Abstract
In Saccharomyces cerevisiae, phosphate acquisition enzymes are regulated by a cyclin-dependent kinase (Pho85), a cyclin (Pho80), the cyclin-dependent kinase inhibitor Pho81, and the helix-loop-helix transcription factor Pho4 (the PHO system). Previous studies in Aspergillus nidulans indicate that a Pho85-like kinase, PHOA, does not regulate the classic PHO system but regulates development in a phosphate-dependent manner. A Pho80-like cyclin has now been isolated through its interaction with PHOA. Surprisingly, unlike PHOA, An-PHO80 does play a negative role in the PHO system. Similarly, an ortholog of Pho4 previously identified genetically as palcA also regulates the PHO system. However, An-PHO81, a putative cyclin-dependent kinase inhibitor, does not regulate the PHO system. Therefore, there are significant differences between the classic PHO system conserved between S. cerevisiae and Neurospora crassa compared with that which has evolved in A. nidulans. Most interestingly, under low phosphate conditions, the An-PHO80 cyclin also promotes sexual development while having a negative effect on asexual development. These effects are independent of the role An-PHO80 has in the classic PHO system. However, in high phosphate medium, An-PHO80 affects development because of deregulation of the PHO system as loss of palcA(Pho4) function negates the developmental defects caused by lack of An-pho80. Therefore, under low phosphate conditions the An-PHO80 cyclin regulates development independently of the PHO system, whereas in high phosphate it affects development through the PHO system. The data indicate that a single cyclin can control various aspects of growth and development in a multicellular organism.
Highlights
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY590766 and AY590767
The PHO80-like cyclin named An-pho80 is the focus of this work
We generated a null allele of the Pho81 ortholog of A. nidulans (AN4310.2, which shares 28% identity with yeast Pho81 and 52% with N. crassa NUC-2; see supplemental Fig. S1) and tested whether the absence of this putative Cyclin-dependent kinases (CDKs) inhibitor would prevent expression of Pi acquisition genes under low Pi conditions. Using both plate assays and protein extracts assays we found no evidence that An-PHO81 plays a role in regulating Pi acquisition enzymes in A. nidulans (Fig. 4, A and B)
Summary
A. nidulans Strains and Medium—The A. nidulans strains used in this study are listed in Table I, and all contain the veA1 marker. Yeast Two-hybrid Screening—A bait plasmid (pH1) was constructed by cloning the phoA cDNA into the EcoRI and BamHI site of the vector pGBKT7 (with c-Myc epitope tag, Clontech). In Vitro Interaction Assay of Proteins—The An-pho cDNA isolated from a yeast two-hybrid screen was subcloned into EcoRI and XhoI sites of vector pGADT7 (with HA epitope tag, from Clontech) resulting in plasmid pGAD36. This plasmid and the bait plasmid pH1 were used to express PHOA and An-PHO80 protein in vitro using the TNT Quick Coupled Transcription/Translation system (Promega). Southern blots were probed by an An-pho80specific DNA fragment or an An-pho flanking sequence using ECL random primer labeling and detection system (Amersham Biosciences). The constructed plasmid was named pAL36 with An-pho C-terminally tagged with two tandem repeats of the HA epitope under control of the inducible alcA promoter
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