Transcriptome analysis reveals the genetic basis of Mandrake cry lethality Karen Thulasi Devendrakumar and Lewis Kurschner Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Published: 13 June 2017. Received: 29 April 2017 | Accepted: 1 June 2017.

Abstract Mandragora mandraka or as it is commonly known, Mandrake is a plant that has been

utilized and studied for millennia by wizards in search of cures for dangerous wizarding maladies. Though considerable magical research has been done on the curative properties of Mandragora, the wizards have overlooked the one deadly characteristic feature of the Mandragora: the adult’s lethal cry. Some research has been done on determining characteristics of this cry and the lethal nature of it . Here, we perform RNA seq and methyl seq to understand the transcriptional differences in the vocal cords of developed and undeveloped Mandragora. We identified two key enzymes: Mandrake cellulose synthase-1 (MmCS1) and cinnamaldehyde dehydrogenase that are highly upregulated in adult vocal cords and we examine their role in the development of cry lethality. Using RNAi and overexpression analysis we examine the key role that MmCS1 plays in development of the cry lethality in Mandragora. Keywords: Harry Potter, Mandragora (Mandrake), epigenetics, vocal cord, frequency, ultrasonic, lethal, plant development, transcriptional activation.

Introduction

Mandragora mandraka is a magical species of the Mandrake plant found in the wizarding world and its non-magical counterpart, Mandragora caulescens, has been traditionally used for medicine and “witchcraft”. M. caulescens contains many important psychotropic active alkaloids which have pain relieving, hypnotic, purgative, sedative, and aphrodisiac uses to name a few.(1) Thus, after the merging of the wizarding world with the non-magical world, researchers began to study magical plants which had similar, non-magical counterparts. Luckily for researchers, magical plants share many of the same molecular properties compared to non-magical plants. For instance, molecular and phylogenetic studies on the genome of M. mandraka, and M. caulescens have revealed many homologous regions.(2) Of course there is much divergence between the species, which can be attributed to M. mandraka’s magical properties, but the similarity allows researchers to employ many of the same laboratory techniques in order to study these magical plants. Previous research on M. mandraka has yielded new medical compounds which have been shown to possess intensive healing properties for diseases such as epilepsy, locked-in syndrome (a condition in which a patient is unable to move or communicate verbally), and persistent vegetative state due to traumatic brain injury.(3) One notable feature of M. mandraka that differs from M. caulescens is their defensive cry. These plants use this cry as a deterrent to a would-be predatory species as it is able to kill them instantly. In juvenile plants this cry is underdeveloped and is only capable of knocking out. Thus, if the M. mandraka is ever harvested in a large-scale operation, research on suppressing this cry must be done. Previously, studies have looked at the properties of this cry and how it is produced (fake refs), but little work has been done to study the development of this cry as it develops in juveniles to adults. In this study, we will refer to Mandrake plants as Mandragora mandraka/ M. mandraka.

Mandrakes, like most tubular plants, have a large below-ground root system that serve as a reservoir for water and nutrients (tuber ref). The shape and structure of these root structures varies at

different ages of the plant’s life cycle but the key feature is that they resemble a rotund humanoid figure. In the center of of what could be considered the “face” of the roots, there is a large opening in which the chamber inside contains the Mandrake’s most notable feature: the vocal cords (fake ref). The below-ground root system leads up to an above ground rosette and the new leaves found in adults can be harvested for their medicinal properties (fake ref). Mandrakes are a dioecious species that have different morphologies for male and female. It is currently unknown how the adult plants reproduce, but it has been theorized that these Mandrakes grow a modified root structure in order to transfer gametes between males and females (fake ref). At the end of the reproductive cycle, the female plants produce seeds in which they shoot out of the top of the rosette through a tube-like structure in order to distribute them in the surrounding area. Mandrakes begin their life cycle when the first roots erupt from the seed. These primary roots are used for rapidly acquiring nutrients in order to build the tubers which emerge after 3 weeks. After 4-5 weeks plants have fully developed the tuber body and have produced a developed rosette. At this point the plants are now considered juveniles as their main body structure has developed but their vocal cords are underdeveloped (fake ref). At 18-20 weeks the plants undergo a process marked by large hormonal changes in which juveniles turn into adults. During this period, Mandrake plants increase in size, their vocal cords develop, and production of alkaloids begins in the leaves. At week 29-31 the plants are considered fully developed adults and their leaves and vocal cords have fully developed. It is at this stage when the cry has transitioned from one that causes fainting to one that is able to kill any would-be predators. The vocal cords are well developed in Mandrake in the juvenile stage, but these cries are not fully developed and cannot kill. They instead are only able to cause fainting in the attacker. In the growth process of Mandragora, at around 29-31 weeks it undergoes drastic changes. This stage is when the Mandragora becomes an adult. Among the various changes that Mandragora undergoes the most significant are the induction of synthesis of medicinal compounds in the leaves and also development of the cry lethality. We predict a huge transcriptional upheaval is responsible for such drastic changes. In this paper we focus on the development of the killer cry. In this study we used RNA- and methyl-seq data to understand what expressional changes occur between juvenile and adult Mandrake plants and how that relates to gene expression. We also determined changes in gene activation due to methylation. Next, we identified the top two upregulated genes and determine their role in development of the cry lethality using RNAi and overexpression analysis.

Materials and Methods

Plant growth conditions, tissue extraction, and tissue homogenization

M. mandraka seeds were obtained from the distributor: Hogsmeade Seed Co. Seeds are soaked in conc. Sulphuric acid (96%) for 10 minutes to perforate the thick seed coat and break the dormancy. The seeds are then soaked in a 30% bleach solution for 5 minutes with constant inverting to remove all potential pathogens (magical pathogens are sturdy enough to survive the acid bath). The seeds were then rinsed 5-10 times with sterile distilled water and transferred to 10 litre soil pots with a potting mix (sungro Sunshine Mix #1). All plants were grown with a 14-h photoperiod (100–150 µmol/m2s) at 21 °C, followed by a 10-h dark period at 20 °C in a Conviron growth room. Juvenile plants were harvested at 20 weeks and adult plants were harvested at 32 weeks. Vocal cord tissue samples of M. mandraka juvenile and adult plants were extracted and then immediately frozen with liquid nitrogen. Tissues were homogenized using liquid nitrogen and a mortar and pestle, and RNA extraction followed.

Mouse housing and treatment

Mice (C57BL/6- 3 weeks of age) sourced from Wizarding Research Supplies inc. License No. 256944 were used. Mice were housed under pathogen-free conditions with a 12 h dark/light cycle and provided with food (standard diet- 8640; Harlan Teklad, Indianapolis, IN) and water. At 8 weeks age they were used in our experiments. The male and female animals were cared for separately. All experiments were approved by the Institutional Animal Care and Use Committee of Hogwarts.

RNA extraction, cDNA library construction, and Illumina deep sequencing RNA from vocal cord samples of both juvenile and adult mandrake plants were extracted

using Trizol reagent (Invitrogen, Carlsbad, CA, USA) (1% agarose gel buffered by Tris–acetate-EDTA was run to indicate the integrity of the RNA.) and subsequently used for mRNA purification and library construction with the TruSeq® DNA Methylation Kit for Illumina (Illumina, USA) following the manufacturer’s instructions. Quality control of the samples were done using a NanoDrop™ One/OneC Microvolume UV-Vis (Thermofisher Scientific, USA) spectrophotometer and an RNA High Sensitivity Assay Kit (Thermofisher Scientific, USA) run on a Qubit™ 3.0 (Thermofisher Scientific, USA) fluorometer. Extent of RNA degradation was determined using a 1.5% denaturing agarose gel. The samples were then sequenced on an Illumina HiSeqTM2500 for 48 h. Normalization of RNA-seq data was done using factor analysis of control genes as per seen in previous literature (ref). Bowtiev2 was then used to perform the alignment and the RNA STAR (ref) tool in Galaxy (ref) was used to map the gapped-reads to the reference genome.

DNA extraction and bisulfite conversion, DNA library construction, and Illumina deep methyl-sequencing

Genomic DNA from vocal cord samples of both juvenile and adult mandrake plants were

extracted using DNeasy Plant Mini Kit (Qiagen, USA) (the DNA was run on a 1% agarose gel buffered by Tris–acetate-EDTA to look at the integrity of the extracted DNA.) and bisulfite conversion as well as library construction was generated with the TruSeq® DNA Methylation Kit for Illumina (Illumina, USA) following the manufacturer’s instructions. Quality control of the samples were done using a NanoDrop™ One/OneC Microvolume UV-Vis (Thermofisher Scientific, USA) spectrophotometer and an RNA High Sensitivity Assay Kit (Thermofisher Scientific, USA) run on a Qubit™ 3.0 (Thermofisher Scientific, USA) fluorometer. The samples were then sequenced on an Illumina HiSeq 4000 for 48 h. Bowtiev2 (ref) was then used to perform the alignment and the MethylDackel tool in Galaxy (ref) was used to map the methylated sequence reads to the reference genome. MEGA7 was used to align the promoter region of our adult and juvenile MmCS1 fragments to the upstream promoter region found in the reference genome (ref).

Agrobacterium Mediated Transformation

Mandragora transformation was carried out using the protocol developed by Bloomsberry et al.(ref) Explants used in agrobacterium mediated transformation was the meristem as it is the most susceptible for Agrobacterium infection (Defence responses are still not developed here). Meristems from 35 week old plants juvenile and adult plants (either the apical or the lateral meristems can be used) and the meristematic dome was cut under a stereoscopic microscope. The dome was then cultured on preculture medium (PCM- 2% Phytagel, 2 ppm Zeatin, 40g/l sucrose) for two days in dark Preculture in the presence of a cytokinin (here Zeatin) leads to cell expansion and primes the cells for transformation. The meristem swelling is seen on the third day at which point the domes are then infected with an Agrobacterium (LBA4404) suspension (0.1 OD, 0.1% Tween 20, 1ppm Acetosyringone) containing the gene of interest. The infected domes are removed from the infection mix and placed on co cultivation medium (CCM- 2% Phytagel, 4 ppm Zeatin, 40g/l sucrose, 1ppm (1µl/l) unicorn serum (ethically harvested)). The addition of unicorn blood makes cells more pervious

to the Agrobacterium owing to the magical, and currently unknown, enzymatic action. After two days on CCM, the domes are transferred to selection media (CCM+50mg/l Hygromycin). After 2 weeks, plantlets regenerate from the dome and they were placed rooted in rooting medium (RM- 2% Phytagel, 5 ppm IBA, 40g/l sucrose) for an additional two weeks. Plantlets were then transferred to sterile potting mix (Sungro Sunshine Mix #1). Single copy transformants were selected and their homozygous progeny were selected and propagated as lines.

RNA isolation and RT-PCR

Total RNA was extracted from harvested leaf tissue (n=3) using an RNeasy plant mini kit (Qiagen) with DNase treatment. cDNA was synthesized using the isolated RNA using a Reverse Transcription System (Promega) according to manufacturer's protocol (Qiagen, USA). RT-qPCR (Real Time- quantitative Polymerase Chain Reaction) was performed using SYBR green as described previously (Kim et al., 2013). cDNA was diluted 20-fold and used for subsequent PCR reaction. MmActin was used for normalization. Vocal cord tissues were biopsied and pooled from plants (n=5) in a line for each sample in a replicate and the ddCt method was used to calculate the relative expression values. Protein Extraction and Immunoblots

To extract total protein from the vocal cord, 2 mg of biopsied samples from each plant (n=5) were ground using 1X PBS and 5X SDS Loading Buffer was added. The samples were then heated for 10 minutes at 95 °C and then centrifuged. Proteins were then loaded onto a 4-12% BT NuPAGE gel (Life Technologies) and run at (how many volts for how long). Proteins were then transferred to a Hybond ECL nitrocellulose membrane (Life Technologies) which was first blocked with 5% skim milk in 1X TBST for 2 h at room temperature followed by incubation with the primary antibody- rabbit anti-GFP (Life Technologies) in 1% skim milk at 4°C overnight. The secondary antibody was horseradish peroxidase-conjugated anti-rabbit IgG (Thermo Fisher). For visualization, SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher) was used. Sample preparation for SEM and imaging

Vocal cord tissue samples were fixed in 5% glutyraldehyde with a 0.05M phosphate buffer (pH 7.1). The samples are fixed overnight at 4°C and specimens were cut into 1mm cubes in the fixative solution. Vocal cord samples were then rinsed three times in the 0.05M phosphate buffer pH 7.1 and secondary fixation was carried out in 1% Osmiumtetroxide/1.5% Potassiumferrocyanide for 1 hour at 4°C a darkroom. The samples were washed in sterile distilled water three times and then slowly dehydrated multiple times using Ethanol at increasing concentrations: 30%, 50%, 70%, 80%, 90% and 100% (15 minutes each; 100% ethanol treatment twice). Specimens were then dried to remove ethanol in a critical point drier and, after drying, specimens were then mounted on stubs and then sputter coated with a 60% gold/40% palladium alloy. Prepared specimens were then imaged using JEOL JSM- 840 Scanning Electron Microscope at 30kV. Mouse lethality experiments and cry frequency analysis The mice (equal number of male and female mice were used- 25 each for each treatment group) were randomly assigned treatments (plant genotype). The mouse lethality experiments were performed by exposing the mice (in mesh cages) to the Mandrake cry from a distance of 3 metres. We used the appropriate PPE and safety protocols to protect ourselves from the Mandrake cries. The plant was uprooted from the pot to induce it to cry. The mice were exposed to the sound for 5 seconds after which the plants are reinserted into the soil which shuts them up immediately. Simultaneously

the frequency range produced is recorded using BioRadFreak® frequency analyzer (BioRad Catalogue Number: AUD199879). Lignin estimation

The lignin was isolated and the composition and the abundance of the components were analyzed using FTIR (Fourier- transform infrared spectroscopy) according to the detailed technique described previously. The approach consists of measuring the absorbance of a solution of whole biomass (vocal cord biopsy tissue) dissolved in the ionic liquid 1-n-butyl-3-methyl imidazolium chloride (ref)

Results Whole transcriptome sequencing of vocal cord tissues

In order to determine what gene expression changes occur in the vocal cords in response to plant development, an RNA-seq experiment was run. Extracted vocal cord tissue from both juvenile (n=5) and adult (n=5) mandrake plants and total RNA was isolated in order to create a cDNA library. Extent of RNA degradation was determined by running each sample out on a gel (Supp. Figure 1.). The ratio of 28s rRNA to 18s rRNA was found to be ~2.3 ±0.12. The 18s RNA purity was determined using the absorbance ratio of 260 nm/280 nm by a NanoDrop™ spectrophotometer. In the 10 samples, the mean absorbance was ~1.96 µg/mL ±0.4 (data not shown). A Qubit test was then used to measure the concentration of the RNA samples. It was found that the RNA samples were all well above the required concentration and could be used for cDNA library preparation and illumina sequencing. In all of our 10 samples, we found that the Phred Q-score was between 30-38 and there was a 99.98% match to the M. mandraka exome (data not shown). Coverage between adult and juvenile M. mandraka vocal cord contigs were then compared in order to determine changes in the transcriptome (Table 1.). It was found that the two most upregulated genes in adult M. mandraka, Mandrake Cellulose Synthase 1 (MmCS1) and Mandrake Lignin Synthase 1 (MLS1), are involved in cellulose and lignin production while the two most downregulated genes, CRE1-like hormone receptor (CLHR) and Auxin-binding Protein 1 (ABP1), are involved in auxin signaling. Subcellular localization of these transcripts was then determined using the Bio-Analytic Resource (BAR) ePlant browser. The top two upregulated genes in adult M. mandraka were found to be expressed only in the vocal cords (Figure 1.) while all other genes of interest were expressed in various plant tissues (data not shown). After changes in gene expression were determined between adult and juvenile plants, it was important to determine what mechanisms may be causing the observed large scale upregulation of cellulose synthase and lignin synthase found in the vocal cords of adult plants. Determining extent of gene repression in juvenile M. mandraka In order to determine the differences in methylation between the juvenile and adult Mandrake plants, a methyl-seq experiment was run. DNA was extracted from the vocal cord tissue in both juvenile (n=5) and adult (n=5) mandrake plants using a kit. Purity of DNA was measured using a NanoDrop™ spectrophotometer. In the 10 samples, the mean absorbance was ~1.81 ±0.4 (data not shown). A Qubit test was then used to measure the concentration of the 10 DNA samples. It was found that the DNA samples were all well above the required concentration and could be used in the bisulfite conversion and library prep kit from Illumina for methyl sequencing. In all of the 10 samples, the Phred Q-score was found to be between 31-38. In the control samples that did not undergo bisulfite conversion (n=4), there was a 99.65% match to the M. mandraka reference genome (data not shown). In the bisulfite converted samples (n=4), there was a 98.77% match to the M. mandraka reference genome (data not shown). We then wanted to compare what sequences were heavily methylated in juvenile plants but was not seen in adults. We found over 19,469 100-5,000 bp regions

of the genome that contained CpGs that were methylated by 20% (DNA histone methyl ref). Of these 19,469 regions, 2,658 had 90% methylation of CpG sites in the promoter region of genes that were found to be upregulated in the adult Mandrake plants (data not shown). In the adult plants, these genes were found to have 20-39% methylation of CpG sites (Table 1.). In the juvenile plants, there was also >80% methylation of CpG sites in the promoter regions of genes found to be upregulated in adult Mandrake plants (data not shown). In the adult plants, these genes were found to have 77-88% methylation of CpG sites (Table 1.). We then performed a multiple sequence alignment on the promoter region found upstream of MmCS1 and found that our juvenile samples had 96% CpG methylation (Figure 2.)

Cellulose synthase and lignin synthase were selected for our study based on the fact that they are most upregulated genes when comparing the RNA seq data from the Adult and the juvenile M. mandraka. To test our hypothesis that the upregulation of cellulose synthase is what is causing the change in the lethality of the mandrake’s cry we performed both overexpression analysis and RNA interference.

RNAi Analysis for determining the role of MmCS1 in vocal cord cellulosic structure

Cellulose synthase is a key plant enzyme that is required for the structural integrity of the Mandrake plants. We found knocking out MmCS1 proved prove fatal as the plants died during the hardening stage of early Agrobacterium transformation (data not shown). Thus, we utilized RNAi as it can generate highly downregulated genes without complete silencing. A vocal cord specific promoter, MmVCsp developed by Xin et al, coupled with our RNAi constructs made it possible to target MmCS1 expression in vocal cord tissue alone.(ref) Two lines (MmCS:RNAi:1 and MmCS:RNAi:2) out of the 13 generated lines were selected for further characterization. The transcript levels of MmCS1 were measured using RT-qPCR and endogenous Actin expression levels were used for normalization of the data. There was little difference in the expression levels in juvenile RNAi transformed plants compared to WT juvenile plants (data not shown). These lines were selected because the level of MmCS1 expression in the adults of these lines was comparable to the levels of CS in WT juvenile plants (Figure 3A). The wild type adults show expression levels fifteen times that of the expression level in the juvenile plants. The expression level in the two selected RNAi lines were comparable to the expression levels in the wild type juvenile plants. The RNAi lines MmCS:RNAi:1 and MmCS:RNAi:2 had MmCS1 expression levels 1.2- and 1.4-fold times the expression of MmCS1 in WT juvenile (Figure 3A).

Vocal cord tissues were biopsied by using a biopsy needle and tissues were prepared for

SEM using the method described by Nova et al.(ref) The prepared samples were sputter coated with a gold-palladium alloy and imaged using an SEM (Figures 3B, 3C and 3D.). The results show a clear difference in the cellulose fiber arrangement. It is seen that in the juvenile plants (Figure 3D.) and the RNAi plants (Figure 3C.) the cellulose structure has a similar loose arrangement whereas in the adult WT plant (Figure 3B.), the cellulose fibers can be seen to be much densely packed. This data suggests MmCS1’s critical role in the process of developing vocal cords.

Overexpression analysis for determining the role of MmCS1 in vocal cord cellulosic structure

Since we studied the impact of downregulating MmCS1 expression in the vocal cords of developing juvenile plants, we wanted to look at the consequences of MmCS1 overexpression. As MmCS1 is required for normal plant function, overexpressing would most likely lead to abnormal plant growth. Hence, similar to the RNAi we expressed the MmCS1 gene driven by a vocal chord specific promoter. The chimeric promoter CaMV35S-MmVCsp was used for targeted overexpression of the

gene. This allowed us to achieve targeted higher expression levels of MmCS1 transcript without causing death of the plant. We analyzed the expression levels of MmCS1 using RT-qPCR in the selected overexpression line CS:OX:31 and CS:OX:34. These lines showed a 10 fold increase in MmCS1 expression levels compared to the expression levels in WT adults (Figure 4A.). This was further confirmed using a western blot which showed a similar 10-fold increase in MmCS1 protein amounts (Figure 4B.). The vocal cord from 32 week old adult overexpression lines were biopsied and imaged using SEM. The micrograph show a clear increase in the density of the cellulose fiber arrangement (Figure 4C.). Frequency dependence of lethality

To study the impact of MmCS1 expression levels on cry lethality, it was important to understand how differential MmCS1 expression levels cause this feature of the Mandrake cry. In these experiments, mice were used to determine how lethal our transgenic plants were. Though the traditional magicians used (barbaric) methods involving house elves in their experiments, we use the murine model in our ours. It has previously been shown that the cries are able to affect all animals with eardrums in a similar manner, and thus, mice would be an accurate model to use.(fake ref) Mandrake cries are also known to reach frequencies which extend into the ultrasonic range and we found that the frequency range of a Mandrake’s cry changes with respect to expression of MmCS1 (Table 2.). WT adults were shown to kill all mice during this experiment. In our RNAi adults, we found that there was a shift in frequency to the lower spectrum and few mice were killed. The frequency ranges present in our RNAi lines were similar to those seen in juvenile plants (data not shown). However, it is seen that in the RNAi line MmCS:RNAi:1 the frequency range extends a little over that of the juvenile and is able to cause two deaths. Our adult OX lines were unable to cause fainting or death in any of the mice tested which can most likely be attributed to the frequency range of the cry being drastically reduced. Here we demonstrated that the frequency is pivotal in determining the cry lethality. Only the cries having frequencies that range into the ultrasonic ranges (above 20,000 hertz) are lethal. These results indicate that MmCS1 expression in the WT adult levels was critical for the development of a lethal cry.

Variation in lignin biosynthetic capabilities Cinnamyl alcohol dehydrogenase (MmCAD) was found to be one of the most upregulated genes in Adult vocal chords. This enzyme is involved in catalyzing the final step of the synthesis of lignin monomers. Lignin is a complex molecule made of several types of molecules and chemical bonds. The best method to quantify lignin is using the FTIR (Fourier- transform infrared spectroscopy) to quantify the various components and bonds present in lignin. We used an RNAi approach to understand the significance of this enzyme in the development of adult vocal cords. This proved to be unsuccessful as lignin was being produced(results not shown). The expression levels of MmCAD were reduced based on our RT-qPCR, but the final concentration of lignin did not vary between differentially expressing individuals (data not shown). The analysis of the relative content of lignin and its constitution reveals no major changes in the composition but shows a definite increase in the adult vocal cord lignin content compared to the lignin content in juvenile plants (Figure 5. and Table 3.). Discussion Whole transcriptome sequencing uncovers upregulated genes related to vocal cord development

As we were interested in running an RNA-seq experiment, it was important to first understand the quality and quantity of our extracted RNA which has been known to affect efficacy of Illumina sequencing. After transcriptome extraction from vocal cord tissues, RNA was run out on a gel to determine any potential degradation (Supp. Figure 1.). When comparing the fluorescence difference between the 28s and the 18s rRNAs, it was apparent that the 28s band was ~2.3 ±0.12 fold brighter than the 18s band (Supp. Figure 1.). This is due to the fact that more nucleotides were fluorescing in this band as it has a longer sequence. However, as these rRNAs are created in a 1:1 ratio in the cell, we expect about a fluorescence ratio of ~2.7. Based on our results, we can conclude that our sample had very little degradation as a ratio of >2.0 is usually indicative of a relatively high quality sample. We then proceeded with measuring the purity of our sample using a NanoDrop™ spectrophotometer. We determined the A260/A280 ratio was ~1.96 µg/mL ±0.4 which indicates a relatively pure sample that is free of DNA (data not shown). We then proceeded with sequencing of our cDNA library. We analyzed our sequencing run using the Illumina Analysis tool and found that our Phred Q-scores were above 30 in 9/10 samples. This indicates that the base calls made in each run were made with very high accuracy (

After we aligned our fragments to the reference genome we wanted to determine what loci were highly methylated in juveniles but not adults. We determined that there were over 19,469 100-5,000 bp regions of the genome that contained CpGs that were methylated by 20% (DNA histone methyl ref). This cutoff was previously determined to be sufficient in detecting methylation of coding and regulatory regions, as roughly 20% of the genome is methylated on CpG sites (DNA methyl ref). Of these 19,469 regions, 2,658 had 90% methylation of CpG sites in the promoter region of genes that were found to be activated in the adult Mandrake plants (data not shown). In the adult plants, these genes were found to have 20-39% methylation of CpG sites (Table 1.). This indicates that genes that were found to be activated in adult plants had low percentages of CpG methylation whereas in juveniles, where these genes were suppressed, there was heavy methylation. We also found that the reverse was apparent as the juvenile plants had >80% methylation of CpG sites in the promoter regions of genes found to be upregulated in adult Mandrake plants (data not shown). In the adult plants, these genes were found to have 77-88% methylation of CpG sites which indicates that genes that are activated in juveniles are in turn repressed in adult plants(Table 1.). These results are further confirmed based on our sequence alignment of the 2 kb promoter region upstream MmCS1 (Figure 2.) As 96% CpGs are methylated in the juvenile plants, this gene is under heavy suppression.

Thus, we can conclude from both of our sequencing experiments that auxin genes are activated and expressed in high quantities in juvenile plants due to the fact in this life stage the plants are rapidly acquiring nutrients in order for growth of their many tissues. This is in preparation for major hormonal changes that will modify certain tissues, such as leaves expressing medicinal compounds and vocal cord strengthening. Once the plant reaches the hormonal, “puberty” stage characterized by tissue development, these auxin genes are methylated to reduce the growth while cytokinin genes are demethylated and upregulated causing downstream effects like vocal cord elongation as well as deposition of cellulose and lignin. Once the plants have reached the adult stage, they are no longer developing or growing, and thus, auxin related gene expression can be greatly downregulated (auxin ref). Interestingly, it would be assumed that once the plants have reached the adult stage, cytokinin would not need to be expressed. However, cytokinin has recently been shown to be involved in cellulose and lignin deposition in M. mandraka(fake ref). Thus, expression would be required as vocal cord cells age and are replaced by new ones. Over- or underexpression of MmCS1 causes changes in the vocal cord cellulose fiber arrangement

Overexpression and RNAi analyses were carried out to better characterize the role that MmCS1 plays in the development of the vocal cord cellulosic structure and how critical it is for the cry’s frequency. Since cellulose synthase is a key enzyme, overexpressing it or under-expressing it in the entire plant would have negative effect on the plant’s growth and development. Hence we used the vocal cord specific promoter for driving the expression of the RNAi construct. This led to the generation of plants in which the adults express MmCS1 transcripts at levels similar to that seen in the WT Juvenile plants (Figure 3.). SEM micrographs of the vocal cord cellulosic structure reveal similarities in the cellulose fiber arrangement in WT juvenile and the RNAi lines (Figure 3C, 3D.). The

cellulose fiber arrangement is seen to be less dense when compared to the density of the cellulose fibers in the WT Adult plants (Figure 3B.). This indicates that the density of the cellulosic fibers may are most likely important for producing ultrasonic pitches.

Our overexpression analysis reveals the effects of enhanced expression of MmCS1 on

cellulose fiber arrangement in vocal cord tissues. Overexpression of MmCS1 had drastic negative effects on the health of the plants and so the expression was driven by CaMV35S-MmVCsp, a chimeric promoter capable of vocal cord specific overexpression in Mandrake. The overexpression lines expressed MmCS1 10 times the expression level in WT adults (Figure 4A, 4B.). The cellulose fiber arrangement indicates a really tight packing in the overexpression lines. This tight packing in the overexpressed lines most likely caused the hardening of the vocal cord fibers to the point where they were unable to vibrate effectively.

To further characterize the role of MmCS1 in causing cry lethality we performed a lethality test

using mice. In both the overexpression lines and in the RNAi lines the lethality of the cry is affected when changing the expression pattern of MmCS1 (Table 2). In WT Juvenile plants and the RNAi lines This shows the deposition of cellulose is a key factor in determining the lethality of a Mandrake’s cry. It can thus be inferred that a specific expression level of MmCS1 is necessary for obtaining the required cellulose content of the vocal cords making a lethal cry possible. It was previously known from the study on the victims of Mandragora and the study of the fluids leaking from their ears that the cause of death was cochlear implosion.(ref). This study conducted by the wizards did not touch upon the possible role of the frequency of sound produced that caused the death. A frequency analyzer was used to determine cry ranges and we see a positive correlation between the percentage of deaths and the frequency range (upper limit) of the sound produced. The overexpression lines show a drastic decrease in the frequency range of the cry produced. The frequency range of the cry produced by the RNAi lines and the juvenile reach the upper limits of the audible frequency range but they can only cause fainting in most cases (Table 2.).

The cellulose structure determines the range of frequencies of the cry produced. In our RNAi

lines the vocal chords were not taut enough to produce sounds in the ultrasonic range. In contrast, it was seen that the high level of MmCS1 in the overexpression lines resulted in vocal chords with really dense arrangement of cellulose fibers. The cords become too taut and almost next to no sound is produced (low frequency range). The overexpression lines are not even able to cause fainting in the mice used which suggests that the frequency of the sound produced determines the killing capacity. It remains unclear how this occurs as these frequencies all occur in nature. As these frequencies are not lethal on their own, it may be a combination of frequencies or it suggests a magical component in causing lethality.(fake ref)

Role of Cinnamyl Alcohol Dehydrogenase in determining cry lethality

Cinnamyl Alcohol Dehydrogenase (MmCAD) is a key enzyme in the lignin biosynthetic

pathway and catalyzes the final step in the biosynthesis of the lignin monomers. The lignin content in WT adult plants is more than that seen in the juvenile plants (Figure 5., Table 3.). Similar to MmCS1 experiments, overexpression and RNAi was carried out to characterize the role of MmCAD in the development of Mandrake cry lethality. It was seen that overexpressing or under-expressing MmCAD did not affect the final levels of lignin in the plants (data not shown). Two potential explanations for the phenomenon are as follows: the step catalyzed by MmCAD is not the rate determining step in lignin biosynthesis and so differential expression of this gene has minimal effects on lignin biosynthesis. It is also possible that MmCAD has homologs that have not yet been characterized and that are able to compensate for the suppressed activity of interfered MmCAD transcript.

Conclusions

In this paper we first investigated the transcriptional differences between adult and juvenile plant vocal cords. We found that genes involved in auxin signalling were repressed in adults while genes involved in cytokinin signaling were upregulated. Two genes which were found to be activated by cytokinin signalling were MmCS1, a gene involved in the cellulose synthesis pathway, and cinnamyl alcohol dehydrogenase, a gene coding for an enzyme in the final step of lignin biosynthetic pathway. We then determined the extent to which the genome was methylated in both juvenile and adults. We found that the promoter region of MmCS1 was highly methylated in juveniles but not adults indicating its activation during development. We then performed RNAi experiments to characterize the phenotype caused by low levels of MmCS1 in adult plants and found that they exhibited underdeveloped vocal cords similar to that seen in juvenile plants. Overexpression lines were generated in order to characterize the effect on the Mandrake’s cry. We found that Mandrake vocal cords that had an increase in MmCS1 expression were rigid and unable to produce lethal or sub-lethal cries; that could also not affect mice. We then determined through RNAi that when Cinnamyl Alcohol Dehydrogenase was underexpressed, there were no changes in lignin deposition in the vocal cords. Phylogenetic analyses comparing vocal cord fibers to those found in hemp and other similar fibrous plants should be undertaken in order to discern the evolutionary processes which allowed for the development of these vocal cords in Mandrakes. We rely on our magic counterparts in order to understand the magical component in the killing since this lethality is seen only in cries containing sounds in the ultrasonic region. These studies would help put together a complete genetic map of vocal cord development. One of the greatest challenges the pharmaceutical industry faces in utilizing Mandrakes for medical purposes is upscaling production and reducing the risk of death due to cries. In this study we have generated a line of Mandrakes that are unable to harm mice due to overexpressed cellulose specifically in the vocal cords. We believe that this may be the beginning of potentially mitigating the risk involved in growing Mandrakes, however, much work needs to be done on reducing cultivation time and overexpression of medicinal compounds in leaves before we see a large increase in output of Mandragora curative potion for disease treatment.

Acknowledgements

We would like to thank the National Sciences and Engineering Research Council of Canada, the Muggle-Wizard Research Council of North America, and the Wizarding Plant Society for the funding of this research. We thank Dr. Neville Longbottom, Professor, Herbology, Hogwarts School of Witchcraft and Wizardry for helping us by sharing his valuable expertise in growing Mandrakes and caring for them. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would also like to thank the class for their questions and suggestions. We would also like to thank Dr. David Ng for his proofreading and technical editing of this paper.

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Figures and Tables Table 1. Top 5 upregulated and downregulated genes in adult M. mandraka

Figure 1: Localization of cellulose synthase and lignin synthase is found to be in the vocal cords of M. mandraka. Yellow shading represents low gene expression while red shading represents high gene expression. Data taken from BAR ePlant viewer.(ref)

Figure 2: Methyl-seq multiple sequence alignment generated from MEGA7 using default parameters (ref). Top sequence is the reference genome. Below is the adult control and then adult bisulfite converted sequence. Further below is the juvenile control and then juvenile bisulfite converted sequence. Stars indicate sequences that aligned 100% at the individual base pair.

Reference Adult Control Adult Bis Juvenile Control Juvenile Bis

Figure 3: A: RT-PCR Relative expression levels of Cellulose Synthase (CS) in denoted genotypes, B, C, D: SEM micrographs of cellulose structure in vocal cord biopsies in the different genotypes, B: WT Adult (32 Week Old), C: Cellulose Synthase RNAi Line No. 1 (32 Week Old), D: WT Juvenile (20 Week Old)

Figure 4: A: Relative Cellulose Synthase expression levels in the denoted genotypes, B: Western blot showing the levels of Cellulose Synthase (CS) and Actin in the denoted genotypes, SEM micrograph of vocal chord biopsy from CS overexpression line CS:OX:31

Table 2: Mandrake genotypes, frequency of cry and the respective mice lethality

Figure 5: FTIR spectra of the lignin isolated from the WT Adult and WT Juvenile Mandrake

Table 3: Peaks in lignin FTIR spectra and the corresponding lignin component

Supplemental Figures

Supp. Figure 1. RNA isolate on a 1.5% denaturing agarose gel with the Millennium RNA Markers to determine transcript size from 0.5 to 9 kb (leftmost lane). The degraded sample was only used as an example of degraded RNA (middle lane). There were 2 bands present at ~5070 bp and ~1869 bp and are indicative of the 28s and the 18s rRNA transcripts respectively. The fluorescent ratio of these transcripts was determined to be ~2.3.