SFB796 - Joint project 03

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Arabidopsis homologs of HsGBP1: role in plant defense?

 

Project summary

The guanylate-binding protein-1 (HsGBP1) belongs to the dynamin superfamily of large GTPases and is among the most significantly induced proteins in cells exposed to interferon (IFN)-?. HsGBP1 plays essential roles in anti-bacterial as well as anti-viral effects of (IFN)-γ, and has recently been shown to remodel F-actin1. Together with six homologs (HsGBP2-7), HsGBP1 constitutes a family of closely related proteins in humans. HsGBP1, HsGBP3 and HsGBP5 are exclusively detected in the cytoplasm, where they display patchy distribution patterns. By contrast, HsGBP2 and HsGBP4 show similar distributions in the cytoplasm, but in addition strongly accumulate in the nucleus.

The aim of this project is to identify homologs of HsGBP1 in Arabidopsis thaliana and to characterize their functions in actin regulation as well as in pathogen defense. Extensive BLAST searches, sequence analyses and bioinformatical domain modelling have resulted in the identification of three A. thaliana homologs of HsGBP1 (AtGBP1a, b & c), which share 20.2 % (AtGBP1a) - 27.2 % (AtGBP1b) identical amino acids with this protein. Publicly available microarray data indicate that AtGBP1a & b are ubiquitously expressed at low levels, whereas AtGBP1c appears to be pollen specific. Interestingly, these data further suggest that AtGBP1b is strongly upregulated upon infection with the bacterial pathogen Pseudomonas syringe pv. tomato (Pto).

Analysis of tobacco pollen tubes and HeLa cells transiently expressing GFP- or FLAG-tagged AtGBP1a, b or c established that in both cell types these A. thaliana proteins display similar distribution patterns, as well as effects on F-actin organization, as human HsGBP1 homologs in Hela cells. These observations are consistent with substantial functional conservation between plant and human GBP1 homologs. To investigate AtGBP1a, b and c functions in more detailed, public collections of A. thaliana T-DNA insertion (Salk, GABI-Kat) and RNAi (AGRIKOLA) lines were searched for mutants, which display reduced expression of one of these proteins. To date, two mutants have been identified, in which the expression of At-GBP1b, which appears to be upregulated upon Pto infection in wild type plants (see above), is reduced by more than 50 % or 95 %, respectively, as determined by qPCR.

Important aims of ongoing research are 1) the generation of additional A. thaliana mutants defective in AtGBP1a, b or c expression based on CRISPR/CAS mediated genome editing, 2) the analysis of effects of reduced At-GBP1b expression in the two identified A. thaliana knock-down lines on Pto proliferation in planta using established syringe infection assays, 3) the characterization of F-actin remodeling by AtGBP1a, b and c in different human cell lines, and 4) detailed structural modelling based on bioinformatics in order to identify conserved active protein domains.

Reference:

  • Ostler, N., N. Britzen-Laurent, A. Liebl, E. Naschberger, G. Lochnit, M. Ostler, F. Forster, P. Kunzelmann, S. Ince, V. Supper, G.J.K. Praefcke, D.W. Schubert, H. Stockinger, C. Herrmann & M. Stürzl.     IFN-γ-induced guanylate binding protein-1 is a novel actin cytoskeleton remodeling factor.     Mol Cell Biol 34, 196-209 (2014).