Genome-wide transcriptional responses to light in S. fimicola

To elucidate the transcriptional regulatory networks in response to light in S.

fimicola, we analyzed blue-light-induced genome-wide transcriptional responses by high-throughput RNA-seq to reveal the transcriptional profiles between the wild type and Sfwc-1^(∆lov) mutant. We aimed to use the data from this approach to identify differentially expressed genes for RT-qPCR verification and future studies. Untreated mycelia (constant darkness sample) and mycelia treated with blue light for 15 and 45 min were labeled WTDD, WT15, and WT45, respectively, for the wild type, and ADD, A15, and A45 for the Sfwc-1^(∆lov) mutant. The N. crassa database was used as reference for predicting putative genes in S. fimicola.

Genome-wide expression profiling of Sordaria wild type and the Sfwc-1^(∆lov) mutant was performed with the Illumina sequencing platform and analyzed with the Trinity program. A total of 62,274 transcript isoforms were de novo assembled and annotated via Blast search. The estimated abundance of these transcripts were quantified at each time point and these was categorized as “expressed genes” under these growth conditions. Using fold change ≥2, FDR p-value <0.005 as the cut-offs for significance, we identified 874 genes differentially expressed in at least one pairwise comparison under illumination or darkness conditions in either S. fimicola wild type or Sfwc-1^(∆lov) mutant. The putative genes were described as SF_ (N. crassa accession number).

Among photoreceptor genes, cytochrome (Sfcryp; SF_NCU00582) and vivid (Sfvivid:

SF_NCU03967) genes were differentially expressed in the wild type upon light induction, and the expression was unaltered in the Sfwc-1^(∆lov) mutant under the same condition (Krobanan et al., 2019). The expression levels of Sfcryp and Sfvivid were confirmed by RT-qPCR analysis and both genes were induced by light via SfWC-1 regulation (Fig. 9). From Venn diagram analysis, 466 putative genes showed differential expression in the wild type, but not in Sfwc-1^(∆lov) mutant, after exposure to light for 15 and 45 min relative to constant darkness (DD) (Fig. 12A; Krobanan et al., 2019).

Moreover, 590 putative genes showed differential regulation between the wild type and Sfwc-1^(∆lov) mutant at each time point (constant darkness and after illumination by blue light for 15 and 45 min) (Fig. 12B; Table S6). In addition to 466 putative genes, we also found that some genes showed differential expression after 15 min relative to 45 min blue light induction. Therefore, a total of 478 putative genes showed significantly differential expression at least two-fold for at least one time point in response to light in the wild type, but not the Sfwc-1^(∆lov) mutant (Krobanan et al., 2019). The differential expression seen in the Sfwc-1^(∆lov) mutant after blue light induction indicates that additional blue light photoreceptor may exist and function. Genes differentially expressed in at least one time point in response to blue light in the wild type but not Sfwc-1^(∆lov) were also subjected to GO analysis for functional annotation to reveal their potential functions. Significantly, enriched GO terms (p ≤ 0.05) were oxidation-reduction processes, oxidoreductase activity, heme binding and single-organism metabolic processes (Fig. 13A, (Krobanan et al., 2019). To better understand the potential pathways regulated by Sfwc-1, the same set of putative genes were further assessed by catagorizing functions using FunCat. The enrichment of specific metabolic

pathways influenced by Sfwc-1 included metabolism of melanin, metabolism involving polyketides, ABC transporters, rhythm (e.g., circadian), metabolism of vitamins, cofactors, and prosthetic groups, lipid, fatty acid and isoprenoid metabolism, detoxification, secondary metabolism and other pathways (Fig 13B; (Krobanan et al., 2019).

Based on the changes in expression (estimated abundance) of 478 putative genes, 306 genes were upregulated and 172 genes were downregulated in the wild type, but not in Sfwc-1^(∆lov) strain, relative to darkness and illumination conditions. Genes which showed differential expression in response to light were further grouped into 4 clusters (Krobanan et al., 2019). Patterns of changes in gene expression in each group were upslope, hill, downslope and valley for cluster 1 to 4 respectively.

Cluster 1 included genes that were typically induced by light in a Sfwc-1 dependent manner and were further subdivided into cluster 1a and 1b based on different levels of estimated abundance. Each sub-cluster showing higher level of RNA at 45 min than at 15 min was considered as main cluster 1, which implied that this cluster may be involved in attenuating light responses in S. fimicola. Cluster 1a included the genes involved in the development of sexual spores such as the constitutively photomorphogenic 9 (COP9) signalosome (SF_NCU08342) and bromodomain protein-1 (SF_NCU08809). The cluster protein-1b was enriched for genes involved in biosynthesis of vitamins, cofactors, and prosthetic groups, secondary metabolism and C-compound and carbohydrate metabolism. This cluster also included genes required for photoperception and response, the well-studied photoadaptation gene Sfvvd (SF_NCU03967) and genes involved in homeostasis and metal ions transporters, such as ferrochelatase (SF_NCU08291), iron-sulfur clusters transporter atm-1 (SF_ NCU05029), oxidative

stress resistance (SF_NCU03145) and peptidyl-prolyl cis-trans isomerase, fkr-3 (SF_NCU04371). In addition, some reported genes directly targeted by WCC including light responsive transcription factors, such as Sffl (fluffy SF_NCU08726), Sfmig-12 TF (SF_NCU09830), and hypothetical protein (SF_NCU01871) and other light responsive genes such as Sfcryp (SF_NCU00582) and UV-endonuclease UVE-1 (SF_NCU08850) were also found in this cluster (Smith et al., 2010; Verma and Idnurm, 2013).

Cluster 2 represented genes that were rapidly induced by light and peaked in expression between 15 min and 45 min in the light, so called early light-induced genes.

Cluster 2 was significantly enriched for genes encoding enzymes for carotenoid biosynthesis including phytoene desaturase (Sfal-1), phytoene synthase (Sfal-2), geranylgeranyl pyrophosphate synthetase (Sfal-3). Some light-responsive transcriptional factor genes including Sfbeak-1 (SF_NCU00097), Sfwc-1 (SF_NCU02356), Sfcsp-1 (SF_NCU02713), hypothetical protein (SF_NCU00275) and other light responsive genes such as Sfcon-6 (conidiation-specific protein 6; SF_NCU08769), Sffrq gene (frq;

SF_NCU02265), Sfbli-4 (SF_NCU08699) were also included in this cluster.

Genes in cluster 3 were repressed by light. Cluster 3 was mainly enriched for genes involved in secondary metabolism, lipid, fatty acid and isoprenoid metabolism and C-compound and carbohydrate metabolism. Interestingly, this cluster also included polyketide synthase-like family genes, SF_NCU04865, SF_NCU02918 and SF_NCU08399, and the genes encoding enzymes involved in the melanin metabolism such as tetrahydroxynaphthalene reductase (SF_NCU06905/SF_ NCU09390), and T gene (Tyrosinase; SF_NCU00776) (Krobanan et al., 2019).

Genes in cluster 4 were repressed in the light by 15 min but their expression was returned to approximately darkness level by 45 min. Although cluster 4 was highly

enriched for genes involved in secondary metabolism and C-compound and carbohydrate metabolism similar to cluster 3, genes related to lipid, fatty acid and isoprenoid metabolism were not found in this cluster. Genes involved in melanin metabolism such as Aspergillus yellowish-green 1 (SF_NCU05821) and scytalone dehydratase (SF_NCU07823), Sfper-1 (per-1) (polyketide synthase; SF_NCU03584) and clock-controlled protein 9 (SF_NCU09559) were also included in this cluster. To investigate and verify light-responsive expression profiles, the wild type and Sfwc-1^(∆lov) mutant were grown for 2 days in constant darkness (DD) and white light (LL), and an additional set of DD samples were exposed to blue light for 15, 30, 45, 60, 120 and 180 min (Fig. 9). The transcript levels of light-responsive genes including Sfbeak-1, Sffrq, and Sfcryp peaked at 15 min and decreased thereafter. However, the light-induced mRNA level of light-responsive genes showed subtle expression changes in the Sfwc-1^(∆lov) mutant (Fig. 9). Because of the known role of wc-1 in the phototropism of perithecial beaks in N. crassa, Neurospora beak-1 is photo-regulated via a WCC mechanism. Lack of bek-1 confers defective perithecial beak development in Neurospora (Colot et al., 2006). Similarly, Sfbeak-1 transcripts were photo-induced in the wild type, whereas sustained low mRNA levels were found in the Sfwc-1^(∆lov) mutant.

In contrast, Sfbeak-1 expression in Sfwc-1^(∆lov) was not affected by light (Fig. 9). This result was consistent with the phenotype of the mutant, with the Sfwc-1^(∆lov) mutant showing a shorter perithecial neck than the wild type. These findings suggest that Sfbeak-1 was regulated by light via Sfwc-1 and may be involved in perithecial beak initiation and elongation in S. fimicola.

3.1.8 Generation of SfWC-1-GFP and nuclear localization