NLS and electroporated into tobacco protoplasts (8). These results clearly indicate that some mechanisms of nuclear transport are common between animal and plant cells.…”

“…Furthermore, experimental evidence indicates the existence of receptors and other cellular factors that mediate nuclear transport. Nuclear transport, although extensively studied in animal and lower eukaryotic systems (3, 20), has only recently been addressed in higher plants (1,2,5,7,8,15,(24)(25)(26).This attention to protein and nucleic acid transport into the nucleus in plants was stimulated not only by a need to understand basic cell biology, but also by the desire to modulate or regulate the expression of genes via genetic engineering.The nucleus (Fig. 1) is surrounded by the nuclear envelope, which includes the inner and outer nuclear membranes and, between them, the perinuclear space.…”

“…Furthermore, experimental evidence indicates the existence of receptors and other cellular factors that mediate nuclear transport. Nuclear transport, although extensively studied in animal and lower eukaryotic systems (3, 20), has only recently been addressed in higher plants (1,2,5,7,8,15,(24)(25)(26).…”

Interaction of the DNA modifying proteins VirD1 and VirD2 of Agrobacterium tumefaciens : Analysis by subcellular localization in mammalian cells

“…The monopartite NLS of the mammalian virus SV40, KKKRK has been shown in several systems to import proteins into plant nuclei: ␤-glucuronidase was transported to tobacco (27) and onion nuclei (28), and T7 RNA polymerase (29) was transported to the nuclei of tobacco protoplasts. Taken together, these data indicate that at least this monopartite NLS, in the context of a variety of proteins, is accepted by the plant import machinery.…”

Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2.

“…The one basic region examined from the Arabidopsis 37-kD TFllD could not redirect GUS. It has also been shown that the SV40 NLS can function in dicot plant cells (Lassner et al, 1991;van der Krol and Chua, 1991). However, no previous studies have identified an NLS in a monocot protein.…”