Bipartite Signal Sequence Mediates Nuclear Translocation of the Plant Potyviral NIa Protein

Carrington, James C.; Freed, Deon D.; Leinicke, Andrew J.

doi:10.2307/3869157

Cited by 75 publications

(117 citation statements)

References 16 publications

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“…In particular, functional plant cell NLSs have been identified in maize in both the anthocyanin regulatory proteins Opaque2 and R (Varagona et al, 1992;Shieh et al, 1993) and the Ac transposase (Boehm et al, 1995), in auxininducible proteins in pea (Abel and Theologis, 1995), and in the GT box-binding transcription factor GT-2 from Arabidopsis (Dehesh et al, 1995). They have also been described in karyophilic proteins encoded by plant pathogens, namely, the NIa protein encoded by tobacco etch virus (Carrington et al, 1991) and the VirD2 and VirE2 proteins encoded by the Ti plasmid of Agrobucterium tumefuciens Howard et al, 1992). Analysis of C1 and R further demonstrate that a11 three consensus NLSs are used in plants (Hicks et al, 1995).…”

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confidence: 97%

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

1996

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For the nuclear replicating bipartite geminiviruses such as squash leaf curl to systemically infect the host requires the active participation of two virus-encoded movement proteins, BR1 and BL1. These act in a cooperative manner to transport the viral singlestranded DNA genome from its site of replication in the nucleus to the cell periphery (A.A. Sanderfoot, S.C. Lazarowitz [1995] Plant Cell 7: 1185-1194). We have proposed that BR1 functions as a nuclear shuttle protein, transporting the viral single-stranded DNA to and from the nucleus as a complex that is recognized by BL1 for movement to adjacent cells. To further investigate this, we expressed BR1 mutants known to affect viral infectivity in Spodoptera frugiperda insect cells and Nicotiana tabacum L. cv Xanthi protoplasts and found these to be defective in either their nuclear targeting or their ability to be redirected to the cell periphery when co-expressed with BL1. Translational fusions to p-glucuronidase and alanine-scanning mutagenesis further demonstrated that the C-terminal 86 amino acids of BR1 contains a domain(s) essential for i t s interaction with B L I and identified two nuclear localization signals within the N-terminal 113 residues of BR1. These nuclear localization signals were precisely located within distinct 16-and 22-peptide segments of BR1. These studies support and extend our model for squash leaf curl virus movement, showing that BR1 has a domain structure, with an N-terminal region required for nuclear targeting and a C-terminal region required for i t s interaction with BL1. Department of Microbiology, University of lllinois 23Regulated trafficking across the nuclear membrane is an essential component of many signal transduction pathways in eukaryotic cells and a fundamental aspect of infection by animal and plant viruses that replicate in the nucleus. The transport of proteins across the nuclear membrane is an energy-requiring process mediated by proteins associated with nuclear pore complexes (Newmeyer and Forbes, 1988). Although nuclear pores have an effective size-exclusion limit of approximately 60 kD, proteins smaller than this appear not to diffuse efficiently across the nuclear membrane (reviewed by Silver, 1991). Karyophilic proteins contain NLSs, short basic regions of approximately 10 to 20 amino acids (reviewed

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confidence: 97%

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

1996

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“…For construction of the pLuc vector for the luciferase assay, we replaced the GFP cassette in pCRGFP vector (Reichel et al 1996) by an NcoI/XbaI fragment of the Promega pGL3 basic vector encoding firefly luciferase. The plant expression vector is a modified pRT vector containing a duplicated CaMV35S enhancer and a tobacco etch virus translational enhancer preceding the ATG start codon (Carrington et al 1991).…”

Section: Reporter Constructs and Assays With Tomato Protoplastsmentioning

confidence: 99%

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato

Mishra¹,

Tripp²,

Winkelhaus³

et al. 2002

Genes Dev.

506

481

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We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a single HsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules. (Pelham 1982;Pelham and Bienz 1982;Nover 1987). Similar to other transcription factors, Hsfs have a modular structure with an N-terminal DNA-binding domain characterized by a central helix-turn-helix motif, an adjacent oligomerization domain with a bipartite heptad pattern of hydrophobic amino acid residues (HR-A/B region), and sequence motifs essential for nuclear import and export (NLS, NES;Wu 1995;Morimoto 1998;Heerklotz et al. 2001;Nover et al. 2001). In many cases, the C-terminal activation domains are characterized by short peptide motifs (AHA motifs) shown to be crucial for the activator function (Döring et al. 2000).Sequencing of the Arabidopsis genome revealed a unique complexity of the Hsf family with 21 members, in contrast to yeast and Drosophila with one Hsf and vertebrates with four Hsfs (Wu 1995;Nover et al. 2001). From analyses of expressed sequence tag (EST) libraries, it is evident that the size of the Hsf family is comparable also in other plants, with 17 Hsfs thus far identified from tomato ESTs . By structural characteristics and phylogenetic comparison, plant Hsfs were assigned to three classes. In Arabidopsis, there are 15

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“…The U2BЉ::GFP cassette was cut out and inserted into the pRTL2 vector (Carrington et al, 1991), creating the pRTL2::U2BЉ::GFP vector. The new U2BЉ::GFP construct was checked by sequence analysis.…”

Section: Construction Of the U2b؆-gfp Fusion Proteinmentioning

confidence: 99%

The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

Boudonck

Dolan

Shaw

1999

MBoC

132

119

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Coiled bodies are nuclear organelles that contain components of at least three RNAprocessing pathways: pre-mRNA splicing, histone mRNA 3Ј-maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3Ј-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2BЉ-green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled body number during the cell cycle. Deletion analysis of the U2BЉ gene construct has shown that the first RNP-80 motif is sufficient for localization to the coiled body. INTRODUCTIONThe coiled body was first described by Ramon y Cajal (1903), who called it the "nucleolar accessory body" because of its association with the nucleolus. This nuclear organelle was later reidentified by electron microscopy and renamed the "coiled body" because of its appearance as loosely packed coiled fibrils (Monneron and Bernhard, 1969). Subsequent studies detected coiled bodies in animal and plant nuclei, showing that it is a conserved structure (Moreno Diaz de la Espina et al., 1980;Seite et al., 1982;Schultz, 1990).Coiled bodies have been shown to contain splicing small nuclear ribonucleoproteins (snRNPs) and small nuclear RNAs (snRNAs), a subset of nucleolar components -including fibrillarin, Nopp140, NAP57, and U3 small nucleolar ribonucleoprotein (U3 snoRNP) -and the protein p80 coilin, which has been widely used as a marker for coiled bodies (Lamond and Earnshaw, 1998;Matera, 1998). The function of coiled bodies is still under debate, but several hypotheses have been proposed that are not necessarily mutually exclusive. Because coiled bodies do not contain DNA, nascent pre-mRNA, heterogeneous nuclear RNPs (hnRNPs), or the SC-35 splicing factor, which is required for splicing in vitro, it has been argued that they are not directly involved in transcription and pre-mRNA splicing . However, it has been speculated that coiled bodies may be sites of splicing factor assembly or recycling, may play a role in histone mRNA 3Ј processing (Gall et al., 1995;Lamond and Earnshaw, 1998), or may be involved in all these activities. As coiled bodies are frequently observed at the nucleolar periphery and also in the nucleoplasm and within nucleoli (Malatesta et al. 1994;Ochs et al., 1994), they may also act as nuclear transport or sorting structures. It has been shown recently that coiled bodies are also involved in processing or transport of small nucleolar RNA (snoRNA) pr...

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Bipartite Signal Sequence Mediates Nuclear Translocation of the Plant Potyviral NIa Protein

Cited by 75 publications

References 16 publications

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

A Viral Movement Protein as a Nuclear Shuttle (The Geminivirus BR1 Movement Protein Contains Domains Essential for Interaction with BL1 and Nuclear Localization)

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato

The Movement of Coiled Bodies Visualized in Living Plant Cells by the Green Fluorescent Protein

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