International Network of Plant Abiotic Stress

COST Action: FA0605

STSM title: Creation of tools for complementation of swi3a/b mutations and lines expressing tagged SWI3 proteins

Reference: : ECOST-STSM-FA0605-171010-003033

STSM dates: from 17-10-2010 to 21-11-2010

Location: Max-Planck Institute for Plant Breeding Research, Carl-von-Linne Weg 10, 50829 Cologne, Germany

 
Host:
Prof. Csaba Koncz

Max-Planck Institute for Plant Breeding Research

koncz@mpiz-koeln.mpg.de

STSM Fellow: Dr. Tomasz Sarnowski, Institute of Biochemistry and Biophysics Polish Academy of Sciences, Pawinskiego 5A, 02-106 Warsaw, Poland 

tsarn@ibb.waw.pl

Purpose of the visit:

During my stay at the Max Planck Institute for Plant Breeding Research in Cologne funded by the COST FA0605 IMPAS program, I performed experiments aiming at the functional characterization of SWI3 subunits of ATP-dependent SWI/SNF-type chromatin remodeling complexes (CRCs) in Arabidopsis thaliana according to the originally proposed goals of the STSM project. These included:

1. Conditional genetic complementation of atswi3a and atswi3b embryo lethal mutations.
2. Creation of cell lines expressing ATSWI3A and ATSWI3D CRC subunits labelled with HIS-STREP-HA and TAP tags

The first aim was to perform genetic complementation of atswi3a and atswi3b embryo lethal mutations using a conditional Cre-lox based expression system established by Heidstra et al. (2004). This technology is based on the use of a heat-inducible CRE recombinase, which precisely excises DNA sequences flanked by lox recognition sites. To genetically complement the embryo lethal atswi3a and atswi3b mutations, the wild type gene copies had to be cloned between the lox sites in the pCB1 binary vector, in which the lox sites are located between a CaMV 35S promoter and a gene encoding the Gal4VP16 transcription activator. Downstream of Gal4VP16, pCB1 carries a GFP reporter, the expression of which is controlled by Gal4-binding upstream activating sequences (UAS).

            Crosses between heterozygous plants carrying the atswi3a and atswi3b mutations and plants carrying the pGa227-HS::CRE construct were performed prior the start of the STMS project, and atswi3a/+ or atswi3b/+ plants carrying the HS-CRE vector were selected. The next step required the preparation of pCB1 vectors that carry the wild type SWI3A and SWI3B genes for genetic complementation of corresponding T-DNA insertion mutations. It was planned that the pCB1 vectors should carry between lox sites DNA fragments from BAC clones that contain full length genomic sequences of ATSWI3A and ATSWI3B genes, in which the stop codons were replaced with coding sequences of the HIS-STREP-HA tag using recombineering. However, the pCB1 vector carries only one restriction site (NotI) enabling insertion of DNA fragments.

            To achieve the proposed goal in the case of ATSWI3A gene, the isolation of high quality BAC DNA was necessary. This was followed by restriction digestion and gel isolation of an approx. 7.5kb fragment carrying the ATSWI3A gene. Subsequently, this fragment was blunt ended using DNA polymerase Klenow fragment followed by addition of a terminal adenosine aiding the cloning of this fragment into the vector pGEM-T easy. After successful ligation, the insert was excised by NotI and ligated into the pCB1 vector. To produce the pCB1 vector carrying the ATSWI3B gene similar approach was undertaken, except for that instead of using a BAC clone, the ATSWI3B gene fused to coding sequences of the HIS-STREP-HA tag was isolated from a previously constructed recombineering gap repair vector pGAP-Kan. Currently, these constructs are being transformed into Agrobacterium C58C1 (pMP90/pJIC Sa-Rep), to transform them into atswi3a+/- and atswi3b+/- seedlings carrying the HS-CRE constuct.

The second goal of teh STSM project was the generation of cell lines for expression of ATSWI3A and ATSWI3D CRC subunits labelled with HIS-STREP-HA and TAP tags

In second experiment, the previously prepared ATSWI3A-HIS-STREP-HA and ATSWI3D-HIS-STREP-HA constructs (as well as their TAP-tag fused versions) were transformed by the help of  Agrobacterium GV3101 (pMP90RK) helper strain into cells of root-derived dark-grown Arabidopsis cell suspension. Stable cell lines carrying the above mentioned constructs are currently under selection and will be subjected to subsequent western blot analysis to monitor the production of tagged ATSWI3A and ATSWI3D proteins, in order to initiate subsequent isolation of CRC complexes and mapping of their positions in the genome by chromatin precipitation.

My stay in the Max-Planck Institute for Plant Breeding Research was successful and very helpful. I will continue the work on the function of SWI/SNF CRCs in plants in the collaboration with the host lab.