Nuclear Transport of Plant Potyviral Proteins

Abstract

We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the 8-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-NIb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.

Highlights and prospects of potyvirus molecular biology

“…It has been shown that the TEV 5' non-coding region can function as an enhancer of translation (Carrington & Freed, 1990). This property could be clue to the likely absence of strong secondary structure, as suggested for the enhancing leaders of other plant viruses (Jobling & Gehrke, 1987;Altmann et al, 1990).…”

Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus

“…Primers containing XhoI and BamHI sites were designed at the 5Ј and 3Ј ends of RACE products and used to amplify the full-length RTM1 cDNA. The full-length cDNA was transferred to the pRTL2 vector (19), which contains a cauliflower mosaic virus 35S promoter and 35S terminator. The expression cassette was excised and subcloned into pSLJ755I5.…”

Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression

“…The authentic 68 kDa GUS protein is localized to the cytoplasm but is targeted to the nucleus when tagged with a nuclear protein or an NLS (Restrepo et al, 1990). Using this system, we have previously demonstrated in transfected protoplasts from cultured tobacco cells the nuclear localization of PS-IAA4-like (Oeller et a/., 1993;Theologis eta/., 1985), early auxin-inducible polypeptides (Abel eta/., 1994).…”

“…We transfected mesophyll protoplasts with the plant expression vector pRTL2-GUS (Restrepo et a/., 1990) and followed the expression of the GUS reporter gene driven by the CaMV 35s promoter. Neither freshly prepared nor mock-transfected Arabidopsis mesophyll protoplasts contained detectable levels of endogenous GUS activity but supported high levels of GUS expression after transfection with pRTL2-GUS.…”