Re-engineering Agrobacterium for T-DNA delivery to chloroplasts

The current bottleneck of plastid transformation in Arabidopsis is the difficulty of obtaining fertile plants from transplastomic tissue culture cells. Tissue culture limitations in Arabidopsis nuclear gene transformation were overcome by using Agrobacterium to directly transform the female gametocyte, and identification of nuclear transgenic events by germinating the resulting seedlings on a selective medium. Our goal is to re-engineer Agrobacterium for T-DNA delivery to chloroplasts to directly transform the plastids in the female gametocyte. T-DNA export from Agrobacterium to plant cells occurs by the type 4 protein secretion machinery. Recently, we obtained proof of concept that proteins can be directly exported from Agrobacterium to chloroplasts. The protein of our choice was the phiC31 phage site-specific integrase (Int), because visitation of the recombinase to chloroplasts created a permanent footprint. We are now working on re-targeting the proteins involved in T-DNA transfer. Side-stepping the tissue culture process will eliminate the need for specialized expertise to practice plastid transformation in Arabidopsis. Therefore, this research will lead to widespread applications of Arabidopsis plastid genome engineering which, combined with the available extensive genomic resources, will have a major impact on basic science and applications in biotechnology. This research is supported by NSF Award IOS-2037155.