Interplay between Amaryllidaceae alkaloids, the bacteriome and phytopathogens in Lycoris radiata.

Alkaloids are a large group of plant secondary metabolites with various structures and activities. It is important to understand their functions in the interplay between plants and the beneficial and pathogenic microbiota. Amaryllidaceae alkaloids (AAs) are unique secondary metabolites in Amaryllidaceae plants. Here, we studied the interplay between AAs and the bacteriome in Lycoris radiata, a traditional Chinese medicinal plant containing high amounts of AAs. The relationship between AAs and bacterial composition in different tissues of L. radiata was studied. In vitro experiments revealed that AAs have varying levels of antimicrobial activity against endophytic bacteria and pathogenic fungi, indicating the importance of AA synthesis in maintaining a balance between plants and beneficial/pathogenic microbiota. Using bacterial synthetic communities with different compositions, we observed a positive feedback loop between bacteria insensitive to AAs and their ability to increase accumulation of AAs in L. radiata, especially in leaves. This may allow insensitive bacteria to outcompete sensitive ones for plant resources. Moreover, the accumulation of AAs enhanced by insensitive bacteria could benefit plants when challenged with fungal pathogens. This study highlights the functions of alkaloids in plant-microbe interactions, opening new avenues for designing plant microbiomes that could contribute to sustainable agriculture.

MYB72-dependent coumarin exudation shapes root microbiome assembly to promote plant health.

Plant roots nurture a tremendous diversity of microbes via exudation of photosynthetically fixed carbon sources. In turn, probiotic members of the root microbiome promote plant growth and protect the host plant against pathogens and pests. In the Arabidopsis thaliana-Pseudomonas simiae WCS417 model system the root-specific transcription factor MYB72 and the MYB72-controlled ß-glucosidase BGLU42 emerged as important regulators of beneficial rhizobacteria-induced systemic resistance (ISR) and iron-uptake responses. MYB72 regulates the biosynthesis of iron-mobilizing fluorescent phenolic compounds, after which BGLU42 activity is required for their excretion into the rhizosphere. Metabolite fingerprinting revealed the antimicrobial coumarin scopoletin as a dominant metabolite that is produced in the roots and excreted into the rhizosphere in a MYB72- and BGLU42-dependent manner. Shotgun-metagenome sequencing of root-associated microbiota of Col-0, myb72, and the scopoletin biosynthesis mutant f6'h1 showed that scopoletin selectively impacts the assembly of the microbial community in the rhizosphere. We show that scopoletin selectively inhibits the soil-borne fungal pathogens Fusarium oxysporum and Verticillium dahliae, while the growth-promoting and ISR-inducing rhizobacteria P. simiae WCS417 and Pseudomonas capeferrum WCS358 are highly tolerant of the antimicrobial effect of scopoletin. Collectively, our results demonstrate a role for coumarins in microbiome assembly and point to a scenario in which plants and probiotic rhizobacteria join forces to trigger MYB72/BGLU42-dependent scopolin production and scopoletin excretion, resulting in improved niche establishment for the microbial partner and growth and immunity benefits for the host plant.

Root transcriptional dynamics induced by beneficial rhizobacteria and microbial immune elicitors reveal signatures of adaptation to mutualists.

Below ground, microbe-associated molecular patterns (MAMPs) of root-associated microbiota can trigger costly defenses at the expense of plant growth. However, beneficial rhizobacteria, such as Pseudomonas simiae WCS417, promote plant growth and induce systemic resistance without being warded off by local root immune responses. To investigate early root responses that facilitate WCS417 to exert its plant-beneficial functions, we performed time series RNA-Seq of Arabidopsis roots in response to live WCS417 and compared it with MAMPs flg22417 (from WCS417), flg22Pa (from pathogenic Pseudomonas aeruginosa) and fungal chitin. The MAMP transcriptional responses differed in timing, but displayed a large overlap in gene identity. MAMP-upregulated genes are enriched for genes with functions in immunity, while downregulated genes are enriched for genes related to growth and development. Although 74% of the transcriptional changes inflicted by live WCS417 overlapped with the flg22417 profile, WCS417 actively suppressed more than half of the MAMP-triggered transcriptional responses, possibly to allow the establishment of a mutually beneficial interaction with the host root. Interestingly, the sector of the flg22417 -repressed transcriptional network that is not affected by WCS417 has a strong auxin signature. Using auxin response mutant tir1afb2afb3, we demonstrate a dual role for auxin signaling in finely balancing growth-promoting and defense-eliciting activities of beneficial microbes in plant roots.

The Age of Coumarins in Plant-Microbe Interactions.

Coumarins are a family of plant-derived secondary metabolites that are produced via the phenylpropanoid pathway. In the past decade, coumarins have emerged as iron-mobilizing compounds that are secreted by plant roots and aid in iron uptake from iron-deprived soils. Members of the coumarin family are found in many plant species. Besides their role in iron uptake, coumarins have been extensively studied for their potential to fight infections in both plants and animals. Coumarin activities range from antimicrobial and antiviral to anticoagulant and anticancer. In recent years, studies in the model plant species tobacco and Arabidopsis have significantly increased our understanding of coumarin biosynthesis, accumulation, secretion, chemical modification and their modes of action against plant pathogens. Here, we review current knowledge on coumarins in different plant species. We focus on simple coumarins and provide an overview on their biosynthesis and role in environmental stress responses, with special attention for the recently discovered semiochemical role of coumarins in aboveground and belowground plant-microbe interactions and the assembly of the root microbiome.

Molecular dialogue between arbuscular mycorrhizal fungi and the nonhost plant Arabidopsis thaliana switches from initial detection to antagonism.

Approximately 29% of all vascular plant species are unable to establish an arbuscular mycorrhizal (AM) symbiosis. Despite this, AM fungi (Rhizophagus spp.) are enriched in the root microbiome of the nonhost Arabidopsis thaliana, and Arabidopsis roots become colonized when AM networks nurtured by host plants are available. Here, we investigated the nonhost-AM fungus interaction by analyzing transcriptional changes in Rhizophagus, Arabidopsis and the host plant Medicago truncatula while growing in the same mycorrhizal network. In early interaction stages, Rhizophagus activated the Arabidopsis strigolactone biosynthesis genes CCD7 and CCD8, suggesting that detection of AM fungi is not completely impaired. However, in colonized Arabidopsis roots, fungal nutrient transporter genes GintPT, GintAMT2, GintMST2 and GintMST4, essential for AM symbiosis, were not activated. RNA-seq transcriptome analysis pointed to activation of costly defenses in colonized Arabidopsis roots. Moreover, Rhizophagus colonization caused a 50% reduction in shoot biomass, but also led to enhanced systemic immunity against Botrytis cinerea. This suggests that early signaling between AM fungi and Arabidopsis is not completely impaired and that incompatibility appears at later interaction stages. Moreover, Rhizophagus-mediated defenses coincide with reduced Arabidopsis growth, but also with systemic disease resistance, highlighting the multifunctional role of AM fungi in host and nonhost interactions.

Microbial small molecules - weapons of plant subversion.

Covering: up to 2018 Plants live in close association with a myriad of microbes that are generally harmless. However, the minority of microbes that are pathogens can severely impact crop quality and yield, thereby endangering food security. By contrast, beneficial microbes provide plants with important services, such as enhanced nutrient uptake and protection against pests and diseases. Like pathogens, beneficial microbes can modulate host immunity to efficiently colonize the nutrient-rich niches within and around the roots and aerial tissues of a plant, a phenomenon mirroring the establishment of commensal microbes in the human gut. Numerous ingenious mechanisms have been described by which pathogenic and beneficial microbes in the plant microbiome communicate with their host, including the delivery of immune-suppressive effector proteins and the production of phytohormones, toxins and other bioactive molecules. Plants signal to their associated microbes via exudation of photosynthetically fixed carbon sources, quorum-sensing mimicry molecules and selective secondary metabolites such as strigolactones and flavonoids. Molecular communication thus forms an integral part of the establishment of both beneficial and pathogenic plant-microbe relations. Here, we review the current knowledge on microbe-derived small molecules that can act as signalling compounds to stimulate plant growth and health by beneficial microbes on the one hand, but also as weapons for plant invasion by pathogens on the other. As an exemplary case, we used comparative genomics to assess the small molecule biosynthetic capabilities of the Pseudomonas genus; a genus rich in both plant pathogenic and beneficial microbes. We highlight the biosynthetic potential of individual microbial genomes and the population at large, providing evidence for the hypothesis that the distinction between detrimental and beneficial microbes is increasingly fading. Knowledge on the biosynthesis and molecular activity of microbial small molecules will aid in the development of successful biological agents boosting crop resiliency in a sustainable manner and could also provide scientific routes to pathogen inhibition or eradication.

Unearthing the genomes of plant-beneficial Pseudomonas model strains WCS358, WCS374 and WCS417.

BACKGROUND: Plant growth-promoting rhizobacteria (PGPR) can protect plants against pathogenic microbes through a diversity of mechanisms including competition for nutrients, production of antibiotics, and stimulation of the host immune system, a phenomenon called induced systemic resistance (ISR). In the past 30 years, the Pseudomonas spp. PGPR strains WCS358, WCS374 and WCS417 of the Willie Commelin Scholten (WCS) collection have been studied in detail in pioneering papers on the molecular basis of PGPR-mediated ISR and mechanisms of biological control of soil-borne pathogens via siderophore-mediated competition for iron. RESULTS: The genomes of the model WCS PGPR strains were sequenced and analyzed to unearth genetic cues related to biological questions that surfaced during the past 30 years of functional studies on these plant-beneficial microbes. Whole genome comparisons revealed important novel insights into iron acquisition strategies with consequences for both bacterial ecology and plant protection, specifics of bacterial determinants involved in plant-PGPR recognition, and diversity of protein secretion systems involved in microbe-microbe and microbe-plant communication. Furthermore, multi-locus sequence alignment and whole genome comparison revealed the taxonomic position of the WCS model strains within the Pseudomonas genus. Despite the enormous diversity of Pseudomonas spp. in soils, several plant-associated Pseudomonas spp. strains that have been isolated from different hosts at different geographic regions appear to be nearly isogenic to WCS358, WCS374, or WCS417. Interestingly, all these WCS look-a-likes have been selected because of their plant protective or plant growth-promoting properties. CONCLUSIONS: The genome sequences of the model WCS strains revealed that they can be considered representatives of universally-present plant-beneficial Pseudomonas spp. With their well-characterized functions in the promotion of plant growth and health, the fully sequenced genomes of the WCS strains provide a genetic framework that allows for detailed analysis of the biological mechanisms of the plant-beneficial traits of these PGPR. Considering the increasing focus on the role of the root microbiome in plant health, functional genomics of the WCS strains will enhance our understanding of the diversity of functions of the root microbiome.

Techniques to Study Common Root Responses to Beneficial Microbes and Iron Deficiency.

Iron (Fe) plays a central role in the vital processes of a plant. The Fe status of a plant influences growth and immunity, but it also dictates interactions of roots with soil microbiota through the production of Fe mobilizing, antimicrobial fluorescent phenolic compounds called coumarins. To adapt to low Fe availability in the soil, plants deploy an efficient Fe deficiency response. Interestingly, this Fe deficiency response is hijacked by root-colonizing microbes in the root microbiome to establish a mutually beneficial relationship. In this chapter, we describe how we cultivate plants and microbes to study the interaction between plants, beneficial rhizobacteria, and the plant's Fe deficiency response. We describe (a) how we study activity and localization of these responses by assessing gene-specific promoter activities using GUS assays, (b) how we visualize root-secreted coumarins in response to Fe deficiency and colonization by beneficial rhizobacteria, and (c) how we prepare our samples for metabolite extraction and reverse-transcriptase quantitative PCR to analyze the expression of marker genes.

Cell-type-specific transcriptomics reveals that root hairs and endodermal barriers play important roles in beneficial plant-rhizobacterium interactions.

Growth- and health-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is such a bacterium that efficiently colonizes roots, modifies the architecture of the root system to increase its size, and induces systemic resistance to make plants more resistant to pests and pathogens. Our previous work suggested that WCS417-induced phenotypes are controlled by root cell-type-specific mechanisms. However, it remains unclear how WCS417 affects these mechanisms. In this study, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. We found that the cortex and endodermis have the most differentially expressed genes, even though they are not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, and mutant analysis suggests that this downregulation facilitates WCS417-driven root architectural changes. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis of WCS417-colonized roots. Using an endodermal barrier mutant, we showed the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Comparison of the transcriptome profiles in the two epidermal cell types that are in direct contact with WCS417-trichoblasts that form root hairs and atrichoblasts that do not-implies a difference in potential for defense gene activation. While both cell types respond to WCS417, trichoblasts displayed both higher basal and WCS417-dependent activation of defense-related genes compared with atrichoblasts. This suggests that root hairs may activate root immunity, a hypothesis that is supported by differential immune responses in root hair mutants. Taken together, these results highlight the strength of cell-type-specific transcriptional profiling to uncover "masked" biological mechanisms underlying beneficial plant-microbe associations.

Unearthing soil-plant-microbiota crosstalk: Looking back to move forward.

The soil is vital for life on Earth and its biodiversity. However, being a non-renewable and threatened resource, preserving soil quality is crucial to maintain a range of ecosystem services critical to ecological balances, food production and human health. In an agricultural context, soil quality is often perceived as the ability to support field production, and thus soil quality and fertility are strictly interconnected. The concept of, as well as the ways to assess, soil fertility has undergone big changes over the years. Crop performance has been historically used as an indicator for soil quality and fertility. Then, analysis of a range of physico-chemical parameters has been used to routinely assess soil quality. Today it is becoming evident that soil quality must be evaluated by combining parameters that refer both to the physico-chemical and the biological levels. However, it can be challenging to find adequate indexes for evaluating soil quality that are both predictive and easy to measure in situ. An ideal soil quality assessment method should be flexible, sensitive enough to detect changes in soil functions, management and climate, and should allow comparability among sites. In this review, we discuss the current status of soil quality indicators and existing databases of harmonized, open-access topsoil data. We also explore the connections between soil biotic and abiotic features and crop performance in an agricultural context. Finally, based on current knowledge and technical advancements, we argue that the use of plant health traits represents a powerful way to assess soil physico-chemical and biological properties. These plant health parameters can serve as proxies for different soil features that characterize soil quality both at the physico-chemical and at the microbiological level, including soil quality, fertility and composition of soil microbial communities.

Evolutionary "hide and seek" between bacterial flagellin and the plant immune system.

Bacterial flagellin is a potent host immune activator. Parys et al. (2021) and Colaianni et al. (2021) dissected effects of flagellin epitope variants on host immune detection and bacterial motility. They report in this issue of Cell Host & Microbe that Arabidopsis-associated bacterial microbiota differentially evolved flg22 variants that allow tunability between motility and defense activation.

Coumarin Communication Along the Microbiome-Root-Shoot Axis.

Plants shape their rhizosphere microbiome by secreting root exudates into the soil environment. Recently, root-exuded coumarins were identified as novel players in plant-microbiome communication. Beneficial members of the root-associated microbiome stimulate coumarin biosynthesis in roots and their excretion into the rhizosphere. The iron-mobilizing activity of coumarins facilitates iron uptake from the soil environment, while their selective antimicrobial activity shapes the root microbiome, resulting in promotion of plant growth and health. Evidence is accumulating that, in analogy to strigolactones and flavonoids, coumarins may act in microbiome-to-root-to-shoot signaling events. Here, we review this multifaceted role of coumarins in bidirectional chemical communication along the microbiome-root-shoot axis.

Transcriptome Signatures in Pseudomonas simiae WCS417 Shed Light on Role of Root-Secreted Coumarins in Arabidopsis-Mutualist Communication.

Pseudomonas simiae WCS417 is a root-colonizing bacterium with well-established plant-beneficial effects. Upon colonization of Arabidopsis roots, WCS417 evades local root immune responses while triggering an induced systemic resistance (ISR) in the leaves. The early onset of ISR in roots shows similarities with the iron deficiency response, as both responses are associated with the production and secretion of coumarins. Coumarins can mobilize iron from the soil environment and have a selective antimicrobial activity that impacts microbiome assembly in the rhizosphere. Being highly coumarin-tolerant, WCS417 induces the secretion of these phenolic compounds, likely to improve its own niche establishment, while providing growth and immunity benefits for the host in return. To investigate the possible signaling function of coumarins in the mutualistic Arabidopsis-WCS417 interaction, we analyzed the transcriptome of WCS417 growing in root exudates of coumarin-producing Arabidopsis Col-0 and the coumarin-biosynthesis mutant f6'h1. We found that coumarins in F6'H1-dependent root exudates significantly affected the expression of 439 bacterial genes (8% of the bacterial genome). Of those, genes with functions related to transport and metabolism of carbohydrates, amino acids, and nucleotides were induced, whereas genes with functions related to cell motility, the bacterial mobilome, and energy production and conversion were repressed. Strikingly, most genes related to flagellar biosynthesis were down-regulated by F6'H1-dependent root exudates and we found that application of selected coumarins reduces bacterial motility. These findings suggest that coumarins' function in the rhizosphere as semiochemicals in the communication between the roots and WCS417. Collectively, our results provide important novel leads for future functional analysis of molecular processes in the establishment of plant-mutualist interactions.

The Soil-Borne Identity and Microbiome-Assisted Agriculture: Looking Back to the Future.

Looking forward includes looking back every now and then. In 2007, David Weller looked back at 30 years of biocontrol of soil-borne pathogens by Pseudomonas and signified that the progress made over decades of research has provided a firm foundation to formulate current and future research questions. It has been recognized for more than a century that soil-borne microbes play a significant role in plant growth and health. The recent application of high-throughput omics technologies has enabled detailed dissection of the microbial players and molecular mechanisms involved in the complex interactions in plant-associated microbiomes. Here, we highlight old and emerging plant microbiome concepts related to plant disease control, and address perspectives that modern and emerging microbiomics technologies can bring to functionally characterize and exploit plant-associated microbiomes for the benefit of plant health in future microbiome-assisted agriculture.

Modulation of the Root Microbiome by Plant Molecules: The Basis for Targeted Disease Suppression and Plant Growth Promotion.

Plants host a mesmerizing diversity of microbes inside and around their roots, known as the microbiome. The microbiome is composed mostly of fungi, bacteria, oomycetes, and archaea that can be either pathogenic or beneficial for plant health and fitness. To grow healthy, plants need to surveil soil niches around the roots for the detection of pathogenic microbes, and in parallel maximize the services of beneficial microbes in nutrients uptake and growth promotion. Plants employ a palette of mechanisms to modulate their microbiome including structural modifications, the exudation of secondary metabolites and the coordinated action of different defence responses. Here, we review the current understanding on the composition and activity of the root microbiome and how different plant molecules can shape the structure of the root-associated microbial communities. Examples are given on interactions that occur in the rhizosphere between plants and soilborne fungi. We also present some well-established examples of microbiome harnessing to highlight how plants can maximize their fitness by selecting their microbiome. Understanding how plants manipulate their microbiome can aid in the design of next-generation microbial inoculants for targeted disease suppression and enhanced plant growth.

Type III Secretion System of Beneficial Rhizobacteria Pseudomonas simiae WCS417 and Pseudomonas defensor WCS374.

Plants roots host myriads of microbes, some of which enhance the defense potential of plants by activating a broad-spectrum immune response in leaves, known as induced systemic resistance (ISR). Nevertheless, establishment of this mutualistic interaction requires active suppression of local root immune responses to allow successful colonization. To facilitate host colonization, phytopathogenic bacteria secrete immune-suppressive effectors into host cells via the type III secretion system (T3SS). Previously, we searched the genomes of the ISR-inducing rhizobacteria Pseudomonas simiae WCS417 and Pseudomonas defensor WCS374 for the presence of a T3SS and identified the components for a T3SS in the genomes of WCS417 and WCS374. By performing a phylogenetic and gene cluster alignment analysis we show that the T3SS of WCS417 and WCS374 are grouped in a clade that is enriched for beneficial rhizobacteria. We also found sequences of putative novel effectors in their genomes, which may facilitate future research on the role of T3SS effectors in plant-beneficial microbe interactions in the rhizosphere.

Rhizosphere-enriched microbes as a pool to design synthetic communities for reproducible beneficial outputs.

Composts represent a sustainable way to suppress diseases and improve plant growth. Identification of compost-derived microbial communities enriched in the rhizosphere of plants and characterization of their traits, could facilitate the design of microbial synthetic communities (SynComs) that upon soil inoculation could yield consistent beneficial effects towards plants. Here, we characterized a collection of compost-derived bacteria, previously isolated from tomato rhizosphere, for in vitro antifungal activity against soil-borne fungal pathogens and for their potential to change growth parameters in Arabidopsis. We further assessed root-competitive traits in the dominant rhizospheric genus Bacillus. Certain isolated rhizobacteria displayed antifungal activity against the tested pathogens and affected the growth of Arabidopsis, and the Bacilli members possessed several enzymatic activities. Subsequently, we designed two SynComs with different composition and tested their effect on Arabidopsis and tomato growth and health. SynCom1, consisting of different bacterial genera, displayed negative effect on Arabidopsis in vitro, but promoted tomato growth in pots. SynCom2, consisting of Bacilli, didn't affect Arabidopsis growth, enhanced tomato growth and suppressed Fusarium wilt symptoms. Overall, we found selection of compost-derived microbes with beneficial properties in the rhizosphere of tomato plants, and observed that application of SynComs on poor substrates can yield reproducible plant phenotypes.

Rhizosphere-Associated Pseudomonas Suppress Local Root Immune Responses by Gluconic Acid-Mediated Lowering of Environmental pH.

The root microbiome consists of commensal, pathogenic, and plant-beneficial microbes [1]. Most members of the root microbiome possess microbe-associated molecular patterns (MAMPs) similar to those of plant pathogens [2]. Their recognition can lead to the activation of host immunity and suppression of plant growth due to growth-defense tradeoffs [3, 4]. We found that 42% of the tested root microbiota, including the plant growth-promoting rhizobacteria Pseudomonas capeferrum WCS358 [5, 6] and Pseudomonas simiae WCS417 [6, 7], are able to quench local Arabidopsis thaliana root immune responses that are triggered by flg22 [8], an immunogenic epitope of the MAMP flagellin [9], suggesting that this is an important function of the root microbiome. In a screen for WCS358 mutants that lost their capacity to suppress flg22-induced CYP71A12pro:GUS MAMP-reporter gene expression, we identified the bacterial genes pqqF and cyoB in WCS358, which are required for the production of gluconic acid and its derivative 2-keto gluconic acid. Both WCS358 mutants are impaired in the production of these organic acids and consequently lowered their extracellular pH to a lesser extent than wild-type WCS358. Acidification of the plant growth medium similarly suppressed flg22-induced CYP71A12pro:GUS and MYB51pro:GUS expression, and the flg22-mediated oxidative burst, suggesting a role for rhizobacterial gluconic acid-mediated modulation of the extracellular pH in the suppression of root immunity. Rhizosphere population densities of the mutants were significantly reduced compared to wild-type. Collectively, these findings show that suppression of immune responses is an important function of the root microbiome, as it facilitates colonization by beneficial root microbiota.