Introduction

Transcription activator like effectors or TAL effectors (TALEs) are DNA binding proteins which are secreted by many species of the pathogenic Xanthonomas genus via a type III secretion system. They gain entry to the host plant through natural openings such as the stomata or hydathodes (glands which secrete water). They are major virulence factors involved in the activation of specific genes that allow colonisation and spread of bacteria in plant species such as rice and cotton. TALEs share a characteristic C-terminus configuration consisting of nuclear localisation signals along with an acidic activating domain which allow them to enter the nucleus and begin affecting gene expression.  The area in which they differ is the central domain which contains 1.5 to 33.5 tandem repeats and is the domain responsible for DNA binding.  (Deng et al., 2012) (Boch, Bonas 2010)

Figure 1. This figure has been adapted from Boch, J., Bonas U., (2010), and shows how the TALE enters the cell, via the type III secretion system. Using this method, the bacterial cell secretes a mixture of effector proteins into the plant cell which disguise the TALEs and allow them to enter the cell undetected. These effector proteins are shown as the red structure attached to the bacteria on the diagram.

Previous studies have found that mutants in the type III secretory proteins have been unable to create disease symptoms in sensitive plants, highlighting the significant role that these effectors have in pathogenicity (Kay, Bonas 2009).  Thus it is important to study TAL effectors to aid our understanding of plant diseases and identify possible mechanisms for control/prevention.  Also, TALEs DNA binding properties can be exploited so that artificially constructed TALEs can be used for gene expression control, displaying their great potential in the field of biotechnology (Boch et al., 2009).

Materials and Methods

Recombinant dHax3 including a histidine tag was cloned into Escherichia Coli using a vector plasmid.  Overexpression was induced through the use of IPTG and cells were harvested after growth.  The protein was then purified through affinity chromatography using nickel resin (affinity for histidine) and a heparin (affinity for positively charged proteins) column. (Deng et al., 2012)

Purified proteins could then be concentrated and put through gel filtration chromatography to prepare for crystallisation.  Several dHax3 fragments were tried for crystallisation, however only the fragment containing residues 270-703 gave a usable diffraction.  Crystals were formed via the hanging-drop vapour-diffusion method and grown to complete size over the course of 3-4 days.  In order to acquire the crystals for the protein bound to DNA, a 17 base pair DNA strand was used with the fragment containing residues 231- 720.  X-ray diffraction data was then collected with the use of a Synchrotron. (Deng et al., 2012)