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.

Rapid systemic accumulation of transcripts encoding a tobacco WRKY transcription factor upon wounding

“…Particle bombardment was performed with the PDS-1000, according to the manufacturer's instructions (Bio-Rad), using gold particles (1.0 lm diameter) coated with plasmids according to the manufacturer's protocol. For transactivation analysis, an appropriate pair of reporter (0.67 lg) and eector (0.67 lg) plasmids was introduced by bombardment, together with the reference pRTL2-GUS vector (provided by J. Carrington) (0.33 lg) carrying two copies of the 35S promoter and a translation leader enhancer sequence (Restrepo et al 1990). The plated cells or the onion leaf were placed at a distance of 6 cm or 9 cm from the stopping screen and bombarded twice per sample under a slight vacuum (28 in.…”

Developmental expression of a turgor-responsive gene that encodes an intrinsic membrane protein

“…N. tabacum (cv. Samsun) was transformed with vector pRTL2 as a control, pRTL2 vector contains the cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region fused to Gus [31]. Seed from transformed tobacco was germinated on Petri dishes containing MS medium plus 50 #g/ml kanamycin [ 16].…”

Biotransformation of an exogenously supplied isoflavonoid by transgenic tobacco cells expressing alfalfa isoflavone reductase

“…The PCR product was subcloned into the NcoI site of vector pSE380 and shown to encode active IFR enzyme in E. coli following induction with IPTG (Paiva et al 1991). The insert was then subcloned into the NcoI site of pRTL2 (Restrepo et al 1990), such that the IFR coding region was in the sense orientation downstream of a cauliflower mosaic virus (CaMV) 35S promoter with duplicated enhancer region and a tobacco etch virus (TEV) 5′ untranslated region upstream of a CaMV 35S 3′ polyadenylation site. Partial digestion with HindIII released a fragment containing the above expression cassette, which was subcloned into the HindIII site in the T-DNA region of the binary vector pGA482.…”