An endogenous inducer of sexual development in Aspergillus nidulans

During development of the homothallic ascomycete Aspergillus nidulans, asexual sporulation is followed by sexual sporulation. We report here the detection of a solvent-extractable activity which inhibits asexual sporulation and stimulates premature sexual sporulation. This activity, called precocious sexual inducer (psi), is overproduced by certain mutants that are blocked in both modes of sporulation. Using partially purified preparations of psi, biological response could be elicited with as little as 50 ng of material. We suggest that psi is a hormone-precursor which is converted to a hormone by normal sporulating strains that respond to psi, but not by the asporogenous mutants that overproduce psi. The stability of psi activity gives promise that the compound can be purified and identified.     

The Aspergillus nidulans phytochrome FphA represses sexual development in red light

Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.     

The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

Differential expression of house-keeping genes of Aspergillus nidulans during sexual development

The rpl3 gene and the rpl37 gene for Aspergillus nidulans ribosomal protein L3 (RPL3) and RPL37, which were identified as located on chromosome I and chromosome III, respectively, were isolated from chromosome-specific cosmid libraries. The nucleotide sequences of both of the rpl3 gene and the rpl37 gene identified the ORFs of 392 amino acids and 92 amino acids, respectively. Both of the two genes were present in a single copy. The expression of both genes together with two other house-keeping genes, the rps16 gene for RPS16 and the gene for gamma-actin, was analyzed during sexual development. All four genes showed nearly identical expression patterns in that each gene expression reached its maximum after 2 h, decreased thereafter, and increased again after 30-40 h of induction of sexual development.     

abaA controls phialide differentiation in Aspergillus nidulans.

Aspergillus nidulans is an ascomycetous fungus that reproduces asexually by forming multicellular conidiophores and uninucleate spores called conidia. Loss of function mutations in the abacus A (abaA) regulatory locus result in formation of aberrant conidiophores that fail to produce conidia. Wild-type conidiophores form two tiers of sterigmata. The first tier, metulae, divide to produce the second tier, phialides. Phialides are sporogenous cells that produce conidia through a specialized apical budding process. We have examined conidiophore development in an abaA- strain at the ultrastructural level. The results showed that in the mutant metulae produce supernumerary tiers of cells with metula-like, rather than phialide-like, properties. Temperature shift experiments with an abaA14ts strain demonstrated that abaA+ function induced phialide formation by the aberrant abacus cells and was continuously required for maintenance of phialide function. In the absence of abaA+ activity, metulae simply proliferated and later developmental steps never occurred. We conclude that abaA+ directs the differentiation of phialides and is continuously required for maintenance of their function.     

A G protein-coupled receptor for UDP-glucose

Uridine 5'-diphosphoglucose (UDP-glucose) has a well established biochemical role as a glycosyl donor in the enzymatic biosynthesis of carbohydrates. It is less well known that UDP-glucose may possess pharmacological activity, suggesting that a receptor for this molecule may exist. Here, we show that UDP-glucose, and some closely related molecules, potently activate the orphan G protein-coupled receptor KIAA0001 heterologously expressed in yeast or mammalian cells. Nucleotides known to activate P2Y receptors were inactive, indicating the distinctly novel pharmacology of this receptor. The receptor is expressed in a wide variety of human tissues, including many regions of the brain. These data suggest that some sugar-nucleotides may serve important physiological roles as extracellular signaling molecules in addition to their familiar role in intermediary metabolism.     

A putative G protein-coupled receptor involved in innate immune defense of Procambarus clarkii against bacterial infection

The immune functions of G protein-coupled receptor (GPCR) were widely investigated in mammals. However, limited researches on immune function of GPCRs were reported in invertebrates. In the present study, the immune functions of HP1R gene, a putative GPCR identified from red swamp crayfish Procambarus clarkii were reported. Expression of HP1R gene was significant up-regulated in response to heat-killed Aeromonas hydrophila challenge. HP1R gene silencing mediated by RNA interference significantly enhanced the susceptibility of red swamp crayfish to A. hydrophila and Vibrio alginolyticus, indicating that HP1R was required for red swamp crayfish to defend against bacterial challenge. In HP1R-silenced crayfish, increased bacterial burden and decreased THC in response to bacterial challenge were observed when compared with control crayfish. No significant difference of proPO gene expression was observed between HP1R-silenced and control crayfish after challenge with heat-killed A. hydrophila. However, PO activity in response to bacterial challenge was significantly reduced in HP1R-silenced crayfish. The results collectively indicated that HP1R was an important immune molecule which was required for red swamp crayfish to defend against bacterial infection.     

Constitutively active G protein-coupled receptor mutants block dictyostelium development

cAR1, a G protein-coupled receptor (GPCR) for cAMP, is required for the multicellular development of Dictyostelium. The activation of multiple pathways by cAR1 is transient because of poorly defined adaptation mechanisms. To investigate this, we used a genetic screen for impaired development to isolate four dominant-negative cAR1 mutants, designated DN1-4. The mutant receptors inhibit multiple cAR1-mediated responses known to undergo adaptation. Reduced in vitro adenylyl cyclase activation by GTPgammaS suggests that they cause constitutive adaptation of this and perhaps other pathways. In addition, the DN mutants are constitutively phosphorylated, which normally requires cAMP binding and possess cAMP affinities that are approximately 100-fold higher than that of wild-type cAR1. Two independent activating mutations, L100H and I104N, were identified. These residues occupy adjacent positions near the cytoplasmic end of the receptor's third transmembrane helix and correspond to the (E/D)RY motif of numerous mammalian GPCRs, which is believed to regulate their activation. Taken together, these findings suggest that the DN mutants are constitutively activated and block development by turning on natural adaptation mechanisms.     

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA⁻ mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA⁻ mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA⁻ mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.