Involvement of the plant cytoskeleton in Agrobacterium transformation
Abstract
Agrobacterium tumefaciens is a soil borne phytopathogen that causes crown gall tumors on plants. The process of tumor formation involves the transfer and integration of a specific DNA segment (T-DNA) from the bacterium into the plant chromosome. VirD2 and VirE2 are bacterial proteins that are involved in this process of transformation. Both VirD2 and VirE2 interact with a plant molecule, α-importin, in vitro. α-Importin interacts with actin and microtubules, both components of the plant cytoskeleton, in vitro and also co-localizes with them in vivo. Hence, with this background, I investigated if the bacterial proteins VirD2 and VirE2 that are components of the T-DNA complex and are transferred to the plant cell interact with either actin or microtubules. For this purpose, I used in vitro co-sedimentation assays to determine if VirD2 and VirE2 interact with filamentous actin (F-actin) and microtubules. Both the virulence proteins interacted with F-actin. VirE2 and VirD2 did not interact with microtubules. I characterized regions of VirD2 and VirE2 required for binding, their dissociation constants, and likely residues important for binding. In order to observe the contribution of the plant cytoskeleton to transformation, I tested the susceptibility of various plant cytoskeletal mutants in a root transformation assay. Plant lines that were mutated in actin genes expressed in the roots were resistant to stable and transient transformation, whereas plant lines mutated in actin expressed predominantly in the pollen were susceptible to transformation. One plant microtubule mutant tested was sensitive to transformation. I also tested the susceptibility of plant actin mutants that had been complemented with one of the actin genes expressed in the roots and found that complementation restored susceptibility to infection. In order to investigate further the importance of the actin cytoskeleton in transformation, I tested the effect of various pharmacological inhibitors of the plant cytoskeleton on the transformation of tobacco BY-2 cells. Inhibitors of the actin cytoskeleton lowered the frequency of transformation, whereas inhibitors directed against microtubules did not. In summary, these results suggest that the plant actin cytoskeleton is involved in the process of Agrobacterium tumefaciens-mediated transformation, whereas microtubules may not be involved.
Degree
Ph.D.
Advisors
Gelvin, Purdue University.
Subject Area
Plant pathology
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