Science, Faculty of
DSpace
UBCV
Quesada-Ramirez, Lucia
Master of Science - MSc
University of British Columbia
Powdery mildew infection, which is caused by the fungal pathogen Golovinomyces ambrosiae (Erysiphaceae), has been extensively characterized in Arabidopsis (Brassicaceae), and other species, but not in Cannabis sativa (Cannabaceae), a commercially important plant species. In this thesis, I explore the response of C. sativa to powdery mildew infection. I used microscopy to examine the phenotypic response and RNA-sequencing to examine the transcriptomic response to powdery mildew. I also examined differences between resistant (AGA10) and susceptible (Purple Kush) genotypes. Microscopy revealed that there are differences between the two genotypes in germination and hypersensitive response (programmed cell death). RNA-sequencing revealed differences in expression of genes encoding enzymes of the phenylpropanoid and monolignol biosynthetic pathways, phytoalexin production, and other plant-pathogen responses. Gene ontology (GO) functional analysis shows that the two genotypes also differ in expression of genes related to carbohydrate metabolic processing and other enzymatic processes. I conclude that a likely main factor behind the AGA10 resistant phenotype is increased reactive oxygen species and hypersensitive response, due to earlier or increased expression of genes such as oxidoreductases, peroxidases, phenylalanine ammonia lyase and trans-cinnamate 4-monooxygenase.
EXPLORING CANNABIS SATIVA’S RESPONSE TO POWDERY MILDEW (GOLOVINOMYCES AMBROSIAE) INFECTION by Lucia Quesada-Ramirez B.Sc., The University of British Columbia, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Genome Science and Technology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2022 © Lucia Quesada-Ramirez, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Exploring Cannabis sativa’s response to powdery mildew (Golovinomyces ambrosiae) infection submitted by Lucia Quesada-Ramirez in partial fulfilment of the requirements for the degree of Master of Science in Genome Science and Technology Examining Committee: Lacey Samuels, Professor, Botany, UBC Co-supervisor Loren Rieseberg, Professor, Botany, UBC Co-supervisor Richard Hamelin, Professor, Department Head, Forest and Conservation Sciences, UBC Supervisory Committee Member Sean Graham, Professor, Botany, UBC Additional Examiner Additional Supervisory Committee Members: Joerg Bohlmann, Professor, Forest and Conservation Sciences, UBC Supervisory Committee Member iii Abstract Powdery mildew infection, which is caused by the fungal pathogen Golovinomyces ambrosiae (Erysiphaceae), has been extensively characterized in Arabidopsis (Brassicaceae), and other species, but not in Cannabis sativa (Cannabaceae), a commercially important plant species. In this thesis, I explore the response of C. sativa to powdery mildew infection. I used microscopy to examine the phenotypic response and RNA-sequencing to examine the transcriptomic response to powdery mildew. I also examined differences between resistant (AGA10) and susceptible (Purple Kush) genotypes. Microscopy revealed that there are differences between the two genotypes in germination and hypersensitive response (programmed cell death). RNA-sequencing revealed differences in expression of genes encoding enzymes of the phenylpropanoid and monolignol biosynthetic pathways, phytoalexin production, and other plant-pathogen responses. Gene ontology (GO) functional analysis shows that the two genotypes also differ in expression of genes related to carbohydrate metabolic processing and other enzymatic processes. I conclude that a likely main factor behind the AGA10 resistant phenotype is increased reactive oxygen species and hypersensitive response, due to earlier or increased expression of genes such as oxidoreductases, peroxidases, phenylalanine ammonia lyase and trans-cinnamate 4-monooxygenase. iv Lay Summary Powdery mildew is a disease that affects cannabis (C. sativa) and is a problem for industrial farming of this species. This disease is caused by a fungal pathogen. Powdery mildew has been studied extensively in other plants but not in Cannabis. I studied powdery mildew at the phenotypic and gene expression levels in a susceptible and resistant variety of Cannabis. After examining phenotypic characteristics with microscopy, I found differences in fungal germination and programmed cell death between the two varieties. I also examined gene expression with RNA-sequencing and found differences in genes involved in plant-pathogen interactions, programmed cell death and metabolism. Based on this, I conclude that the resistance phenotype is most likely due to increased programmed cell death through enhanced expression of genes associated with this process. v Preface The Cannabis sativa mother plants used in this project were grown and maintained by Dr. Pauline Duriez and Aurora Cannabis staff. The vegetative clones were transplanted by Dr. Pauline Duriez and me. Dr. Pauline Duriez identified the pathogen causing powdery mildew as Golovinomyces ambrosiae. I inoculated the leaves, did microscopy sampling, imaging and quantification, extracted RNA and did subsequent bioinformatic analysis. vi Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ............................................................................................................................... iv Preface .............................................................................................................................................v Table of Contents ......................................................................................................................... vi List of Tables ................................................................................................................................ ix List of Figures .................................................................................................................................x List of Abbreviations ................................................................................................................... xi Acknowledgements ..................................................................................................................... xii Chapter 1: Introduction ................................................................................................................1 1.1 Overview ......................................................................................................................... 1 1.2 C. sativa farming ............................................................................................................. 1 1.3 Powdery mildew ............................................................................................................. 2 1.3.1 The life cycle of Golovinomyces spp. ................................................................ 3 1.3.2 Management practices ....................................................................................... 4 1.4 Differential gene expression in response to powdery mildew ........................................ 5 1.5 Plant defenses.................................................................................................................. 6 1.5.1 Callose deposition .............................................................................................. 7 1.5.2 Phenolics ............................................................................................................ 8 1.5.3 Plant signaling pathways .................................................................................... 8 1.5.4 Hypersensitive response ..................................................................................... 9 1.6 Research objectives ...................................................................................................... 10 vii Chapter 2: Materials and methods ............................................................................................12 2.1 Plant materials and growth conditons ........................................................................... 12 2.2 Experimental design and set up .................................................................................... 12 2.3 Pathogen identification ................................................................................................. 13 2.4 Inoculation .................................................................................................................... 13 2.5 Plant RNA extraction ................................................................................................... 14 2.6 Fixation and staining for microscopy ........................................................................... 14 2.7 Light microscopy .......................................................................................................... 15 2.8 Scanning electron microscopy (SEM) .......................................................................... 16 2.9 Transcriptome sequencing ............................................................................................ 16 2.10 Gene Ontology (GO) enrichment analysis .................................................................... 17 Chapter 3: Results........................................................................................................................18 3.1 Microscopy .................................................................................................................. 18 3.1.1 Morphology .............................................................................................................. 19 3.1.2 Quantification .......................................................................................................... 22 3.2 RNA-sequencing ........................................................................................................... 28 3.2.1 Identifying differentially expressed genes ................................................................ 28 3.2.2 Top 20 differentially expressed genes ...................................................................... 29 3.2.3 Gene Ontology analysis ............................................................................................ 35 Chapter 4: Discussion ..................................................................................................................42 4.1 Summary of findings .................................................................................................... 42 4.2 Germination rates and penetration rates ...................................................................... 42 4.3 Hypersensitive response, reactive oxygen species and phenolics ............................... 43 viii 4.4 Phenolics and antimicrobial properties ........................................................................ 44 4.5 Phytoalexins and germination suppression ................................................................... 45 4.6 Cell wall and carbohydrate differences between genotypes ......................................... 46 4.7 Timing as a possible key factor in AGA10 resistance .................................................. 47 Bibliography .................................................................................................................................49 ix List of Tables Table 1. Top 20 DEGs in response to powdery mildew infection in Cannabis AGA10 (resistant) genotype ....................................................................................................................................... 30 Table 2. Top 20 DEGs in response to powdery mildew infection in Cannabis Purple Kush (susceptible) genotype ................................................................................................................. 32 Table 3. Top 20 DEGs between the two genotypes, AGA10 (resistant) and Purple Kush (susceptible) during powdery mildew infection ........................................................................... 34 x List of Figures Figure 1. The life cycle of Golovinomyces orontii in Arabidopsis thaliana ................................. 4 Figure 2. Scanning electron microscopy images of Purple Kush Cannabis leaves highly infected with powdery mildew .................................................................................................................. 19 Figure 3. Light microscopy image of powdery mildew on Cannabis leaves, AGA10 genotype 21 Figure 4. Number of germination events for the two genotypes ................................................ 22 Figure 5. Germination rates for the two genotypes ..................................................................... 23 Figure 6. Number of penetration events for the two genotypes .................................................. 24 Figure 7. Penetration rates for the two genotypes ....................................................................... 25 Figure 8. Hypersensitive response events in AGA10 genotype .................................................. 26 Figure 9. Hypersensitive response events for the two genotypes ............................................... 27 Figure 10. Number of differentially expressed genes (DEGs) during powdery mildew infection of Cannabis leaves compared to leaves without infection ........................................................... 29 Figure 11. Top 10 Biological Process and Molecular Function GO terms for AGA10 (resistant) genotype ....................................................................................................................................... 37 Figure 12. Top 10 Biological Process and Molecular Function GO terms for Purple Kush (susceptible) genotype ................................................................................................................. 39 Figure 13. Top 10 Biological Process and Molecular Function GO terms between the two genotypes ..................................................................................................................................... 41 xi List of Abbreviations ABC – ATP-binding cassette ATP – Adenosine triphosphate CAD – Cinnamic acid dehydrogenase C4H – Cinnamate-4-hydroxylase DAPI – 4′,6-diamidino-2-phenylindole DEG – Differentially expressed genes GO – Gene ontology HR – Hypersensitive response ITS – Internal transcribed spacer NCBI – National Center for Biotechnology Information PAL – Phenylalanine ammonia lyase RNA – Ribonucleic acid RNA-seq – RNA-sequencing ROS – Reactive oxygen species SEM – Scanning electron microscopy xii Acknowledgements I would like to express my deepest gratitude to Dr. Lacey Samuels for welcoming me into her lab, her support, patience, guidance and mentorship. I will be forever grateful for her warmth, words of encouragement, and advice. My deepest appreciation also goes to Dr. Loren Rieseberg for welcoming me into his lab, and his invaluable support, guidance and mentorship. I want to give special thanks to Dr. Sean Graham for his irreplaceable support, mentorship, and helping me navigate obstacles during this program. Words cannot express my gratitude. I am also grateful to my committee members, Dr. Jöerg Bohlmann and Dr. Richard Hamelin for their input and support. I want to extend my sincere thanks to Dr. Peter Stirling and Dr. Jöerg Bohlmann for their continued support and helping me through barriers during this program. I am very thankful to Dr. Pauline Duriez for her guidance, and her friendship. Lastly, I would like to thank Dr. Zhe Cai for his help and patience with trouble-shooting bioinformatics issues. 1 Chapter 1: Introduction 1.1 Overview In this thesis I explore the response of Cannabis sativa (Cannabaceae) to powdery mildew infection at the transcriptomic and phenotypic levels. Previous studies characterized powdery mildew infection in other systems, such as Arabidopsis (Brassicaceae); however, information is lacking for C. sativa. The main goals of my thesis are to investigate the processes underlying powdery mildew infection in C. sativa by using complementary techniques, and to identify differences in these processes between susceptible and resistant genotypes. I hypothesize that the functions of differentially expressed genes detected by RNA-seq are correlated with phenotypic differences in the plants, and that the early resistance of the resistant genotype is due to hypersensitive response. To put this work in context, I introduce C. sativa as an important industrial crop, and powdery mildew as a current threat to production. The most common pathogen associated with powdery mildew disease in C. sativa is the ascomycete fungus Golovinomyces ambrosiae (Erysiphaceae), so understanding of its life cycle is important to interpret my data. Additionally, I discuss the use of RNA-seq as a tool to elucidate the transcriptional reprogramming behind the response to powdery mildew. Finally, I briefly review the plant defenses to biotic stress that are known from other species and predicted to occur in this system. 1.2 Cannabis sativa farming Cannabis sativa is grown for many purposes, including fiber, narcotics, oilseed, and biofuel (reviewed by Small, 2015). It is an annual plant that grows vegetatively until induced to flower 2 by photoperiod (Zhang et al., 2021). In Canada, C. sativa is grown in outdoor facilities, indoor facilities such as greenhouses, and in the field with plastic mulch (Punja, 2021). At the industrial level, female stock plants, referred to as mother plants, are propagated vegetatively as clones by placing them on rockwool and inducing them to root (Punja, 2021). Flowering is induced by photoperiod on these clones, and the inflorescences are then harvested (Punja, 2021). 1.3 Powdery mildew Powdery mildew is a very common disease in cultivated C. sativa; it is among the most common fungal pathogens (Scott & Punja, 2020). Powdery mildew in C. sativa looks like white patches on the adaxial surface of leaves, stems and inflorescences, and leads to lower yield and plant vigor (Scott & Punja, 2020). Even when there is not a complete loss of production, powdery mildew causes quality reduction, and is considered to be a significant post-harvest contaminant (Punja et al., 2019). Powdery mildew disease is caused by multiple fungal taxa in the genus Golovinomyces; in C. sativa, these were previously attributed to G. spadiceus and G. cichoracearum sensu lato (Pépin et al., 2018, Szarka et al., 2019, Farinas & Peduto Hand, 2020, Maymon et al., 2020). However, both of those species are now classified as G. ambrosiae, which was recently amended to include a broader host range (Qiu et al., 2020). Golovinomyces is a threat to production, and has been found in industrial and greenhouse environments (Wiseman et al., 2021). Additionally, Golovinomyces was found to be the third most abundant pathogen in medicinal marijuana in northern California (Thompson et al., 2017). 3 1.3.1 The life cycle of Golovinomyces spp. Different Golovinomyces species infect different host plants. Golovinomyces ambrosiae is often responsible for powdery mildew in C. sativa, but Arabidopsis is usually infected by Golovinomyces orontii. The life cycle of Golovinomyces orontii (Figure 1) has been characterized in Arabidopsis (Kuhn et al., 2016). In Arabidopsis, spores develop a short germ tube that turns into an appressorium roughly six hours after inoculation (Kuhn et al., 2016). The appressorium penetrates a epidermal cell, and at the site of penetration, papillae are formed (Kuhn et al., 2016). Papillae are structures formed below the appressorium (Kuhn et al., 2016) that contain callose, reactive oxygen species, and phenolics, among other components (Assaad et al., 2004; Collins et al., 2003; Koh et al., 2005). The haustorium serves as a hyphal feeding structure and, in Arabidopsis, is formed after cell wall penetration at 12–14 hours post-inoculation (Kuhn et al., 2016). Surrounding the haustorium, an extra-haustorial membrane forms as a hub for effector secretion and nutrient uptake (Kuhn et al., 2016). The extra-haustorial membrane is thought to be formed by either invagination and remodeling of the plant (host) plasma membrane, or to be built early during penetration through vesicle secretion of components specific to the extra-haustorial membrane (Koh et al., 2005). Host protein RPW2.8 confers resistance to powdery mildew, and is located in the extra-haustorial membrane, but not the host membrane, supporting the second model (Wang et al., 2009). Secondary hyphae form between 24- and 48-hours post-inoculation, these then give rise to new appressoria that go on to infect additional cells (Kuhn et al., 2016). Approximately three to seven days post-inoculation, conidiophores are formed, a form of hyphae capable of producing spores (Kuhn et al., 2016). 4 In C. sativa, the major landmarks of Golovinomyces infection have not yet been characterized; my first objective was therefore to examine these landmarks using optical microscopy. 1.3.2 Management practices Current management practices for powdery mildew in C. sativa include deleafing, applying humidity levels that are not favourable for fungal growth, and using reduced risk chemicals (pesticides designed to have lower toxicity levels) and fungicides (Scott & Punja, 2020). Fungicides and reduced risk chemicals have been demonstrated to be efficient in controlling the Figure 1. The life cycle of Golovinomyces orontii in Arabidopsis thaliana. The major landmarks with respective timepoints are shown. Taken from Figure 2 with permission of the American Society for Plant Biologists (Kuhn et al., 2016). hpi=hours post inoculation, dpi=days post inoculation 5 fungus to varying degrees (Scott & Punja, 2020). However, Health Canada limits the use of fungicides due to health concerns related to inhalation of chemical residues (Punja, 2021). Other strategies to manage powdery mildew in C. sativa include strategic environmental control and use of high-powered UV-C clamps (Scott & Punja, 2020). Harnessing plant resistance (R) genes is another possible avenue for dealing with powdery mildew; however, resistance genes tend to be highly specific and to work on only a narrow range of pathogens (Kourelis & van der Hoorn, 2018). Resistance genes work by conferring resistance through an R gene that is complementary to a pathogen avirulence gene, known as the “gene-for-gene” model (Flor, 1971; Staskawicz et al., 1995). Partial resistance has also been documented due to loss of function and deletion mutations in susceptibility (S) genes, such as mildew resistance locus O (MLO) (Kusch & Panstruga, 2017). Originally discovered in barley, recessive mutations to the mlo confer resistance to powdery mildew (Jørgensen, 1992). The main difference between MLO resistance compared to that of R genes, is that MLO genes are not race-specific (Brown, 2015; Lyngkjaer & Carver, 2000). MLO genes have been found in most economically important species and are now considered a “universal” method for acquiring resistance (Kusch & Panstruga, 2017). 1.4 Differential gene expression in response to powdery mildew Analysis of RNA-sequencing and differential expression in response to powdery mildew has been done in multiple systems. For example, in apple, differentially expressed genes in response to powdery mildew infection were found to include transcription factors, pathogenesis-related proteins, and phytohormone signaling genes (Tian et al., 2019). Similarly in pumpkin, genes found to be differentially expressed in response to powdery mildew were those related to plant 6 defense, transcription factors and hormone-signal transduction pathways (Guo et al., 2018). A study of RNA-seq in C. sativa in response to powdery mildew was recently done (Pépin et al., 2021). They used the Pineapple Express genotype and looked at differential expression of mlo genes to further elucidate this gene family in C. sativa. Naturally occurring resistance to G. ambrosiae was also recently reported in cannabis through an resistance (R) gene that follows dominant, single-gene inheritance (Mihalyov & Garfinkel, 2021). While mlo genes are important candidate genes, other genes are likely also involved in the response to infection and biotic stress. In my thesis project I plan to identify differentially expressed genes in response to powdery mildew infection and conduct follow-up gene ontology and pathway analysis. I will also look at the response to infection using optical microscopy to explore infection at the phenotypic level. The aim is to provide a more comprehensive overview of the response to powdery mildew infection in C. sativa. This will lead to the development of more tools to manage powdery mildew, in addition to exploiting susceptibility or resistance genes. 1.5 Plant defenses Plant defenses to biotic stress include physical barriers (cuticle waxes, trichomes), chemical defenses, and responses triggered by pattern recognition receptors (PRRs) and plant resistance (R) proteins (reviewed by Iqbal et al., 2021). Pattern recognition receptors are triggered by pathogen-associated molecular patterns (PAMPs) (Monaghan & Zipfel, 2012, Iqbal et al., 2021). These molecular patterns, such as chitin, are released by the fungus or pathogen in question, and activate immune signaling (Boller & Felix, 2009, Iqbal et al., 2021). The plant then mounts a 7 defense response to these events, such as accumulating defense-related proteins and depositing callose (Iqbal et al., 2021). 1.5.1 Callose deposition Callose is a β-1,3-glucan, an amorphous cell-wall polymer that is triggered in the response by plants to both abiotic and biotic stressors (reviewed by Ellinger & Voigt, 2014). While growth and environmental conditions affect the plant’s ability to deposit callose, callose deposition is an established immune response to biotic stress (Luna et al., 2011). During biotic stress, callose deposition occurs mostly in between the cell wall and plasma membrane, and this is believed to serve a direct protective function against the pathogen in question (Nishimura et al., 2003, Ellinger & Voigt, 2014). These sites of callose deposition and resulting cell-wall thickening are termed papillae (Ellinger & Voigt, 2014). Callose synthases involved in barrier formation and biotic stress include Glucan Synthase-like (GSL) 5, GSL6, GSL7, GSL11 and GSL12 (Jacobs et al., 2003, Nishimura et al., 2003, Luna et al., 2011, Ellinger & Voigt, 2014). Callose synthases are tightly regulated temporally and spatially (Ellinger & Voigt, 2014). These regulatory mechanisms potentially include transcriptional regulation, phosphorylation, complex formation and callose synthase transport (Ellinger & Voigt, 2014). Callose deposition in papillae is prevalent during fungal attack, as in epidermal cells during powdery mildew infection (Jacobs et al., 2003; Nishimura et al., 2003; Zimmerli et al., 2004; Koh et al., 2005; Nielsen et al., 2012; Ellinger & Voigt, 2014). In Arabidopsis, the callose synthase GSL5 is relocated to the plasma membrane for callose deposition at the site of fungal penetration (Ellinger et al., 2013). Callose-mediated fungal resistance relies on a few key factors, such as precise timing of callose synthesis, and cell-wall strengthening acting as a barrier against pathogen-secreted molecules (Nielsen et al., 2012, Ellinger et al., 2013, Naumann et al., 2013, Eggert et al., 2014). 8 1.5.2 Phenolics Phenolics are compounds derived from the amino acids phenylalanine or tyrosine via the general phenylpropanoid pathway (reviewed by Dixon et al., 2002). Phenolics are a highly diverse group and include many compounds, which range from lignin to pigments like anthocyanins and small molecules like salicylic acid (Dixon et al., 2002). A subset of this large class of compounds are involved in plant defenses against pathogens and biotic stress; these compounds tend to accumulate early on during infection and serve to decelerate pathogen growth (reviewed by Chowdhary et al., 2021). Phenolic responses include the synthesis of lignin, and accumulation of phenols, such as phenylpropanoids and benzoic acids within cell walls (reviewed by Nicholson & Hammerschmidt, 1992). Host accumulation of polymerized phenols may be responsible for autofluorescence seen at the sites of infection (Nicholson & Hammerschmidt, 1992). Phenolic compounds in plants can have the seemingly dual function of being antioxidants and causing cytotoxicity (reviewed by Shalaby & Horwitz, 2014). In fungal infections, phenolic compounds are capable of inducing fungal enzyme degradation (Shalaby & Horwitz, 2014). Phenolics such as cinnamic acid, benzoic acid and salicylic acid also seemingly have anti-microbial and anti-fungal properties (Shalaby & Horwitz, 2014). 1.5.3 Plant signaling pathways Salicylic acid and jasmonic acid play a role in plant defense signaling, these signaling interactions are highly specific to each pathogen (reviewed by Beckers & Spoel, 2006). Additionally, the interplay between these two compounds has its own role in plant defenses (Beckers & Spoel, 2006). Salicylic acid has mostly been observed as a response to biotrophs (Beckers & Spoel, 2006). It is thought to be positively regulated by NPR1 (non-expressor of PR 9 genes) and is involved in systemic acquired resistance, which primes the plant to resist subsequent pathogen infections (Beckers & Spoel, 2006). Jasmonic acid signaling also happens in response to biotic stress, but usually during herbivory and in response to necrotrophy (Beckers & Spoel, 2006). Salicylic acid and jasmonic acid can act synergistically or antagonistically (Beckers & Spoel, 2006). 1.5.4 Hypersensitive response Plant hypersensitive response refers to programmed cell death as a direct response to pathogen penetration (reviewed by Balint‐Kurti, 2019), and was first observed by Stakman (1915) in association with disease resistance to Puccinia graminis. Hypersensitive response is induced by a range of pathogens, usually fungi that have a biotrophic lifestyle (Balint‐Kurti, 2019). Most cases of hypersensitive response are associated with R genes, and this type of dominant resistance is observed when an R gene co-exists with an avirulence gene of the corresponding pathogen (Balint‐Kurti, 2019; Kourelis & van der Hoorn, 2018). Hypersensitive response is thought to be initiated by oxidative and nitric oxide burst early in the infection process (Delledonne et al., 2001). Processes such as transcriptional reprogramming, cell-wall fortification and ion fluctuation are also associated with hypersensitive response (Balint‐Kurti, 2019). These known plant defense responses should be considered as possible mechanisms taking place in C. sativa powdery mildew infection. Among the differentially expressed genes in C. sativa during powdery mildew infection using RNA-seq, I predict that genes related to processes, such as callose deposition, phenolic synthesis, and salicylic acid signaling, will be represented. Additionally, I predict differences in differential expression in these genes between the resistant and susceptible genotypes. For example, if the resistant genotype shows 10 differentially expressed genes involved in oxidative burst and cell-wall fortification, this would suggest that hypersensitive response may contribute to the resistance observed in this genotype. 1.6 Research objectives Powdery mildew has been studied in multiple species and has been characterized extensively in Arabidopsis. There have been multiple reports of powdery mildew in C. sativa, but these reports have mostly consisted of pathogen identification. The only RNA-seq study of powdery mildew C. sativa (Pépin et al., 2018) so far was focused on the MLO gene family. Additionally, RNA-seq and differential expression analysis currently have only been done on genotypes susceptible to powdery mildew. For my thesis I carried out light microscopy, SEM and RNA-seq and differential gene expression analysis, with the aim of using these techniques in a complementary manner. Light microscopy can give insights into what occurs at the phenotypic level during powdery mildew infection, while RNA-seq monitors changes at the transcriptional level. Comparison of both susceptible resistant genotypes can give some insight into the differences at the phenotypic and transcriptional levels that are responsible for limiting powdery mildew infection. My thesis objectives are as follows: Objective 1: Generate qualitative data to gain a snapshot overview of infection. This work was done using optical microscopy. The first biological experiment was used to get a sense of suitable conditions for growing powdery mildew in C. sativa, and to get a sense of the timing of this to inform Objective 3. After carrying out this first objective, 48 h post-inoculation was 11 selected as the timepoint for RNA-seq. Higher humidity and lower light intensity were found to be more favourable for powdery mildew growth. Objective 2: Determine which genes are important in the Cannabis powdery mildew infection process. This work was carried out with RNA-seq. Using inoculated vs non-inoculated control samples, the aim was to identify which host plant genes are related to the response to infection. Differentially expressed genes result between the two conditions. I did further analysis to compare differences between susceptible and resistant genotype. Objective 3: Identify the phenotypic and transcriptomic differences in susceptible and resistant plant responses during powdery mildew infection. This goal of this objective is to get a more comprehensive view of the process by correlating the two techniques. With optical microscopy, I imaged susceptible and resistant genotypes to determine differences in the phenotypic responses of plant to the fungal attack. I collected additional replicates for microscopy under this objective. Using RNA-seq, I also identified transcriptomic differences in the plant responses to the fungal attack between susceptible and resistant genotypes. 12 Chapter 2: Materials and Methods 2.1 Plant materials and growth conditions Cannabis sativa mother plants were grown vegetatively in soil, in one-gallon pots, following an 18 h light, 6 h dark cycle. The soil used was 60-75% Canadian Sphagnum peat moss, perlite, and dolomite limestone. Plants were kept at a temperature of 22C and at 70% relative humidity. The mother plants were irrigated every day by flooding table and draining after 10 minutes. 2.2 Experimental design and set up Powdery mildew (Golovinomyces ambrosiae) grows on the epidermal surface of leaves, so cannabis leaves are the tissue of interest in this project. The C. sativa genotypes used in this project are Purple Kush, which is susceptible to infection, and AGA10, which is resistant. Vegetative clones were taken from mother plants: the size of the branches taken was between 10 and 20 cm. These were placed in rockwool and induced to root by dipping in hormone rooting powder. After rooting, the clones were then transplanted to soil (as above), into 1-L pots. The plants were placed in quarantine chambers to avoid external contamination. One quarantine chamber was designated for inoculated plants and the other for non-inoculated plants. The chambers were situated side by side and have the same conditions. Each pot was watered once a week with 100 mL of water and Peter’s excel fertilizer and kept at a light intensity of 10 mol/m2/s. Both genotypes were kept in each chamber for two months. 13 2.3 Pathogen Identification Naturally occurring powdery mildew was brought to the Aurora Cannabis facility at UBC through a contaminated vegetative clone from the Aurora Cannabis facility in Comox, BC. The pathogen causing powdery mildew was identified using internal transcribed spacer (ITS) sequencing. The ITS regions were amplified and sequenced, and the sequences were then BLASTed against the NCBI database. The sequences were a 100% similarity match to Golovinomyces ambrosiae, Golovinomyces cichoracearum, Golovinomyces latisporus and Golovinomyces spadiceus. These species, with the exception of G. latisporus are now classified as G. ambrosiae under an amended, broader host range (Qiu et al., 2020). 2.4 Inoculation Infected leaves were used as a source of Golovinomyces ambrosiae conidiospores to inoculate Cannabis plants. The source plants were 10 weeks old and had been infected with powdery mildew for eight weeks. The inoculation was done on the three middle leaflets of the compound leaves by gently pressing and rubbing highly infected leaves against them. Each inoculated leaf was marked for collection. The plants inoculated for microscopy and RNA-seq were eight weeks old after transplantation. The first round of inoculations was done with a relative humidity of 40% and was not successful. Conditions were modified by increasing the relative humidity to 80% and decreasing the light intensity to 10 lux, this resulted in successful inoculation. 14 2.5 Plant RNA extraction Leaves were sampled at 48 h post-inoculation by plucking them and using a leaf puncher. Three leaf disks from three different leaves of the same plant were pooled to make one replicate and placed into an Eppendorf 2 mL tube. Three replicates, representing three plants, were collected and frozen in liquid Nitrogen and stored at -80°C. The leaf tissue was ground using the tissue lyser GenoGrinder. RNA extractions were carried out following manufacturer instructions with the Qiagen RNeasy kit, using RLC buffer and eluting RNA in RNase-free water. Following extraction, the RNA was treated with Turbo free DNase enzyme (Invitrogen) to ensure there was no genomic DNA remaining in the samples. RNA integrity was ascertained by running a 1% agarose gel and checking for distinct and clear 28S and 18S rDNA bands. 2.6 Fixation and staining for microscopy First, preliminary experiments to determine staining conditions, to test for timing and productive conidiospore germinations. Leaf samples from Purple Kush and AGA10 plants were collected at 4, 8, 12 and 24 h post inoculation (hpi). The experiment was repeated twice to establish timing of infection. Using this information, Purple Kush and AGA10 plants were sampled for the microscopy data reported in Chapter 3 in two replicate experiments. In the first experiment, three leaf disks came from one plant and in the second experiment, four leaf disks from one plant were sampled. Each disk came from a different leaf, for a total of seven disks, representing seven leaves. Leaves were sampled at 48 h post-inoculation by plucking them and using a leaf puncher. Each leaf disk that was plucked was placed into an Eppendorf 2 mL tube. The samples were fixed in ethanol:acetic acid (3:1) for light microscopy, and 2% glutaraldehyde in 50 mM of PIPES buffer pH 7.0 for scanning electron microscopy (SEM). For light microscopy, the samples 15 were dually stained with 0.1% aniline and 0.1% trypan blue following the methodology by Bhadauria et al. (2010). Aniline blue stains callose and trypan blue stains chitin and yeast glucan, so the stain combination shows fungal infection structures and host plant defense reactions (Bhadauria et al., 2010). 2.7 Light microscopy After fixation and staining, inoculated leaf discs were imaged using an Olympus BX53 microscope with 4X, 20X and 40X objectives and CellSens software. Samples were mounted in 30% glycerol. Leaf sections were first scanned using the overview function at the 4X objective. By this means, it was possible to locate a specific field of view on the overall structure of the respective leaf. For each leaf sample, five fields of view were acquired using brightfield microscopy. Images of each respective field of view were also acquired using a DAPI filter to check for aniline blue fluorescence. For each field of view, the number of germinated spores in brightfield was counted to use as a proxy for germination rate. The number of germinated spores adjacent to plant cells with aniline blue fluorescence was also quantified, as a proxy for penetration rate. The number of hypersensitive cell responses, where an entire epidermal cell was fluorescent due to phenolic autofluorescence, was also counted for each genotype. A total of seven samples per genotype was used, and five fields of view were captured for each sample, for a total of 35 fields of view per genotype. A two-tailed, two-sampled student t-test was used to determine if differences between the two genotypes for quantification metrics were statistically significant. Non-inoculated controls were checked under brightfield and DAPI filters at 20X to document 16 background fluorescence levels of leaves with no powdery mildew growing on them, and to help ensure non-inoculated plants were not contaminated with inadvertent powdery mildew infection. 2.8 Scanning electron microscopy (SEM) Fixed samples were post-fixed in osmium tetroxide (OsO4), followed by a dehydration series and critical point drying. Samples were then sputter coated with gold. Imaging was done with Hitachi S-2600 Variable Pressure SEM. 2.9 Transcriptome sequencing The DNase-treated RNA was sent to the Biomedical Research Centre at the University of British Columbia for mRNA sequencing. A stranded, polyA library preparation was done, followed by paired-end read sequencing (read length 59 bp). Adaptor trimming was done by the sequencing centre. RNA-seq reads were checked for quality with FastQC (Andrew, 2010). The reads were mapped with the gapped aligner STAR (Dobin et al., 2013) to the Cannabis CS10 assembly reference genome (Genbank assembly accession: GCA_900626175.2), following ENCODE parameters. Reads were quantified with the python-based program HTSeq (Anders et al., 2015). Gene expression analysis between the two conditions (susceptible vs. resistant) was done with the R Bioconductor package DESeq2 (Love et al., 2014) to identify differentially expressed genes (DEGs). The p-value used as a cutoff was 0.05, adjusted for multiple test correction using the Benjamini-Hochberg procedure. Genes with low expression levels are automatically removed by DESeq2 before testing for significance. 17 2.10 Gene ontology (GO) enrichment analysis The DEGs identified for each condition were then used for downstream gene ontology (GO) analysis. Cannabis sativa, like nearly all non-model plants, does not have official GO annotations. In this case, to be able to carry out GO analysis, an annotation file that maps genes to GO terms is required. This was retrieved from GOMAP_Cannabis_sativa_NCBI-cs10_January_2020. 1, 1.0. CyVerse Data Commons (Lawrence-Dill, 2020). Gene Ontology analysis was done with the TopGO R Bioconductor package (Alexa et al., 2006). Overrepresentation analysis was carried for each of the three GO subcategories: Biological Process, Molecular Function and Cellular Component, using Fisher’s test. The top 10 most enriched GO terms for each subontology were then plotted using the enrichment_barplot function. 18 Chapter 3: Results 3.1 Microscopy I observed phenotypic changes in Cannabis leaves in response to powdery mildew using light microscopy and SEM imaging, and generated qualitative data to gain an overview of infection. Originally, samples were collected at 4, 8, 12 and 24 h post inoculation (hpi), fixed and stained for optical microscopy. In two replicate experiments, minimal germination rate was seen in the first 24 h with these conditions. Given the low germination rate with those conditions, lower light and higher humidity were assessed to see if growth was favored under these conditions. While I did not quantify germination between the original and modified conditions, the modified conditions showed a visible and qualitative increase in germination. I therefore used a single timepoint of 48 h post-infection moving forward. The light microscopy samples were dually stained with 0.05% aniline blue and 0.05% trypan blue following the methodology of Bhadauria et al. (2010). Aniline blue stains callose and trypan blue stains chitin and yeast glucan, so the combination shows fungal infection structures and host plant defense reactions (Bhadauria et al., 2010). Seven leaf disks were sampled per genotype, and for each leaf disk, images were acquired for five different fields of view. In total, thirty-five field of views were acquired per genotype for quantification purposes. Each field of view has an area of 0.32 mm2. Uninfected controls of both genotypes were sampled and stained, and images were acquired, to ensure the germination observed was the result of inoculation and not contamination. 19 I generated the SEM images using a highly infected leaf sample. SEM images show the surface details of powdery mildew infection on the Cannabis leaf in a more intuitive way than light microscopy, and help to better visualize the fungal structures. 3.1.1 Morphology Powdery mildew forms a hyphal network on the Cannabis leaf surface during infection. SEM images of heavily infected Purple Kush Cannabis leaves are shown in Figure 2. A hyphal network is visible, and secondary hyphae and conidiophores are also present. Figure 2. Scanning electron microscopy images of Purple Kush Cannabis leaves highly infected with powdery mildew. T=trichome, H=hypha, C=conidiophore T H C T H H T 20 I also acquired brightfield images of leaves infected with powdery (see Figure 3, which shows these leaves at 48 hpi for the AGA10 genotype); these images show the germinating spores on the leaf epidermal cells (the spores are stained blue). Germinating spores have a germ tube protruding from the spore. Hyphae are also visible in these samples and are also stained blue. Fluorescence imaging using DAPI shows callose deposition at sites of penetration. The controls show no contamination (brightfield) and no fluorescence (DAPI), confirming that fluorescence observed in infected samples is due to callose deposition. 21 Figure 3. A. Light microscopy image of powdery mildew on Cannabis leaves, AGA10 genotype. The blue stained structures are spores and hyphae. Germinated spores have a germ tube protruding from the spore. B. Fluorescence image of powdery mildew on Cannabis leaves, AGA10 genotype. Callose plugs look like fluorescent round spots at the site of penetration. C-D. Control images of non-infected and stained leaves, AGA10 genotype. The controls show there was no powdery mildew contamination. No spores or fungal structures are observed (C), and no fluorescence (D) is seen in these control samples. This confirms that the fluorescence seen in the infected samples is due to callose deposition in response to powdery mildew. T=trichome, S=spore, GT=germ tube, H=hyphae, CP=callose plug S GT T H CP T A. B. C. D. 22 3.1.2 Quantification To test whether the spores on the surface of the leaves had similar levels of germination for the two genotypes, I counted the number of germinated spores per field of view for each genotype. A germination event is defined as a spore with a protruding germ tube. The mean number of germinated spores per field of view for AGA10 (resistant) is 4.71 (n=35), and for Purple Kush (susceptible) 6.34 is (n=35). Figure 4 shows the distribution and mean germination events for each of the two genotypes. A two-sample, two-tailed student t-test was carried out and the difference in germination events between the two genotypes was found to be statistically significant (p-value 0.03). This shows that fewer spores germinated in AGA10 compared to Purple Kush. Figure 4. Number of germination events for the two genotypes. Cannabis leaves of each genotype were infected with powdery mildew, fixed, and stained. The number of germinated spores per field of view were counted for a total 35 fields of view per genotype. The violin plot shows the distribution, and the rhombus shows the mean number of germination events. Overall, fewer germination events were observed in AGA10 (resistant) compared to Purple Kush (susceptible). The difference was found to be statistically significant (P = 0.03). 23 Because the difference in number of germination events could be explained if there were fewer spores on AGA10 compared to Purple Kush, I also calculated the germination rate between the two genotypes by dividing the number of germinated spores by the total number of spores present in a field of view. The mean germination rate for AGA10 was 27.8 (n = 14) and 57.8 (n = 14) for Purple Kush; this difference was found to be statistically significant (P = 0.001; Figure 5). In addition to having fewer germination events, the spores are germinating at a lower rate in AGA10 compared to Purple Kush. Figure 5. Germination rates for the two genotypes. Cannabis leaves of each genotype were infected with powdery mildew, fixed, and stained. The germination rates were calculated per field of view for a total of 14 fields of view per genotype. The violin plot shows the distribution, and the rhombus shows the mean germination rate. Overall, the germination rate observed in AGA10 (resistant) was lower than the rate observed in Purple Kush (susceptible). The difference was found to be statistically significant (P = 0.001). 24 Because not all the spores that germinate also penetrate the epidermal leaf cells, penetration events were counted. Penetration events can be determined by looking for aniline blue fluorescence at the location of a germinated spore under DAPI filter. The mean number of penetration events for AGA10 was 0.71 (n=35) and for Purple Kush 1.54 (n=35), summarized in Figure 6. The difference in penetration events between the two genotypes was also not found to be statistically significant using the conventional p-value of 0.05 (p = 0.06), but the differences between the means and variability shown in the violin plot indicate further sampling of the penetration events may reveal differences. Figure 6. Number of penetration events for the two genotypes. Cannabis leaves of each genotype were infected with powdery mildew, fixed, and stained. The number of penetrating spores per field of view were counted for a total 35 fields of view per genotype. The violin plot shows the distribution, and the rhombus shows the mean number of penetration events. While less penetration events were observed in AGA10 (resistant) compared to Purple Kush (susceptible), the difference was (P=0.06), close to the conventional p-value cut-off of 0.05. 25 While there is no significant difference in the number of penetration events between the two genotypes, the penetration rate was calculated to see if there was a significant difference in rates. The penetration rate was calculated by dividing the number of penetration events by the number of germination events. The mean penetration rate for AGA10 is 14.29 (n=35) and for Purple Kush 22.05 (n=35). Figure 7. Penetration rates for the two genotypes. Cannabis leaves of each genotype were infected with powdery mildew, fixed, and stained. The penetration rates were calculated per field of view for a total of 35 fields of view per genotype. The violin plot shows the distribution, and the rhombus shows the mean penetration rate. While the rate of penetration events was lower in AGA10 (resistant) compared to Purple Kush (susceptible), the difference in penetration rates was not found to be statistically significant (P = 0.17). 26 I also observed Hypersensitive response (HR), or programmed cell death events during powdery mildew infection. Occurrences of fluorescence around the perimeter of an epidermal cell due to lignification were considered a HR event, e.g., see HR events in an infected AGA10 sample in Figure 8; all the HR events present in this sample are circled in red. In the two replicate experiments, I observed hypersensitive response multiple times in AGA10 and only once in Purple Kush (Figure 9). The difference was found to be significant between the two genotypes (p-value 0.002). This shows that hypersensitive response is happening more frequently in AGA10 compared to Purple Kush. Figure 8. Hypersensitive response events in AGA10 genotype. Fluorescence around the perimeter of an epidermal cell due to lignification signals a HR event during powdery mildew infection. Each event is circled in red. 27 Figure 9. Hypersensitive response events for the two genotypes. Cannabis leaves of each genotype were infected with powdery mildew, fixed, and stained. The number of HR events per field of view were counted for a total 35 fields of view per genotype. Overall, more HR events were observed in AGA10 (resistant) compared to Purple Kush (susceptible). The difference was found to be statistically significant (P = 0.002). 28 3.2 RNA-Sequencing I chose 48 h post inoculation as the RNA-seq timepoint because a clear phenotypic response was observed with microscopy at this timepoint, and so this timing should ensure an observable robust transcriptional response correlating with phenotype. After screening RNA-sequencing reads from Cannabis leaves for quality control, I aligned them to the Cannabis CS10 assembly reference genome, followed by read quantification performed with HTSeq (Anders et al., 2015). 3.2.1 Identifying differentially expressed genes To test whether the phenotypes observed by microscopy reflect changes in gene expression, I performed differential expression analysis between control and infected samples for the two genotypes, AGA10 (resistant) and Purple Kush (susceptible). Additionally, differentially expressed genes (DEGs) were identified between the two genotypes during infection. To identify these genes, first all the DEGs between the two genotypes from infected plants and from non-infected (control) plants were identified, then the genes that overlapped both conditions were removed from the list, because DEGs identified from control plants are not directly involved in infection. This way only the genes that were differentially expressed between the two genotypes during infection were kept and analyzed further. For the resistant AGA10 variety, 541 genes were found to be differentially expressed during infection compared to control conditions, and for the sensitive Purple Kush variety, 1558 genes were found to be differentially expressed during infection (Figure 10). A total of 3335 genes were found to be differentially expressed between the two genotypes during infection. 29 3.2.2 Top 20 differentially expressed genes The next step was to explore whether the gene expression reflected Cannabis plant responses to powdery mildew infection. After ranking the DEGs by statistical significance (p-value), the top 20 DEGs for each genotype and between genotypes were examined. Genes with low expression levels are automatically removed by DESeq2 before testing for significance (Love et al., 2014). Gene descriptions for each gene were retrieved using Entrez Direct (Kans, 2013). Table 1 shows the top DEGs ordered by p-value for AGA10. The gene ID, gene description and p-value are shown. Figure 10. Number of differentially expressed genes (DEGs) during powdery mildew infection of Cannabis leaves compared to leaves without infection. Two varieties were tested: Purple Kush (susceptible) and AGA10 (resistant). Following 48 h post inoculation with conidiospores, three leaves from one plant were pooled to make a replicate. Three replicates per genotype were analyzed. 30 Gene ID Gene Description Direction p-value LOC115721103 germin-like protein subfamily 2 member 1 upregulated 1.20 e-18 LOC115708777 cytochrome P450 71A22-like (putative indoleacetaldoxime dehydratase) upregulated 5.15e-17 LOC115719463 trans-cinnamate 4-monooxygenase/ cinnamate 4-hydroxylase (C4H) upregulated 8.29e-12 LOC115700724 protein COBRA upregulated 2.43e-10 LOC115709995 aquaporin PIP2-2 upregulated 6.31e-09 LOC115697719 aquaporin PIP1-1 upregulated 8.38e-09 LOC115696650 peroxidase 15 upregulated 1.00e-08 LOC115707956 shikimate O-hydroxycinnamoyltransferase (HCT) upregulated 1.19e-08 LOC115699054 ribonucleoside-diphosphate reductase small chain upregulated 1.52e-08 LOC115701280 phenolic glucoside malonyltransferase 1 upregulated 3.19e-08 LOC115724148 probable glutathione peroxidase 2 upregulated 3.43e-08 LOC115706668 phenylalanine ammonia-lyase 1 (PAL1) upregulated 1.26e-07 LOC115702938 sugar transporter ERD6-like 7 upregulated 1.37e-07 LOC115719391 pleiotropic drug resistance protein 2-like upregulated 1.44e-07 LOC115713020 cinnamyl alcohol dehydrogenase 1 (CAD1) upregulated 5.25e-07 LOC115711929 uncharacterized LOC115711929 upregulated 5.29e-07 LOC115697432 auxin-repressed 12.5 kDa protein downregulated 5.99e-07 LOC115711343 S-adenosylmethionine synthase 3 upregulated 6.80e-07 LOC115720696 pleiotropic drug resistance protein 2 upregulated 6.80e-07 LOC115717039 uncharacterized LOC115717039 upregulated 7.67e-07 Table 1. Top 20 DEGs in response to powdery mildew infection in Cannabis AGA10 (resistant) genotype. DEGs were identified by comparing read counts from infected leaves to uninfected leaves, harvested 48 h post inoculation. The genes are ordered by p-value. The gene description for each gene is listed. Whether genes were upregulated or downregulated relative to the uninfected control is listed in the directionality column. 31 Within this list, there are several DEGs that can be related in an obvious manner to powdery mildew infection. These include genes previously reported to be upregulated in fungal infections, such as Germin-like protein subfamily 2 member 1 (LOC115721103). This gene encodes a glycoprotein involved in plant response to stress. In cotton, germin-like proteins have been found to be involved in fungal pathogen response (Pei et al., 2019). Other DEGs could be related to the production of soluble phenolic defense compounds, such as cytochrome P450 71A22-like (LOC115708777) a putative indoleacetaldoxime biosynthetic gene. In Arabidopsis, the P450 family of cytochrome enzymes are involved in the production of camalexins (Glawischnig, 2006), and although this class of phytoalexins are restricted to the Brassicaceae, the upregulation of this enzyme could relate to an unknown phytoalexin biosynthesis. Key enzymes of the general phenylpropanoid pathway are also differentially expressed in the resistant AGA10 variety during infection, such as phenylalanine ammonia-lyase1, PAL1 (LOC115706668) and cinnamate 4-hydroxylase, C4H (LOC115719463) (Dixon et al., 2002). Significant enzymes in the monolignol biosynthetic pathway, such as shikimate O-hydroxycinnamoyltransferase, HCT (LOC115707956) and cinnamyl alcohol dehydrogenase 1, CAD1 (LOC115713020) that catalyzes the last step of monolignol biosynthesis were differentially expressed in AGA10 (Dixon et al., 2002). The induction of monolignol biosynthetic genes in the resistant AGA10 variety suggests that pathogen-triggered lignification could be occurring in this variety. In addition to biosynthetic pathway genes, ABC (ATP-binding cassette) transporters were upregulated in response to pathogen attack. These are members of the pleiotropic drug resistance-type subfamily of ABC transporters, which has been reclassified into the ABCG subfamily (Verrier et al., 2009). The differentially expressed pleiotropic drug resistance protein 32 2-like (LOC115719391) and pleiotropic drug resistance protein 2 (LOC115720696) are orthologs of Arabidopsis thaliana AtABCG30. ABCG transporters from several species have been reported in other studies of plant responses to pathogens by transporting anti-microbial compounds (Do et al., 2021). In addition, a gene encoding an auxin-repressed 12.5 kDa protein (LOC115697432) was downregulated, which is possibly significant as these proteins are reported to respond to hormones that are involved in the response to biotic stress (Souza et al., 2019). In contrast to the strong differential regulation of the phenylpropanoid pathway and monolignol biosynthesis in the resistant AGA 10, the sensitive Purple Kush variety did not have phenolic metabolic genes in the top 20 DEGs. In this case, multiple DEGs relevant to plant defense were detected (Table 2). Gene ID Gene Description Direction p-value LOC115699396 endochitinase EP3 upregulated 1.29e-64 LOC115722579 proline-rich protein 4 upregulated 6.58e-36 LOC115708023 endochitinase EP3 upregulated 1.03e-30 LOC115707933 BURP domain-containing protein BNM2A upregulated 1.02e-23 LOC115722890 phosphoenolpyruvate carboxylase kinase 1 upregulated 2.90e-23 LOC115706309 pathogenesis-related protein PR-1 type-like downregulated 5.52e-23 LOC115708243 germin-like protein subfamily 3 member 1 downregulated 5.81e-23 LOC115707511 WRKY DNA-binding transcription factor 70 upregulated 1.96e-22 LOC115714544 putative ABC transporter C family member 15 upregulated 2.70e-22 LOC115700763 uncharacterized LOC115700763 upregulated 4.14e-21 LOC115696784 wall-associated receptor kinase 2 upregulated 1.75e-20 LOC115701247 carbonic anhydrase 2 downregulated 2.11e-20 LOC115701200 protein SPIRAL1-like 3 downregulated 9.89e-19 LOC115697714 wall-associated receptor kinase 2-like upregulated 7.16e-18 LOC115724459 subtilisin-like protease SBT5.3 upregulated 1.02e-17 LOC115704755 probable inactive purple acid phosphatase 29 upregulated 5.06e-17 Table 2. Top 20 DEGs in response to powdery mildew infection in Cannabis Purple Kush (susceptible) genotype. DEGs were identified by comparing read counts from infected leaves to uninfected leaves, harvested 48 h post inoculation. The genes are ordered by p-value. The gene description for each gene is listed. Whether genes were upregulated or downregulated relative to the uninfected control is listed in the directionality column. 33 Chitinases (LOC115699396 and LOC115708023) are enzymes involved in fungal pathogen attack (Punja & Zhang, 1993). Chitin is one of the major components of fungal cell walls. Pathogenesis-related protein PR-1 type-like (LOC115706309) is involved in response to microbial pathogen attack (Breen et al., 2017). Germin-like protein subfamily 3 member 1 (LOC115708243), like germin-like protein subfamily 2 member 1 (LOC115721103) in AGA10, encodes a glycoprotein involved in plant response to stress. Wall-associated receptor kinase 2 (LOC115696784) and wall-associated receptor kinase 2-like (LOC115697714) encode cell wall-associated kinases. In Arabidopsis these proteins are bound to the cell wall and bind pectins; this role has been demonstrated to correlate with pathogen response (Kohorn & Kohorn, 2012). Beta-glucosidase BoGH3B (LOC115719163) is a beta-glucosidase enzyme. During herbivore and pathogen attack, these proteins activate phytoanticipins, secondary metabolites that act as chemical defenses in plants, and so, these enzymes are known as chemical activators (Morant et al., 2008). LOC115719524 uncharacterized LOC115719524 upregulated 1.76e-16 LOC115721769 uncharacterized LOC115721769 upregulated 1.30e-15 LOC115719163 beta-glucosidase BoGH3B upregulated 4.29e-15 LOC115711534 purple acid phosphatase 3 upregulated 2.54e-14 34 Finally, Table 3 shows the top DEGs between the two genotypes during powdery mildew infection ordered by p-value. The gene ID, gene description and p-value are shown. DEGs between the two genotypes that are relevant to powdery mildew are noted in Table 3. Glycine-rich cell wall structural protein 2 (LOC115708235) belongs to a class of glycine-rich proteins that are thought to be implicated in plant defense. Specifically, a glycine-rich protein has Gene ID Gene Description Direction p-value LOC115723393 uncharacterized LOC115723393 upregulated 4.50e-32 LOC115714277 purple acid phosphatase 15 downregulated 1.40e-31 LOC115708235 glycine-rich cell wall structural protein 2 downregulated 2.15e-30 LOC115696650 peroxidase 15 upregulated 4.21e-29 LOC115712355 uncharacterized LOC115712355 upregulated 7.41e-29 LOC115716339 uncharacterized LOC115716339 downregulated 1.60e-24 LOC115717157 probable pectinesterase/pectinesterase inhibitor 61 upregulated 4.71e-24 LOC115719463 trans-cinnamate 4-monooxygenase upregulated 1.40e-22 LOC115711343 S-adenosylmethionine synthase 3 upregulated 3.11e-22 LOC115721891 7-ethoxycoumarin O-deethylase upregulated 2.20e-20 LOC115706668 phenylalanine ammonia-lyase 1 upregulated 2.42e-20 LOC115695648 classical arabinogalactan protein 1-like upregulated 3.49e-20 LOC115720967 pathogenesis-related protein PR-4 upregulated 2.77e-19 LOC115711284 SNF1-related protein kinase regulatory subunit gamma-1 downregulated 4.43e-19 LOC115725612 E3 ubiquitin-protein ligase At4g11680 downregulated 8.70e-19 LOC115700724 protein COBRA upregulated 4.26e-18 LOC115715133 alpha-xylosidase 1 upregulated 4.81e-18 LOC115715828 NAC domain-containing protein 71 downregulated 3.56e-16 LOC115704921 glutamyl-tRNA(Gln) amidotransferase subunit A upregulated 4.25e-16 LOC115715710 cyclic nucleotide-gated ion channel 1 upregulated 4.37e-16 Table 3. Top 20 DEGs between the two genotypes, AGA10 (resistant) and Purple Kush (susceptible), during powdery mildew infection. DEGs were identified by comparing read counts between AGA10 and Purple Kush, harvested 48 h post inoculation, and then removing genes that were also differentially expressed between the two genotypes during control (uninfected) conditions. The genes are ordered by p-value. The gene description for each gene is listed. Whether AGA10 genes were upregulated or downregulated relative to Purple Kush is listed in the directionality column. 35 been shown to block systemic spread of a virus in tobacco through callose deposition (Ueki & Citovsky, 2002). Trans-cinnamate 4-monooxygenase (LOC115719463) and Phenylalanine ammonia-lyase 1 (LOC115706668) are differentially expressed between the two genotypes. Because these two proteins are involved in the phenylpropanoid pathway, this suggests differences in phenolic compound biosynthesis between the two genotypes. Classical arabinogalactan protein 1-like (LOC115695648) is part of a protein family that are present in the cell wall and have many functions, including response to biotic stress (Hromadová et al., 2021). Arabinogalactan proteins have been demonstrated to play a suppressive role in early microbial pathogen infection (Cannesan et al., 2012), which could be associated with the resistant phenotype of AGA10. Pathogenesis-related protein PR-4 (LOC115720967) belongs to the same family of proteins as pathogenesis-related protein PR-1 type-like (LOC115706309) in Purple Kush, and has a role in microbial infection response. NAC domain-containing protein 71 (LOC115715828) encodes a transcription factor containing a domain known to be involved in response to biotic stress, and responds to auxin, ethylene and jasmonate (Asahina et al., 2011). 3.2.3 Gene ontology analysis The DEGs identified for each condition were then used for downstream gene ontology (GO) analysis. Overrepresentation analysis was carried out using the TopGO R Bioconductor package (Alexa et al., 2006), using Fisher’s test. The analysis was done for each of the three GO subcategories: Biological Process, Molecular Function and Cellular Component. The top 10 most significant GO terms were plotted for the Biological process and Molecular Function subontologies for AGA10, Purple Kush and between genotypes. The terms for Cellular Component were not specific or informative, and so are not considered further. 36 Figure 11 shows the top 10 Biological Process and Molecular Function GO terms for the AGA10 genotype, the terms are ordered by p-value and the number of genes associated with each term is shown. The Biological Process GO terms with the most genes were: biological process, followed by polysaccharide metabolic process and polysaccharide metabolic process. The production of callose and papillae in response to infection may be explained by the GO term polysaccharide metabolic process, while polysaccharide catabolic process and carbohydrate catabolic process can be explained by the presence of genes involved in degradation of fungal compounds, like chitinases. The molecular function GO terms with the most genes were: catalytic activity, followed by ion binding (metal ion binding and cation binding), and oxidoreductase activity. These GO terms encompass many enzymes that are differentially expressed in AGA10 during infection. 37 Figure 11. Top 10 Biological Process and Molecular Function GO terms for AGA10 (resistant) genotype. Overrepresentation analysis was done for each GO subontology using the DEGs in response to powdery mildew infection previously identified. The 10 most significant GO terms are shown for the two subontologies. The gene count is shown for each enriched term. Terms are ordered and colour-coded by p-value. 38 Figure 12 shows the top 10 Biological Process and Molecular Function GO terms for Purple Kush genotype, the terms are ordered by p-value and the number of genes associated with each term is shown. The Biological Process GO terms with the most genes are: response to stimulus, signaling, and cellular communication. These GO terms are directly related to biotic stress response and are terms that encompass genes and processes involved in plant defense. The Molecular Function GO terms with the most genes are catalytic activity and transferase activity. Similarly to AGA10, these GO terms encompass the many enzymes that are differentially expressed during infection in Purple Kush. 39 Figure 12. Top 10 Biological Process and Molecular Function GO terms for Purple Kush (susceptible) genotype. Overrepresentation analysis was done for each GO subontology using the DEGs in response to powdery mildew infection previously identified. The 10 most significant GO terms are shown for the two subontologies. The gene count is shown for each enriched term. Terms are ordered and colour-coded by p-value. 40 Figure 13 shows the top 10 Biological Process and Molecular Function GO terms between the two genotypes, the terms are ordered by p-value and the number of genes associated with each term is shown. The Biological Process GO terms with the most genes are cellular carbohydrate metabolic process and carbohydrate metabolic process, suggesting a difference in carbohydrate production between the two genotypes. This could potentially be due to a difference in production of callose and other cell wall carbohydrates. The Molecular Function GO terms with the most genes are catalytic activity and transferase activity. These are likely due to differences in enzymatic activity between the two genotypes. 41 Figure 13. Top 10 Biological Process and Molecular Function GO terms between the two genotypes. Overrepresentation analysis was done for each GO subontology using the DEGs identified between the two genotypes during to powdery mildew infection. The 10 most significant GO terms are shown for the two subontologies. The gene count is shown for each enriched term. Terms are ordered and colour-coded by p-value. 42 Chapter 4: Discussion 4.1 Summary of findings I investigated the phenotypic and transcriptomic responses during powdery mildew infection on Cannabis leaves in a resistant (AGA10) and a susceptible (Purple Kush) genotype, and the differences in the responses to infection between these two genotypes. Light microscopy revealed that the two genotypes differ significantly in conidiospore germination rates on the leaf surfaces and the robustness of hypersensitive response (HR) in the leaf cells, but they do not differ significantly in fungal penetration rates into the leaves of the two varieties. While RNA-Seq of the leaves of both Cannabis genotypes presented a transcriptomic response consistent with the literature, the resistant genotype differed from the susceptible one in expression of genes encoding enzymes of the phenylpropanoid and monolignol biosynthetic pathways, phytoalexin production, and other plant-pathogen responses. GO functional analysis showed the two genotypes also differ in expression of genes related to carbohydrate metabolic processing and other enzymatic processes. 4.2 Germination rates and penetration rates Infection is less widespread in AGA10 compared to Purple Kush, reflecting its resistant phenotype. The germination rate of the fungal spores on the surfaces of AGA10 leaves was statistically significantly lower than spore germination on leaves of Purple Kush. If fewer fungal spores germinate, it follows that AGA10 should have less infection events overall. The resistance of AGA10 then, may be in part due to the plant influencing the conidiospores on the leaf surface to produce a lower spore germination rate. As discussed below, the transcriptomic analysis leads 43 to several hypotheses on why this may be so, including phytoalexins and ROS production that may lead to the lower spore germination on AGA10. In contrast, there was no statistically significant difference in fungal penetration into the Cannabis leaf between the two genotypes. While the difference in penetration between the genotypes was not statistically significant, it may be of biological importance. It is worth noting that the difference in germination events between genotypes was on the borderline of significance (P = 0.06); however, this was not the case for the difference in penetration rates (P = 017). More replicates and a larger sample size could help elucidate whether the difference in penetration is meaningful. Overall, these data suggest that once the spores germinate in AGA10, they are effectively penetrating into the epidermal cells as the same rate as in Purple Kush. If the genotypes significantly differ during germination, but not during penetration, the likely limiting step in infection is germination. This suggests that a mechanism causing the resistant phenotype seen in AGA10 occurs early during the infection process. 4.3 Hypersensitive response, reactive oxygen species and phenolics Another feature of the powdery mildew defense response in AGA10 is increased hypersensitive response, which includes rapid programmed cell death, lignification and production of reactive oxygen species (ROS) (Balint‐Kurti, 2019). Phenolics are overexpressed and accumulated during plant hypersensitive response (Sedlarova & Lebeda, 2001). This is followed by deposition of callose and lignin, which in turn activates pathogenesis-related proteins (reviewed by Lattanzio et al., 2006). Reactive oxygen species (ROS) also play a role in conferring resistance against pathogens (Tripathy & Oelmüller, 2012). ROS are generated before cell death during HR, and the oxidative burst they cause is thought to trigger localized cell death (Doke, 1983; Zurbriggen 44 et al., 2010). Peroxidase 15 (LOC115696650) is differentially expressed between the two genotypes, and oxidoreductase activity is a top enriched GO term for AGA10. The role of ROS and phenolic accumulation in HR, could explain the oxidoreductases and phenolic-involved genes (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase) being differentially expressed and the higher levels of HR observed in AGA10, relative to Purple Kush. This finding correlates phenotypic and transcriptomic responses, and suggests a plausible mechanism for AGA10 resistance. It is worth noting that only one event of HR was observed in Purple Kush. This suggests that HR is not occurring at a high enough level in the susceptible genotype to limit infection spread, or at the very least that HR is happening at a later stage (after 48 hours) during the infection process in Purple Kush. 4.4 Phenolics and antimicrobial properties Successful plant defense against pathogens and concentration of antimicrobial compounds, including phenolics, have been found to be correlated (González-Lamothe et al., 2009). For example, the phenolics salicylic acid, cinnamic acid, syringic acid and catechol have a strong growth inhibitory effect on the bacterial pathogen Pectobacterium (Joshi et al., 2015); these authors also found that different bacterial strains have varying sensitivities to antimicrobial phenolics. For example, while some strains were highly sensitive to cinnamic, salicylic and coumaric acids, others were more tolerant (Joshi et al., 2015). Joshi et al. (2015) also found that the phenolic compounds that act as antimicrobials are mostly non-flavonoids, and can be categorized into cinnamic acid vs. benzoic acid derivatives. As reviewed by Hättenschwiler & Vitousek (2000), ferulic acid, gallic acid and flavonoids have shown growth inhibition of fungal 45 pathogens. These compounds inhibit spore germination and hyphal growth in saprophytic fungi (Hättenschwiler & Vitousek, 2000). In apple, hydroxycinnamic acids, dihydrochalcones and flavonoids accumulate in response to scab fungus (Venturia inaequalis) primarily at the site of infection (Mikulič Petkovšek et al., 2009; Slatnar et al., 2012). It is possible that this antimicrobial response is happening even earlier in AGA10 and with a more robust intensity, accounting for the resistant phenotype observed. Notably, the genes trans-cinnamate 4-monooxygenase (LOC115719463) and phenylalanine ammonia-lyase 1 (LOC115706668), which are required for phenylpropanoid production, are differentially expressed between the two genotypes. Additionally, the gene cinnamyl alcohol dehydrogenase (LOC115713020), required for cinnamic acid production, is one of the top 20 DEGs in AGA10, but not in Purple Kush. In Arabidopsis, knocking out phenylalanine ammonia lyase and cinnamyl alcohol dehydrogenase results in a reduction of resistance to pathogens (Huang et al., 2010, Tronchet et al., 2010). This suggests these three genes are key players in pathogen resistance. The fact that these three genes are among the top 20 DEGs for AGA10 is further suggestive that they are limiting pathogen spread and conferring subsequent resistance seen in AGA10. 4.5 Phytoalexins and germination suppression Phytoalexins are compounds secreted by plants that serve as antimicrobials and help defend against pathogenic microorganisms (reviewed by Jeandet, 2015). In Camelina sativa (Brassicaceae), the phytoalexin camalexin has been found to inhibit fungal growth, specifically by inhibiting germination and germ-tube growth (Jejelowo et al., 1991). Since camalexins inhibit 46 germination and germ-tube growth, a similar phenomenon could be occurring in the cannabis strain AGA10, contributing be to the lower powdery mildew germination rate that I observed. Camalexins are synthesized from tryptophan by cytochrome P450 enzyme via the intermediate indole-3-acetaldoxime, which is also a precursor of other indolics such as the auxin indole-3-acetic acid (Zhou et al., 2020). While camalexins have not been found in Cannabis, given that AGA10 genotype exhibits increased transcription of the putative indole-3-acetaldoxime dehydratase cytochrome P450 71A22-like (LOC115708777), and that the GO terms indole-containing compound biosynthetic process and indolalkylamine biosynthetic process are enriched, it is possible that AGA10 is producing phytoalexins, indolic compounds or other small molecules with similar properties that function like camalexins, and that have not been yet identified. This would be useful to study in future. Thus, the molecular product resulting from the putative cytochrome P450 71A22-like could be contributing to the resistant phenotype of AGA10, when there is increased or earlier stage production compared to Purple Kush. In rice and Arabidopsis, phytoalexins are regulated by kinases, specifically by MAPK cascades (Kishi-Kaboshi et al., 2010). Kinase activity is an enriched GO term between the two genotypes. This could potentially mean that phytoalexins are being produced at a different rate or regulated differently in the two genotypes. 4.6 Cell wall and carbohydrate differences between genotypes Host cell-wall remodeling happens during pathogen infection through cell-wall degrading enzymes and other processes (reviewed by Bellincampi et al., 2014). Cellulose synthases are involved in cell-wall integrity and disease resistance (Bellincampi et al., 2014), so differences in cell-wall composition/structuring between genotypes, could be a factor in explaining resistance. 47 For example, cellular carbohydrate metabolic process and carbohydrate metabolic process differ between the two genotypes according to the GO analysis. This could mean that the genotypes differ in their cell-wall composition and/or production of callose and papillae during penetration. However, given that the penetration rate did not differ significantly between the two genotypes, differences in callose deposition and papillae do not seem to be directly involved in the resistant phenotype (AGA10). Furthermore, there are no genes directly related to cell-wall integrity in the top 20 DEGs between the two genotypes. For these reasons, differences in phenolics and phytoalexins in AGA10 compared to Purple Kush may be a more likely explanation for the resistant genotype. Arabinogalactans are also worth considering, as they have been found to play an early role in suppressing infection. In pea, arabinogalactans were found to suppress germination of the Oomycota fungal pathogen Aphanomyces euteiches during early stages of infection (Cannesan et al., 2012). LOC115695648 (classical arabinogalactan protein 1-like) is differentially expressed between the two genotypes and could also be suppressing germination early in AGA10. 4.7 Timing as a possible key factor in AGA10 resistance The timing of application of biological control agents has been shown to have a significant effect on defense against Sclerotinia sclerotiorum (Savchuk & Dilantha Fernando, 2004). Similarly, timing in fungicide application has been shown to influence disease severity in wheat cultivars (Arif et al., 2021). It is possible that timing is a key factor in the resistant AGA10 phenotype. The genes involved in defense compounds, such as phytoalexins and phenolics, that are among the top DEGs and GO terms in AGA10 at 48 h post-inoculation, are also normally secreted during infection in plants that are not phenotypically resistant. Additionally, only one HR event 48 was observed in Purple Kush. The intense HR phenotype observed in AGA10 could be due to either higher occurrences of HR in AGA10 relative to Purple Kush, or to HR happening earlier in AGA10 than in Purple Kush. Looking at HR phenotype and accompanying transcriptomic response in Purple Kush at more time points after 48 h post-inoculation would help clarify this. If a similar phenotypic and transcriptomic response to AGA10 is observed, this would suggest timing is a key factor in AGA10 resistance. Future work could also include knockdowns of phytoalexin and phenolic genes in AGA10. If resistance decreases along with the lower expression of these genes, this would suggest that differences in expression of these genes, rather than timing, is the key factor causing resistance. 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Thesis/Dissertation
eng
Genome Science and Technology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Samuels, Anne Lacey, 1962-
Rieseberg, Loren H.
Exploring Cannabis sativa's response to powdery mildew (Golovinomyces ambrosiae) infection
Text