Putative RhoGAP proteins orchestrate vegetative growth, conidiogenesis and pathogenicity of the rice blast fungus Magnaporthe oryzae

• Magnaporthe oryzae encodes eight putative RhoGAP proteins.
• MoRga1 regulates conidial morphology and appressorial formation.
• MoLrg1 is required for conidiogenesis, vegetative growth and pathogenicity.
• Both MoRga1 and MoLrg1 interact with MoRac1 and MoCdc42.
• LIM domain, RhoGAP domain and Arginine 1032 are all essential for MoRga1 function.

Rho GTPases, acting as molecular switches, are involved in the regulation of diverse cellular functions. Rho GTPase activating proteins (Rho GAPs) function as negative regulators of Rho GTPases and are required for a variety of signaling processes in cell development. But the mechanisms underlying Rho GAPs in Rho-mediated signaling pathways in fungi are still elusive. There are eight RhoGAP domain-containing genes annotated in the Magnaporthe oryzae genome. To understand the function of these RhoGAP genes, we generated knockout mutants of each of the RhoGAP genes through a homologous recombination-based method. Phenotypic analysis showed that growth rate of aerial hyphae of the Molrg1 deletion mutant decreased dramatically. The ΔMolrg1 mutant showed significantly reduced conidiation and appressorium formation by germ tubes. Moreover, it lost pathogenicity completely. Deletion of another Rho GAP (MoRga1) resulted in high percentage of larger or gherkin-shaped conidia and slight decrease in conidiation. Appressorial formation of the ΔMoRga1 mutant was delayed significantly on hydrophobic surface, while the development of mycelial growth and pathogenicity in plants was not affected. Confocal fluorescence microscopy imaging showed that MoRga1-GFP localizes to septal pore of the conidium, and this localization pattern requires both LIM and RhoGAP domains. Furthermore, either deleting the LIM or RhoGAP domain or introducing an inactivating R1032A mutation in the RhoGAP domain of MoRga1 caused similar defects as the Morga1 deletion mutant in terms of conidial morphology and appressorial formation, suggesting that MoRga1 is a stage-specific regulator of conidial differentiation by regulating some specific Rho GTPases. In this regard, MoRga1 and MoLrg1 physically interacted with both MoRac1-CA and MoCdc42-CA in the yeast two-hybrid and pull-down assays, suggesting that the actions of these two GAPs are involved in MoRac1 and MoCdc42 pathways. On the other hand, six other putative Rho GAPs (MoRga2 to MoRga7) were dispensable for conidiation, vegetative growth, appressorial formation and pathogenicity, suggesting that these Rho GAPs function redundantly during fungal development. Taking together, Rho GAP genes play important roles in M. oryzae development and infectious processes through coordination and modulation of Rho GTPases.