The VirD2 pilot protein of
            <i>Agrobacterium</i>
            -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

Bako, L.; Umeda, Masaaki; Tiburcio, Antonio F.; Schell, Jeff; Koncz, Csaba

doi:10.1073/pnas.1733208100

Cited by 85 publications

(63 citation statements)

References 62 publications

(82 reference statements)

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“…Cells were harvested by centrifugation and then resuspended and sonicated in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 1 mM phenylmethylsulfonylfluoride, 0.1 mM benzamidine, 10 µg/mL aprotinin, and 10 µg/mL leupeptin, pH 8.0). The cell lysate was subjected to centrifugation (Sorvall HB-4 rotor; 16,500g for 30 min at 4°C), and the cleared extract was used for affinity purification on glutathione-Sepharose 4B (Amersham Biosciences) as described (Umeda et al, 1998;Bakó et al, 2003). E. coli BL21 (DE3) pLysS cells carrying pET201-Trx-CDKDs-His 6 , pET201-Trx-CyclinH;1-His 6 , and pET201-Trx-CDKF;1-His 6 (see Supplemental Methods 1 online) were disrupted by sonication in lysis buffer, and upon centrifugation the cleared extracts were affinity purified on nickel-nitriloacetic acid agarose (Qiagen) according to the manufacturer's instruction.…”

Section: Purification Of Arabidopsis Proteins Expressed In Escherichimentioning

confidence: 99%

CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II

et al. 2012

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Phosphorylation of conserved Y 1 S 2 P 3 T 4 S 5 P 6 S 7 repeats in the C-terminal domain of largest subunit of RNA polymerase II (RNAPII CTD) plays a central role in the regulation of transcription and cotranscriptional RNA processing. Here, we show that Ser phosphorylation of Arabidopsis thaliana RNAPII CTD is governed by CYCLIN-DEPENDENT KINASE F;1 (CDKF;1), a unique plant-specific CTD S 7 -kinase. CDKF;1 is required for in vivo activation of functionally redundant CYCLIN-DEPENDENT KINASE Ds (CDKDs), which are major CTD S 5 -kinases that also phosphorylate in vitro the S 2 and S 7 CTD residues. Inactivation of CDKF;1 causes extreme dwarfism and sterility. Inhibition of CTD S 7 -phosphorylation in germinating cdkf;1 seedlings is accompanied by 39-polyadenylation defects of pre-microRNAs and transcripts encoding key regulators of small RNA biogenesis pathways. The cdkf;1 mutation also decreases the levels of both precursor and mature small RNAs without causing global downregulation of the protein-coding transcriptome and enhances the removal of introns that carry premicroRNA stem-loops. A triple cdkd knockout mutant is not viable, but a combination of null and weak cdkd;3 alleles in a triple cdkd123* mutant permits semidwarf growth. Germinating cdkd123* seedlings show reduced CTD S 5 -phosphorylation, accumulation of uncapped precursor microRNAs, and a parallel decrease in mature microRNA. During later development of cdkd123* seedlings, however, S 7 -phosphorylation and unprocessed small RNA levels decline similarly as in the cdkf;1 mutant. Taken together, cotranscriptional processing and stability of a set of small RNAs and transcripts involved in their biogenesis are sensitive to changes in the phosphorylation of RNAPII CTD by CDKF;1 and CDKDs.

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Section: Purification Of Arabidopsis Proteins Expressed In Escherichimentioning

confidence: 99%

CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II

et al. 2012

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“…However, in a recent study it was concluded that VIP1 is not important for AMT or VirE2 subcellular localization (Shi et al, 2014). VirD2 may also have a role in the targeting to the chromatin as Bakó et al (2003) reported that VirD2 bound to the TATA-box-binding protein in transformed Arabidopsis thaliana cells. In our study, we showed that both in vitro as well as in vivo, VirD2 binds to the yeast histone proteins (H2A, H2B, H3 and H4) (Figs 2 and 3) and that VirD2 translocated from Agrobacterium to yeast also binds to histone proteins (Fig.…”

Section: Discussionmentioning

confidence: 96%

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

et al. 2015

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Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 59 end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His 6 -tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA.

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“…Using yeast two-hybrid and plant-based BiFC systems, scientists have identified several plant proteins that interact with VirD2. Prominent among these plant proteins are members of the importin a family, which play a role in nuclear targeting of VirD2/T-strand complexes (Ballas and Citovsky, 1997;Bhattacharjee et al, 2008), and several different cyclophillin proteins whose role in transformation is not yet known Bakó et al, 2003).…”

Section: Screening An Arabidopsis Cdna Library For Proteins That Intementioning

confidence: 99%

“…The first of these (clone 130-14O) is cyclophilin ROC3 (At2g16600). Using yeast two-hybrid analyses, Deng et al (1998) and Bakó et al (2003) previously identified several Arabidopsis cyclophilins, including ROC3, as proteins that interact with VirD2. Finally, we identified the clone 130-6O whose protein product strongly interacts with nYFP-VirD2.…”

Section: Screening An Arabidopsis Cdna Library For Proteins That Intementioning

confidence: 99%

“…In addition to investigating interactions between two known proteins, one tagged with the transcription factor DNA binding domain (the bait protein) and the other with the transcription activation domain (the prey protein), the yeast twohybrid system has been widely used to identify interacting prey proteins encoded by a cDNA library. Plant scientists have frequently taken advantage of this technique to identify novel proteins that interact with a known bait protein (Ballas and Citovsky, 1997;Bakó et al, 2003;Hwang and Gelvin, 2004;Tao et al, 2004).…”

Section: Introductionmentioning

confidence: 99%

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Screening a cDNA Library for Protein–Protein Interactions Directly in Planta

Lee

Hsu

et al. 2012

Plant Cell

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Screening cDNA libraries for genes encoding proteins that interact with a bait protein is usually performed in yeast. However, subcellular compartmentation and protein modification may differ in yeast and plant cells, resulting in misidentification of protein partners. We used bimolecular fluorescence complementation technology to screen a plant cDNA library against a bait protein directly in plants. As proof of concept, we used the N-terminal fragment of yellow fluorescent protein-or nVenus-tagged Agrobacterium tumefaciens VirE2 and VirD2 proteins and the C-terminal extension (CTE) domain of Arabidopsis thaliana telomerase reverse transcriptase as baits to screen an Arabidopsis cDNA library encoding proteins tagged with the C-terminal fragment of yellow fluorescent protein. A library of colonies representing ;2 3 10 5 cDNAs was arrayed in 384-well plates. DNA was isolated from pools of 10 plates, individual plates, and individual rows and columns of the plates. Sequential screening of subsets of cDNAs in Arabidopsis leaf or tobacco (Nicotiana tabacum) Bright Yellow-2 protoplasts identified single cDNA clones encoding proteins that interact with either, or both, of the Agrobacterium bait proteins, or with CTE. T-DNA insertions in the genes represented by some cDNAs revealed five novel Arabidopsis proteins important for Agrobacterium-mediated plant transformation. We also used this cDNA library to confirm VirE2-interacting proteins in orchid (Phalaenopsis amabilis) flowers. Thus, this technology can be applied to several plant species.

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The VirD2 pilot protein of Agrobacterium -transferred DNA interacts with the TATA box-binding protein and a nuclear protein kinase in plants

Cited by 85 publications

References 62 publications

CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II

CDKF;1 and CDKD Protein Kinases Regulate Phosphorylation of Serine Residues in the C-Terminal Domain of Arabidopsis RNA Polymerase II

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

Screening a cDNA Library for Protein–Protein Interactions Directly in Planta

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