Morphotype Transition and Sexual Reproduction Are Genetically Associated in a Ubiquitous Environmental Pathogen
Figure 5
Pum1 directs hyphal initiation and extension partially through a novel hypha-specific extracellular protein Fas1.
(A) FAS1, FAD1, and DHA1 are the genes encoding extracellular proteins overlapped in the regulons of Znf2 and Pum1. (B) Fas1 recapitulates the role of Pum1 in unisexual hyphal production. The fas1Δ mutant, like the pum1Δ mutant, showed reduced abundance of aerial hyphae. (C) The indicated strains were grown on V8 agar plate containing 200 µM BCS in the dark at 22°C. Images of the colonies were photographed after 5 days of incubation. Released Fas1 proteins were detected by colony immune-blot. Only overexpression of Fas1 with intact signal peptide led to a more robust filamentation and longer hyphae. Thus, the secretion of Fas1 is crucial for its biological function. (D) Fas1-mCherry is observed on hyphal surface and in vesicles at 72 hrs post unisexual mating stimulation. (E) The FAS1 expression level was positively regulated by Pum1 based on the transcriptional analysis of its transcript level in the pum1Δ mutant and the PGPD1-PUM1 strain by qPCR. The deletion or the overexpression of CFL1 did not significantly affect the FAS1 expression level. (F and G) Pum1 increased the intensity of Fas1 expression and reduces its stochasticity. The average intensity of Fas1-mCherry fluorescence was dramatically reduced in the absence of Pum1, but not Cfl1. The deletion of PUM1 led to a much higher variability in the Fas1-mCherry level among hyphal cells during unisexual filamentation (Inset of Figure? 5G). The frequencies of Fas1-mCherry's mean intensity in the hyphal population were plotted for the wildtype strain, the cfl1Δ mutant, and the pum1Δ mutant (software: ZEN 2011).