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
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Copyright
By
Dianna Mojica
2021
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Table of Contents
Streptomyces, an unusual bacterial genus Streptomyces as symbionts in interactions with plants and animals Streptomyces scabies, a well-established and worldwide occurring plant pathogen Literature Cited
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10 12 16 22 22 23 26 26 26 28 29
Chapter 2 Abstract Introduction Materials and Methods Bacterial strains and growth conditions used in this study Thaxtomin A production in response to living or dead radish plant material Thaxtomin A production in response to radish root exudates Cellulase activity and thaxtomin A production in response to cellulose S. scabies cannot distinguish between a living and a dead host plant to start thaxtomin A
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Literature Cited Figures Chapter 3 Conclusion Literature Cited
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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.
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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.
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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 nonmotile; 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
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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
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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
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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 Grampositive 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, antihypertension, 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
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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). The aerial mycelium further progresses into the formation of chains of single-celled spores (Bodek et al. 2017). Mature spores can withstand harsh physical and chemical environmental conditions and hence function in survival. The spores can be disseminated easily and when conditions are favorable, they will germinate starting another developmental cycle (McCormick and Flardh 2011).
Bacteria within the Streptomyces genus are non-motile. However, recently some
Streptomyces species have been shown to be able to perform exploratory growth, a form of rapid spreading of cells (Jones et al. 2017). As mentioned before, Streptomyces have a development cycle like fungi in that Streptomyces grow as a substrate and aerial mycelium and produce spores.
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It has been identified that under stressful conditions such as nutrient depletion and competing neighboring fungi, some Streptomyces can produce explorer cells that resemble the cells in their vegetative hyphae except that these explorer cells are non-branching (Jones et al. 2017). The explorer cells disassociate from the colony and can rapidly traverse solid surfaces while releasing airborne signals to communicate with other Streptomyces species to induce them to also explore other environments for optimal growth (Jones et al. 2017). The first studies done to unravel the mechanisms by which exploratory growth is performed showed that exploratory growth is analogous to the microbial movement known as sliding, yet more research is needed to determine if and how exploratory growth differs from sliding (Jones and Elliot 2018). Sliding is defined as the passive spreading of a colony through expansive forces created by cell division and using surfactants, exopolysaccharides, or surface proteins, to help facilitate this uniform spreading (Henrichsen 1972, Kinsinger et al. 2003, Hölscher and Kovács 2017). It is advantageous as a bacterium to be able to move with benefits such as increased efficiency of nutrient acquisition, avoidance of toxic substances, the ability to translocate to preferred hosts, access to optimal colonization sites within these hosts, and dispersal in the environment (Ottemann and Miller 1997).
Streptomyces as symbionts in interactions with plants and animals
The secondary metabolites produced by Streptomyces are very diverse and have many different functions and applications (Chater et al. 2009, Seipke et al. 2012). This has made it possible for species to engage in complex symbiotic relationships with other organisms such as insects, invertebrates, and plants. For instance, the female beewolf, Philanthus triangulum, harbors Streptomyces endosymbionts to protect her larvae during development (Kroiss et al. 2010). The beewolf secretes a white substance from her antenna glands that contains antifungals produced by
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Streptomyces. The larvae take up the substance and incorporate it into their cocoons, hereby protecting them from fungal infections during development (Kroiss et al. 2010). The ancestors of Acromyrmex attine ants have evolved to feed on fungi. However, certain fungi can be pathogenic to attine ants and to protect themselves, the ants form a symbiotic relationship with Streptomyces species that produce the antifungals candicidin and antimycins (Seipke et al. 2012).
A particularly emerging field of study looks at the mutualistic relationships that
Streptomyces species form with plants. Most Streptomyces species are free-living saprophytes in the soil and many species can colonize the nutrient-rich rhizosphere, phyllosphere, or even live in the interior of plants (endophytes) (Rey and Dumas 2016). Hence, due to the unique properties of Streptomyces species mentioned above, these bacteria have a lot of potential as biofertilizers and biocontrol agents. Streptomyces can enhance soil fertility by producing enzymes that break down complex substrates into simple mineral forms making them readily available to plants (Vurukonda et al. 2018). Phosphorus, essential for plant growth, is highly reactive and forms complexes with elements in the soil that cannot be taken up by the plants. Certain Streptomyces species are capable of solubilizing phosphate complexes into monobasic and dibasic forms making phosphorus available for plant absorption (Rani et al. 2018). Plant development can also be modulated by phytohormones produced by Streptomyces. Several species can synthesize auxin, which promotes cell division, elongation, embryogenesis, and emergence of lateral roots (Rani et al. 2018).
Streptomyces have also been recognized as suitable antagonists against soilborne and airborne plant pathogenic organisms. As mentioned before, Streptomyces species are known to produce a wide array of antibacterial and antifungal agents (Jeong et al. 2004; Vurukonda et al. 2018). Moreover, several species can secrete hydrolytic enzymes such as chitinases, glucanases, proteases, and lipases that can lyse the cells of devastating fungal pathogens (Compant et al. 2005).
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Streptomyces as well as their purified metabolites are used as soil or foliar treatments. For example,
S. lydicus, S. violaceusniger, S. griseoviridis, and S. saraceticus are used in six commercial
biocontrol products used to control fungal and bacterial soil diseases (Rey and Dumas 2016). Streptomyces species can also protect plants from harmful organisms by inducing natural plant defenses, for example through the production of nitrous oxide (Kinkel et al. 2012).
Among the many Streptomyces species only about a dozen engage in a harmful way with plants as plant pathogens. The best-known pathogenic species are S. scabies, S. acidiscabies, S.
turgidiscabies, and S. ipomoeae (Loria et al. 1997; Li et al. 2019). S. scabies is the oldest known
streptomycete pathogen and is found worldwide. As for S. turgidiscabies and S. acidiscabies, they are more recent emergent pathogens that were first found in Japan and the northeastern United States, respectively, and their pathogenic abilities are thought to be received through horizontal gene transfer of a genomic region containing the virulence genes of S. scabies (Loria et al. 1997, Bignell et al. 2010, Huguet-Tapia et al. 2016). These three species infect a plant’s underground structures causing scab lesions on various root and tuber crops leading to tissue necrosis as well as root and shoot stunting. Although they are neither tissue nor host specific, they are most notorious due to the damage they can cause to potato tubers (Solanum tuberosum), a disease called potato common scab (Loria et al. 1997, Bignell et al. 2010, 2013). In contrast, S. ipomoeae strictly infects sweet potato (Ipomoea batatas) causing necrosis of the sweet potato fibrous roots (Loria et
al. 1997, Guan et al. 2012).
Streptomyces scabies, a well-established and worldwide occurring plant pathogen
S. scabies is the oldest and best-studied plant pathogenic species within the genus
Streptomyces (Bignell et al. 2013, Huguet-Tapia et al. 2016). S. scabies causes common scab on
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various tuber and root crops and is responsible for significant losses in the potato industry worldwide (Loria et al. 1997). S. scabies induces harmful scabby lesions on potato tubers, which do not necessarily reduce crop yield and the consumption of scab-infected tubers is not threatening to human health, but the lesions greatly affect the market value of the potatoes leading to significant economic losses for the growers (Loria et al. 1997, Loria et al. 2008, Lerat et al. 2009).
Although S. scabies is well-recognized to infect potato tubers, the bacteria, in fact, are neither host nor tissue specific. They do, however, only infect actively growing plant tissue with a preference of underground plant structures (Loria et al. 2008). This is due to their pathogenicity determinant, the phytotoxin thaxtomin A. Thaxtomin A inhibits the cellulose synthase complex that is active during plant growth. Blocking this enzyme that builds the cellulose plant cell wall during cell elongation and duplication of growing tissue will lead to cell lysis and death which helps the pathogens to penetrate and infect underground plant tissue (Loria et al. 2008). Since the target of thaxtomin A inhibits the plants cellulose synthase complex that is active in all plants during plant growth, it is capable to cause disease to any plant if the plant is actively growing, causing root and shoot stunting, radial swelling, and tissue necrosis (Bischoff et al. 2009, Li et al. 2019).
The biosynthesis of thaxtomin A is an energy demanding process using large, specialized enzyme complexes (non-ribosomal peptide synthases or NRPSs) and is therefore tightly regulated (Francis et al. 2015; Joshi et al. 2007; Loria et al. 2008). The cello-oligosaccharides cellobiose and cellotriose, subunits of the major plant cell wall component cellulose, have been shown to act as signals that induce thaxtomin A production. Upon import via a specific cellobiose/cellotriosespecific ATP-binding cassette (ABC) transporter, these plant-derived molecules induce thaxtomin A production (Jourdan et al. 2016), not only through interaction with TxtR, the pathway-specific
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activator of the thaxtomin A biosynthetic cluster (Joshi et al. 2007), but mainly by relieving inhibition of transcription by the cellulose utilization regulator CebR (Francis et al. 2015). The main function of which is to control the expression of the transporter genes and cellulosedegrading enzymes (cellulases).
The gene cluster coding for CebR and the cellobiose/cellotriose-specific ABC transporter is present in all Streptomyces species, including the non-pathogenic species, because these cellooligosaccharides can be used by the bacteria as a carbon source (Schlösser et al. 1999; 2000). The polysaccharide cellulose itself does not trigger thaxtomin A production (Johnson et al. 2007; Wach et al. 2007) and cannot be degraded by S. scabies despite the large number of cellulase-encoding genes in its genome, more than many of the efficient decomposers within the genus (Medie et al. 2012). This points to an evolutionary adaptation of the pathogen to avoid self-triggering of toxin production through feeding on dead plant material hereby releasing cello-oligosaccharides from the plant cell walls (Jourdan et al. 2017). It would indeed be more energy-efficient for S. scabies to remain in a saprophytic state in the vicinity of decaying dead plant material and to only start the production of thaxtomin A when the bacterium is in the vicinity of a living root. Although it is assumed that S. scabies has evolved to be able to avoid this self-triggering of thaxtomin A production, no research has yet established if this is true. Hence, it is still unclear if and how S. scabies can distinguish between cello-oligosaccharides derived from dead plant material and cellooligosaccharides derived from actively growing plants (Jourdan et al. 2017).
This study aimed to substantiate the hypothesis that S. scabies can distinguish between dead and living plants by evaluating thaxtomin A production upon inoculation of strain 87-22. And more specifically, the potential role of root exudates secreted by actively growing plants as a signal to identify a living host was investigated. Broadly, we aimed to gain more insight into the
