The purpose of my research was to develop a system in which to transport foreign proteins from Agrobacterium to plant cells. Agrobacterium can genetically transform plants by transferring single stranded DNA in accordance with VirE2 and VirD2 proteins (this combination is also known as a T-complex). My system is aimed toward expression and export of foreign proteins from Agrobacterium to plant cells. The system I developed included fusing different proteins together in Agrobacterium expression plasmids. The initial plasmid of use was pE2431; it contains spectomyocin resistance as well as a virB promoter (which can be turned on and off). An additional plasmid named pSL301 was used as an intermediate plasmid in order to perform particular fusions that were impossible to perform directly in pE2431. Using different combinations of proteins I was hoping to determine whether the export signal (found at the end of a VirE2 protein) was solely responsible for protein transport between Agrobacterium and plants and can also be used for the technical export and tagging of various foreign proteins.

The proteins that I designed a transport system for were: GFP (green fluorescent protein, foreign protein), VIP1 (plant foreign protein), VP16 (synthetic foreign protein), VirE2, and the export signal. VP16 allowed for an alternative method of monitoring the export results. In case the proteins transported were too minimal to be seen VP16 would allow for amplification of their signal by if necessary. The controls in the experiment were both the VirE2 and the GFP. The VirE2 will definitely be transported and the GFP will hopefully allow me to see the protein's expression and ideally its actual transport using a confocal microscope. Primers were designed to isolate the specific nucleotides of the proteins that I would need. These included stop codons if I was designing a final clone (one that would not be fused to another) as well as continuous open reading frames in order to continue fusing the clone to another. Each clone was a respective section of the above mentioned proteins. My transport system included a final amount of 17 fused clones. Most having previously been separate clones (that would later be fused together), thus making the total needed clones number 21.

My hypothesis wondering the significance of the export signal in protein transport is still under testing as I hope to finish the experiment during the beginning of the upcoming year. This study was supported by the Simons foundation and a grant from BRDC.