Functional Studies Of Agrobacterium Effector VirD5 In The Process Of Agrobacterium Mediated Plant Genetic Transformation

Agrobacterium-mediated transformation serves as an important model system for studying host-pathogen recognition and delivery of macromolecules into target cells.In the process of the interaction between Agrobacterium and plant cells, Agrobacterium express many virulence proteins in order to accompolish the infection to host cells. Virulence proteins of Agrobacterium VirE2, VirE3, VirD2, VirF and VirD5 were found to be able to enter the plant cell nucleus. The functions of VirE2, VirE3, VirD2 and VirF have been studied extensively. However, the function of VirD5 is largely unknown.Agrobacterium-mediated transformation. is an extremely complex and elaborate process, including multiple steps and involving a number of Agrobacterium and host factors. It is of great importance to further illustrate the interaction between Agrobacterium and plants and reveal the details of transforming process.This study aims to explore the functions of Agrobacterium effector VirD5 in the process of Agrobacterium-mediated plant genetic transformation by using RT-PCR, subcellular localization, yeast two-hybrid, Bimolecular fluorescence complementation and GST pull down. The main results are as follows:1 VirD5 was found to locate on the VirD operon, controlled by the pVirD promoter by using VirD5-specific reverse transcription primer and RT-PCR.2 Expression of VirE2 is known to be induced by acetosyringone. Using VirE2 as a control in the experiment of semi-quantitative RT-PCR, the expression of VirD5 was also found to be induced by acetosyringone.3 VirD5 was linked to pGBKT7 and pGADT7 vectors, and then transformed into the competent cells of yeast. It showed transcription activation activity and slight specific DNA binding activity in yeast cells.4 To determine the transcriptional activation region of Agrobacterium VirD5, various truncated fragments of VirD5 were linked to pGBKT7 and transformed into yeast competent cells. The transcription activation region of VirD5 was determined to be in the 1-102 amino acid region of the N-terminal.5 Protoplast transformation experiments showed that Agrobacterium VirD5 can enter the nucleus of plant cells. Bioinformatics analysis showed that it contains three potential eukaryotic NLS locus in VirD5(173KRKR176,769KKDLEAKSVGVRRKKKE785, 820RRVYDPRDRAQDKAFKR836). Subcellular localization experiments demonstrated that the separated N and C terminal of VirD5 both can enter the nucleus of plant cells.6 According to the results of 4 and 5, it can be speculated that VirD5 may play the role of transcription factors in the nucleus of plant cells. As a rule, transcription factors take action in the forms of dimer or multimer. BiFC and GST pull down experiments demonstrated that VirD5 itself can interact to form a dimer or multimer.7 Arabidopsis VirE2 interacting protein 1(At VIP1) plays an important role in bringing T-complex into the plant nucleus and T-DNA integration in the process of Agrobacterium-mediated plant transformation. BiFC and GST pull down experiments determined that VirD5 can interact with AtVIP1.