RacA and Cdc42 regulate polarized growth and microsclerotium formation in the dimorphic fungus Nomuraea rileyi

Small GTPases, RacA and Cdc42, act as molecular switches in fungi, regulating cell signaling, cytoskeletal organization, polar growth and reactive oxygen species (ROS) generation, the latter by influencing the activity of the NADPH oxidase complex. In this study, the racA and cdc42 genes from Nomuraea rileyi were cloned and shown to encode 218 and 184 amino acid proteins, respectively. To determine the functions of racA and cdc42, gene-silencing mutants (racARM, cdc42RM and racA&cdc42RM, respectively) were generated using RNA silencing technology. In racARM and cdc42RM, the conidial and microsclerotium (MS) yields, ROS production and virulence were reduced, the hyphal extension rate was decreased and the dimorphic switch was delayed. On the other hand, the double-silencing mutants showed growth retardation and virtually no conidia, MS or ROS production. The transcription levels of the noxA and noxR genes that regulate ROS generation were reduced in the three RNAi-silenced strains. Interestingly, when compared with the controls, racARM exhibited thicker hyphae and bigger conidia; moreover, the MS produced by racARM were bigger than those of the control and smaller than those of cdc42RM. Thus RacA and Cdc42 appear to share some essential functions in N. rileyi, including hyphal growth, conidiation, MS formation, ROS generation and virulence. Yet RacA appears to play a more pivotal role in the polar growth of N. rileyi.