Chapter 1 introduction
1.8 History of Sordaria fimicola
Sordaria fimicola, a coprophilous homothallic ascomycete commonly found in animal dung, has long been used to study genetic recombination and chromosome segregation (Lamb et al., 1998; Olive, 1956). S. fimicola is easily cultivated and has a short life cycle in the laboratory. In response to light illumination, S. fimicola displays carotenoid pigmentation and phototropism of perithecial beaks (under unilateral light)
as well as directional ascospore discharge (Ingold, 1958; Ingold and Hadland, 1959).
Moreover, the rhythm of spore discharge has been previously reported (Ingold and Dring, 1957; Kramer and Long, 1970). Collectively, these reports have provided information about the effect of light on the life cycle of S. fimicola from the vegetative stage to reproductive stages, although phenotype based studies were predominantly emphasized (Ingold and Dring, 1957). More recently, a gene delivery system and fluorescence microscopy studies have firstly been reported in S. fimicola (Krobanan and Shen, 2018). In this study, I aim to analyze the light regulation pathway of S. fimicola.
There are some advantages using S. fimicola over other fungal systems. Firstly, it shows a short life cycle from ascospore germination to spore discharge (from perithecia).
Secondly, it is self-fertile (homothallic), indicating it does not require another mating partner. Lastly, it no longer produces massive aerial hyphae containing asexual spores (conidia). Thus, these features make it easily to observe the pre-fruiting (ascogonia) and protoperthica structure and to prevent spore dispersal into the environment. A main objective of this study is to investigate and clarify light dependent phenotypic responses in S. fimicola using molecular and genetic techniques. To address these issues, we generated a Sfwc-1(∆lov) and a ΔSfvvd mutant and then compared phenotypes and physiological changes related to those of the wild type. Moreover, we also constructed green fluorescent protein (GFP)-tagged SfWC-1 and SfVVD to observe protein localization in both dark and light conditions. GFP-tagged SfWC-1 fluorescent signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. GFP-tagged SfVVD signals were barely observed in dark but showed a strong fluorescent in both cytoplasm and nuclear under light induction. Genome-wide expression analysis revealed the light-induced differential gene expression and cellular
response upon light stimulation in S. fimicola. Our studies focused on the putative blue light photoreceptor in a model ascomycete and contributed to a better understanding of the photoregulatory functions and networks mediated by evolutionarily conserved photoreceptors across diverse fungal phyla.
Chapter 2
Materials and Methods
2.1 Cultivation of organisms
Escherichia coli and Agrobacterium tumefaciens
Escherichia coli DH5-Alpha strain was used for cloning and propagation of recombinant plasmids and Agrobacterium tumefaciens (AGL-1 and EHA105) strains were used as the host strain to deliver the target gene to S. fimicola by Agrobacterium mediated protoplast transformation. E. coli and A. tumefaciens strains were routinely cultured on solid Luria Bertani (LB) medium and in liquid LB medium with shaking at 200 rpm and incubated at 37°C and 28°C, respectively. Bacterial strains harboring targeted plasmids were selected on LB medium supplemented with antibiotics either 100 μg/ml ampicillin or 50 µg/ml kanamycin (Sambrook and Russell, 2001).
Sordaria fimicola
Sordaria fimicola (Roberge ex Desm.) Ces. & De Not. (BCRC 33665) was obtained from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan and used as the wild type strain in this study. S. fimicola was grown on malt extract agar I medium (Blakeslee's Formula, 2% (w/v) glucose, 2% (w/v) malt extract (Himedia, India), 0.1 % (w/v) peptone, 1.5% agar) and malt extract agar III (2% (w/v) malt extract (Himedia, India) and 1.5% agars) to induce sexual reproductive structures.
The compositions of malt extract agar I and III media were provided by BCRC website (http://www.bcrc.firdi.org.tw/).
2.2 Preparation and transformation of competent microorganisms Escherichia coli
The target plasmids were introduced into Escherichia coli (DH5α) RBC HIT competent cells (Bioman Scientific. Taiwan). Transformation procedure was carried out according to manufacturer’s protocol with modifications. Briefly, plasmid DNA was added to the competent cells and then incubated on ice for 30 min. After heat shock at 42°C for 30-45s, the mixture was immediately incubated on ice for 2 min followed by the addition of 900 µl liquid LB and then incubated for 1 h at 37°C on the rotator 100 rpm. The recovered mixture was plated on solid LB medium containing either 100 µg/ml ampicillin or 50 µg/ml kanamycin.
Agrobacterium tumefaciens
In order to transform the plasmid into A. tumefaciens strains, the electroporation method was done as previous described (Krobanan and Shen, 2018). To prepare the electro-competent of A. tumefaciens, AGL-1 and EHA105 strains were grown on LB agar medium at 28°C for 2 days. A single colony was inoculated into 50 ml LB and incubated at 28°C with agitation 220 rpm for overnight or until an OD600 of 2.0. The cultures were re-grown into LB liquid and incubated at 28°C with 220 rpm for overnight or an OD600 of 0.6-0.9. The pellet was collected by centrifuging for 10 min at 4,000 rpm 4°C and then washed with 10 ml of 10% ice-cold glycerol 2 times. After final centrifugation for 10 min at 4,000 rpm at 4°C, the pellet was re-suspended in 2-3 ml of 10% ice-cold glycerol and aliquot stored in 100 µl for long-term preservation at -80°C.
For the transformation, 50-100 µl competent cells were mixed with 200-500 ng DNA and then put on ice for 20 min. The reaction mixture was electroporated by using Gene-Pulser-Xcell Electroporation System (Bio-Rad, Hercules, CA, USA) with the condition
of 2.54 kV, 25µF capacity and 200-ohm resistance. After electroporation, 800 μl of fresh LB liquid was added into reaction mixture and the diluted bacteria was plated on LB solid agar containing either 100 µg/ml ampicillin or 50 µg/ml kanamycin (Minz and Sharon, 2010).
Sordaria fimicola
In order to generate S. fimicola protoplasts, the mycelial plugs were grown in 100 ml malt extract broth I or complete liquid medium containing 1% glucose,
100 rpm. Recovered mycelia were harvested by centrifugation and subsequently washed with protoplast buffer (0.01 M Tris, 0.01 M MgSO4·7H2O, 1 M KCl, pH 7.0 or 13 mM Na2HPO4, 45 mM KH2PO4, 0.6 M KCl, pH 6.0). Protoplasts were generated by digesting with lysing buffer composed of 20 mg/ml of VinoTaste® Pro enzyme (Novozymes, Denmark), 2 mg/ml β-D-glucanase (InterSpex Product, USA), 5 mg/ml Driselase (InterSpex Product, USA), and 0.47 µl/ml of β-glucuronidase (Sigma, USA) in protoplast buffer at 28 °C for 12 h. To obtain protoplast cells, the lysate solution was filtered through Miracloth (Calbiochem, Germany), washed with protoplast buffer, collected by low-speed (1000–1200 rpm) centrifugation for 15 min at 4 °C, and adjusted to a final concentration of 107–108 cells/ml (Krobanan and Shen, 2018; Walz and Kuck, 1995).
The A. tumefaciens EHA105 strain was used as the host strain to deliver the target gene to S. fimicola according to a slightly modified Agrobacterium mediated protoplast transformation protocol (Bundock et al., 1995; Idnurm et al., 2004; Minz and Sharon, 2010). First, Agrobacterium was subjected to plasmid electroporation. The Agrobacterium strain was pre-cultured in LB liquid medium supplemented with 100 µg/ml of kanamycin at 28oC for 48 h. Bacterial cells were washed in minimal medium and then re-suspended in induction medium (minimal medium supplemented with 100 µM acetosyringone) until reaching an optical density of 0.15 at 660 nm. The culture was further incubated with shaking for 6 h at 28°C, and then 200 µl of A. tumefaciens cells (in protoplast buffer) was added to 200 µl of the S. fimicola protoplasts obtained as described above (107–108 cells/ml). The mixtures were plated on induction medium agar (IMA: containing 5 mM glucose and 200 µM acetosyringone). After incubation at 28°C for 48 h, the induction medium agar plates were washed with 5 ml of malt extract I or CM liquid medium, and 1 ml was plated onto corn meal agar medium supplemented with 200 µg/ml cefotaxime (Sigma, USA) and 100 µg/ml hygromycin B (or 400 µg/ml G418) (Bioman scientific, Taiwan) and incubated at 28°C for 2 days. The colonies of the transformants were transferred to malt extract agar I meal agar medium containing 100 µg/ml hygromycin B (or 400 µg/ml G418).
2.3 Morphological investigation ofS. fimicola
The sexual fruiting body phototropism and zonation analysis
To determine and characterize the perithecial phototropism, the mycelial plugs were inoculated near the edge of malt extract agar III (15-cm Petri dish plate) at 28oC for 21 days. The cultures were placed into a fungal growth chamber that allowed light to
pass through only from one direction. The light sources used in this experiment were phototropism was observed under a stereomicroscope and a proportion of perithecial beaks displaying phototropism relative to direction of light source were determined by counting the number of perithecia approximately 1-cm2 agar plugs. The perithecial beaks were photographed using Leica Z16 APO (5x) system equipped with Leica DFC490 digital camera of 8 megapixel CCD. The images were stacked by using Helicon Focus 6 software (Helicon Soft, Ukraine).
Observation of perithecial development
To induce sexual reproductive structures and observe perithecial development, S.
fimicola mycelial plugs were grown in the center of malt extract agar III (6-cm Petri dish plates) and irradiated with uniform constant fluorescent white light at 28oC for 14 days. The same growth regions on the plates were cut out at days 1, 3, 5, 7 and 14 and then examined by light microscopy. The number of fruiting bodies were scored by counting the perithecia formed on the medium surface under a stereomicroscope at day 7. The constant darkness sample was used as a control. The fruiting body developmental structure images were obtained using an Olympus BX41 (Olympus, Japan) compound microscope connected with Canon EOS 600D Digital SLR Camera (Canon, Japan). The images were analyzed and stacked by using Helicon Focus 6. The culture plate images were captured using a Nikon COOLPIX P300 CMOS Digital Camera (Nikon, Japan).
Observation of carotenoid pigmentation and growth inhibition
To examine hyphae pigmentation, Sordaria strains were culture on 6-cm malt extract agar I plates and incubated at 28oC in constant darkness for 48 h. constant dark conditions for 48 hr. All plates of each strain were continuously grown for another 48 h with different illumination exposure time for 3, 6, 12, 24 and 48 h toward the end of cultivation to produce time course sample. To see the influence of light on fungal growth, the Sordaria mycelia plugs were grown on the center of 9-cmmalt extract agar I plate under light/dark cycles (L/D: 0h/24h, 3h/21h, 6h/18h, 12h/12h, and 24h/0h) for 8 days. The constant darkness sample was used as a control for all experiments. Plate pictures were taken by using a Nikon COOLPIX P300 CMOS Digital Camera (Nikon, Japan).
2.4 Molecular methods
Plasmid Isolation from E. coli and A. tumefaciens
E. coli and A. tumefaciens strains were grown on LB broth for overnight at 37°C and 28°C, respectively, 100 rpm. Cells were collected by centrifuging at 4000 rpm for 15 min. Plasmids were isolated using either the Qiaprep Spin Miniprep Kit (Qiagen, Germany) or Bioman (Bioman scientific, Taiwan) performed according to the manufacturer’s protocol.
Isolation of genomic DNA and RNA from S. fimicola
For PCR screening and Southern blot analysis, genomic DNA (gDNA) of S.
fimicola was extracted by using slight modified CTAB (Cetyl trimethylammonium bromide) method according to previous study (Pitkin et al., 1996). In detail, the Sordaria strains were grown on cellophane-covered malt extract agar I medium and
mycelia on cellophane were peeled and dipped into liquid nitrogen. The fine power of mycelia pad was obtained by using a mortar and pestle with liquid nitrogen. The fine powder was homogenized in 500 µl DNA extraction buffer (l00 mM Tris/HCl, pH 7.5, 2% hexadecyltrimethylammonium bromide (CTAB), 1.4 M NaCl, 20 mM ethylenediaminetetraacetic acid (EDTA), 2% polyvinylpyrrolidone (PVP-40) and 1%
mercaptoethanol) and then incubated for 1 h at 65oC. Cell lysate was removed with the same volume of phenol:chloroform (1:1) and centrifuged at 13,200rpm, 4oC for 15 min.
The upper (aqueous) was transferred to a new tube and mixed with the same volume of chloroform. After centrifugation, the aqueous layer was transferred into a new 1.5 ml tube. The DNA was precipitated by adding isopropanol and incubated for 15 min at 25
°C. And collected by centrifuging at 14,000 rpm, 4 °C for 10 min. The purified pellet was finally washed with 70% ethanol and collected by centrifugation. The DNA pellet was air-dried and re-suspended in double distilled water.
Sordaria mycelial plugs were cultured on cellophane-covered malt extract agar I plate at 28oC in constant darkness for 2 days or until the colony reached the edge of 6-cm plate. After mycelia reached the edge, dark-grown samples were harvested for analysis as the constant darkness control, and the remaining dark-grown samples were irradiated with LED blue light to produce time course samples. The plates grown under constant white light for 2 days were used as a constant illumination (LL) sample. For light induction experiments, mycelial samples were exposed to blue light for 15, 30, 45 (for Sfwc-1), 60, 120, 180 and 360 (for Sfvvd) min. The illuminated samples were harvested and immediately dipped into liquid nitrogen before storage at -80°C. An LED red light-strip of 650 nm (0.5-1 µmol/(m2s), Taiwan HiPoint Corp.) was used for sample handling to mimic dark conditions. For total RNA isolation, frozen mycelial samples
were disrupted by grinding in liquid nitrogen and then extracted by Trizol reagent (Invitrogen, USA). All the procedures were performed according to manufacturer’s protocol. The fine powder was suspended with 1 ml Trizol and then stranded in room temperate (25°C) for 15 min. Upper supernatant was obtained by centrifuging for 10 min at 4°C, 13,000 rpm and the supernatant was transferred to a new 1.5 ml tube before adding 0.2 ml chloroform. The mixture was incubated for 7 min at 25°C and then centrifuged for 15 min at 4°C, 13.000 rpm. Approximately 450 µl of clean upper phase was transferred and precipitated by isopropanol for 10 min at room temperature (RT).
The purified RNA pellets collected by centrifugation was obtained by washing with 70% ethanol and centrifuged at 4°C 13,000 rpm for 15 min. The air dried RNA pellet was re-suspended in 100 µl sterile diethyl pyrocarbonate (DEPC)-treated RNAase free water. DNA or cDNA was amplified by using BioTaq DNA Polymerase (Bioman Scientific, Taiwan). The high fidelity amplification was carried out using KAPA HiFi DNA Polymerase (KAPA Biosystems, USA) and performed according to manufacturer’s protocol. The reactions were performed by using cycler machines including Biometra TGradient Thermocycler (Biometra GmbH, Germany) and Applied Biosystems GeneAmp® PCR System 9700 (Applied Biosystems, USA).
Purification of nucleic acids
Prior to transformation or ligation experiments, DNA fragments were needed to be removed the salt (buffer) from nucleic acid solution. PCR product and DNA fragment on agarose gels were purified using the QIAquick PCR Purification kit or QIAquick Gel Extraction Kit (Qiagen, Germany), respectively, according to the manufacture’s protocol.
Ligation and cloning of DNA fragments
The ligation reaction involved use of T4 DNA ligase (New England Biolabs, UK) and DNA Ligation Kit, Mighty Mix (Takara Bio, Japan) performed according to the manufacture’s protocol. Moreover, the purified vectors were dephosphorylated using Alkaline Phosphatase Calf Intestinal (CIP) (New England Biolabs, UK). For cloning purpose, Zero Blunt TOPO® PCR Cloning Kit (Invitrogen, USA) and pGEM®-T Easy Vector Systems (Promega Corporation, USA) were used according to the manufacturer’s recommendations.
Gel electrophoresis of nucleic acids
In order to separate DNA fragments, the PCR reactions were mixed with 6x loading dye prior to load onto a 0.8-1.5% agarose gel (1 % (W/V) agarose in 0.5x TBE (Tris/Borate/EDTA) buffer) (Sambrook and Russell, 2001). DNA fragments were separated by using a horizontal gel chamber (i-MyRun.N ¸Cosmo Bio, Japan) in 0.5x TBE buffer by using voltage of 80-100 V. The DNA gel was stained with 1 μg/ml ethidium bromide solution and visualized by UV trans-illumination. Standard DNA ladder was obtained from Cosmo Bio (Cosmo Bio, Japan).
The RNA gel electrophoresis procedure was carried out according to a protocol (Sambrook and Russell, 2001). Air dried RNA sample (2-5µg) were re-suspended in RNA loading solution and incubated at 65°C for 10 min. Then, RNA fragment were separated on agarose gel using voltage of 80-100 V. The 1x MOPS ((3-(N-morpholino)propanesulfonic acid) was used as electrophoresis buffer. Similar to DNA, RNA was visualized by UV trans-illumination.
Southern blotting and hybridization membrane (Hybond-N+, Amersham Pharmacia Biotech, Freiburg, Germany), cross-linked by using UV stratalinker at 1200 mJoules (mJ) and subjected to hybridization at 55-60oC, depending on the Tm of DNA probe, overnight. The non-radioactive probe labeling and detections system involved the use of digoxigenin (DIG) DNA Labeling and detection Kit (Roche, Germany), respectively, and performed according to the manufacturer’s protocol. The CDP-Star nucleic acid chemiluminescence reagent (Perkin Elmer, USA) was used as a substrate to detect the signal on X-ray film. All procedures and reagents were prepared as described (Sambrook and Russell, 2001). The Southern blot probes were obtained by an amplification of S. fimicola genomic DNA with the primer pair WC1398/WC1340 (for Sfwc-1) and WC2246/WC2600 (for Sfvvd) (Table 3).
Synthesis of cDNA and quantitative real-time PCR Synthesis of cDNA
RNA concentration and quality were evaluated with a NanoDrop ND-1000 spectrophotometer UV-VIS (Thermo Fisher Scientific, USA) and by denaturing gel electrophoresis, respectively. To remove gDNA contamination, TURBO™ DNase, RNAase-free (Thermo Fisher Scientific, USA) was used according to manufacturer’s protocol. Reverse transcription was performed with MultiScribe reverse transcriptase (Thermo Fisher Scientific, USA) according to the supplier’s recommended procedures.
Quantitative real-time PCR
For quantitative real time PCR experiments, the KAPA SYBR FAST qPCR kit master mix (2X) for ABI Prism (KAPA Biosystems, USA) was used for detection. The quantification of gene expression was conducted by the StepOne real-time PCR system (Thermo Fisher Scientific, USA). Real-time PCRs were performed as follows: 12.5 µl of 2 X FAST SYBR green PCR master mixes, 1 µl of 2.5 µM each primer, and 5 µl of 1 ng/µl cDNA template in total volume of 25 µl. S. fimicola actin gene (WC1791/WC1792) was used for normalizing gene expression. The expression of light-treated samples was relative to constant darkness sample. The primers used was listed in Table 3.
2.5 Analytical procedures
Oligonucleotide synthesis and DNA sequencing
The oligonucleotide primers used in this study were provided by PURIGO Biotechnology (Taiwan) and DNA sequencing was serviced by Techcomm (College of Life Science, National Taiwan University, Taiwan). All primers were shown in Table 3.
The nucleotide sequences were blasted search in public server NCBI. The nucleotide sequence alignments were analyzed by using the ClustalX2 program (Larkin et al., 2007), Geneious (Guindon et al., 2010) and DNASTAR software (Burland, 2000) and visualized using Gene DOC software.
Phylogenetic analysis
The amino acid and nucleotide sequences of white collar-1 and vivid orthologue were identified by the National Center for Biotechnology Information Search database (NCBI) NCBI BLAST alignment. The deduced amino acid of white collar-1 and vivid
orthologous genes from other organisms were obtained from NCBI, Kyoto Encyclopedia of Genes and Genomes (KEGG) database and DOE joint Genome Institute. Amino acid alignments were performed using MUSCLE alignment method (Edgar, 2004), and the divergent regions were removed using Gblocks (Castresana, 2000; Talavera and Castresana, 2007). The phylogenetic tree of WC-1 was constructed with the MEGA7 program with the neighbor-joining method (Kumar et al., 2016).
Preparation of RNA sequencing
For transcriptome analysis, the S. fimiocla wild type and Sfwc-1(∆lov) mutant were grown on malt extract agar I in constant darkness for 2 days. Some of the culture samples were exposed to blue light for 15 and 45 min and immediately dipped in liquid nitrogen. RNA extraction of frozen mycelium was carried out using Trizol reagent according to the manufacturer’s protocol. RNA concentration, quality and integrity were evaluated by using the Nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA samples showing RNA integrity > 7.0 and ratio of 28S:18S rRNA >1.4, as well as without DNA contamination underwent RNA sequencing. The cDNA was synthesized from mRNA by using the TruSeq Stranded mRNA Library Prep kit (Illumina, USA) and then sequenced with Illumina NextSeq 500.
Transcriptome de novo assembly and data analysis
The transcriptome assembly from short-read (average 140 nt) RNA-Seq data was performed at the GenoInfo Core Facility (C1) of National Core Facility Program of Biotechnology, NCFPB. The high-quality RNA-seq reads were then de novo assembled into a transcript sequence by using the Trinity platform with the strand-specific mode and the jaccard_clip option to avoid artifact transcript fusion in a high-gene density
genome (Grabherr et al., 2011). The quantified gene and isoform abundances (transcript abundance estimation) from mapping RNA-seq of de novo assembled transcripts were obtained by using RNA-seq by expectation-maximization (RSEM) software with the default setting of Bowtie. Then differentially expressed genes (DEGs) were identified by using the edgeR Bioconductor package of the Trinity platform. The normalized RSEM-estimated abundances weighted by Trimmed Mean of M values (TMM) method was used for pairwise comparison of each of the sample pairs for enriched (upregulated) or depleted (downregulated) gene expression using edgeR (Haas et al., 2013). Fold change in expression ≥2 and false discovery rate (FDR) p value <0.005 were the thresholds for significant differential expression. All these Perl scripts were performed in the Trinity platform. The filtered transcript reads were compared by using NCBI tblastx against the N. crassa genome database retrieved from the Broad Institute with the default setting (Camacho et al., 2009). The tblastx output presented the N. crassa gene ID and description under the filtering criteria of blast E-value of 1e-3 (and 1e-5 in supplementary data) for further analysis. Ward's hierarchical clustering and the Venn diagram of DEGs showing log (Fold change) of 1 were generated by using the limma and ggplot packages in R studio (Loraine et al., 2015). To identify the metabolic pathways mediated by Sfwc-1 in response to illumination, gene annotation and pathway mapping involved use of the BlastKOALA server and Kyoto Encyclopedia of Genes
genome (Grabherr et al., 2011). The quantified gene and isoform abundances (transcript abundance estimation) from mapping RNA-seq of de novo assembled transcripts were obtained by using RNA-seq by expectation-maximization (RSEM) software with the default setting of Bowtie. Then differentially expressed genes (DEGs) were identified by using the edgeR Bioconductor package of the Trinity platform. The normalized RSEM-estimated abundances weighted by Trimmed Mean of M values (TMM) method was used for pairwise comparison of each of the sample pairs for enriched (upregulated) or depleted (downregulated) gene expression using edgeR (Haas et al., 2013). Fold change in expression ≥2 and false discovery rate (FDR) p value <0.005 were the thresholds for significant differential expression. All these Perl scripts were performed in the Trinity platform. The filtered transcript reads were compared by using NCBI tblastx against the N. crassa genome database retrieved from the Broad Institute with the default setting (Camacho et al., 2009). The tblastx output presented the N. crassa gene ID and description under the filtering criteria of blast E-value of 1e-3 (and 1e-5 in supplementary data) for further analysis. Ward's hierarchical clustering and the Venn diagram of DEGs showing log (Fold change) of 1 were generated by using the limma and ggplot packages in R studio (Loraine et al., 2015). To identify the metabolic pathways mediated by Sfwc-1 in response to illumination, gene annotation and pathway mapping involved use of the BlastKOALA server and Kyoto Encyclopedia of Genes