Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

Loo, Suzanne Wolterink-van; Escamilla-Ayala, Abril; Hooykaas, Paul J. J.; Heusden, G. Paul H. van

doi:10.1099/mic.0.083410-0

Cited by 11 publications

(7 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It has been shown that the agrobacterium proteins VirE2, which coats the T‐DNA, and VirD2, which attaches to 5’ end of the T‐DNA and mediates nuclear entry, both associate with cellular histones (Lacroix et al ., ; Wolterink‐van Loo et al . ). As MAR are thought to influence chromatin structure, the association of T‐DNA with histones suggests a possible mechanism by which MAR function in vectors delivered by agroinfiltration.…”

Section: Discussionmentioning

confidence: 97%

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Diamos

Mason

2018

Plant Biotechnology Journal

115

View full text Add to dashboard Cite

SummaryPlants represent a promising platform for the highly scalable production of recombinant proteins. Previously, we identified the tobacco extensin terminator lacking its intron as an element that reduced transcript read‐through and improved recombinant protein production in a plant‐based system. In this study, we systematically compared nonreplicating plant expression vectors containing over 20 commonly used or newly identified terminators from diverse sources. We found that eight gene terminators enhance reporter gene expression significantly more than the commonly used 35S and NOS terminators. The intronless extensin terminator provided a 13.6‐fold increase compared with the NOS terminator. Combining terminators in tandem produced large synergistic effects, with many combinations providing a >25‐fold increase in expression. Addition of the tobacco Rb7 or TM6 matrix attachment region (MAR) strongly enhanced protein production when added to most terminators, with the Rb7 MAR providing the greatest enhancement. Using deletion analysis, the full activity of the 1193 bp Rb7 MAR was found to require only a 463‐bp region at its 3’ end. Combined terminators and MAR together provided a >60‐fold increase compared with the NOS terminator alone. These combinations were then placed in a replicating geminiviral vector, providing a total of >150‐fold enhancement over the original NOS vector, corresponding to an estimated yield of 3–5 g recombinant protein per kg leaf fresh weight or around 50% of the leaf total soluble protein. These results demonstrate the importance of 3’ flanking regions in optimizing gene expression and show great potential for 3’ flanking regions to improve DNA‐based recombinant protein production systems.

show abstract

Section: Discussionmentioning

confidence: 97%

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Diamos

Mason

2018

Plant Biotechnology Journal

115

View full text Add to dashboard Cite

show abstract

“…VirD2 contains NLS that guide the T‐complex to the nucleus of the host cell (Ziemienowicz et al ., ). Ectopically expressed GFP−VirD2 has a nuclear localization in both yeast and plant cells (Citovsky et al ., ; Wolterink‐van Loo et al ., ). In this study we found a similar localization for both GFP 11 ‐ and phiLOV2.1‐tagged VirD2 ectopically expressed in yeast (Figures and S1).…”

Section: Discussionmentioning

confidence: 97%

“…The translocated VirD2 is most likely located in the nucleus, but because of the low amount of translocated VirD2 we were unable to determine the exact subcellular localization. Using the BiFC approach translocated VirD2 was shown to interact with yeast nuclear histone proteins (Wolterink-van Loo et al, 2015). phiLOV2.1ÀVirD2 translocated to A. thaliana root cells was found in the nucleus (Figure 4), whereas in N. tabacum leaves it was found in dot-shaped structures close to or at the cell membrane ( Figure 6).…”

Section: Discussionmentioning

confidence: 99%

Application of phiLOV2.1 as a fluorescent marker for visualization of Agrobacterium effector protein translocation

et al. 2018

Self Cite

View full text Add to dashboard Cite

Agrobacterium tumefaciens can genetically transform plants by translocating a piece of oncogenic DNA, called T-DNA, into host cells. Transfer is mediated by a type IV secretion system (T4SS). Besides the T-DNA, which is transferred in a single-stranded form and at its 5' end covalently bound to VirD2, several other effector proteins (VirE2, VirE3, VirD5, and VirF) are translocated into the host cells. The fate and function of the translocated proteins inside the host cell are only partly known. Therefore, several studies were conducted to visualize the translocation of the VirE2 protein. As GFP-tagged effector proteins are unable to pass the T4SS, other approaches like the split GFP system were used, but these require specific transgenic recipient cells expressing the complementary part of GFP. Here, we investigated whether use can be made of the photostable variant of LOV, phiLOV2.1, to visualize effector protein translocation from Agrobacterium to non-transgenic yeast and plant cells. We were able to visualize the translocation of all five effector proteins, both to yeast cells, and to cells in Nicotiana tabacum leaves and Arabidopsis thaliana roots. Clear signals were obtained that are easily distinguishable from the background, even in cases in which by comparison the split GFP system did not generate a signal.

show abstract

“…One option to solve this problem could be a mechanism that targets the T-DNA to DSBs that are, for example, created by DNA topoisomerases during transcription or replication. It is interesting to note that VirD2 has been shown to interact with yeast histones (Wolterink-van Loo et al, 2015). One may speculate that histone phosphorylation upon DSB detection (Waterworth et al, 2011) might serve as a signal to guide VirD2 and the T-strand to DSBs.…”

Section: Plant Repair Pathway For T-dna Integrationmentioning

confidence: 99%

The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double-Strand Break Repair-Based Insertion Mechanism

et al. 2015

View full text Add to dashboard Cite

Transformation by Agrobacterium tumefaciens, an important tool in modern plant research, involves the integration of T-DNA initially present on a plasmid in agrobacteria into the genome of plant cells. The process of attachment of the agrobacteria to plant cells and the transport of T-DNA into the cell and further to the nucleus has been well described. However, the exact mechanism of integration into the host's DNA is still unclear, although several models have been proposed. During confirmation of T-DNA insertion alleles from the GABI-Kat collection of Arabidopsis thaliana mutants, we have generated about 34,000 sequences from the junctions between inserted T-DNA and adjacent genome regions. Here, we describe the evaluation of this dataset with regard to existing models for T-DNA integration. The results suggest that integration into the plant genome is mainly mediated by the endogenous plant DNA repair machinery. The observed integration events showed characteristics highly similar to those of repair sites of double-strand breaks with respect to microhomology and deletion sizes. In addition, we describe unexpected integration events, such as large deletions and inversions at the integration site that are relevant for correct interpretation of results from T-DNA insertion mutants in reverse genetics experiments.

show abstract

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones

Cited by 11 publications

References 48 publications

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Chimeric 3’ flanking regions strongly enhance gene expression in plants

Application of phiLOV2.1 as a fluorescent marker for visualization of Agrobacterium effector protein translocation

The Structural Features of Thousands of T-DNA Insertion Sites Are Consistent with a Double-Strand Break Repair-Based Insertion Mechanism

Contact Info

Product

Resources

About