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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

CRAGE-RB-PI-seq reveals transcriptional dynamics of plant-associated bacteria during root colonization

Plant roots release a wide array of metabolites into the rhizosphere, shaping microbial communities and their functions. While metagenomics has expanded our understanding of these communities, little is known about the physiology of their members in host environments. Transcriptome analysis via RNA sequencing is a common approach to learning more, but its use has been challenging because of low bacterial biomass and interference from plant RNA. To overcome this, we developed a randomly-barcoded promoter-library insertion sequencing (RB-PI-seq) combined with chassis-independent recombinase-assisted genome engineering (CRAGE). Using Pseudomonas simiae WCS417 as a model rhizobacterium, this method enabled targeted amplification of barcoded transcripts, bypassing plant RNA interference and allowing measurement of thousands of promoter activities during Arabidopsis root colonization. Our analysis revealed temporally resolved transcriptional regulation, including those associated with cell growth, chemotaxis, plant immune suppression, biofilm formation, and stress responses, reflecting the coordinated physiological adaptation to the root environment. Additionally, we discovered that transcriptional activation of xanthine dehydrogenase and a lysozyme inhibitor is crucial for evading plant immune systems. This framework is scalable to other bacterial species and provides new opportunities for understanding rhizobacterial gene regulation in native environments.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Impacts of Soil Microbiome Variations on Root Colonization by Fungi and Bacteria and on the Metabolome of Populus tremula × alba

Trees depend on beneficial interactions between roots and soil microbes for their nutrition and protection against stresses. The soil microbiome provides the main reservoir of microbes for root colonization and is subject to natural variations that can affect its composition. It is not clear whether the tree’s root system is able to buffer the natural variations occurring in the soil microbiome to capture a stable and effective microbiome or whether these variations affect its microbiome to impact its physiology. To address this question, we planted cuttings of Gray Poplar (Populus tremula × alba clone 717-1B4) in natural soil taken from a poplar stand under the same tree over two consecutive years and grew them in a greenhouse. We analyzed the soil and root microbiomes by high throughput Illumina MiSeq sequencing of fungal rDNA internal transcribed spacer and bacterial 16S rRNA amplicons and we characterized the root metabolome by gas chromatography-mass spectrometry. Soil and root microbial communities significantly shifted over the 2 years. A modification of the balance between endophytes, saprophytes, and mycorrhizal fungi occurred in the roots as well as a replacement of some dominant operational taxonomic units by others. These modifications were correlated with a significant alteration of the levels of about 10% of primary and secondary metabolites, suggesting that natural fluctuations in soil microbial communities can have a profound impact on tree root metabolism and physiology. Tree roots functioning may thus be indirectly strongly affected by the effects of future extreme climatic variations on the soil microbiome.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Laser ablation tomography for visualization of root colonization by edaphic organisms

Soil biota have important effects on crop productivity, but can be difficult to study in situ . Laser ablation tomography (LAT) is a novel method that allows for rapid, three-dimensional quantitative and qualitative analysis of root anatomy, providing new opportunities to investigate interactions between roots and edaphic organisms. LAT was used for analysis of maize roots colonized by arbuscular mycorrhizal fungi, maize roots herbivorized by western corn rootworm, barley roots parasitized by cereal cyst nematode, and common bean roots damaged by Fusarium . UV excitation of root tissues affected by edaphic organisms resulted in differential autofluorescence emission, facilitating the classification of tissues and anatomical features. Samples were spatially resolved in three dimensions, enabling quantification of the volume and distribution of fungal colonization, western corn rootworm damage, nematode feeding sites, tissue compromised by Fusarium , and as well as root anatomical phenotypes. Owing to its capability for high-throughput sample imaging, LAT serves as an excellent tool to conduct large, quantitative screens to characterize genetic control of root anatomy and interactions with edaphic organisms. Additionally, this technology improves interpretation of root–organism interactions in relatively large, opaque root segments, providing opportunities for novel research investigating the effects of root anatomical phenes on associations with edaphic organisms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2019

In-Situ Metabolomic Analysis of Setaria viridis Roots Colonized by Beneficial Endophytic Bacteria

Over the past decades, crop yields have risen in parallel with increasing use of fossil-fuel derived nitrogen (N) fertilizers, but with concomitant negative impacts on climate and water resources. There is a need for more sustainable agricultural practices, and biological nitrogen fixation (BNF) could be part of the solution. A variety of nitrogen-fixing, epiphytic and endophytic plant growth promoting bacteria (PGPB) are known to stimulate plant growth. However, compared to the rhizobium-legume symbiosis, little mechanistic information is available as to how PGPB affect plant metabolism. Therefore, we investigated the metabolic changes in roots of the model grass species Setaria viridis upon endophytic colonization by Herbaspirillum seropedicae SmR1 (fix + ) or a fix - mutant strain (SmR54), compared to uninoculated roots. Endophytic colonization of the root is highly localized and, hence, analysis of whole root segments dilutes the metabolic signature of those few cells impacted by the bacteria. Therefore, we utilized in situ laser ablation electrospray ionization mass spectrometry (LAESI-MS) to sample only those root segments at or adjacent to the sites of bacterial colonization. We report netabolites involved in purine, zeatin, and riboflavin pathways were significantly more abundant in inoculated plants while metabolites indicative of nitrogen, starch, and sucrose metabolism were reduced in roots inoculated with the fix - strain or uninoculated, presumably due to N limitation. Interestingly, compounds, involved in indole-alkaloid biosynthesis were more abundant in the roots colonized by the fix - strain, perhaps reflecting a plant defense response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mycorrhizal effector PaMiSSP10b alters polyamine biosynthesis in Eucalyptus root cells and promotes root colonization

Summary Pathogenic microbes are known to manipulate the defences of their hosts through the production of secreted effector proteins. More recently, mutualistic mycorrhizal fungi have also been described as using these secreted effectors to promote host colonization. Here we characterize a mycorrhiza‐induced small secreted effector protein of 10 kDa produced by the ectomycorrhizal fungus Pisolithus albus , PaMiSSP10b. We demonstrate that PaMiSSP10b is secreted from fungal hyphae, enters the cells of its host, Eucalyptus grandis, and interacts with an S‐adenosyl methionine decarboxylase (AdoMetDC) in the polyamine pathway. Plant polyamines are regulatory molecules integral to the plant immune system during microbial challenge. Using biochemical and transgenic approaches we show that expression of PaMiSSP10b influences levels of polyamines in the plant roots as it enhances the enzymatic activity of AdoMetDC and increases the biosynthesis of higher polyamines. This ultimately favours the colonization success of P. albus . These results identify a new mechanism by which mutualistic microbes are able to manipulate the host´s enzymatic pathways to favour colonization.

Plett, Jonathan M.↗
DOE OSTI2024

Catabolic pathway acquisition by rhizosphere bacteria readily enables growth with a root exudate component but does not affect root colonization

Horizontal gene transfer (HGT) is a fundamental evolutionary process that plays a key role in bacterial evolution. The likelihood of a successful transfer event is expected to depend on the precise balance of costs and benefits resulting from pathway acquisition. Most experimental analyses of HGT have focused on phenotypes that have large fitness benefits under appropriate selective conditions, such as antibiotic resistance. However, many examples of HGT involve phenotypes that are predicted to provide smaller benefits, such as the ability to catabolize additional carbon sources. We have experimentally simulated the consequences of one such HGT event in the laboratory, studying the effects of transferring a pathway for catabolism of the plant-derived aromatic compound salicyl alcohol between rhizosphere isolates from the Pseudomonas genus. We find that pathway acquisition enables rapid catabolism of salicyl alcohol with only minor disruptions to the existing metabolic and regulatory networks of the new host. However, this new catabolic potential does not confer a measurable fitness advantage during competitive growth in the rhizosphere. We conclude that the phenotype of salicyl alcohol catabolism is readily transferable but is selectively neutral under environmentally relevant conditions. We propose that this condition is common and that HGT of many pathways will be self-limiting because the selective benefits are small.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor‐like kinase

Summary Soil‐borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole‐genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor‐like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor‐root colonization by the ectomycorrhizal fungus Laccaria bicolor . By introducing PtLecRLK1 into a perennial grass known to be a non‐host of L. bicolor , switchgrass ( Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro‐PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non‐host to a host of L. bicolor . Furthermore, transcriptomic and proteomic analyses on inoculated‐transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down‐regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N‐containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non‐host to enhance plant productivity and fitness on marginal lands.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Identification of a diguanylate cyclase expressed in the presence of plants and its application for discovering candidate gene products involved in plant colonization by Pantoea sp. YR343

Microbial colonization of plant roots is a highly complex process that requires the coordination and regulation of many gene networks, yet the identities and functions of many of these gene products have yet to be discovered. Pantoea sp. YR343, a gamma-proteobacterium isolated from the rhizosphere of Populus deltoides , forms robust biofilms along the root surfaces of Populus and possesses plant growth-promoting characteristics. In this work, we identified three diguanylate cyclases in the plant-associated microbe Pantoea sp. YR343 that are expressed in the presence of plant roots. One of these diguanylate cyclases, DGC2884, localizes to discrete sites in the cells and its overexpression results in reduced motility and increased EPS production and biofilm formation. We performed a genetic screen by expressing this diguanylate cyclase from an inducible promoter in order to identify candidate gene products that may be involved in root colonization by Pantoea sp. YR343. Further, we demonstrate the importance of other domains in DGC2884 to its activity, which in combination with the genes identified by transposon mutagenesis, may yield insights into the mechanisms of plant association as well as the activity and regulation of homologous enzymes in medically and agriculturally relevant microbes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Lectin Receptor-like Kinase Signaling during Engineered Ectomycorrhiza Colonization

Mutualistic association can improve a plant’s health and productivity. G-type lectin receptor-like kinase (PtLecRLK1) is a susceptibility factor in Populus trichocarpa that permits root colonization by a beneficial fungus, Laccaria bicolor. Engineering PtLecRLK1 also permits L. bicolor root colonization in non-host plants similar to Populus trichocarpa. The intracellular signaling reprogramed by PtLecRLK1 upon recognition of L. bicolor to allow for the development and maintenance of symbiosis is yet to be determined. In this study, phosphoproteomics was utilized to identify phosphorylation-based relevant signaling pathways associated with PtLecRLK1 recognition of L. bicolor in transgenic switchgrass roots. Our finding shows that PtLecRLK1 in transgenic plants modifies the chitin-triggered plant defense and MAPK signaling along with a significant adjustment in phytohormone signaling, ROS balance, endocytosis, cytoskeleton movement, and proteasomal degradation in order to facilitate the establishment and maintenance of L. bicolor colonization. Moreover, protein–protein interaction data implicate a cGMP-dependent protein kinase as a potential substrate of PtLecRLK1.

ectomycorrhiza colonization↗
DOE OSTI2022

Grassland ecosystem type drives AM fungal diversity and functional guild distribution in North American grasslands

Nutrient exchange forms the basis of the ancient symbiotic relationship that occurs between most land plants and arbuscular mycorrhizal (AM) fungi. Plants provide carbon (C) to AM fungi and fungi provide the plant with nutrients such as nitrogen (N) and phosphorous (P). Nutrient addition can alter this symbiotic coupling in key ways, such as reducing AM fungal root colonization and changing the AM fungal community composition. However, environmental parameters that differentiate ecosystems and drive plant distribution patterns (e.g., pH, moisture), are also known to impact AM fungal communities. Identifying the relative contribution of environmental factors impacting AM fungal distribution patterns is important for predicting biogeochemical cycling patterns and plant-microbe relationships across ecosystems. Here, to evaluate the relative impacts of local environmental conditions and long-term nutrient addition on AM fungal abundance and composition across grasslands, we studied experimental plots amended for 10 years with N, P, or N and P fertilizer in different grassland ecosystem types, including tallgrass prairie, montane, shortgrass prairie, and desert grasslands. Contrary to our hypothesis, we found ecosystem type, not nutrient treatment, was the main driver of AM fungal root colonization, diversity, and community composition, even when accounting for site-specific nutrient limitations. We identified several important environmental drivers of grassland ecosystem AM fungal distribution patterns, including aridity, mean annual temperature, root moisture, and soil pH. This work provides empirical evidence for niche partitioning strategies of AM fungal functional guilds and emphasizes the importance of long-term, large scale research projects to provide ecologically relevant context to nutrient addition studies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

X-ray fluorescence and XANES spectroscopy revealed diverse potassium chemistries and colocalization with phosphorus in the ectomycorrhizal fungus Paxillus ammoniavirescens

Ectomycorrhizal (ECM) fungi play a major role in forest ecosystems and managed tree plantations. Particularly, they facilitate mineral weathering and nutrient transfer towards colonized roots. Among nutrients provided by these fungi, potassium (K) has been understudied compared to phosphorus (P) or nitrogen (N). The ECM fungus Paxillus ammoniavirescens is a generalist species that interacts with the root of many trees and can directly transfer K to them, including loblolly pine. However, the forms of K that ECM fungi can store is still unknown. Here, we used synchrotron potassium X-ray fluorescence (XRF) and K-edge X-ray Absorption Near Edge Structure (XANES) spectroscopy on P. ammoniavirescens growing in axenic conditions to investigate the K chemistries accumulating in the center and the edge of the mycelium. We observed that various K forms accumulated in different part of the mycelium, including K-nitrate (KNO 3 ), K-C-O compounds (such as K-tartrate K 2 (C 4 H 4 O 6 ) and K-oxalate (K 2 C 2 O 4 )), K-S and K-P compounds. Saprotrophic fungi have been shown to excrete carboxylic acids, which in turn play a role in soil mineral weathering. Our finding of several K counter-ions to carboxylic acids may suggest that, besides their direct transfer to colonized roots, K ions can also be involved in the production of compounds necessary for sourcing nutrients from their surrounding environment by ECM fungi. Additionally, this work reveals that XANES spectroscopy can be used to identify the various forms of K accumulating in biological systems.

Ectomycorrhizal symbiosis↗
DOE OSTI2026

Nutrient limitation shapes functional traits of mycorrhizal fungi and phosphorus-cycling bacteria across an elevation gradient

In nutrient-limited high-elevation ecosystems, plants rely on arbuscular mycorrhizal (AM) fungi to provide mineral phosphorus (P) in the form of phosphate (PO43-). AM fungi gather these nutrients from phosphorus-cycling bacteria (PCBs) that can mineralize PO43- from organic matter and solubilize mineral-bound P. How climate, soil factors, and nutrient limitation influence AM fungi and PCB assembly remains unclear. We collected soil from montane meadows across a 1,000-m elevation gradient on three replicate mountainsides and analyzed AM fungal marker genes, P-cycling genes from shotgun metagenomes, and edaphic measurements. High-elevation soils had nearly 50-fold less soil PO₄³⁻ and 60% more AM fungal hyphae than low-elevation soils. AM fungal turnover was linked to changes in pH, organic carbon, and PO₄³-. The composition of 198 P-cycling genes was influenced by the AM fungal community structure. Drivers of individual PCB functional genes, including pH and organic carbon, varied with gene phylogeny. We found a trade-off in P-cycling strategies across elevation: P-rich, low-elevation soils supported root-colonizing AM fungi and organic P-mineralizing bacteria. P-poor, high-elevation soils were dominated by stress-tolerant AM fungi and mineral P-solubilizing bacteria. Our results suggest that AM fungi and PCB community turnover across elevation are both shaped by pH, organic carbon, and P availability. With continued climate warming, the structure and function of mountaintop ecosystems might shift to resemble lower elevations, disrupting long-established and specialized microbial assemblages, with consequences for P-cycling dynamics and the total P available to plant communities.IMPORTANCEPhosphorus (P) limits plant productivity in high-elevation ecosystems, yet the microbial networks that mobilize P, including arbuscular mycorrhizal (AM) fungi and phosphorus-cycling bacteria (PCBs), remain under-characterized in these nutrient-poor soils. We show that across a 10,00-m elevation gradient, AM fungi and P-cycling gene assemblages shift predictably with pH, organic carbon, and phosphate availability. Higher elevations, with less available P, select for stress-tolerant AM fungal taxa and PCB strategies geared toward mineral solubilization, while low-elevation sites favor root colonization by AM fungi and organic P mineralization. These results suggest that nutrient limitation can constrain microbial community assembly in consistent ways across landscapes. High mountain soils are low in P and rely on a network of underground AM fungi and PCB to deliver nutrients to plants. This study shows how those underground relationships reorganize with elevation and how climate change could collapse long-standing microbial strategies by pushing high-elevation ecosystems toward lowland conditions. As soils warm and dry, the microbial scaffolding that supports alpine plant life may become increasingly unstable.

arbuscular mycorrhizal fungi↗
DOE OSTI2023

Fine scale sampling reveals early differentiation of rhizosphere microbiome from bulk soil in young Brachypodium plant roots

Abstract For a deeper and comprehensive understanding of the composition and function of rhizosphere microbiomes, we need to focus at the scale of individual roots in standardized growth containers. Root exudation patterns are known to vary along distinct parts of the root even in juvenile plants giving rise to spatially distinct microbial niches. To address this, we analyzed the microbial community from two spatially distinct zones of the developing primary root (tip and base) in young Brachypodium distachyon grown in natural soil using standardized fabricated ecosystems known as EcoFABs as well as in more conventional pot and tubes. 16S rRNA based community analysis showed a strong rhizosphere effect resulting in significant enrichment of several OTUs belonging to Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria. However, microbial community composition did not differ between root tips and root base or across different growth containers. Functional analysis of bulk metagenomics revealed significant differences between root tips and bulk soil. The genes associated with different metabolic pathways and root colonization were enriched in root tips. On the other hand, genes associated with nutrient-limitation and environmental stress were prominent in the bulk soil compared to root tips, implying the absence of easily available, labile carbon and nutrients in bulk soil relative to roots. Such insights into the relationships between developing root and microbial communities are critical for judicious understanding of plant-microbe interactions in early developmental stages of plants.

Acharya, Shwetha M.↗
DOE OSTI2021

Colonization of Naive Roots from Populus tremula × alba Involves Successive Waves of Fungi and Bacteria with Different Trophic Abilities

ABSTRACT Through their roots, trees interact with a highly complex community of microorganisms belonging to various trophic guilds and contributing to tree nutrition, development, and protection against stresses. Tree roots select for specific microbial species from the bulk soil communities. The root microbiome formation is a dynamic process, but little is known on how the different microorganisms colonize the roots and how the selection occurs. To decipher whether the final composition of the root microbiome is the product of several waves of colonization by different guilds of microorganisms, we planted sterile rooted cuttings of gray poplar obtained from plantlets propagated in axenic conditions in natural poplar stand soil. We analyzed the root microbiome at different time points between 2 and 50 days of culture by combining high-throughput Illumina MiSeq sequencing of the fungal ribosomal DNA internal transcribed spacer and bacterial 16S rRNA amplicons with confocal laser scanning microscopy observations. The microbial colonization of poplar roots took place in three stages, but bacteria and fungi had different dynamics. Root bacterial communities were clearly different from those in the soil after 2 days of culture. In contrast, if fungi were also already colonizing roots after 2 days, the initial communities were very close to that in the soil and were dominated by saprotrophs. They were slowly replaced by endophytes and ectomycorhizal fungi. The replacement of the most abundant fungal and bacterial community members observed in poplar roots over time suggest potential competition effect between microorganisms and/or a selection by the host. IMPORTANCE The tree root microbiome is composed of a very diverse set of bacterial and fungal communities. These microorganisms have a profound impact on tree growth, development, and protection against different types of stress. They mainly originate from the bulk soil and colonize the root system, which provides a unique nutrient-rich environment for a diverse assemblage of microbial communities. In order to better understand how the tree root microbiome is shaped over time, we observed the composition of root-associated microbial communities of naive plantlets of poplar transferred in natural soil. The composition of the final root microbiome relies on a series of colonization stages characterized by the dominance of different fungal guilds and bacterial community members over time. Our observations suggest an early stabilization of bacterial communities, whereas fungal communities are established following a more gradual pattern.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2010

Mutualism in a Reduced Gravity Environment (MuRGE)

Mutualism in a Reduced Gravity Environment (MuRGE) is a ground research study to determine the feasibility of assessing fungi-plant (Piriformospora indica-Arabidopsis thaliana) interactions in microgravity. Seeds from the plant Arabiddospsis thaliana (At) will be grown in the presence of Piriformospora indica (Pi) an endophytic Sebacinacae family fungus. Pi is capable of colonizing the roots of a wide variety of plant species, including non-mycorrhizal hosts like At, and promoting plant growth similarly to AMF (arbusuclar mychorrizal fungi) unlike most AMF, Pi is not an obligate plant symbiont and can be grown in the absence of a host. In the presence of a suitable plant host, Pi can attach to and colonize root tips. Interaction visualization is accomplished with strong autofluorescence in the roots, followed by root colonization via fungal hyphae, and chlamydospore production. Increased root growth can be observed even before root colonization is detectable. In addition, Pi chlamydospores generated from axenic culture in microgravity will be used to inoculate roots of At grown in 1g to determine the effect of microgravity upon the inherent virulence or beneficial effects. Based on recent reports of increased virulence of S. typhimurium, P. aeruginosa, and S. Pneumoniae in reduced gravity, differences in microbial pathogenic responses and host plant systemic acquired resistance are expected. The focus of this project within MuRGE involved the development P. indica culture media evaluation and microscopy protocol development. High, clean spore harvest yields for the detection of fungi-plant interactions microscopically was the immediate goal of this experiment.

Haire, Timothy C.↗