Chapter 2 Materials and methods
2.8 Localization study
To construct a nuclear fluorescence plasmid, the PR27p-H2B-mCherry plasmid was amplified by fusion polymerase chain reaction (PCR) using KAPA HiFi DNA Polymerase (KAPA Biosystems, USA) according to the manufacturer's protocol. The PR27 promoter and Trpc terminator fragments were amplified with the primer pairs WC1218/WC2902 and WC2905/WC2906, respectively. S. fimicola histone H2B cDNA and mCherry fragments were then obtained by amplification with WC2903/WC2904 and WC601/WC602, respectively. The fusion fragment of PR27p-H2B and mCherry-TrpCt was obtained by amplification with WC1218/WC2904 and WC601/WC2906, respectively. These two fragments were purified, mixed and amplified with primers
WC1218 and WC2906 to generate the PR27p-H2B-mCherry-TrpCt fragment. The overlap-extension PCR product was cloned into the pCR-BluntII-TOPO vector and subsequently subcloned into pCAMBIA::hph(S) by cutting with SpeI and SmaI to finally obtain pCAMBIA::hph::RP27p-H2B-mCherry-TrpCt. The pCAMBIA::hph(S) plasmid was generated by cutting with XhoI and SacI and ligating with a 1.4-kb hph cassette from pCR::hph derived from the pCB1004 vector. Information for all the strains, plasmids and oligonucleotide sequences were listed in Tables 1, 2 and 3, respectively.
Cloning of vectors for GFP-SfWC-1
For cellular localization of SfWC-1, the Sfwc-1-gfp construct was generated by the overlap extension PCR approach. First, the 5’ region containing the 2-kb promoter and coding sequence of Sfwc-1 was amplified with the primer pair WC1620/WC1698 from S. fimicola genomic DNA. The primers WC1538/WC1539 were used to amplify the GFP fragment. These two PCR fragments were purified and amplified with the primer pair WC1620/WC1539 to generate the Sfwc-1-gfp fragment. The fusion product was purified and then cloned into the pCR-BluntII-TOPO vector to construct the pCR::Sfwc-1-gfp plasmid. This construct was cut with SmaI and HindIII and subcloned into pCAMBIA::neo to generate the pCAMBIA::neo::Sfwc-1-gfp plasmid. The H2B-mCherry tagged strain was obtained from a previous study as a control (Krobanan and Shen, 2018).
The construction of GFP-SfVVD plasmid
For cellular localization of SfVVD, the Sfvvd-gfp vector was generated by the overlap extension PCR approach. The 5’ region containing the 3.5-kb promoter and Sfvvd coding region were firstly amplified with the primer pair WC2537/WC2538 and
WC2539/WC2540 from wild-type genomic DNA. Then, these two PCR fragments were purified and amplified with the primer pair WC2537/WC2540 to generate the Sfvvdp-flag-Sfvvd(orf) fragment. Next, the primers WC1538/WC1539 were used to amplify the GFP fragment. The Sfvvdp-flag-Sfvvd(orf) and GFP fragment were purified and amplified with the primer pair WC2537/WC1539 to generate Sfvvd-gfp fragment. The overlapped product was purified and then cloned into the pCR-BluntII-TOPO vector to construct the pCR::Sfvvd-gfp plasmid. The pCR::Sfvvd-gfp vector was subcloned into pCAMBIA::neo to generate the pCAMBIA::neo::Sfvvd-gfp plasmid. The H2B-mCherry tagged strain was obtained from a previous study as a control (Krobanan and Shen, 2018).
Microscopic investigations
The strains harboring fluorescent tags either SfWC-1::GFP or H2B::mCherry or both were grown on malt extract agar I coated-glass slides for 16–18 h in the darkness at 28oC. Vegetative hyphae were transferred and immediately exposed to LED blue light (20–35 µmol/(m2s) for 15 and 60 min. Samples grown in constant darkness were used as controls. The live cell images were visualized on a spinning-disk confocal imaging system (Revolution WD Andor, UK) with a Ti-E inverted microscope (Nikon, USA).
Images were obtained by using a PlanApo 100X/1.45 oil objective lens and an iXON Ultra 888 EMCCD camera (Andor, UK). Imaging was controlled by using MetaMorph (Molecular Devices, USA). Fluorescence images were acquired with standard filter sets of 488 nm and 561 nm for GFP and mCherry, respectively.
Moreover, the Sordaria strains containing fluorescent tags either SfVVD::GFP or H2B::mCherry or of SfVVD::GFP, H2B::mCherry were used to exam the protein localization. Similarly, Sordaria mycelia plugs was grown malt extract agar I
coated-glass slides for 14–18 h in the constant darkness at 28oC. Then, dark-grown vegetative hyphae were transferred and immediately illuminated with to LED blue light (20–35 µmol/(m2s) for 30, 60, 120 min, respectively. The samples in constant darkness were used as control. The live-cell images were examined an inverted Leica laser scanning confocal microscope TCS SP5 with HCX PL APO lambda blue 63.0x oil objective (NA= 1.40; Leica Microsystems, Germany).
Chapter 3 Results
3.1 Characterization of white collar-1 orthologue (Sfwc-1) in S. fimicola 3.1.1 Light-regulated perithecial zonation and beak direction in S. fimicola
To understand the effects of illumination on sexual reproduction, S. fimicola was cultured on malt extract agar medium III and placed in a customized growth chamber where it was illuminated with unidirectional light. The zonation pattern depicted the rhythmic feature of perithecial formation on agar plates was observed under light/dark cycles and not seen under both constant illumination and constant darkness conditions (Fig. 1). Perithecia were abundantly produced under illumination conditions as compared with the darkness condition. Furthermore, wild-type perithecia formed beaks that bent toward the light, whereas wild-type perithecia in dark-grown cultures mostly produced very short beaks (Fig. 1). Moreover, illumination also induced carotenoid pigment production in S. fimicola. Mycelial colonies produced carotenoid pigment after exposure to light for 3 h and a more intense orange color was displayed upon illumination for 6 h (Fig. 2A). As expected, the illumination periods determined the patterns and beak direction of perithecial development in S. fimicola.
3.1.2 Identification and cloning of Sfwc-1 gene in the S. fimicola genome
Carotenoid pigmentation and perithecial beak phototropism in S. fimicola were strictly light dependent, similar to previously observed in N. crassa, which indicated the presence of photoreceptor gene(s) in S. fimicola genome (Harding and Melles, 1983;
Harding and Turner, 1981). Carotenoid pigmentation and perithecial beak phototropism
(Harding and Melles, 1983; Harding and Turner, 1981). To understand the functional roles of the blue-light photoreceptor in S. fimicola, we initially cloned a partial fragment of the putative blue-light receptor gene using primers designed based on the wc-1 gene of N. crassa. Based on Southern blot analysis, a 6.3-kb sequence composed of 2.1 kb upstream, a 3.4-kb open reading frame (ORF), and 0.8 kb downstream of the putative wc-1 gene was obtained by the inverse PCR approach. The corresponding gene was designated Sfwc-1 for S. fimicola wc-1. Sfwc-1 contains a single 68-bp intron by comparing the sequences between genomic DNA and cDNA. The ORF of the Sfwc-1 gene contains 3,434 base pairs and encodes a predicted protein containing 1,121 amino acids. The predicted domain structures of the SfWC-1 protein contained an LOV domain, two PAS domains, and a GATA type zinc finger DNA binding domain, which might support the light-sensing function of SfWC-1 as a white collar-1 blue-light photoreceptor similar to N. crassa WC-1 (Fig. 3A) (Belozerskaya et al., 2012; Chen and Loros, 2009; Crosson et al., 2003).
The amino acid sequence of SfWC-1 was compared with that of other fungi.
SfWC-1 shared 91.0% similarity and 88.1% identity with entire N. crassa WC-1 protein and 99.1% similarity and 98.3% identity within the LOV domain. The SfWC-1 amino acid sequence also shared 89.5% similarity and 86.0% identity with entire putative WC-1 of S. macrospora and 99.WC-1% similarity and 98.3% identity within the LOV domain.
Moreover, SfWC-1 showed 65.7% similarity and 55.2% identity to Fusarium fujikuroi (Gibberella fujikuroi mating population C) WcoA, an ortholog of N. crassa wc-1;
however, the LOV domain of both proteins shared 94.8% similarity and 87.0 % identity.
Sequence similarity and identity analysis were calculated by using EMBOSS protein alignment (http://www.ebi.ac.uk/Tools/psa/emboss_water/). An alignment of LOV
domains identified strictly conserved a flavin chromophore-binding site located in the LOV domain of SfWC-1 (Fig 4). Notably, studies of N. crassa indicated the distinct roles of LOV domain for light sensing and signal transduction (Fig 4) (Crosson et al., 2003). Furthermore, phylogenetic studies of fungal WC-1-like proteins supported that SfWC-1 protein was well separated in the major taxonomic clades of Ascomycota, Basidiomycota, and Mucoromycotina and tightly grouped within a clade formed by Sordariomycetes with high bootstrap values (Fig. 3B). Moreover, to show its conserved roles in light response, we examined Sfwc-1 gene expression in the wild type. Sfwc-1 was strongly induced after illumination, and its mRNA level quickly peaked at 15 min upon light treatment and decreased thereafter (Fig. 3C). Therefore, the Sfwc-1 gene appears to be a conserved fungal WC-1 ortholog regulated by light and potentially functions as a light-sensing protein in S. fimicola.
3.1.3 Generation of the disruption mutants of the Sfwc-1 gene
Because the LOV domain is critical for the function of WC-1 protein, we generated the Sfwc-1 mutant strains by replacing the LOV domain with a hygromycin B resistance cassette to investigate the roles of Sfwc-1 gene in S. fimicola. A. tumefaciens EHA105 and AGL-1 strains containing the disruption cassette were used to transform S.
fimicola protoplasts in order to generate homologous replacement of the Sfwc-1 allele, so called Sfwc-1(∆lov) mutant (Fig. 5A). We obtained 25 hygromycin B-resistant transformants, which were further analyzed by PCR. Four potential homologous recombinants underwent homokaryon purification by isolating ascospores from the cross between putative Sfwc-1(∆lov) mutants and the wild type. To verify a homologous integration event, two Sfwc-1(∆lov) progeny strains, A10-13 and E19-7, were selected and confirmed by Southern blot analysis. A single expected band of 4.5 kb without the
3.6-kb wild-type fragment was confirmed. These results indicated that the LOV domain of Sfwc-1 gene in both strains was replaced by the hygromycin B resistance cassette (Fig.
5B).
To confirm the functional role of SfWC-1, the Sfwc-1(∆lov) mutant was complemented back with the functional Sfwc-1 wild-type cassette. The Agrobacterium AGL-1 strain harboring the complementation construct was used to transform A10-13 and E19-7 protoplasts to generate complementation strains. The complementation transformants were analyzed by PCR and confirmed by Southern blot analysis. Two complementation strains derived from A10-13 mutant, A10-13AN2 and A10-13AN5, were selected and displayed the presence of two bands representing the homologous replacement (4.5 kb) and wild-type (3.6 kb) alleles (Fig. 5B). Furthermore, the absence of the Sfwc-1 transcript corresponding to the region deleted in the Sfwc-1(∆lov) mutant was proven by real-time PCR. Briefly, S. fimicola wild-type, Sfwc-1(∆lov) mutant, and complementation strains were cultured in constant darkness for 2 days and then exposed to blue light for 15 and 30 min. The cultures were harvested and subjected to RNA extraction. Forward and reverse primers respectively located within deleted (LOV domain) part and downstream of LOV domain region were designed for real-time PCR to examine Sfwc-1 gene expression. Constant darkness samples were used as a control. In the wild-type and complementation strain, Sfwc-1 expression was significantly increased upon illumination exposure for 15 min and decreased thereafter upon light treatment for 30 min (Fig. 5C). Unlike the wild-type and complementation strain, we failed to detect Sfwc-1 transcript in the Sfwc-1(∆lov) mutant (Fig. 5C). This indicated that the deletion of the Sfwc-1 LOV domain in S. fimicola (Sfwc-1(∆lov)) was successful.
3.1.4 Sfwc-1 is essential for the phototropism of perithecial beak
It was previously shown that N. crassa wc-1 mutant stains showed the defect of phototrophic response of perithecial beak (Harding and Melles, 1983). To investigate the phenotypes of the Sfwc-1(∆lov) mutant in sexual development, S. fimicola strains were grown on malt extract agar III under unilateral constant illumination and a 12-h light-dark photoperiod. Under the 12L:12D regimes, the wild-type but not Sfwc-1(∆lov) mutant strain showed perithecial zonation and complementation of the Sfwc-1 gene into the Sfwc-1(∆lov) strain restored the zonation pattern similar to that seen in the wild type (Fig.
6A-C). Furthermore, positive phototropism of perithecial beaks in the Sfwc-1(∆lov) mutant was lost under unidirectional illumination (Fig. 6B). After 21 days under the same conditions, more than 76% of perithecial beaks in the wild-type showed a positive orientation toward the light source (Fig. 6A; Table 4). Perithecial beaks in Sfwc-1(∆lov) were shorter than those of the wild type, with less than 5% positively phototropic orientation under same illumination conditions (Fig. 6B; Table 4). The beak length and phototropism of perithecia were phenotypically restored to the wild-type features in the complementation strains (Fig. 6C; Table 4). These results confirm that the Sfwc-1 gene affected the formation and phototropic behavior of the sexual fruiting body in S.
fimicola, confirming that peritheical zonation and phototropism of perithecial beak were governed by Sfwc-1 via light dependent regulation.
3.1.5 Disruption of the Sfwc-1gene reduces protoperithecial formation
To determine the effect of photoreceptor disruption on self-fertility in S. fimicola, strains were grown on 6-cm malt extract agar III plates under uniformly constant illumination and darkness. After 7 days, the wild type and the complementation strains
produced more perithecia under illumination than in darkness. In contrast, the Sfwc-1(∆lov) mutant showed greatly decreased number of fruiting bodies under illumination condition and was a similar amount of perithecia in the darkness (Fig. 7A). To better evaluate the difference, we quantified the number of fruiting bodies formed on agar plates by manually counting under a dissection microscope. After a 7-day incubation under constant illumination, the number of fruiting bodies in the Sfwc-1(∆lov) mutantwas reduced by up to 213- and 207-fold relative to the wild type and complementation strain, respectively. Under darkness conditions, the wild type and complementation strain produced substantially less perithecia, with a slight reduction (< 2-fold) observed in the Sfwc-1(∆lov) mutant(Fig. 7B). These results indicate that fruiting-body formation in S.
fimicola was photo-induced and the Sfwc-1 gene played an important role in this process.
To more precisely determine the developmental defects of the Sfwc-1(∆lov) mutant, we observed sexual structures of the strains over 14 days. Under the same conditions described previously, early-differentiated sexual structures including ascogonial coils were observed in all tested strains after 24-h incubation; however, the Sfwc-1(∆lov) strain seemed to produce much fewer ascogonial coil structures as compared with the other two strains (Fig. 8A-C). The wild type and complementation strain showed incipient protoperithecia or protoperithecium–perithecium transition structures at about 72 h post-incubation and these structures further differentiated into small immature perithecia up to 200 µm in width after 5 days incubation. In contrast, the Sfwc-1(∆lov) mutantshowed developmental defects at these stages. Protoperithecia were barely seen, and instead, clumps of hyphae or tiny protoperithecia were observed after 3 days incubation (Fig.
8B). Many of these structures were delayed or unable to further develop into perithecia, which resulted in a structure of 50 µm in width after 5 days incubation. After 7 days
incubation, the wild type and complementation strain formed black flask-shaped perithecia containing abundant asci with an eight-ascospore linear arrangement under illumination conditions; however, development in the darkness was delayed and immature perithecia were observed at the same period (Fig. 8A and C). In contrast, the Sfwc-1(∆lov) mutant at the same stage showed only a few immature perithecia with asci in various stages of development under both illumination and darkness conditions.
Moreover, after 2 weeks incubation, the wild type and complementation strain showed elongated beaks and mature ascospore discharge, whereas the Sfwc-1(∆lov) mutant mostly formed immature perithecia. The few mature perithecia produced by the mutant were further examined for the rosette of asci, and mature asci with black eight ascospores were observed in limited amounts (Fig. 8B). Thus, disruption of the Sfwc-1 gene significantly compromised the sexual and fruiting body development in S. fimicola, which indicates that Sfwc-1 played important roles in the light-regulated sexual development.
Among early light-induced transcription factors (TFs) in Neurospora, sub-1, a GATA type family TF gene, is essential for WCC-dependent light induction of numerous late light-inducible genes (Chen et al., 2009; Sancar et al., 2015; Wu et al., 2014). The sub-1 mutants exhibited defects in protoperithecia formation and altered expression of numerous genes, which indicates that SUB1 acts as a global regulator of light-induced transcription (Chen et al., 2009; Colot et al., 2006; Sancar et al., 2015). To correlate the phenotypic data with the gene potentially involved in the sexual development cascade, we determined S. fimicola sub-1 (Sfsub-1) mRNA levels in the wild type and Sfwc-1(∆lov) mutant under different conditions of illumination (Fig. 9). The mRNA level of Sfsub-1 was photo-induced in the wild type and showed the highest expression with
60-min illumination. However, Sfsub-1 mRNA level in light-treated samples was similar to that under the darkness condition in the Sfwc-1(∆lov) mutant (Fig. 9). Therefore, Sfsub-1 was light-induced in a Sfwc-1 dependent manner. These results indicate that SfWC-1 was required for the light-activated sexual developmental process by activating other regulatory genes such as Sfsub-1, which suggests that a SfWC-1–mediated light-responsive network was important for proper development.
3.1.6 Light-induced carotenoid pigmentation via Sfwc-1 gene regulation
Wild-type mycelia of S. fimicola exhibited an orange pigment after light induction as previously described (Ingold and Hadland, 1959). Phenotypically, carotenoid pigment was light-induced in the wild type and complementation strain but was unaltered in the Sfwc-1(∆lov) mutant, even with continued illumination for 6 h (Fig.
2B). The Sfwc-1 photoreceptor gene might be required to regulate genes involved in the carotenoid biosynthesis pathway. In Neurospora, albino genes (al-1, al-2, and al-3) encoding enzymes responsible for carotenoid biosynthesis are light-induced in hyphae and regulated by WCC (Belozerskaya et al., 2012; Iigusa et al., 2005). To better understand the role of Sfwc-1 in the carotenoid biosynthesis pathway, we analyzed the mRNA expression profiles of putative genes involved in carotenogenesis, Sfal-1 (SF_NCU00552), Sfal-2 (SF_NCU00585), Sfal-3 (SF_NCU01427), and Sfcao-2 (SF_NCU11424) in the wild type and Sfwc-1(∆lov) mutant under light induction. The mRNA levels of Sfal-1 and Sfal-2 peaked 30 min after exposure to blue light and decreased after longer exposure to blue light (Fig. 10). Moreover, Sfal-3 encodes for the enzyme responsible for the synthesis of geranylgeranyl diphosphate, a precursor molecule of several biochemical pathways involving the carotenoid biosynthesis pathway (Iigusa et al., 2005; Sandmann et al., 1993). The mRNA level of Sfal-3 peaked
at 15 min and decreased thereafter. However, the expression of Sfcao-2, probably required for a final step of the carotenoid biosynthesis pathway, gradually increased and peaked at 60 min, and decreased after long-term exposure to blue light for 120 min (Fig.
10). In contrast, the mRNA levels of these genes in the Sfwc-1(∆lov) mutant showed no significant changes with light induction and were much lower in the mutant than the wild type and similar to that in constant darkness. These findings agreed with the light-induced carotenoid pigmentation of mycelia found in the wild type and undetectable accumulation of carotenoid pigment in Sfwc-1(∆lov) mycelia (Fig. 2). Hence, carotenoid biosynthesis genes in S. fimicola were light-induced via Sfwc-1 regulation, similar to that found in N. crassa (Belozerskaya et al., 2012; Lee et al., 2003).
Besides the effect on carotenoid pigmentation, light also affects pigment production in sexual reproductive structures via melanin biosynthesis pathways. Several genes and enzymes required for melanization of sexual structure have been reported (Chinnici et al., 2014; Wang et al., 2014). Based on initial screening of transcriptome results, we identified the genes encoding enzymes involved in the melanin biosynthesis, including scytalone dehydratase (SF_NCU07823), Sfper-1 (per-1) (polyketide synthase;
SF_NCU03584), tetrahydroxynaphthalene reductase (SF_NCU06905/ SF_NCU09390), and T gene (Tyrosinase; SF_NCU00776) (Krobanan et al., 2019). Their transcript levels were downregulated under illumination relative to darkness in the wild type whereas subtle changes were observed in the Sfwc-1(∆lov) mutant, indicating that genes involved in melanin production may be regulated by light via Sfwc-1 (Fig. 11, Krobanan et al., 2019). Moreover, a putative polyketide synthase-like gene was found in S. fimicola and it was homologous to N. crassa polyketide synthase-4 (pks-4; NCU08399). We also examined the transcript level of this Sfpks gene in response to illumination. Sfpks
transcript remained at a basal level in constant darkness for both wild type and mutant strains. Interestingly, Sfpks expression was down-regulated under illumination conditions both in the wild type and mutant strains, but the Sfwc-1(∆lov) mutant displayed a different expression pattern. The role and regulation of this Sfpks gene in S. fimicola requires further investigation (Fig. 9).
3.1.7 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
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