Silja Seemann (Universität Hamburg)
Silja Janina Seemann's Master thesis was awarded with the Prize for the Best Plant Science Master Thesis, which was carried out at Hamburg University in the year 2025 with the title:
Identification of amino acid residues critical for the activity, dimerisation, and regulation of the small GTPase, Arabidopsis immune-associated nucleotide-binding protein 12 (AtIAN12), by site-directed mutagenesis
How small GTPases of the IAN family are regulated in Arabidopsis
In this study, one specific amino acid was found to be crucial for dimerisation and GTPase activity of AtIAN12. Previously AtIAN12 was shown to belong to the special subgroup of small GTPases that are activated by nucleotide dependent dimerisation (GADs).
Small GTPases play crucial roles in the immune system of animals and plants; however, their regulatory functions in plants remain insufficiently characterised. One specific subgroup are the so-called GTPases activated by nucleotide-dependent dimerisation (GADs), which include the Arabidopsis immune-associated nucleotide-binding protein 12 (AtIAN12), as first described in the previous doctoral thesis in our group (Pokhrel, 2022).
In this Master´s thesis, the functions of two key amino acids were investigated with respect to their role in the activity, dimerisation, and regulation of the GTPase. Mutant AtIAN12 variants were generated by site-directed mutagenesis, and complementary in vitro analyses included yeast two-hybrid assays to examine dimerisation, enzymatic analyses to quantify the GTPase activity, and in silico predictions of the GDP binding site within the AtIAN12 dimer. The combined results indicated that one acidic amino acid residue was essential for dimerisation, GTPase activity, and nucleotide binding, whereas the neighbouring residue did not play any functional role. In addition, size exclusion chromatography coupled with multi-angle laser light scattering (SEC-MALLS) was employed to characterise the oligomeric state of AtIAN12 in vitro, providing clear evidence for dimer formation next to monomers. Small-angle X-ray scattering (SAXS) served as the initial structural analysis of AtIAN12 and showed consistency with the predicted structural model.
Overall, this study identified one specific key residue required for the regulatory role of AtIAN12 as a GAD and provided new insights into its activation mechanism, nucleotide binding, and structural properties, thereby opening up new possibilities for determining its precise physiological function in Arabidopsis.
___
Silja Seemann conducted this work at the Institute for Plant Science and Microbiology at the University of Hamburg in the department of Plant Biochemistry and Infection Biology in the working group of Prof. Dr. Sigrun Reumann.