Involvement of Arabidopsis thaliana importin α isoforms in the Agrobacterium-mediated transformation process

Saikat Bhattacharjee, Purdue University

Abstract

Plant transformation by Agrobacterium tumefaciens has been widely used to genetically engineer plants for new and improved traits. Considering the economic importance of this tool, an understanding of plant factors involved in the transformation process is essential for further improvement of this technique. Successful transformation of plants by Agrobacterium requires active nuclear import of the T-complex. The T-complex is proposed to consist of a transferred single-stranded DNA (T-DNA) covalently attached to a molecule of VirD2 at the 5′-end, and coated with the non-specific single-stranded DNA-binding protein VirE2. Both VirD2 and VirE2 have plant-functional nuclear localization signal sequences (NLSs) that may employ importin α proteins for nuclear import in plant, animal, and fungal cells. In Arabidopsis thaliana, nine members constitute the importin a family. I performed yeast two-hybrid and in vitro protein-protein interaction assays which demonstrated that multiple, but not all, Arabidopsis importin α members interact with VirD2 and VirE2. VirD2 and VirE2 interact with importin α via residues including and adjoining the NLSs. Using a gel mobility shift assay, I showed that ssDNA-bound VirE2 molecules retain the ability to bind importin α in vitro. Although multiple Arabidopsis importin α proteins interact with both VirD2 and VirE2, T-DNA disruption of only importin AtImpa-4, but not other importin a members, resulted in a rat (resistant to Agrobacterium -mediated transformation) phenotype of the host plants. This result suggests a vital role for AtImpa-4 in the transformation process. Interestingly, over-expression of VirD2/VirE2 interacting importin α members, but not of the VirD2/VirE2 non-interacting members, in the AtImpa-4 mutant background increases the transformation susceptibility of many of the derived transgenic lines. Transgenic Arabidopsis lines expressing translational gusA fusions from promoters of different Arabidopsis importin a genes display overlapping, although discrete, expression patterns. Taken together, these results suggest that Arabidopsis importin α isoforms may differentially participate in Agrobacterium-mediated transformation. The involvement of specific importin α proteins may be prescribed by the native expression pattern, native expression levels, and/or selective cargo-recognition of NLS domains of Vir proteins constituting the T-complex.

Degree

Ph.D.

Advisors

Gelvin, Purdue University.

Subject Area

Plant pathology

COinS