Investigating the role of exudates in recruiting Streptomyces bacteria to the Arabidopsis thaliana root microbiome
Sarah F. WorsleyMichael C. MaceyJake NewittElaine PatrickDouglas W. YuBarrie WilkinsonJ. Colin MurrellMatthew I. Hutchings
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Arabidopsis thaliana has a diverse but consistent root microbiome, recruited in part by the release of fixed carbon in root exudates. Here we focussed on the recruitment of Streptomyces bacteria, which are well established plant-growth-promoting rhizobacteria and which have been proposed to be recruited to A. thaliana roots by the release of salicylic acid. We generated high quality genome sequences for eight Streptomyces endophyte strains and showed that although some strains do enhance plant growth, they are not attracted to, and do not feed on, salicyclic acid. We used 13CO2 DNA-stable isotope probing to determine which bacteria are fed by the plants in the rhizo- and endosphere and found that streptomycetes did not feed on root exudates in vivo, despite the fact that they can use exudate as sole carbon and nitrogen sources in vitro. We confirmed increased root colonisation by streptomycetes in plants that constitutively produce salicylic acid, but these plants exhibited a pleiotropic phenotype of early senescence and weak growth. We propose that streptomycetes are attracted to the rhizosphere by root exudates but can be outcompeted for this food source by more abundant proteobacteria and most likely feed off unlabelled complex organic matter.Keywords:
Exudate
Endophyte
Colonisation
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Abstract Streptomyces bacteria are ubiquitous in soils and are well-known for producing secondary metabolites, including antimicrobials. Increasingly, they are being isolated from plant roots and several studies have shown they are specifically recruited to the rhizosphere and the endosphere of the model plant Arabidopsis thaliana . Here we test the hypothesis that Streptomyces bacteria have a beneficial effect on A. thaliana growth and could potentially be used as plant probiotics. To do this, we selectively isolated streptomycetes from surface washed A. thaliana roots and generated high quality genome sequences for five strains which we named L2, M2, M3, N1 and N2. Re-infection of A. thaliana plants with L2, M2 and M3 significantly increased plant biomass individually and in combination whereas N1 and N2 had a negative effect on plant growth, likely due to their production of polyene natural products which can bind to phytosterols and reduce plant growth. N2 exhibits broad spectrum antimicrobial activity and makes filipin-like polyenes, including 14-hydroxyisochainin which inhibits the Take-all fungus, Gaeumannomyces graminis var. tritici . N2 antifungal activity as a whole was upregulated ~2-fold in response to indole-3-acetic acid (IAA) suggesting a possible role during competition in the rhizosphere. Furthermore, coating wheat seeds with N2 spores protected wheat seedlings against Take-all disease. We conclude that at least some soil dwelling streptomycetes confer growth promoting benefits on A. thaliana while others might be exploited to protect crops against disease. Importance It is vital that we reduce our reliance on agrochemicals and there is increasing interest in using bacterial strains to promote plant growth and protect against disease. Our study follows up reports that Arabidopsis thaliana specifically recruits Streptomyces bacteria to its roots. In particular, we test the hypothesis that these bacteria can offer benefits to their A. thaliana hosts and that strains isolated from these plants might be used as probiotics. We isolated Streptomyces strains from surface washed A. thaliana roots and genome sequenced five phylogenetically distinct strains. Genome mining and bioassays indicated that all five strains have plant growth promoting properties, including production of IAA, siderophores and ACC deaminase activity. Three strains significantly increased A. thaliana growth in vitro and when applied in combination in soil. Another produces potent filipin-like antifungal metabolites and we used it as a seed coating to protect germinating wheat seeds against the fungal pathogen Gaeumannomyces graminis var. tritici (wheat Take-all fungus). We conclude that introducing an optimal combination of Streptomyces strains into the root microbiome can provide significant benefits to plants.
Phyllosphere
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Actinobacteria are abundant in soil and well-known for producing antimicrobial compounds. Increasingly, members of this phylum are also found to form symbiotic relationships, for example with plants and insects, and provide protection against host infection. However, it remains poorly understood how Actinobacteria can be selectively recruited to the host microbiome from the environment.
Acromyrmex echinatior leafcutter ants transmit Pseudonocardia bacteria between generations and also recruit Streptomyces species to their cuticular microbiome. RNA Stable Isotope Probing (SIP) experiments demonstrated that ants supply carbon-based resources to their cuticular bacteria. In turn, RNA-sequencing showed that genes encoding Pseudonocardia secondary metabolites, including bacteriocins and terpenes, were expressed in vivo on the ant cuticle. This suggests that publicly available host resources fuel interference competition between microbial species on the cuticle, which in turn selects for antibiotic-producing bacteria.
In addition to leafcutter ants, Actinobacteria are known to be abundant in plant roots. Several plant-growth-promoting and antibiotic-producing Streptomyces bacteria were isolated from the root microbiome of Arabidopsis thaliana. Root exudates are hypothesised to play a major role in root microbiome assembly and DNA SIP, coupled with Illumina sequencing showed that these were utilised by many bacterial genera. However, Streptomyces appeared to be outcompeted for resources by fast-growing Proteobacteria, despite the fact that streptomycete isolates could grow on purified root exudates in the absence of competition. We found no evidence that the plant defense phytohormone salicylic acid selectively recruits Streptomyces to the plant root microbiome, which contradicts the conclusions made by previous published studies and suggests that they make use of other resources.
Overall, this research demonstrates that host-nutrients, coupled with priority effects, can help to define competitive outcomes within the host microbiome. Understanding factors that influence the establishment of protective bacteria has implications for the development of more consistent biocontrol strategies.
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The rhizosphere is an active interface where plants and microorganisms (pathogenic, beneficial and neutral) establish a complex and varied molecular dialogue, however knowledge of the functional mechanisms mediating interactions is still limited. Plants invest a significant proportion of their photosynthetically fixed carbon in maintaining the rhizosphere microbiome via root exudation and in return beneficial microbes provide profitable functions to the plant. The potential of naturally occurring soil microorganisms to control phytopathogens and to promote plant growth is well documented, but the functional mechanisms governing the reciprocal signaling between microbial communities and plants are not well understood. The aim of the studies described in this thesis was to gain insight into the functional basis of interactions between the fungal root pathogen Rhizoctonia solani and root associated antagonistic bacteria of the genus Serratia in the rhizosphere of Brassica napus.
Transcriptomic responses of the oilseed rape pathogen R. solani, to the plant-associated and pathogen- antagonistic bacteria Serratia proteamaculans S4 and S. plymuthica AS13, were studied using RNA-sequencing. The results demonstrate a major shift in the fungal gene expression with simultaneous alterations in primary metabolism, activation of defense and attack mechanisms and distortions in hyphal morphology.
Stable isotope probing coupled with high throughput sequencing allowed the description of the composition of bacterial and fungal communities in the rhizosphere soil and the roots of B. napus and the identification of active taxa capable of assimilating recently fixed plant carbon. Our results support the idea of active selection of microbial communities from the more diverse rhizosphere environment by the roots. Furthermore, the data confirm the potential of some active genera (Streptomyces, Rhizobium, Clonostachys and Fusarium) to be used as microbial inoculants for improved productivity and health of oilseed rape.
Patterns of gene expression in B. napus exposed to factorial combinations of R. solani and S. proteamaculans S4 were examined in-vitro using RNA-sequencing. Plants inoculated with R. solani only were almost dead at 240h post-inoculation and massive transcriptional reprogramming was observed, whereas the presence of S4 modulated the transcriptional responses and resulted in healthy plants. With R. solani present, we observed an interplay between stress and defense involving salicylic acid, jasmonic acid, ethylene and abscisic acid as common regulators. Induced systemic resistance when S4 present potentially depends on jasmonic acid, auxin and salicylic acid. Downregulation of stress-related and upregulation of defense-related genes were associated with transcriptional responses suggesting floral induction and plant development.
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Abstract Humic acids (HAs) stimulate the growth of several plant species by regulating their hormonal and redox metabolisms. Nevertheless, studies on the relationship of these substances with the plant-associated microbiota are almost nonexistent. Here, we hypothesized that the effect of HAs occurs in parallel with the regulation of the plant-associated bacterial community. Our results show the positive influence of HAs on the growth of rice and its stimulation of the root system. Metataxonomics revealed that the structure and composition of root bacterial communities were affected upon the application of HAs. Chitinophaga and Mucilaginibacter were the predominant genera in HA-treated roots. These bacteria produce enzymes that degrade compounds like those present in the wall of fungi, oomycetes, and nematode eggs. Pseudomonas and the Gp 1 group of Acidobacteria, both siderophore-producers and plant-growth promoters were also enriched, although with lower abundances. Given these results, we suggest that plants recruit these microorganisms in response to the stress caused by the HA-root interaction. For the first time, our findings indicate that HA-stimulated plants adopt the ecological strategy of recruiting members of the bacterial community that are candidates for the suppression of pathogens and, therefore, involved in plant defense.
Acidobacteria
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Plants are master regulators of rhizosphere ecology, secreting a complex mixture of compounds into the soil, collectively termed plant root exudate. Root exudate composition is highly dynamic and functional, mediating interactions between plants and a wide range of beneficial / harmful soil organisms. Exudate composition is under selective pressure to diversify in response to pathogen perception, whilst maintaining interactions with beneficial organisms. However, crop domestication has exerted significant and unintended changes to crop root exudate composition, and we know very little about genotype - phenotype linkages that pertain to root exudates and rhizosphere interactions. Better understanding could enable the modulation of root exudate composition for crop improvement by promoting positive, and impeding negative, interactions. Root expressed transporters modulate exudate composition and could be manipulated towards the rational design of beneficial root exudate profiles. Using Virus Induced Gene silencing (VIGS), we demonstrate that knockdown of two root-expressed ABC transporter genes in tomato cv. Moneymaker, ABC-G33 and ABC-C6 , alters the composition of semi-volatile compounds in collected root exudates. Root exudate chemotaxis assays demonstrate that knockdown of each transporter gene triggers the repulsion of economically relevant Meloidogyne and Globodera spp. plant parasitic nematodes, which are attracted to control treatment root exudates. Knockdown of ABC-C6 inhibits egg hatching of Meloidogyne and Globodera spp ., relative to controls. Knockdown of ABC-G33 has no impact on egg hatching of Meloidogyne spp. but has a substantial inhibitory impact on egg hatching of G. pallida . ABC-C6 knockdown has no impact on the attraction of the plant pathogen Agrobacterium tumefaciens , or the plant growth promoting Bacillus subtilis , relative to controls. Silencing ABC-G33 induces a statistically significant reduction in attraction of B. subtilis , with no impact on attraction of A. tumefaciens . ABC-C6 represents a promising target for breeding or biotechnology intervention strategies as gene knockdown (-64.9%) leads to the repulsion of economically important plant parasites and retains attraction of the beneficial rhizobacterium B. subtilis . This study exposes the link between ABC transporters, root exudate composition, and ex planta interactions with agriculturally and economically relevant rhizosphere organisms, paving the way for an entirely new approach to rhizosphere engineering and crop protection.
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The rhizosphere is the critical interface between plant roots and soil where beneficial and harmful interactions between plants and microorganisms occur. Although microorganisms have historically been studied as planktonic (or free-swimming) cells, most are found attached to surfaces, in multicellular assemblies known as biofilms. When found in association with plants, certain bacteria such as plant growth promoting rhizobacteria not only induce plant growth but also protect plants from soil-borne pathogens in a process known as biocontrol. Contrastingly, other rhizobacteria in a biofilm matrix may cause pathogenesis in plants. Although research suggests that biofilm formation on plants is associated with biological control and pathogenic response, little is known about how plants regulate this association. Here, we assess the biological importance of biofilm association on plants.
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Streptomyces species are saprophytic soil bacteria that produce a diverse array of specialized metabolites, including half of all known antibiotics. They are also rhizobacteria and plant endophytes that can promote plant growth and protect against disease. Several studies have shown that streptomycetes are enriched in the rhizosphere and endosphere of the model plant Arabidopsis thaliana . Here, we set out to test the hypothesis that they are attracted to plant roots by root exudates, and specifically by the plant phytohormone salicylate, which they might use as a nutrient source. We confirmed a previously published report that salicylate over-producing cpr5 plants are colonized more readily by streptomycetes but found that salicylate-deficient sid2-2 and pad4 plants had the same levels of root colonization by Streptomyces bacteria as the wild-type plants. We then tested eight genome sequenced Streptomyces endophyte strains in vitro and found that none were attracted to or could grow on salicylate as a sole carbon source. We next used 13 CO 2 DNA stable isotope probing to test whether Streptomyces species can feed off a wider range of plant metabolites but found that Streptomyces bacteria were outcompeted by faster growing proteobacteria and did not incorporate photosynthetically fixed carbon into their DNA. We conclude that, given their saprotrophic nature and under conditions of high competition, streptomycetes most likely feed on more complex organic material shed by growing plant roots. Understanding the factors that impact the competitiveness of strains in the plant root microbiome could have consequences for the effective application of biocontrol strains.
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Plant Physiology
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