Article Figures & Data
- FIG 1
Expression of N. crassa phytochrome genes phy-1 and phy-2. (A) Expression level of phytochromes across asexual development, assessed using gene expression microarrays (60). (B) Expression of phytochromes during sexual development using transcriptome sequencing (62). Expression levels are presented in fold change relative to the lowest time point, which was normalized to 1. Error bars represent 95% confidence intervals.
- FIG 2
Wild-type and phytochrome knockout strains all respond dynamically to light, but respond differently, especially for genes involved in sexual reproduction. Disruption of phytochromes affected expression of early sexual development genes in response to light but did not markedly affect expression of conidiation genes. (A) The conidiation genes con-6, con-8, and con-10, showed similar expression profiles among wild-type and mutant strains. (B) Highly correlated upregulation of expression of sexual development-related genes occurred immediately following light stimulation in the Δphy-2 KO strain. The log2 ratio of expression level was estimated based on analysis of microarray data (7). Expression is depicted under conditions of constant darkness (D) and following light exposure (5 to 240 min [LL5 to LL240]).
- FIG 3
Effects of light on sexual development in N. crassa. (A) Abundance of protoperithecia in constant darkness or exposed to red light, and white light for the Δphy-1, Δphy-2, Δphy-1 Δphy-2, and wild-type strains after 6 days of incubation. Error bars represent standard deviations. (B to D) The Δphy-2 strain was cultured for 6 days under constant red light and exhibited early sexual development of protoperithecia and perithecia. (B) Protoperithecia and early perithecia formed on the Δphy-2 strain side. (C) The asymmetry was even more evident after the cultures were placed under 3 days of constant white light for an additional 3 days. (D) The wild-type side of the crossing plate begins to mature at 6 days after shifting to continuous white light.
- FIG 4
Evolution of phytochromes in fungi. Homologs were color coded for phy-1 (red), phy-2 (green), and fphA (blue), and sequences were numbered as in Table S2B in the supplemental material. (A) Maximum likelihood phylogeny of fungal phytochromes. Branches with strong support (BP of >85% or BPP of >0.98) are in boldface. Species with multiple copies of phytochromes are marked with stars. A circular tree shows a distribution of phytochromes in major ascomycetous groups. (Phylogenetic details are provided in Fig. S6 in the supplemental material.) (B) Excerpts of amino acid alignments of the PAS, GAF, and PHY domains. The cysteine sites putatively functioning in chromophore binding are indicated by arrows. PHY-1 of N. crassa was used as a reference for positions of the cysteine sites. Fast-evolving phytochromes are marked with the symbol #.
- TABLE 1
Transcriptomic data analyzed for this study
Expt Species Conditions Accession no. Reference Asexual development Neurospora crassa Bird medium, constant light, wild type GSE26209 60 Sexual development N. crassa Carrot medium, constant light, wild type GSE41484 62 N. tetrasperma Carrot medium, constant light, wild type GSE60255 63 N. discreta Carrot medium, constant light, wild type GSE60266 63 Fusarium graminearum Carrot medium, constant light, wild type GSE61865 64 F. verticillioides Carrot medium, constant light, wild type GSE61865 64 Light response N. crassa Bird medium, dark and light conditions, wild-type and KO strains GSE8932 7
Supplemental Material
Figure S1
Expression level of phytochrome genes phy-1 and phy-2, measured by transcriptomic sequencing in Neurospora and Fusarium species during sexual development from protoperithecium (0 h) to fully matured perithecia (144 h after crossing) on carrot agar (63, 64). Expression levels are presented as fold change relative to the lowest time point, which was normalized to 1. Error bars represent 95% confidence intervals. (A) N. tetrasperma. (B) N. discreta. (C) Only phy-1 is present in Fusarium graminearum (fg) and F. vercillioides (fv). Download Figure S1, TIF file, 0.7 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
Expression level of genes’ responses to blue light, measured by transcriptomic sequencing in N. crassa during sexual development from protoperithecium (0 h) to fully matured perithecia (144 h after crossing) on carrot agar (62). Expression levels are presented as fold change relative to the lowest time point, which was normalized to 1. Genes are color coded, and error bars of 95% confidence intervals are too small to exhibit. Download Figure S2, TIF file, 0.6 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Knockout mutants of phytochromes affected expression levels genome-wide. Expression levels were estimated from published microarray results (7) using BAGEL (see Table S1 in the supplemental material). Inferences of expression were made for constant darkness (D), short light exposure (S [incorporating time points from 10, 15, and 30 min]), medium light exposure (M [incorporating time points from 45 and 60 to 90 min]), and long light exposure (L [incorporating time points from 120 to 240 min]) for genotypes of the wild-type, Δphy-1, Δphy-2, and Δphy-1 Δphy-2 strains. (A) Number of genes (y axis) showing a significant difference in expression level (P < 0.05) between genotypes under each light condition. (B) Number of genes showing a significant difference in expression (P < 0.05), including both higher and lower expression, between genotypes under different light conditions. Download Figure S3, TIF file, 1.3 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Expression level was estimated from microarray results in the 2009 article by Chen et al. (7) using BAGEL. Inferences of expression were made for constant darkness (D), short light exposure (S [incorporating time points from 10, 15, and 30 min]), medium light exposure (M [incorporating time points from 45, 60, and 90 min]), and long light exposure (L [incorporating time points from 120 and 240 min]) for genotypes of wild-type, Δphy-1, Δphy-2, and Δphy-1 Δphy-2 strains. Table S1, XLSX file, 1.5 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Supplemental information for gene expression and gene identification used in this study. (A) Functional enrichment analysis for genes significantly differentially expressed between genotypes under various light conditions, based on results in Table S1 in the supplemental material. (B) GenBank accession numbers for sequences analyzed and presented in Fig. 4. (C) JGI protein identifications for proteins analyzed and presented in Fig. S6 in the supplemental material. Table S2, PDF file, 0.3 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S6
Phylogeny of phytochromes in ascomycetes. Amino acid sequences were achieved from JGI fungal genome databases from eight major classes of ascomycetes (see Table S2C in the supplemental material), and homologs were color coded for phy-1 (red), phy-2 (green), and fphA (blue). The alignment has 116 sequences and 2,738 aligned positions. (A) Fifty percent majority consensus bootstrap tree based on 1,000 replicates of heuristic search for maximum parsimony trees using PAUP 4.0*. There were no significant conflicts in topology and branch support among maximum parsimony and neighbor joining analyses using PAUP 4.0* and maximum likelihood analyses using RAxML 8 (substitution matrix WAG with fixed base frequencies). (B) Consensus tree based on the last 8,000 trees sampled per 1,000 generations in Bayesian analyses using MrBayes 3.2 (2 runs, 10,000,000 generations, 4 chains, and mixed state frequencies with substitution rates). Branches with a Bayesian posterior probability higher than 98% are marked with *. Download Figure S6, TIF file, 1.2 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Crosses between Δphy-1 and Δphy-2 strains of N. crassa and sexual development under red light. Protoperithecia and perithecia were apparently more abundant on the phy-2 sides along the crossing zones. Cultures were photographed 6 days after inoculation. (A) View from above the culture plate. (B) View through the bottom of the plate. Download Figure S4, TIF file, 1.7 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
Impacts of light on different N. crassa cultures. (A) Crosses and sexual development in the N. crassa wild type on SCM and carrot medium under five different light conditions. While both media showed similar impacts on sexual development under different light conditions, morphological development is observed much easier on SCM plates. Incidentally, the finding that the phenotype of a sexual development-related gene does not change between carrot agar and SCM is consistent with the finding that developmental gene expression does not change much between these media (61). (B) Crosses were performed between wild-type mat A and mat a strains. From left to right, every triplet of crossing was under complete dark, constant white light, and red light conditions separately. Cultures were photographed 6 days after inoculation. (C) Noisy phenotypes of phytochrome knockouts under the blue light condition. Triplet crosses between phy-1 and phy-2 knockout strains and between knockout and wild-type strains of N. crassa and sexual development under blue light. The culture was photographed 6 days after inoculation. Protoperithecia and perithecia were slightly more abundant on the phy-1 sides along the crossing zones. (D) Three representative images (of the 30 hygromycin-resistant progeny from a WT × Δphy-2 strain cross on hygromycin SCM plates cultured under red light) demonstrate cosegregation of the removal of inhibition to initiation of sexual development and deletion of phy-2. Download Figure S5, PDF file, 2 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S7
Maximum likelihood phylogeny of sensory and output modules of ascomycetous phytochromes, rooted with basidiomycetous phytochromes. Strongly supported branches (BP of >85%) are represented by thick lines. Weakly supported branches (BP of <70%) are collapsed. Species with multiple copies of phytochromes are indicated by stars. Segments of several extremely long branches were deleted and replaced with “—//—” to optimize figure legibility. (A) Sensory module, including the PAS, GAF, and PHY domains. (B) Output module, including the HK and RR domains. Download Figure S7, TIF file, 0.8 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S8
The presence of the hph cassette in the target locus of the Δphy-1 and Δphy-2 strains was verified by PCR genotyping. The existence of targeting genes was verified by PCR products of primer pairs covering internal region of the gene (phy-1 or phy-2) and covering the 3′ and 5′ ends of the gene and its fringe regions. Correct replacements of targeting genes by the hph cassette are verified by PCR products of primers covering the 3′ and 5′ ends of the hph cassette and fringe regions of targeting genes. The crossing background of mus-51 and/or mus-52 was checked for knockout strains, and the gene coding for actin was used as a standard reference. Download Figure S8, TIF file, 0.4 MB.
Copyright © 2016 Wang et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
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, PDF - Figure sf5, PDF
- Figure sf6, TIF - Figure sf6, TIF
- Figure sf7, TIF - Figure sf7, TIF
- Figure sf8, TIF - Figure sf8, TIF
- Table st1, XLSX - Table st1, XLSX
- Table st2, PDF - Table st2, PDF
