It takes a plant village to raise a microbiome.

In this issue of Cell Host and Microbe, Meyer et al. explore the effects of host history on the inheritance of the plant microbiome. They find that transmission from the same plant species resulted in microbiota specialization, while transmission from a different species resulted in host generalism.

PSKR1 balances the plant growth-defence trade-off in the rhizosphere microbiome.

Microbiota benefit their hosts by improving nutrient uptake and pathogen protection. How host immunity restricts microbiota while avoiding autoimmunity is poorly understood. Here we show that the Arabidopsis phytosulfokine receptor 1 (pskr1) mutant displays autoimmunity (plant stunting, defence-gene expression and reduced rhizosphere bacterial growth) in response to growth-promoting Pseudomonas fluorescens. Microbiome profiling and microbiota colonization showed that PSKR1-mediated reduction in bacterial growth and stunting is largely specific to Pseudomonas. Transcriptional profiling demonstrated that PSKR1 regulates the growth-defence trade-off during Pseudomonas colonization: PSKR1 upregulates plant photosynthesis and root growth but suppresses salicylic-acid-mediated defences. Genetic epistasis experiments showed that pskr1 stunting and restriction of bacterial growth are salicylic acid dependent. Finally, we showed that Pseudomonas, but not other bacteria, induces PSKR1 expression in roots, suggesting that Pseudomonas might manipulate plant signalling to promote its colonization. Our data demonstrate a genetic mechanism to coordinate beneficial functions of the microbiome while preventing autoimmunity.

Amino Acid Availability Determines Plant Immune Homeostasis in the Rhizosphere Microbiome.

Microbes possess conserved microbe-associated molecular patterns (MAMPs) that are recognized by plant receptors to induce pattern-triggered immunity (PTI). Despite containing the same MAMPs as pathogens, commensals thrive in the plant rhizosphere microbiome, indicating they must suppress or evade host immunity. Previous work found that bacterial-secreted gluconic acid is sufficient to suppress PTI. Here, we show that gluconic acid biosynthesis is not necessary for immunity suppression by the beneficial bacterial strain Pseudomonas simiae WCS417. We performed a forward genetic screen with EMS-mutagenized P. simiae WCS417 and a flagellin-inducible CYP71A12pro:GUS reporter as a PTI readout. We identified a loss of function mutant in ornithine carbamoyltransferase argF, which is required for ornithine conversion to arginine, that cannot suppress PTI or acidify the rhizosphere. Fungal pathogens use alkalization through production of ammonia and glutamate, and arginine biosynthetic precursors, to promote their own growth and virulence. While a ΔargF mutant has a growth defect in the rhizosphere, we found that restoring growth with exogenous arginine resulted in rhizosphere alkalization in a mutant that cannot make gluconic acid, indicating that arginine biosynthesis is required for both growth and acidification. Furthermore, blocking bacterial arginine, glutamine, or proline biosynthesis through genetic mutations or feedback inhibition by adding corresponding amino acids, resulted in rhizosphere alkalization. Untargeted metabolomics determined that ornithine, an alkaline molecule, accumulates under conditions associated with rhizosphere alkalization. Our findings show that bacterial amino acid biosynthesis contributes to acidification by preventing accumulation of ornithine and the resulting alkalization. IMPORTANCE Understanding how microbiota evade and suppress host immunity is critical to our knowledge of how beneficial microbes persist in association with a host. Prior work has shown that secretion of organic acids by beneficial microbes is sufficient to suppress plant immunity. This work shows that microbial amino acid metabolism is not only critical for growth in the plant rhizosphere microbiome, but also for regulation of plant rhizosphere pH, and, consequentially, regulation of plant immunity. We found that, in the absence of microbial glutamate and arginine metabolism, rhizosphere alkalization and microbial overgrowth occurs. Collectively, our findings suggest that, by regulating nutrient availability, plants have the potential to regulate their immune homeostasis in the rhizosphere microbiome.

Origins of symbiosis: shared mechanisms underlying microbial pathogenesis, commensalism and mutualism of plants and animals.

Regardless of the outcome of symbiosis, whether it is pathogenic, mutualistic or commensal, bacteria must first colonize their hosts. Intriguingly, closely related bacteria that colonize diverse hosts with diverse outcomes of symbiosis have conserved host-association and virulence factors. This review describes commonalities in the process of becoming host associated amongst bacteria with diverse lifestyles. Whether a pathogen, commensal or mutualist, bacteria must sense the presence of and migrate towards a host, compete for space and nutrients with other microbes, evade the host immune system, and change their physiology to enable long-term host association. We primarily focus on well-studied taxa, such as Pseudomonas, that associate with diverse model plant and animal hosts, with far-ranging symbiotic outcomes. Given the importance of opportunistic pathogens and chronic infections in both human health and agriculture, understanding the mechanisms that facilitate symbiotic relationships between bacteria and their hosts will help inform the development of disease treatments for both humans, and the plants we eat.

Engineering plant microbiomes by integrating eco-evolutionary principles into current strategies.

Engineering plant microbiomes has the potential to improve plant health in a rapid and sustainable way. Rapidly changing climates and relatively long timelines for plant breeding make microbiome engineering an appealing approach to improving food security. However, approaches that have shown promise in the lab have not resulted in wide-scale implementation in the field. Here, we suggest the use of an integrated approach, combining mechanistic molecular and genetic knowledge, with ecological and evolutionary theory, to target knowledge gaps in plant microbiome engineering that may facilitate translatability of approaches into the field. We highlight examples where understanding microbial community ecology is essential for a holistic understanding of the efficacy and consequences of microbiome engineering. We also review examples where understanding plant-microbe evolution could facilitate the design of plants able to recruit specific microbial communities. Finally, we discuss possible trade-offs in plant-microbiome interactions that should be considered during microbiome engineering efforts so as not to introduce off-target negative effects. We include classic and emergent approaches, ranging from microbial inoculants to plant breeding to host-driven microbiome engineering, and address areas that would benefit from multidisciplinary approaches.

The ColR/S two-component system is a conserved determinant of host association across Pseudomonas species.

Members of the bacterial genus Pseudomonas form mutualistic, commensal, and pathogenic associations with diverse hosts. The prevalence of host association across the genus suggests that symbiosis may be a conserved ancestral trait and that distinct symbiotic lifestyles may be more recently evolved. Here we show that the ColR/S two-component system, part of the Pseudomonas core genome, is functionally conserved between Pseudomonas aeruginosa and Pseudomonas fluorescens. Using plant rhizosphere colonization and virulence in a murine abscess model, we show that colR is required for commensalism with plants and virulence in animals. Comparative transcriptomics revealed that the ColR regulon has diverged between P. aeruginosa and P. fluorescens and deleting components of the ColR regulon revealed strain-specific, but not host-specific, requirements for ColR-dependent genes. Collectively, our results suggest that ColR/S allows Pseudomonas to sense and respond to a host, but that the ColR-regulon has diverged between Pseudomonas strains with distinct lifestyles. This suggests that conservation of two-component systems, coupled with life-style dependent diversification of the regulon, may play a role in host association and lifestyle transitions.

Gastric acid and escape to systemic circulation represent major bottlenecks to host infection by Citrobacter rodentium.

The gastrointestinal (GI) environment plays a critical role in shaping enteric infections. Host environmental factors create bottlenecks, restrictive events that reduce the genetic diversity of invading bacterial populations. However, the identity and impact of bottleneck events on bacterial infection are largely unknown. We used Citrobacter rodentium infection of mice, a model of human pathogenic Escherichia coli infections, to examine bacterial population dynamics and quantify bottlenecks to host colonization. Using Sequence Tag-based Analysis of Microbial Populations (STAMP) we characterized the founding population size (Nb') and relatedness of C. rodentium populations at relevant tissue sites during early- and peak-infection. We demonstrate that the GI environment severely restricts the colonizing population, with an average Nb' of only 12-43 lineages (of 2,000+ inoculated) identified regardless of time or biogeographic location. Passage through gastric acid and escape to the systemic circulation were identified as major bottlenecks during C. rodentium colonization. Manipulating such events by increasing gastric pH dramatically increased intestinal Nb'. Importantly, removal of the stomach acid barrier had downstream consequences on host systemic colonization, morbidity, and mortality. These findings highlight the capability of the host GI environment to limit early pathogen colonization, controlling the population of initial founders with consequences for downstream infection outcomes.

Surviving the host: Microbial metabolic genes required for growth of Pseudomonas aeruginosa in physiologically-relevant conditions.

Pseudomonas aeruginosa, like other pathogens, adapts to the limiting nutritional environment of the host by altering patterns of gene expression and utilizing alternative pathways required for survival. Understanding the genes essential for survival in the host gives insight into pathways that this organism requires during infection and has the potential to identify better ways to treat infections. Here, we used a saturated transposon insertion mutant pool of P. aeruginosa strain PAO1 and transposon insertion sequencing (Tn-Seq), to identify genes conditionally important for survival under conditions mimicking the environment of a nosocomial infection. Conditions tested included tissue culture medium with and without human serum, a murine abscess model, and a human skin organoid model. Genes known to be upregulated during infections, as well as those involved in nucleotide metabolism, and cobalamin (vitamin B12) biosynthesis, etc., were required for survival in vivo- and in host mimicking conditions, but not in nutrient rich lab medium, Mueller Hinton broth (MHB). Correspondingly, mutants in genes encoding proteins of nucleotide and cobalamin metabolism pathways were shown to have growth defects under physiologically-relevant media conditions, in vivo, and in vivo-like models, and were downregulated in expression under these conditions, when compared to MHB. This study provides evidence for the relevance of studying P. aeruginosa fitness in physiologically-relevant host mimicking conditions and identified metabolic pathways that represent potential novel targets for alternative therapies.

BosR: A novel biofilm-specific regulator in Pseudomonas aeruginosa.

Biofilms are the most common cause of bacterial infections in humans and notoriously hard to treat due to their ability to withstand antibiotics and host immune defenses. To overcome the current lack of effective antibiofilm therapies and guide future design, the identification of novel biofilm-specific gene targets is crucial. In this regard, transcriptional regulators have been proposed as promising targets for antimicrobial drug design. Therefore, a Transposon insertion sequencing approach was employed to systematically identify regulators phenotypically affecting biofilm growth in Pseudomonas aeruginosa PA14 using the TnSeq analysis tools Bio-TraDIS and TRANSIT. A screen of a pool of 300,000 transposon insertion mutants identified 349 genes involved in biofilm growth on hydroxyapatite, including 47 regulators. Detection of 19 regulatory genes participating in well-established biofilm pathways validated the results. An additional 28 novel prospective biofilm regulators suggested the requirement for multiple one-component transcriptional regulators. Biofilm-defective phenotypes were confirmed for five one-component transcriptional regulators and a protein kinase, which did not affect motility phenotypes. The one-component transcriptional regulator bosR displayed a conserved role in P. aeruginosa biofilm growth since its ortholog in P. aeruginosa strain PAO1 was also required for biofilm growth. Microscopic analysis of a chromosomal deletion mutant of bosR confirmed the role of this regulator in biofilm growth. Overall, our results highlighted that the gene network driving biofilm growth is complex and involves regulators beyond the primarily studied groups of two-component systems and cyclic diguanylate signaling proteins. Furthermore, biofilm-specific regulators, such as bosR, might constitute prospective new drug targets to overcome biofilm infections.

Sticky Pi is a high-frequency smart trap that enables the study of insect circadian activity under natural conditions.

In the face of severe environmental crises that threaten insect biodiversity, new technologies are imperative to monitor both the identity and ecology of insect species. Traditionally, insect surveys rely on manual collection of traps, which provide abundance data but mask the large intra- and interday variations in insect activity, an important facet of their ecology. Although laboratory studies have shown that circadian processes are central to insects' biological functions, from feeding to reproduction, we lack the high-frequency monitoring tools to study insect circadian biology in the field. To address these issues, we developed the Sticky Pi, a novel, autonomous, open-source, insect trap that acquires images of sticky cards every 20 minutes. Using custom deep learning algorithms, we automatically and accurately scored where, when, and which insects were captured. First, we validated our device in controlled laboratory conditions with a classic chronobiological model organism, Drosophila melanogaster. Then, we deployed an array of Sticky Pis to the field to characterise the daily activity of an agricultural pest, Drosophila suzukii, and its parasitoid wasps. Finally, we demonstrate the wide scope of our smart trap by describing the sympatric arrangement of insect temporal niches in a community, without targeting particular taxa a priori. Together, the automatic identification and high sampling rate of our tool provide biologists with unique data that impacts research far beyond chronobiology, with applications to biodiversity monitoring and pest control as well as fundamental implications for phenology, behavioural ecology, and ecophysiology. We released the Sticky Pi project as an open community resource on https://doc.sticky-pi.com.

Putrescine and Its Metabolic Precursor Arginine Promote Biofilm and c-di-GMP Synthesis in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.

The Pseudomonas aeruginosa whole genome sequence: A 20th anniversary celebration.

Toward the end of August 2000, the 6.3 Mbp whole genome sequence of Pseudomonas aeruginosa strain PAO1 was published. With 5570 open reading frames (ORFs), PAO1 had the largest microbial genome sequenced up to that point in time-including a large proportion of metabolic, transport and antimicrobial resistance genes supporting its ability to colonize diverse environments. A remarkable 9% of its ORFs were predicted to encode proteins with regulatory functions, providing new insight into bacterial network complexity as a function of network size. In this celebratory article, we fast forward 20 years, and examine how access to this resource has transformed our understanding of P. aeruginosa. What follows is more than a simple review or commentary; we have specifically asked some of the leaders in the field to provide personal reflections on how the PAO1 genome sequence, along with the Pseudomonas Community Annotation Project (PseudoCAP) and Pseudomonas Genome Database (pseudomonas.com), have contributed to the many exciting discoveries in this field. In addition to bringing us all up to date with the latest developments, we also ask our contributors to speculate on how the next 20 years of Pseudomonas research might pan out.

Pseudomonas Strains Induce Transcriptional and Morphological Changes and Reduce Root Colonization of Verticillium spp.

Phytopathogenic Verticillia cause Verticillium wilt on numerous economically important crops. Plant infection begins at the roots, where the fungus is confronted with rhizosphere inhabiting bacteria. The effects of different fluorescent pseudomonads, including some known biocontrol agents of other plant pathogens, on fungal growth of the haploid Verticillium dahliae and/or the amphidiploid Verticillium longisporum were compared on pectin-rich medium, in microfluidic interaction channels, allowing visualization of single hyphae, or on Arabidopsis thaliana roots. We found that the potential for formation of bacterial lipopeptide syringomycin resulted in stronger growth reduction effects on saprophytic Aspergillus nidulans compared to Verticillium spp. A more detailed analyses on bacterial-fungal co-cultivation in narrow interaction channels of microfluidic devices revealed that the strongest inhibitory potential was found for Pseudomonas protegens CHA0, with its inhibitory potential depending on the presence of the GacS/GacA system controlling several bacterial metabolites. Hyphal tip polarity was altered when V. longisporum was confronted with pseudomonads in narrow interaction channels, resulting in a curly morphology instead of straight hyphal tip growth. These results support the hypothesis that the fungus attempts to evade the bacterial confrontation. Alterations due to co-cultivation with bacteria could not only be observed in fungal morphology but also in fungal transcriptome. P. protegens CHA0 alters transcriptional profiles of V. longisporum during 2 h liquid media co-cultivation in pectin-rich medium. Genes required for degradation of and growth on the carbon source pectin were down-regulated, whereas transcripts involved in redox processes were up-regulated. Thus, the secondary metabolite mediated effect of Pseudomonas isolates on Verticillium species results in a complex transcriptional response, leading to decreased growth with precautions for self-protection combined with the initiation of a change in fungal growth direction. This interplay of bacterial effects on the pathogen can be beneficial to protect plants from infection, as shown with A. thaliana root experiments. Treatment of the roots with bacteria prior to infection with V. dahliae resulted in a significant reduction of fungal root colonization. Taken together we demonstrate how pseudomonads interfere with the growth of Verticillium spp. and show that these bacteria could serve in plant protection.

FERONIA restricts Pseudomonas in the rhizosphere microbiome via regulation of reactive oxygen species.

Maintaining microbiome structure is critical for the health of both plants and animals. By re-screening a collection of Arabidopsis mutants affecting root immunity and hormone crosstalk, we identified a FERONIA (FER) receptor kinase mutant (fer-8) with a rhizosphere microbiome enriched in Pseudomonas fluorescens without phylum-level dysbiosis. Using microbiome transplant experiments, we found that the fer-8 microbiome was beneficial. The effect of FER on rhizosphere pseudomonads was largely independent of its immune scaffold function, role in development and jasmonic acid autoimmunity. We found that the fer-8 mutant has reduced basal levels of reactive oxygen species (ROS) in roots and that mutants deficient in NADPH oxidase showed elevated rhizosphere pseudomonads. The addition of RALF23 peptides, a FER ligand, was sufficient to enrich P. fluorescens. This work shows that FER-mediated ROS production regulates levels of beneficial pseudomonads in the rhizosphere microbiome.

Maintaining Symbiotic Homeostasis: How Do Plants Engage With Beneficial Microorganisms While at the Same Time Restricting Pathogens?

This article is part of the Top 10 Unanswered Questions in MPMI invited review series.That plants recruit beneficial microbes while simultaneously restricting pathogens is critical to their survival. Plants must exclude pathogens; however, most land plants are able to form mutualistic symbioses with arbuscular mycorrhizal fungi. Plants also associate with the complex microbial communities that form the microbiome. The outcome of each symbiotic interaction-whether a specific microbe is pathogenic, commensal, or mutualistic-relies on the specific interplay of host and microbial genetics and the environment. Here, we discuss how plants use metabolites as a gate to select which microbes can be symbiotic. Once present, we discuss how plants integrate multiple inputs to initiate programs of immunity or mutualistic symbiosis and how this paradigm may be expanded to the microbiome. Finally, we discuss how environmental signals are integrated with immunity to fine-tune a thermostat that determines whether a plant engages in mutualism, resistance to pathogens, and shapes associations with the microbiome. Collectively, we propose that the plant immune thermostat is set to select for and tolerate a largely nonharmful microbiome while receptor-mediated decision making allows plants to detect and dynamically respond to the presence of potential pathogens or mutualists.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Mechanisms in plant-microbiome interactions: lessons from model systems.

The use of genetically tractable plant-microbe pairs has driven research in plant immunity and mutualistic symbiosis. Clear functional readouts for the outcomes of symbiosis or immunity have facilitated forward genetic screening and identification of signals, molecules and mechanisms that determine the outcome of these interactions. Plants also associate with beneficial microbial communities that form the microbiome. However, the complexity of the microbiome, combined with relatively subtle effects on plant growth and immunity, has impeded forward genetic screening to identify plant and bacterial genes that shape the microbiome. As a result, microbiome research has relied largely on reverse genetics approaches, based on what is known about plant nutrient uptake and immunity, to identify mechanisms in plant-microbiome research. Here we revisit the features of reductionist model systems that have made them so powerful for studying plant-microbe interactions, and how modeling microbiome research after these systems can propel discovery of novel mechanisms.

Commensal Pseudomonas fluorescens Strains Protect Arabidopsis from Closely Related Pseudomonas Pathogens in a Colonization-Dependent Manner.

Plants form commensal associations with soil microorganisms, creating a root microbiome that provides benefits, including protection against pathogens. While bacteria can inhibit pathogens through the production of antimicrobial compounds in vitro, it is largely unknown how microbiota contribute to pathogen protection in planta. We developed a gnotobiotic model consisting of Arabidopsis thaliana and the opportunistic pathogen Pseudomonas sp. N2C3, to identify mechanisms that determine the outcome of plant-pathogen-microbiome interactions in the rhizosphere. We screened 25 phylogenetically diverse Pseudomonas strains for their ability to protect against N2C3 and found that commensal strains closely related to N2C3, including Pseudomonas sp. WCS365, were more likely to protect against pathogenesis. We used comparative genomics to identify genes unique to the protective strains and found no genes that correlate with protection, suggesting that variable regulation of components of the core Pseudomonas genome may contribute to pathogen protection. We found that commensal colonization level was highly predictive of protection, so we tested deletions in genes required for Arabidopsis rhizosphere colonization. We identified a response regulator colR, and two ColR-dependent genes with predicted roles in membrane modifications (warB and pap2_2), that are required for Pseudomonas-mediated protection from N2C3. We found that WCS365 also protects against the agricultural pathogen Pseudomonas fuscovaginae SE-1, the causal agent of bacterial sheath brown rot of rice, in a ColR-dependent manner. This work establishes a gnotobiotic model to uncover mechanisms by which members of the microbiome can protect hosts from pathogens and informs our understanding of the use of beneficial strains for microbiome engineering in dysbiotic soil systems. IMPORTANCE Microbiota can protect diverse hosts from pathogens, and microbiome dysbiosis can result in increased vulnerability to opportunistic pathogens. Here, we developed a rhizosphere commensal-pathogen model to identify bacterial strains and mechanisms that can protect plants from an opportunistic Pseudomonas pathogen. Our finding that protective strains are closely related to the pathogen suggests that the presence of specific microbial taxa may help protect plants from disease. We found that commensal colonization level was highly correlated with protection, suggesting that competition with pathogens may play a role in protection. As we found that commensal Pseudomonas were also able to protect against an agricultural pathogen, this system may be broadly relevant for identifying strains and mechanisms to control agriculturally important pathogens. This work also suggests that beneficial plant-associated microbes may be useful for engineering soils where microbial complexity is low, such as hydroponic, or disturbed agricultural soils.