Modification of plant Rac/Rop GTPase signalling using bacterial toxin transgenes

Abstract

SUMMARYBacterial protein toxins which modify Rho GTPase are useful for the analysis of Rho signalling in animal cells, but these toxins cannot be taken up by plant cells. We demonstrate in vitro deamidation of Arabidopsis Rop4 by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1) and glucosylation by Clostridium difficile toxin B. Expression of the catalytic domain of CNF1 caused modification and activation of co-expressed Arabidopsis Rop4 GTPase in tobacco leaves, resulting in hypersensitive-like cell death. By contrast, the catalytic domain of toxin B modified and inactivated co-expressed constitutively active Rop4, blocking the hypersensitive response caused by over-expression of active Rops. In transgenic Arabidopsis, both CNF1 and toxin B inhibited Rop-dependent polar morphogenesis of leaf epidermal cells. Toxin B expression also inhibited Rop-dependent morphogenesis of root hairs and trichome branching, and resulted in root meristem enlargement and dwarf growth. Our results show that CNF1 and toxin B transgenes are effective tools in Rop GTPase signalling studies.

The Endoplasmic Reticulum Is a Reservoir for WAVE/SCAR Regulatory Complex Signaling in the Arabidopsis Leaf

“…A recent report describes a clever strategy to generate ROP loss-of-function lines that used the ectopic expression of ROP-specific bacterial toxins. There was a strong association between inducible expression of the toxins and the appearance of trichomes with severe trichome swelling and reduced branch number phenotypes (Singh et al, 2012). Although the exact mechanism of ROP-dependent control of W/SRC remains to be determined, the results described above in combination with the detection of direct interactions between the ROPGEF SPK1, active forms of ROP, and W/SRC subunits (Basu et al, , 2008Uhrig et al, 2007) strongly suggest that W/SRC is a ROP effector complex.…”

A family of ROP proteins that suppress actin dynamics and are essential for polarized growth and cell adhesion

“…In fact, in rop1/rop6 double-mutant plants, the frequency of root hair branching is increased, but root hairs are still polarized and grow to similar lengths as in the wild type (Venus and Oelmüller, 2013). When ROP loss-of-function was engineered previously, by expressing a bacterial toxin transgene in A. thaliana that is known to inhibit ROP4 in vitro, plants formed few, if any, root hairs (Singh et al, 2013). Although this is a severe phenotype, the specificity of the effects of the transgene in vivo requires confirmation.…”

The Arabidopsis ROP-activated receptor-like cytoplasmic kinase RLCK VI_A3 is involved in control of basal resistance to powdery mildew and trichome branching

“…In addition, a recent study showed that bacterial toxin-mediated changes in RAC/ROP activity cause failure of trichome branching (Singh et al 2013). Taken together, the development of leaf pavement cells and trichome morphogenesis require similar processes of cytoskeleton organization.…”