Versatile cloning vectors for transient gene expression and direct gene transfer in plant cells

Töpfer, Reinhard; Schell, J.; Steinbiß, Hans‐Henning

doi:10.1093/nar/16.17.8725

Cited by 142 publications

(80 citation statements)

References 3 publications

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“…Standard procedures were used for cloning (Ausubel et al 1993;Sambrook and Russell 2001). Plant expression vectors are derivatives of pRT101 (Töpfer et al 1988). For convenient deletions or combinations of functional parts of HsfA1 and HsfA2, we generated unique SalI sites in different regions of the cDNAs introduced by site-directed mutagenesis (Treuter et al 1993;Döring et al 2000).…”

Section: General Materials and Methodsmentioning

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.

509

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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Section: General Materials and Methodsmentioning

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.

509

481

View full text Add to dashboard Cite

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“…Plasmid constructs for Hsf expression in plant cells are based on the pRT series of vectors (30). Constructs for Hsfs A1, A2, and A3 were described before (10,21).…”

Section: Methodsmentioning

confidence: 99%

Role of Heat Stress Transcription Factor HsfA5 as Specific Repressor of HsfA4

Baniwal

Chan

Scharf

et al. 2007

Journal of Biological Chemistry

141

117

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“…Plant expression vectors were derived from the pRT series of vectors (Töpfer et al 1988). Modified versions providing a Myc epitope for N-terminal immunotag fusions were described before (Kirschner et al 2000;Siddique et al 2003).…”

Section: Constructs For Transient Protein Expression In Plant Protoplmentioning

confidence: 99%

“…The ACD is flanked by mostly unstructured variable N-and C-terminal extensions leading to sHsp monomer molecular sizes ranging from~16 to 42 kDa. Another characteristic feature of sHsps is that in their native state, they typically exist as multimeric complexes of 8 to 24 or more subunits, although dimeric forms have also been described (Kim et al 1998a;van Montfort et al 2001a;Stamler et al 2005). X-ray crystal structures are available for two oligomeric sHsps, the 24-subunit Hsp16.5 from Methanococcus jannaschii and the dodecamer Hsp16.9 from wheat (Triticum aestivum), and they show that despite different quaternary structures and oligomeric sizes, the monomers have a very similar fold, and both oligomers are built from the same basic dimer involving strand exchange of β6 into the ACD β-sandwich of the reciprocal monomers (Kim et al 1998b;van Montfort et al 2001b).…”

Section: Introductionmentioning

confidence: 99%

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

Siddique

Gernhard

Koskull‐Döring

et al. 2008

Cell Stress and Chaperones

216

215

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Versatile cloning vectors for transient gene expression and direct gene transfer in plant cells

Cited by 142 publications

References 3 publications

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

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

Role of Heat Stress Transcription Factor HsfA5 as Specific Repressor of HsfA4

The plant sHSP superfamily: five new members in Arabidopsis thaliana with unexpected properties

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