Unraveling the First Step in the Streptomyces Scabies Pathology: How Does the Pathogen Sense It Is in the Vicinity of a Living R
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Unraveling the first step in the Streptomyces scabies pathology: How does the pathogen sense it is in the vicinity of a living root? By: Dianna Mojica A thesis submitted to the Department of Biology at California State University, Bakersfield in partial fulfilment for the degree of Master of Science in Biology Spring 2021 ii Copyright By Dianna Mojica 2021 i Table of Contents Acknowledgements v Abstract vi Keywords viii List of Figures ix Chapter 1 8 Introduction 8 Streptomyces, an unusual bacterial genus 8 Streptomyces as symbionts in interactions with plants and animals 10 Streptomyces scabies, a well-established and worldwide occurring plant pathogen 12 Literature Cited 16 Chapter 2 22 Abstract 22 Introduction 23 Materials and Methods 26 Bacterial strains and growth conditions used in this study 26 Thaxtomin A production in response to living or dead radish plant material 26 Thaxtomin A production in response to radish root exudates 28 Cellulase activity and thaxtomin A production in response to cellulose 29 S. scabies cannot distinguish between a living and a dead host plant to start thaxtomin A production 30 Literature Cited 39 Figures 43 Chapter 3 48 Conclusion 48 Literature Cited 50 iv Acknowledgements I thank my thesis advisor Dr. Francis for donating her time and sharing her knowledge on Streptomyces scabies as well as guiding me on how to write this thesis. I also thank Dr. Stokes and Dr. Keller for agreeing to be part of my thesis committee in such short notice. v Abstract Streptomyces is the largest genus within the phylum Actinobacteria. The genus contains Gram-positive bacteria with a complex life cycle resembling that of fungi and are distinctly recognized for their diverse secondary metabolites that have pharmaceutical, agricultural and industrial properties. From the more than 800 known Streptomyces species, only about a dozen are plant pathogens. S. scabies is the best-studied plant pathogen within this group with a wide host range, but it is most known due to the significant economic losses it causes to potato industry worldwide. Potato common scab is characterized by harmful scabby lesions which decrease the market value of the potato tubers. S. scabies produces the phytotoxin thaxtomin A as its main virulence factor which inhibits the cellulose synthase complex found in actively growing plants resulting in plant cell lysis, thus allowing the S. scabies to enter its host. Biosynthesis of thaxtomin A requires large, specialized enzyme complexes and is induced by cellobiose and cellotriose. These cello-oligosaccharide inducers of thaxtomin A are derived from cellulose, the most abundant polymer in the soil that can be derived from living and dead plant material. Since the target of thaxtomin A is the cellulose synthase complex, which is active in living plants, and because thaxtomin A production comes at a high energy cost, it was investigated whether S. scabies can distinguish between dead degraded plant wall subunits and from those of a living plant. Although previous studies hypothesized that S. scabies can identify a living host, our results show that there is still toxin production when the bacteria were inoculated on dried radish plants. The cellulase activity of S. scabies was also investigated, as S. scabies contains a large number of cellulase genes, even more than the average successful saprophyte. This can be challenging as degradation of cellulose would lead to self-triggering of thaxtomin A production. vi Results showed that cellulose is a poor nutrient source when S. scabies was allowed to grow with the development of hyphae on agar medium. The cellulase genes have a repressor, CebR, that inhibits transcription when cellobiose and cellotriose are not there to release the repressor from its binding site. However, although the S. scabies 87-22 ΔcebR mutant, which has the gene for the cellulase utilization repressor deleted, started growth sooner than the wild type upon inoculation on cellulose, it did not produce more thaxtomin A than the wild type. So, S. scabies has managed to limit self-triggering of thaxtomin A production due to cellulose degradation, but it has yet to evolve further to be able to identify a living from a dead host effectively use its main virulence factor thaxtomin A and become an even more efficient pathogen. Most of the known soil-borne plant pathogens are motile and thus can actively move towards a host plant. S. scabies is considered non-motile and therefore relies on chance to reach a host. Recent studies, however, have shown motility in bacteria that were once classified as non- motile; even certain Streptomyces species were observed to cover a larger surface area under nutrient limiting conditions, a form of motility known as exploratory growth. Considering exploratory growth is a recent discovery of motility within the Streptomyces genus, not all species have been evaluated. It was investigated whether S. scabies can perform exploratory growth and/or other forms of motility such as twitching and gliding. For both motility studies, colony size was examined to determine motility. Colony expansion on low agar concentrations indicative of sliding or twitching motility as well as exploratory growth on nutrient-poor medium were not observed for S. scabies 87-22. Elucidating some of the processes regarding plant recognition and making connection to the plant host are crucial to complete our knowledge on the S. scabies pathology. It’s striking to discover that S. scabies cannot move towards or identify a living host even though biosynthesis of vii its virulence factor is tightly regulated and energy demanding. However, S. scabies has evolved to minimize its cellulase genes to prevent self-triggering. Interestingly, this study gives insight in some of the evolutionary processes on how S. scabies is transitioning to become more energy efficient while relying on the use of subunits of a commonly found polymer in the soil as a signal to being plant invasion. Keywords Plant pathogenicity, Streptomyces scabies, thaxtomin A, root exudates, plant sensing, motility viii List of Figures Chapter 2 Figure 2.1. HPLC chromatograms at 380 nm of methanol extracted samples from plant material inoculated with S. scabies 87-22………………………………………………………………….42 Figure 2.2. Thaxtomin A production by S. scabies 87-22 when inoculated on germinated radish seeds or dead whole radish plants………………………………………………………………...43 Figure 2.3. Growth (A) and thaxtomin A production (B) of S. scabies 87-22 on Basal medium with 1% cellulose or cellobiose as the only carbon source………………………………………..44 Figure 2.4. Growth (A) and thaxtomin A production (B) of S. scabies 87-22 (wild type) and the ΔcebR mutant on Basal media with 1% cellulose as the only carbon source……………………...45 Figure 2.5. Exploratory growth assay showing S. venezuelae (positive control), S. coelicolor (negative control), and S. scabies 87-22 on YP (nutrient poor) and YPD (nutrient rich) medium...46 Figure 2.6. Motility assay of S. scabies 87-22 when inoculated on different media with a lower agar concentration (0.25%). Pictures were taken at two weeks post inoculation…………………47 ix Chapter 1 Introduction Streptomyces, an unusual bacterial genus Streptomyces is the largest genus within the phylum Actinobacteria with an estimated 848 species and 38 subspecies (Law et al. 2019). The genus contains aerobic filamentous Gram- positive bacteria with a high G +C content in their DNA (Anderson and Wellington 2001, Barka et al. 2016). Streptomycetes are particularly ubiquitous in the soil and have been described as soil chemists due to the synthesis of diverse secondary metabolites (Barka et al. 2016). They are distinctly recognized to produce the volatile compound geosmin described as the aroma of moist soil (Jiang et al. 2007). Moreover, streptomycetes are renowned for their production of diverse bioactive secondary metabolites with pharmaceutical, agricultural, and other industrial applications. This makes Streptomyces the most studied genus within the Actinobacteria (Barka et al. 2016, Law et al. 2019). Most streptomycetes also synthesize various hydrolyzing molecules that can degrade recalcitrant substrates, such as cellulose and chitin, which are among the most abundant polymers in soil. Only a limited number of organisms can degrade these substrates, making streptomycetes some of the most successful nutrient cycling (saprophytic) prokaryotes (Loria et al. 2006, McCormick and Flardh 2011, Law et al. 2019). Streptomyces synthesize many antimicrobial compounds such as antibacterial, antifungal, and antiviral compounds. Several of their secondary metabolites also have anti-tumor, anti- hypertension, and immunosuppressant properties (Loria et al. 2006, Mahajan and Balachandran 2012, Procopio et al. 2012, Lapaz et al. 2019,). In nature, the production of antibiotics can act as 8 a defense mechanism for Streptomyces against competing microbes in the often unfavorable and harsh soil environment (McCormick and Flardh 2011, Law et al. 2019). Streptomyces is Greek for “twisted fungi”, which holds true when looking at their development and colony morphology (Law et al. 2019). They are among the few prokaryotes with a complex life cycle consisting of spore formation and development of vegetative and aerial hyphae (Jones and Elliot 2018). When conditions such as temperature, availability of nutrients, and moisture are favorable, a dormant spore germinates into a germ tube that forms an outgrowth of one or more hyphal structures penetrating the soil. The hyphae form a network known as substrate mycelium that helps anchor the colony and absorbs nutrients (McCormick and Flardh 2011, Procopio et al. 2012). Environmental cues such as nutrient depletion trigger Streptomyces to transition its development from substrate mycelium to aerial mycelium (Jones and Elliot 2018). Aerial hyphae grow upward into the air away from the substrate mycelium. Lysis of the substrate mycelium often provides nutrients enabling the development of these aerial hyphae. The transition from substrate to aerial mycelium coincides with the production and secretion of antibiotics and other secondary metabolites (McCormick and Flardh 2011).