Tomato Root Development: Characterizing the Potential Role of Tomato ROP-GTPases SlROP4 and SlROP9

Studying root development in tomato (Solanum lycopersicum) might help in the improving of tomatoes survival under extremities of abiotic stresses and root born fungal pathogens. Plant Rho-like small GTPases (ROPs-Rho of plants) function as crucial molecular signal switches in regulating diverse cellular and developmental processes. Based on amino acid sequences there are 9 ROP homologs found in tomato genome which designated as SlROP1-9. SlROP4 (Solyc01g111560.2.1) is homologous to Arabidopsis type-I ROPs AtROP2, AtROP4, AtROP6; while SlROP9 (Solyc03g114070.2.1) is homologous to Arabidopsis type-II ROPs AtROP10 and AtROP11. To study the function of SlROP4 and SlROP9, we generated loss-of-function mutants in their genes using CRISPR/Cas9 genome editing. M82. Several mutant alleles in each gene have been identified and are currently characterized. In parallel, we have generated estradiol-induced transgenic tomato lines expressing GFP- fused proteins of wild type, constitutively active (CA) and dominant negative (DN) mutants of SlROP4 and SlROP9. Expression of the recombinant proteins in roots was verified following induction with 17-β estradiol. Spectral analysis was utilized to discern between autofluorescence and GFP signals. Analysis of subcellular protein distribution demonstrated that wile wild type and constitutively active forms of GFP-ROP4 were localized primarily in the plasma membrane the dominant negative forms of GFP-ROP4 and GFP-ROP9 accumulated in intracellular membranes. To examine the effect ROP4 and ROP9 in tissue and cell organization, plants were cleared and their primary and secondary cell walls were stained with calcofluor white and basic fuchsine, respectively. Preliminary analysis indicates that expression GFP-rop4 CA increased the cell division zone while expression of GFP-rop4 DN and GFP-rop9 DN enhanced root differentiation. Taken together our studies indicate that ROPs have essential function in regulation of tomato root development.