MOLECULAR-CLONING AND CHARACTERIZATION OF RHO, A RAS-RELATED SMALL GTP-BINDING PROTEIN FROM THE GARDEN PEA

The rho proteins, members of the ras superfamily of small GTP-binding proteins, play a central role in the modulation of cellular functions involving the actin cytoskeleton such as in the establishment of cell polarity and morphology. As a first step in elucidating signal transdu ction pathways leading to processes mediated by the actin cytoskeleton in plants, we initiated cloning and characterization of rho proteins from pea. One rho-related, partial cDNA clone of 167 bp was isolated u tilizing a polymerase chain reaction-based cloning strategy, using deg enerate primers that correspond to conserved domains within the rho pr oteins. A full-length cDNA was isolated by screening a pea cDNA librar y using the 167-bp cDNA as a probe. The Rho1Ps cDNA contains an open r eading frame encoding a polypeptide (Rho1Ps) of 197 amino acids that s hows 45-64% sequence identity to members of the rho family and about 3 0% identity to other members of the ras superfamily. In addition to th e nucleotide-binding and GTPase domains, Rho1Ps shares conserved resid ues and motifs unique to the rho proteins. Purified Rho1Ps protein exp ressed in Escherichia coli retains specific GTP-binding activity. Thes e data indicate that Rho1Ps encodes a small GTP-binding protein of the rho family. The Rho1Ps transcript is expressed in all organs of pea s eedlings, being more abundant in root tips and apical buds. DNA gel bl ot analyses show that the rho proteins in pea are encoded by a multige ne family.