The Adenylate-Forming Enzymes AfeA and TmpB Are Involved in Aspergillus nidulans Self-Communication during Asexual Development

Abstract

Aspergillus nidulans asexual sporulation (conidiation) is triggered by different environmental signals and involves the differentiation of specialized structures called conidiophores. The elimination of genes flbA-E, fluG, and tmpA results in a fluffy phenotype characterized by delayed conidiophore development and decreased expression of the conidiation essential gene brlA. While flbA-E encode regulatory proteins, fluG and tmpA encode enzymes involved in the biosynthesis of independent signals needed for normal conidiation. Here we identify afeA and tmpB as new genes encoding members the adenylate-forming enzyme superfamily, whose inactivation cause different fluffy phenotypes and decreased conidiation and brlA expression. AfeA is most similar to unknown function coumarate ligase-like (4CL-Lk) enzymes and consistent with this, a K544N active site modification eliminates AfeA function. TmpB, identified previously as a larger homolog of the oxidoreductase TmpA, contains a NRPS-type adenylation domain. A high degree of synteny in the afeA-tmpA and tmpB regions in the Aspergilli suggests that these genes are part of conserved gene clusters. afeA, tmpA, and tmpB double and triple mutant analysis as well as afeA overexpression experiments indicate that TmpA and AfeA act in the same conidiation pathway, with TmpB acting in a different pathway. Fluorescent protein tagging shows that functional versions of AfeA are localized in lipid bodies and the plasma membrane, while TmpA and TmpB are localized at the plasma membrane. We propose that AfeA participates in the biosynthesis of an acylated compound, either a p-cuomaryl type or a fatty acid compound, which might be oxidized by TmpA and/or TmpB, while TmpB adenylation domain would be involved in the activation of a hydrophobic amino acid, which in turn would be oxidized by the TmpB oxidoreductase domain. Both, AfeA-TmpA and TmpB signals are involved in self-communication and reproduction in A. nidulans.

Fungal Morphogenesis, from the Polarized Growth of Hyphae to Complex Reproduction and Infection Structures

“…TmpA is a plasma membrane oxidoreductase (279), while AfeA is an adenylate-forming enzyme related to fatty acyl-CoA synthetases and the plant 4-coumarate ligase-like enzyme At4g05160, involved in fatty acid activation and jasmonic acid biosynthesis. AfeA is localized in the plasma membrane, and like the oxylipin biosynthesis enzyme fatty acid dioxygenase PpoA, it is also localized in lipid bodies (280). The role of AfeA in oxylipin metabolism is further supported by the fact that afeA-null mutants show a clearly altered oxylipin profile (M. Reverberi and J. Aguirre, unpublished data).…”

“…One approach aimed at understanding what controls brlA expression consisted of the isolation of fluffy mutants that macroscopically show a proliferation of aerial hyphae and decreased conidiation (279)(280)(281). This led to the identification and characterization of the brlA upstream activators FluG, FlbA, FlbB, FlbC, FlbD, FlbE, TmpA, AfeA, and TmpB (262)(263)(264).…”

“…fluG overexpression induces brlA activation and conidiation in liquid-submerged cultures, while fluG-null mutants fail to produce an extracellular signal (284, 285) identified as a diorcinol-dehydroaustinol adduct that rescues fluG mutant conidiation defects (286). TmpB contains adenylation and oxidoreductase domains, an architecture shared by the bimodular ChNPS12/ETP nonribosomal peptide synthetases (NRPSs), and its inactivation produces a fluffy phenotype in the center of the colony but not in the periphery (280). This and the fact that dehydroaustinol is produced through a polyketide synthase (PKS)-mediated pathway (287) show that both PKSs and NRPSs are involved in the production of different sporulation signals.…”

“…The inactivation of the gmcA gene, encoding a putative glucosemethanol-choline oxidoreductase, also results in a clearly fluffy phenotype but only at alkaline pH. Colony cross-feeding experiments showed that GmcA is necessary for the synthesis of a metabolite that can be transmitted extracellularly (289), different from the sporulation signals produced through the FluG and the AfeA-TmpA pathways (279,280). The regulation of conidiation by secondary-metabolism-derived signals is conserved in other fungi, such as Fusarium fujikuroi (290).…”

“…The enzymes AfeA and TmpA are part of a single pathway that might be involved in oxylipin biosynthesis and is required for brlA expression and conidiation (280). TmpA is a plasma membrane oxidoreductase (279), while AfeA is an adenylate-forming enzyme related to fatty acyl-CoA synthetases and the plant 4-coumarate ligase-like enzyme At4g05160, involved in fatty acid activation and jasmonic acid biosynthesis.…”