Figure 1.
(A) Colonies of WT (RNIW5) and multi-copy vosA (MvosA) strains grown on solid MM at 37°C for 3 d together with the close-up views (lower panel). (B) A phylogenetic tree of proteins similar to VosA generated by MegAlign in Lasergene v7.0 (DNASTAR). ClustalV method was used for protein alignment. An: A. nidulans, Af: A. fumigatus, Ao: A. oryzae, Ap: A. parasiticus, Ci: Coccidioides immitis, Mg: Magnaporthe grisea; Cg: Chaetomium globosum; Nc: Neurospora crassa; Um: Ustilago maydis; Cn: Cryptococcus neoformans; Gz: Gibberella zeae. (C) Levels of vosA mRNA throughout the lifecycle of WT. Numbers indicate the time (h) of incubation in liquid MM (Veg) and post-asexual (Asex) or sexual (Sex) developmental induction. C and Ac represent conidia and ascospores. Last lane shows elevated vosA mRNA level in the MvosA colony grown on solid MM for 3 d. (D) Levels of the VosA protein throughout the lifecycle of a vosA(p)::VosA::FLAG strain (TNI10.34.1). Arrows indicate two protein bands. (E) Levels of AfvosA mRNA during the lifecycle of A. fumigatus WT.
Figure 2.
Phenotypes resulting from ΔvosA.
(A) Photographs of the colonies of WT (FGSC26), ΔvosA (RNI10.2) and complemented (ΔvosA+vosA-FLAG; TNI10.34.1) strains grown on solid MM for 3 d. (B) Viability of the conidia of WT and ΔvosA strains grown at 37°C for 2, 5, 10 and 20 days. (C) Photomicrographs of 20 d old conidia, 3 month old cleistothecia and ascospores of ΔvosA and WT strains. (D) TEM images of 15 d old conidiophores, 3 d and 8 d old conidia, and 24 d old ascospores of ΔvosA and WT strains.
Figure 3.
Requirement of VosA for trehalose accumulation and stress tolerance.
(A) The amount of trehalose (pg) per conidium in the freshly collected 2 d old conidia of WT (FGSC4, veA+ and FGSC26, veA1) and ΔvosA (RNI14.1, ΔvosA; veA+ and RNI10.2, ΔvosA; veA1) strains (triplicate). No trehalase treatment served as a negative control. (B) Levels of tpsA, tpsC, orlA, treA and treB transcripts in WT (FGSC26) and ΔvosA (RNI10.2) strains. (C–D) Tolerance of WT (filled bar) and the ΔvosA mutant (open bar) to heat (C) or oxidative (D) stress.
Figure 4.
VosA is a negative regulator of development.
(A) Hyperactive conidiation caused by ΔvosA (photographed at 24 h in liquid MM). The arrows indicate conidiophores. (B) Levels of vosA, brlA, rodA, abaA, yA, wetA and wA transcripts in WT (FGSC26) and ΔvosA (RNI10.2) strains in liquid MM (Veg) and post developmental induction (Asex). A dot between the ΔvosA Veg 18 and 24 lanes in abaA hybridization is an artifact. (C) Photographs of the colonies of WT (FGSC26) and vosA overexpression (TNI9.1) strains grown on non-inducing (MMG) and inducing (MMT + YE) medium at 37°C for 3 d. (D) Northern blot for levels of vosA and brlA mRNA in control (TJA53.1) and alcA(p)::vosA (TNI9.1) strains.
Figure 5.
VosA is a potential TF and localizes in the nucleus of mature conidia.
(A) Designated transformants were spotted in serial dilutions on Ura- MM (u-) and Ura-His- MM (u-h-) with various concentrations of 3AT. Yeast colonies were photographed after incubation at 30°C for 48 h. Mean LacZ activity (+/−SD; triplicate) is shown. (B) A simplified diagram of a conidiophore. (C) Control (gpdA[p]::RFP; TNI20.1) and vosA(p)::VosA::RFP (TNI13.3) strains were incubated on solid MM at 37°C for 24 and 48 h. Conidiophores were fixed and stained with Hoechst 33258 and images of DIC, RFP, Hoechst and the merge of RFP and Hoechst are shown. Yellow arrow indicates newly formed conidia and white arrow indicates mature conidia. Note that VosA mainly localizes in the nucleus of mature conidia.
Figure 6.
Model for regulation of conidiation (see text).