The RHO1-specific GTPase-activating protein LRG1 regulates polar tip growth in parallel to Ndr kinase signaling in Neurospora.

Regulation of Rho GTPase signaling is critical for cell shape determination and polarity. Here, we investigated the role of LRG1, a novel member of the GTPase-activating proteins (GAPs) of Neurospora crassa. LRG1 is essential for apical tip extension and to restrict excessive branch formation in subapical regions of the hypha and is involved in determining the size of the hyphal compartments. LRG1 localizes to hyphal tips and sites of septation via its three LIM domains. The accumulation of LRG1 as an apical cap is dependent on a functional actin cytoskeleton and active growth, and is influenced by the opposing microtubule-dependent motor proteins dynein and kinesin-1. Genetic evidence and in vitro GTPase assays identify LRG1 as a RHO1-specific GAP affecting several output pathways of RHO1, based on hyposensitivity to the glucan inhibitor caspofungin, synthetic lethality with a hyperactive beta1,3-glucan synthase mutant, altered PKC/MAK1 pathway activities, and hypersensitivity to latrunculin A. The morphological defects of lrg-1 are highly reminiscent to the Ndr kinase/RAM pathway mutants cot-1 and pod-6, and genetic evidence suggests that RHO1/LRG1 function in parallel with COT1 in coordinating apical tip growth.