Abstract
Coprinopsis cinerea has seven homologs of the Aspergillus nidulans transcription factor NsdD. Of these, CcNsdD1 and CcNsdD2 from C. cinerea show the best identities of 62 and 50% to A. nidulans NsdD, respectively. After 4 days of constant darkness cultivation, CcnsdD2, but not CcnsdD1, was upregulated on the first day of light/dark cultivation to induce fruiting bodies, and overexpression of CcnsdD2, but not CcnsdD1, produced more fruiting bodies under a light/dark rhythm. Although single knockdown of CcnsdD2 did not affect fruiting body production due to upregulation of its homolog CcnsdD1, the double-knockdown CcNsdD1/NsdD2-RNAi transformant showed defects in fruiting body formation under a light/dark rhythm. Knockdown of CcnsdD1/nsdD2 led to the differentiation of primary hyphal knots into sclerotia rather than secondary hyphal knots under a light/dark rhythm, similar to the differentiation of primary hyphal knots into sclerotia of the wild-type strain under darkness. The CcNsdD2-overexpressing transformant produced more primary hyphal knots, secondary hyphal knots, and fruiting bodies under a light/dark rhythm but only more primary hyphal knots and sclerotia under darkness. RNA-seq revealed that some genes reported previously to be involved in formation of hyphal knots and primordia, cyclopropane-fatty-acyl-phospholipid synthases cfs1-3, galectins cgl1-3, and hydrophobins hyd1-3 were downregulated in the CcNsdD1/NsdD2-RNAi transformant compared to the mock transformant. ChIP-seq and electrophoretic mobility shift assay demonstrated that CcNsdD2 bound to promoter regulatory sequences containing a GATC motif in cfs1, cfs2, cgl1, and hyd1. A molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions is proposed. IMPORTANCE The model mushroom Coprinopsis cinerea exhibits remarkable photomorphogenesis during fruiting body development. This study reports that the C. cinerea transcription factor CcNsdD2 promotes primary hyphal knot formation by upregulating cfs1, cfs2, cgl1, and hyd1. Although the induction of CcnsdD2 is not under direct control of light and photoreceptors, the CcNsdD2-mediated developmental fates of the primary hyphal knots depend on the following light/dark rhythm cultivation or dark cultivation after full growth of mycelia in the constant dark cultivation. This study provides new insight into the molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions. In addition, the result that overexpression of CcnsdD2 induced more secondary hyphal knots, primordia, and fruiting bodies under light/dark rhythm cultivation conditions has potential applied value in the edible mushroom industry.