Cfl1 and Pum1 represent two major circuits downstream of Znf2 in directing aerial hyphae formation.

<p>(A) Transcriptional analysis by qPCR indicates that alterations of Cfl1 or Pum1 do not affect the transcript level of each other. The expression level of genes in the wildtype strain XL280 was arbitrarily set as 1 for comparison. (B) The production or the release of Cfl1 proteins was not drastically affected in the <i>pum1</i>Δ mutant. The expression of Cfl1 proteins was abolished in the <i>znf2</i>Δ mutant and enhanced when the compatible mating partner was present. The mCherry-labeled Cfl1 was used to measure the Cfl1 protein level by fluorescence microscopy and released Cfl1 proteins was detected by colony immunoblot. (C) Pum1 is dispensable for the expression or the processing of Cfl1. By comparison, the Cfl1 protein expression was below the detectable level in the <i>znf2</i>Δ mutant. This served as a negative control. The asterisk indicates the non-specific band, which is also shown in control strain XL280 without mCherry. (D) Double deletion of <i>CFL1</i> and <i>PUM1</i> resulted in a much more severe defect in aerial filamentation during bisexual mating compared to either of the single deletion. (E) Pum1 and Cfl1 represent two major downstream regulatory branches of Znf2 in initiating filamentation. (F) Pum1 is also involved in hyphal extension. The deletion of <i>PUM1</i> resulted in shorter filaments during bisexual mating. Such a phenotype was not observed in the <i>cfl1</i>Δ mutant. Scale bar: 20<i> µ</i>m.