Identification and functional characterization of the putative members of the CTDK-1 kinase complex as regulators of growth and development in Aspergillus nidulans and Aspergillus fumigatus

Abstract

ABSTRACT Asexual spores are the main vehicle used by fungi to disperse to new niches. The Eurotiomycete Aspergillus nidulans is the main reference for the study of the genetic/molecular control of asexual development. In this species, Flb proteins control the expression of the master gene brlA, and thus, loss-of-function mutations in flb (upstream developmental activation [UDA]) genes block brlA transcription and, consequently, the production of conidiophores, the structures bearing asexual spores known as conidia. However, the aconidial phenotype of specific flb mutants, such as that of the Δ flbB strain, is reverted under salt-stress conditions. Previously, we generated a collection of second-site mutants of Δ flbB unable to conidiate on culture medium supplemented with NaH 2 PO 4 (0.65 M). Here, we identified a Gly347Stop mutation within flpA as responsible for the FLIP57 phenotype and characterized the role of the putative cyclin FlpA and the remaining putative components of the C-terminal domain kinase-1 (CTDK-1) complex in A. nidulans and Aspergillus fumigatus . FlpA, Stk47, and FlpB are necessary (i) for timely germination, (ii) in the transition from metulae to phialides (the cells generating conidia) during conidiophore development, and (iii) for the development of sexual structures (cleistothecia) in A. nidulans . The three proteins are nuclear, and the nucleoplasmic localization of Stk47 depends on the activity of FlpA, which correlates with the retention of Stk47 by FlpA in pull-down assays. Overall, this work links the putative CTDK-1 complex of aspergilli with growth and developmental control. Identification of a mutation in flpA as inhibitor of conidiation in A. nidulans and functional characterization of FlpA, Stk47 and FlpB as putative members of the C-terminal domain kinase complex CTDK-1 in A. nidulans and A. fumigatus . IMPORTANCE Aspergillus fumigatus has been included by the World Health Organization in the priority list of fungal pathogens because (i) it causes 90% of invasive aspergillosis cases, with a high mortality rate, and (ii) infections are becoming increasingly resistant to azole antifungals. A. nidulans is an opportunistic pathogen and a saprotroph which has served during the last 80 years as a reference system for filamentous fungi. Here, we characterized the role in morphogenesis and development of the putative transcriptional cyclin/kinase complex CTDK-1 in both aspergilli. The null mutants of the corresponding genes showed delayed germination, aberrant conidiophore development, and inhibition of cleistothecia production. While in higher eukaryotes this complex is formed only by a cyclin and a kinase, the fungal complex would incorporate a fungal-specific third component, FlpB, which would enable the interaction between the kinase (Stk47) and the cyclin (FlpA) and may be used as a target for antifungals.