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Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium

Abstract

Genetic transformation of plant cells by Agrobacterium represents a unique case of trans-kingdom DNA transfer1. During this process, Agrobacterium exports its transferred (T) DNA and several virulence (Vir) proteins into the host cell2, within which T-DNA nuclear import is mediated by VirD2 (ref. 3) and VirE2 (ref. 4) and their host cell interactors AtKAP-α5 and VIP1 (ref. 6), whereas its integration is mediated mainly by host cell proteins7,8,9. The factors involved in the uncoating of T-DNA from its cognate proteins, which occurs before integration into the host genome, are still unknown. Here, we report that VirF—one of the few known exported Vir proteins whose function in the host cell remains unknown—is involved in targeted proteolysis of VIP1 and VirE2. We show that VirF localizes to the plant cell nucleus and interacts with VIP1, a nuclear protein. VirF, which contains an F-box motif10, significantly destabilizes both VIP1 and VirE2 in yeast cells. Destabilization of VIP1 in the presence of VirF was then confirmed in planta. These results suggest that VIP1 and its cognate VirE2 are specifically targeted by the VirF-containing Skp1–Cdc53-cullin–F-box complex for proteolysis. The critical role of proteasomal degradation in Agrobacterium-mediated genetic transformation was also evident from inhibition of T-DNA expression by a proteasomal inhibitor.

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Figure 1: Specific interaction between VirF and VIP1 in yeast and plant cells.
Figure 2: VirF-mediated and Skp1-dependent destabilization of VIP1 and VirE2 in yeast cells.
Figure 3: VirF-mediated destabilization of VIP1 in planta.
Figure 4: The effect of 26S proteasome inhibition on T-DNA expression.

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Acknowledgements

We thank S. Gelvin for VirF and Agrobacterium strains, T. Durfee for ASK1, and M. Goodin for pGDR. We are also grateful to R. Sternglanz and A. Neiman for their suggestions and discussion. The work in our laboratory is supported by grants from the National Institutes of Health, National Science Foundation, US Department of Agriculture, US–Israel Bi-national Science Foundation (BSF), and US–Israel Bi-national Agricultural Research and Development Fund (BARD) to V.C., and by grants from BARD and Human Frontiers Science Program to T.T.

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Tzfira, T., Vaidya, M. & Citovsky, V. Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431, 87–92 (2004). https://doi.org/10.1038/nature02857

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