Dayakar V. Badri

Rhizosphere chemical dialogues: plant–microbe interactions

Every organism on earth relies on associations with its neighbors to sustain life. For example, plants form associations with neighboring plants, microflora, and microfauna, while humans maintain symbiotic associations with intestinal microbial flora, which is indispensable for nutrient assimilation and development of the innate immune system. Most of these associations are facilitated by chemical cues exchanged between the host and the symbionts. In the rhizosphere, which includes plant roots and the surrounding area of soil influenced by the roots, plants exude chemicals to effectively communicate with their neighboring soil organisms. Here we review the current literature pertaining to the chemical communication that exists between plants and microorganisms and the biological processes they sustain.

Rhizosphere microbiome assemblage is affected by plant development

There is a concerted understanding of the ability of root exudates to influence the structure of rhizosphere microbial communities. However, our knowledge of the connection between plant development, root exudation and microbiome assemblage is limited. Here, we analyzed the structure of the rhizospheric bacterial community associated with Arabidopsis at four time points corresponding to distinct stages of plant development: seedling, vegetative, bolting and flowering. Overall, there were no significant differences in bacterial community structure, but we observed that the microbial community at the seedling stage was distinct from the other developmental time points. At a closer level, phylum such as Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and specific genera within those phyla followed distinct patterns associated with plant development and root exudation. These results suggested that the plant can select a subset of microbes at different stages of development, presumably for specific functions. Accordingly, metatranscriptomics analysis of the rhizosphere microbiome revealed that 81 unique transcripts were significantly (P<0.05) expressed at different stages of plant development. For instance, genes involved in streptomycin synthesis were significantly induced at bolting and flowering stages, presumably for disease suppression. We surmise that plants secrete blends of compounds and specific phytochemicals in the root exudates that are differentially produced at distinct stages of development to help orchestrate rhizosphere microbiome assemblage.

Root Exudation of Phytochemicals in Arabidopsis Follows Specific Patterns That Are Developmentally Programmed and Correlate with Soil Microbial Functions

Plant roots constantly secrete compounds into the soil to interact with neighboring organisms presumably to gain certain functional advantages at different stages of development. Accordingly, it has been hypothesized that the phytochemical composition present in the root exudates changes over the course of the lifespan of a plant. Here, root exudates of in vitro grown Arabidopsis plants were collected at different developmental stages and analyzed using GC-MS. Principle component analysis revealed that the composition of root exudates varied at each developmental stage. Cumulative secretion levels of sugars and sugar alcohols were higher in early time points and decreased through development. In contrast, the cumulative secretion levels of amino acids and phenolics increased over time. The expression in roots of genes involved in biosynthesis and transportation of compounds represented in the root exudates were consistent with patterns of root exudation. Correlation analyses were performed of the in vitro root exudation patterns with the functional capacity of the rhizosphere microbiome to metabolize these compounds at different developmental stages of Arabidopsis grown in natural soils. Pyrosequencing of rhizosphere mRNA revealed strong correlations (p<0.05) between microbial functional genes involved in the metabolism of carbohydrates, amino acids and secondary metabolites with the corresponding compounds released by the roots at particular stages of plant development. In summary, our results suggest that the root exudation process of phytochemicals follows a developmental pattern that is genetically programmed.

Application of natural blends of phytochemicals derived from the root exudates of Arabidopsis to the soil reveal that phenolic-related compounds predominantly modulate the soil microbiome.

Background: Roots can alter the rhizosphere microbial composition presumably by the secretion of root exudates. Results: Natural blends of phytochemicals present in the root exudates can modulate the soil microbiome in the absence of the plant. Conclusion: Different groups of compounds impact soil microbe composition at various levels. Significance: Identifying natural mixes of compounds that could positively influence plant-microbiome interactions can increase crop yield and sustainability. The roots of plants have the ability to influence its surrounding microbiology, the so-called rhizosphere microbiome, through the creation of specific chemical niches in the soil mediated by the release of phytochemicals. Here we report how these phytochemicals could modulate the microbial composition of a soil in the absence of the plant. For this purpose, root exudates of Arabidopsis were collected and fractionated to obtain natural blends of phytochemicals at various relative concentrations that were characterized by GC-MS and applied repeatedly to a soil. Soil bacterial changes were monitored by amplifying and pyrosequencing the 16 S ribosomal small subunit region. Our analyses reveal that one phytochemical can culture different operational taxonomic units (OTUs), mixtures of phytochemicals synergistically culture groups of OTUs, and the same phytochemical can act as a stimulator or deterrent to different groups of OTUs. Furthermore, phenolic-related compounds showed positive correlation with a higher number of unique OTUs compared with other groups of compounds (i.e. sugars, sugar alcohols, and amino acids). For instance, salicylic acid showed positive correlations with species of Corynebacterineae, Pseudonocardineae and Streptomycineae, and GABA correlated with species of Sphingomonas, Methylobacterium, Frankineae, Variovorax, Micromonosporineae, and Skermanella. These results imply that phenolic compounds act as specific substrates or signaling molecules for a large group of microbial species in the soil.

An ABC Transporter Mutation Alters Root Exudation of Phytochemicals That Provoke an Overhaul of Natural Soil Microbiota

Root exudates influence the surrounding soil microbial community, and recent evidence demonstrates the involvement of ATP-binding cassette (ABC) transporters in root secretion of phytochemicals. In this study, we examined effects of seven Arabidopsis (Arabidopsis thaliana) ABC transporter mutants on the microbial community in native soils. After two generations, only the Arabidopsis abcg30 (Atpdr2) mutant had significantly altered both the fungal and bacterial communities compared with the wild type using automated ribosomal intergenic spacer analysis. Similarly, root exudate profiles differed between the mutants; however, the largest variance from the wild type (Columbia-0) was observed in abcg30, which showed increased phenolics and decreased sugars. In support of this biochemical observation, whole-genome expression analyses of abcg30 roots revealed that some genes involved in biosynthesis and transport of secondary metabolites were up-regulated, while some sugar transporters were down-regulated compared with genome expression in wild-type roots. Microbial taxa associated with Columbia-0 and abcg30 cultured soils determined by pyrosequencing revealed that exudates from abcg30 cultivated a microbial community with a relatively greater abundance of potentially beneficial bacteria (i.e. plant-growth-promoting rhizobacteria and nitrogen fixers) and were specifically enriched in bacteria involved in heavy metal remediation. In summary, we report how a single gene mutation from a functional plant mutant influences the surrounding community of soil organisms, showing that genes are not only important for intrinsic plant physiology but also for the interactions with the surrounding community of organisms as well.

Altered Profile of Secondary Metabolites in the Root Exudates of Arabidopsis ATP-Binding Cassette Transporter Mutants

Following recent indirect evidence suggesting a role for ATP-binding cassette (ABC) transporters in root exudation of phytochemicals, we identified 25 ABC transporter genes highly expressed in the root cells most likely to be involved in secretion processes. Of these 25 genes, we also selected six full-length ABC transporters and a half-size transporter for in-depth molecular and biochemical analyses. We compared the exuded root phytochemical profiles of these seven ABC transporter mutants to those of the wild type. There were three nonpolar phytochemicals missing in various ABC transporter mutants compared to the wild type when the samples were analyzed by high-performance liquid chromatography-mass spectrometry. These data suggest that more than one ABC transporter can be involved in the secretion of a given phytochemical and that a transporter can be involved in the secretion of more than one secondary metabolite. The primary and secondary metabolites present in the root exudates of the mutants were also analyzed by gas chromatography-mass spectrometry, which allowed for the identification of groups of compounds differentially found in some of the mutants compared to the wild type. For instance, the mutant Atpdr6 secreted a lower level of organic acids and Atmrp2 secreted a higher level of amino acids as compared to the wild type. We conclude that the release of phytochemicals by roots is partially controlled by ABC transporters.

Potential impact of soil microbiomes on the leaf metabolome and on herbivore feeding behavior

It is known that environmental factors can affect the biosynthesis of leaf metabolites. Similarly, specific pairwise plant-microbe interactions modulate the plants metabolome by stimulating production of phytoalexins and other defense-related compounds. However, there is no information about how different soil microbiomes could affect the plant growth and the leaf metabolome. We analyzed experimentally how diverse soil microbiomes applied to the roots of Arabidopsis thaliana were able to modulate plant growth and the leaf metabolome, as assessed by GC-MS analyses. Further, we determined the effects of soil microbiome-driven changes in leaf metabolomics on the feeding behavior of Trichopulsia ni larvae. Soil microbiomes differentially impacted plant growth patterns as well as leaf metabolome composition. Similarly, most microbiome-treated plants showed inhibition to larvae feeding, compared with unamended control plants. Pyrosequencing analysis was conducted to determine the soil microbial composition and diversity of the soils used in this study. Correlation analyses were performed to determine relationships between various factors (soil microbial taxa, leaf chemical components, plant growth patterns and insect feeding behavior) and revealed that leaf amino acid content was positively correlated with both microbiome composition and insect feeding behavior.

Activation of the Jasmonic Acid Plant Defence Pathway Alters the Composition of Rhizosphere Bacterial Communities

Jasmonic acid (JA) signalling plays a central role in plant defences against necrotrophic pathogens and herbivorous insects, which afflict both roots and shoots. This pathway is also activated following the interaction with beneficial microbes that may lead to induced systemic resistance. Activation of the JA signalling pathway via application of methyl jasmonate (MeJA) alters the composition of carbon containing compounds released by roots, which are implicated as key determinants of rhizosphere microbial community structure. In this study, we investigated the influence of the JA defence signalling pathway activation in Arabidopsis thaliana on the structure of associated rhizosphere bacterial communities using 16S rRNA gene amplicon pyrosequencing. Application of MeJA did not directly influence bulk soil microbial communities but significant changes in rhizosphere community composition were observed upon activation of the jasmonate signalling pathway. Our results suggest that JA signalling may mediate plant-bacteria interactions in the soil upon necrotrophic pathogen and herbivorous insect attacks.

Transcriptome analysis of Arabidopsis roots treated with signaling compounds: a focus on signal transduction, metabolic regulation and secretion

Gene expression in response to signaling molecules has been well studied in the leaves of the model plant species Arabidopsis thaliana. However, knowledge of gene expression and metabolic regulation at the root level is limited. Here, the signaling compounds salicylic acid (SA), methyl jasmonate (MeJA) and nitric oxide (NO) were applied exogenously to induce various defense responses in roots, and their effect was studied using a combination of genomic, molecular and biochemical approaches. Genes involved in defense signaling/activation, cellular redox state, metabolism, transcription factors and membrane transport were altered in expression following treatment with SA, MeJA and NO. In addition, it was found that SA-, MeJA- and NO-elicited roots increased the root exudation of phytochemicals compared with the roots of nontreated control plants. Transport systems likely to be involved in the root exudation of phytochemicals, including the MATE, ABC, MFS, amino acid, sugar and inorganic solute transporters, showed altered expression profiles in response to treatments. Overall, significant differences were found in the signaling compound-elicited expression profiles of genes in roots vs those in leaves. These differences could be correlated to the underground nature of roots and their exposure to higher microbial inoculum rates under natural conditions.

Root secretion of defense-related proteins is development-dependent and correlated with flowering time.

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.