Article Figures & Data
- FIG 1
A. fumigatus isolates induce pigmentation or conidiation in response to light. Conidia of the indicated strain were point inoculated onto GMM (plate pictures) or RPMI 1640 (graphs) and incubated for 48 h either in constant darkness or under constant white light illumination. Conidiation values are normalized to the dark-grown sample of the indicated strain. Enumeration of conidia was performed in triplicate, and data were statistically analyzed by Student’s t test (*, P ≤ 0.05).
- FIG 2
The photoconidiation response is blue light and lreA dependent. (A) Conidia were point inoculated onto RPMI 1640 plates and incubated for 6 days in an alternating 12-h-light/12-h-dark photocycle (left column). Alternatively, plates were transferred to constant darkness following an initial 2-day incubation in the 12-h-light/12-h-dark environment (right column). (B) Conidia were spread across RPMI 1640 plates and incubated for 48 h either in the dark or under constant illumination under red or blue LEDs. Conidial counts are normalized to the respective dark-grown sample. Experiments were performed in triplicate, and total conidia were compared by Student’s t test (**, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant). (C) Conidia were spread across RPMI 1640 plates and incubated for 48 h either in constant darkness or under constant white light illumination. Experiments were performed in triplicate, and error bars represent the mean conidial counts (±standard deviation). Groups were compared by Student’s t test (*, P ≤ 0.05). (D) Scheme of LreA-dependent regulation of asexual development in A. nidulans versus A. fumigatus.
- FIG 3
The photosensory control of germination and cell wall homeostasis differs between Af293 and CEA10. (A) Conidia were inoculated into GMM broth and incubated at 37°C for 8 h either in constant darkness or under constant illumination (blue plus red LEDs). A minimum of 100 conidia were scored for the presence or absence of a germ tube in each group, and light and dark samples were compared by the chi-square test (*, P ≤ 0.05). (B) Conidia of the indicated CEA10 genotype were spot inoculated onto GMM plus Congo red agar and incubated at 37°C for 48 h in constant darkness or under constant illumination (blue plus red LEDs).
- FIG 4
Virulence is variable across isolates and is independent of either the overt light response or lreA. (A) All animals were immunosuppressed with a single dose of triamcinolone acetonide (day −1) and inoculated intranasally with 1.0 × 106 conidia of the indicated isolate (CEA10, n = 8; Af293, n = 11; DCF-3 and DCF-6, n = 12). (B) Left, CEA10 background. The wild-type curve is from the same experiment as that depicted in panel A; the ΔlreA group was run concurrently with the WT and under the same protocol. Survival curves are not statistically significant (P = 0.16). Right, DCF-3 background. Groups of 10 CD-1 mice were immunosuppressed with two doses of Kenalog (days −1 and +3) and inoculated intranasally with the indicated genotype. Two independently isolated ΔlreA mutants are shown. WT versus ΔlreA-1, P = 0.14; WT versus ΔlreA-2, P = 0.03. All statistical analyses were performed with the log rank test.
Supplemental Material
Table S1
Strains used in this study Table S1, DOCX file, 0.1 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S1
Photopigmentation and photoconidiation responses of additional A. fumigatus isolates. Conidia of the indicated strain were point inoculated onto GMM (plate pictures) or RPMI 1640 (graphs) and incubated for 48 h either in constant darkness or under constant white light illumination. Conidiation values are normalized to the dark-grown sample of the indicated strain. Enumeration of conidia was performed in triplicate, and data were statistically analyzed by Student’s t test (*, P ≤ 0.05). Download Figure S1, TIF file, 50.2 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S2
(A) The photoconidiation response is more pronounced on RPMI 1640 medium (R) than on GMM (G). Experiments were performed in triplicate, and groups were compared by Student’s t test (*, P ≤ 0.05). (B) The overall conidiation levels are variable across the strains. Values are normalized to the Af293 DD sample. (C) The effect of light on growth rate is minimal for each of the tested isolates. Conidia were point inoculated on GMM and incubated for 3 days at 37°C in constant darkness or under constant white light illumination. Download Figure S2, TIF file, 66.9 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Table S2
Summary of photoresponsive behaviors in the analyzed isolates. Photopigmenting isolates are shaded. Table S2, DOCX file, 0.1 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S3
(A) RT-PCR analysis of lreA expression. Strains were incubated in the dark for 48 h and then transferred to constant white light illumination for the indicated time points. (B) Schematic depiction of the split-marker deletion strategy of lreA is shown as well as an RT-PCR demonstrating loss of the lreA transcript in the putative knockouts. hph, hygromycin phosphotransferase gene. (C) Schematic depiction of the split-marker deletion strategy of fphA is shown as well as an RT-PCR demonstrating loss of the fphA transcript in either the CEA10 WT or ΔlreA background. (D) RT-PCR demonstrating the transcript of either lreA (top) or fphA (bottom) in the respective complemented strain. Download Figure S3, TIF file, 40.8 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S4
Light decreases germination rates in most A. fumigatus isolates. Conidia were inoculated into GMM and incubated for 8 h either in constant darkness or under constant white light illumination. A minimum of 300 conidia were scored for the presence or absence of a germ tube in each group, and light and dark samples were compared by the chi-square test (*, P ≤ 0.05). Download Figure S4, TIF file, 9 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S5
FphA protein sequence alignment based on Sanger sequencing from the indicated isolates. Download Figure S5, TIF file, 52.1 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S6
Additional phenotypes of the CEA10 photoreceptor mutants. (A) Pigmentation. Conidia were point inoculated into GMM plates and incubated at 37°C for 48 h either in constant darkness or under constant illumination (blue plus red LEDs). Several independent isolates of the ΔfphA mutation are shown and are distinguished numerically (e.g., 5-1, 7-2). (B) Congo red sensitivity. Conidia were point inoculated onto GMM plates supplemented with the indicated concentration of Congo red. Plates were then incubated for 48 h at 37°C for 48 h either in constant darkness or under constant illumination (blue plus red LEDs). Download Figure S6, TIF file, 26.5 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S7
Deletion of lreA in the Af293 background does not attenuate virulence. (A) Single-dose steroid model. Groups of 12 CD-1 mice were immunosuppressed with Kenalog-10 (day −1) and inoculated intranasally with 2.0 × 106 conidia of the indicated genotype. (B) Multidose steroid model. Groups of 16 CD-1 mice were immunosuppressed with two doses of Kenalog-10 (days −1 and +3) and inoculated intranasally with 1.5 × 106 conidia of the indicated genotypes. Survival curves are not statistically different (P = 0.22, log rank test). (C) Chemotherapy (neutropenia model). Groups of 17 CD-1 mice were immunosuppressed with cyclophosphamide (days −2 and +3) and Kenalog-10 (day −1) and inoculated intranasally with 2.0 × 106 conidia of the indicated genotypes. Survival curves are not statistically different (P = 0.4, log rank test). Download Figure S7, TIF file, 32.4 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Figure S8
VeA protein alignment based on Sanger sequencing from the indicated isolates. Download Figure S8, TIF file, 63.3 MB.
Copyright © 2016 Fuller et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
Additional Files
Supplementary Data
Supplementary Data
- Figure sf1, TIF - Figure sf1, TIF
- Figure sf2, TIF - Figure sf2, TIF
- Figure sf3, TIF - Figure sf3, TIF
- Figure sf4, TIF - Figure sf4, TIF
- Figure sf5, TIF - Figure sf5, TIF
- Figure sf6, TIF - Figure sf6, TIF
- Figure sf7, TIF - Figure sf7, TIF
- Figure sf8, TIF - Figure sf8, TIF
- Table st1, DOCX - Table st1, DOCX
- Table st2, DOCX - Table st2, DOCX
