p21-activated kinase is involved in the sporulation, pathogenicity, and stress response of Arthrobotrys oligospora under the indirect regulation of Rho GTPase-activating protein

The p21-GTPase-activated protein kinases (PAKs) participate in signal transduction downstream of Rho GTPases, which are regulated by Rho GTPase-activating proteins (Rho-GAP). Herein, we characterized two orthologous Rho-GAPs (AoRga1 and AoRga2) and two PAKs (AoPak1 and AoPak2) through bioinformatics analysis and reverse genetics in Arthrobotrys oligospora, a typical nematode-trapping (NT) fungus. The transcription analyses performed at different development stages suggested that Aopaks and Aorga1 play a crucial role during sporulation and trap formation, respectively. In addition, we successfully deleted Aopak1 and Aorga1 via the homologous recombination method. The disruption of Aopak1 and Aorga1 caused a remarkable reduction in spore yield and the number of nuclei per cell, but did not affect mycelial growth. In Delta Aopak1 mutants, the trap number was decreased at 48 h after the introduction of nematodes, but nematode predatory efficiency was not affected because the extracellular proteolytic activity was increased. On the contrary, the number of traps in Delta Aorga1 mutants was significantly increased at 36 h and 48 h. In addition, Aopak1 and Aorga1 had different effects on the sensitivity to cell-wall-disturbing reagent and oxidant. A yeast two-hybrid assay revealed that AoPak1 and AoRga1 both interacted with AoRac, and AoPak1 also interacted with AoCdc42. Furthermore, the Aopaks were up-regulated in Delta Aorga1 mutants, and Aorga1 was down-regulated in Delta Aopak1 mutants. These results reveal that AoRga1 indirectly regulated AoPAKs by regulating small GTPases.