CHAPTER 3 Isolation of Arabidopsis proteins that interact with the Agrobacterium virulence protein VirF

The Agrobacterium tumefaciens VirF protein is directly translocated into host cells via the VirB/VirD4 transport system during the infection process. It is a host range determinant required for full virulence on certain host plants, and absence of virF in Agrobacterium leads to attenuated virulence on such hosts but can be complemented by in planta expression of VirF. The precise function of VirF in the host cell is not clear but VirF contains an F-box domain by which it interacts with the Arabidopsis thaliana homologues of the yeast SKP1 protein (ASK1 and ASK2). SKP1 and F-box proteins (FBPs) are subunits of the SCF-Ubiquitin protein ligases (E3 ligases) that play an essential role in many cellular processes by recruiting specific proteins for ubiquitination and subsequent degradation by the 26S proteasome. F-box proteins are the key element of the SCF-complex, as they are responsible for the recognition of the proteins to be degraded. To identify target proteins of VirF we performed a yeast two-hybrid screen with VirF as bait using an A. thaliana cDNA library. Putative interactors of VirF (PIFs) were identified; GST pull down assays confirmed for five selected proteins direct binding to VirF in vitro. These five PIFs represent a Lon protease like, a putative pirin like protein, 3-deoxy-7-phosphoheptulonate 7-phosphate (DAHP) synthase 2 (DHS2), the vacuolar H+ ATPase subunit B3 (VHA-B3) and branched-chain amino-transferase (BCAT4). VirF may enhance virulence by stimulating the targeted degradation of some or all of these proteins. How this could promote virulence is being discussed. Isolation of Arabidopsis protein interactors of VirF 62 INTRODUCTION The capacity of Agrobacterium tumefaciens to induce tumorigenesis in a wide range of host plants involves the translocation of a segment of oncogenic DNA called the transferred DNA (T-DNA) and several effector or virulence proteins, which are encoded by genes located in the vir-region on the Ti plasmid, into host cells. Specific phenolic compounds, such as acetosyringone, which are released upon wounding of plant cells, activate the VirA/VirG two-component regulatory system that subsequently induces the expression of the vir genes (Reviewed by Hansen and Chilton, 1999; Gelvin, 2003; 2006; McCullen and Binns, 2006). Proteins encoded by the virB and virD4 genes form a transport apparatus, also called type IV secretion system (T4SS), that spans the bacterial envelope and through which protein and DNA are transferred from the bacterium into the plant cell (Christie, 2004). The T-DNA is derived from the T-region of the Ti-plasmid, which is flanked by an imperfect 24 bp direct DNA repeat. The VirD2 protein introduces a nick at the border repeats and possibly via a replacement mechanism a single stranded form of the T-region called T-strand is released. VirD2 remains attached to the 5‟ of the T-strand, and pilots the Tstrand through the T4SS into the host cell (reviewed by Hansen and Chilton, 1999; Gelvin 2003). The VirE2 protein is an effector protein transferred into host cells (Vergunst et al., 2000; Schrammeijer et al., 2001), where it binds to the T-strand, protecting the T-strand against nucleolytic attack (Ziemienowicz et al., 2001). By the presence of nuclear localization signals (NLS) both in VirD2 and VirE2, and interactions of these proteins with plant proteins, the T-complex is directed to the cell nucleus where it becomes integrated in the host genome (Zambryski, 1992; Ballas and Citovsky, 1997; Pelczar et al., 2004). Use of the Cre reporter assay for protein translocation (CRAfT) directly demonstrated transfer of the effector proteins VirF, VirE2, VirE3, VirD2 and VirD5 to plant and yeast cells via the VirB/VirD4 channel even in absence of T-DNA transfer (Vergunst et al., 2000; Schrammeijer et al., 2003; Vergunst et al., 2003; Vergunst et al., 2005; Chapter 2). Although the function of VirE2 and VirD2 in planta has been clarified to a large extent, the function of additional translocated virulence proteins such as VirF, VirE3 and VirD5 has not been clearly defined yet. Previous extracellular complementation tests demonstrated that the attenuated virulence of virF mutant strains on N. glauca was restored by co-infection with A. tumefaciens helper strains, lacking the T region but expressing the VirF protein (Otten et al., 1984). Full virulence of the virF mutant was also seen after infection of N. glauca plants expressing the VirF protein (Regensburg-Tuïnk and Hooykaas, 1993). These data suggested that VirF has a function inside the plant host. Like VirF, VirE3 is a host range factor; although a virE3 deletion only has a minor effect on virulence on specific host plants, it dramatically aggravates the effect of a virF mutation. (Schrammeijer, 2001, García-Rodríguez, Schrammeijer and Hooykaas, 2006). Later findings showed that VirE3 interacts with specific plant nuclear proteins, and it was suggested that VirE3 may be a transcriptional activator of genes involved in tumor formation (García-Rodríguez, Schrammeijer and Hooykaas, 2006). On the other hand VirE3 was shown to interact with VirE2 and it was suggested that VirE3 could fulfill a task similar to that of the plant VirE2 interacting protein (VIP1), mediating the import of VirE2 into the nucleus (Lacroix et al., 2005).