RHO1 and RHO2 share partially overlapping functions in the regulation of cell wall integrity and hyphal polarity in Neurospora crassa

Abstract

SummaryRho proteins are key regulators of cellular morphogenesis, but their function in filamentous fungi is poorly understood. By generating conditional rho-1 mutants, we dissected the function of the essential GTPase RHO1 in cell polarization and maintenance of cell wall integrity in Neurospora crassa. We identified NCU00668/RGF1 as RHO1-specific exchange factor, which controls actin organization and the cell wall integrity MAK1 MAP kinase pathway through the direct interaction of active RHO1 with the formin BNI1 and PKC1 respectively. The activity of RGF1 is controlled by an intramolecular interaction of its DEP and GEF domains that blocks the activation of the GTPase. Moreover, the N-terminal region including the DEP domain of RGF1 interacts with the plasma membrane sensor NCU06910/WSC1, potentially to activate the cell wall integrity pathway. RHO1 also functions as regulatory subunit of the glucan synthase. N. crassa possesses a second GTPase, RHO2, that is highly homologous to RHO1. RHO2 is of minor importance for growth and does not interact with BNI1. Conditional rho-1;rho-2 double mutants display strong synthetic growth and cell polarity defects. We show that RHO2 does not regulate glucan synthase activity and the actin cytoskeleton, but physically interacts with PKC1 to regulate the cell wall integrity pathway.

Role of the Guanine Nucleotide Exchange Factor Rom2 in Cell Wall Integrity Maintenance of Aspergillus fumigatus

“…On the other hand, this localization may also reflect a potential role of Rho1 during septum formation, which is evident in S. cerevisiae and S. pombe (3,17). The Rom2-GFP localization to the hyphal tips is in good agreement with the recently characterized subcellular localization pattern of its homologue NcRGF1 in N. crassa and the predominant localization of its interacting Rho GTPase Rho1 to the hyphal tips (7,16).…”

“…In S. cerevisiae, deletion of ScROM2 results in an increased basal phosphorylation of ScMpk1 (35). In N. crassa, a heterokaryon deletion of Ncrgf-1 also results in an increased basal phosphorylation of the MpkA homologue NcMAK1 (16). According to our results and on the basis of similar observations made in other fungi, we propose that alter- native stress signaling mechanisms can activate the downstream Bck1-Mkk2-MpkA CWI MAP kinase module independently of Rom2 in mutants with chronic cell wall stress.…”

“…When the present study was initiated, the relevance of the DEP domain present in Rom2 and its homologues in other species was unknown. Very recently, the research group of Stephan Seiler showed that the DEP domain of NcRGF1, a Rom2 homologue in Neurospora crassa that acts as a specific GEF for NcRHO1, negatively selfregulates its GEF activity (16). We speculated that a similar selfinhibitory mechanism could also exist in Rom2.…”

“…remains to be demonstrated. Very recently, components of the CWI signaling pathway were also investigated in the filamentous fungus Neurospora crassa (15,16). Overall, it appears that the principal components are also conserved in filamentous fungi; however, the importance of the individual components for fungal physiology, antifungal drug susceptibility, and pathogenicity may differ and have different implications depending on the medical relevance of the organism.…”

The Plasma Membrane Proton Pump PMA-1 Is Incorporated into Distal Parts of the Hyphae Independently of the Spitzenkörper in Neurospora crassa

“…The homogenate was centrifuged at 5,000 ϫ g for 5 min at 4°C. The total extract was loaded onto a sucrose gradient as described previously (33). Density (Abbe refractometer; Carl Zeiss, Göttingen, Germany) and absorbance (280 nm, 6505 UV-visible [UV-Vis] spectrophotometer; Jenway) were determined for each fraction.…”