Research Article

A specialized metabolic network selectively modulates Arabidopsis root microbiota

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Science  10 May 2019:
Vol. 364, Issue 6440, eaau6389
DOI: 10.1126/science.aau6389

Mix of metabolites tunes root microbiota

Uncharacterized biosynthetic genes in plant genomes suggest that plants make a plethora of specialized metabolites. Huang et al. reconstructed three biosynthetic networks from the small mustard plant Arabidopsis thaliana. Promiscuous acyltransferases and dehydrogenases contributed to metabolite diversification. The plant may use these specialized metabolites to modulate the microbiota surrounding its roots. Disruption of the pathways and intervention with purified compounds caused changes in the root microbiota.

Science, this issue p. eaau6389

Structured Abstract

INTRODUCTION

Specialized metabolism is a feature of plant evolution and adaption. Plant-specialized metabolites have ecological functions, mediating interactions between plants and their environments. Although microbes can have diverse effects on plant growth and fitness, how plants assemble and modulate their microbiota remains unclear. Understanding the factors and mechanisms underlying this process will open up avenues for engineering plant microbiota for sustainable agriculture. Plants are estimated to use ~20% of their photosynthesized carbon to make root-derived organic molecules. However, whether (and if so, which) specialized metabolites can direct the assembly of specific root microbiota is not known.

RATIONALE

Triterpenes are plant-specialized metabolites that have functions in plant defense and signaling and also have antimicrobial activities. They are one of the largest and most structurally diverse families of plant natural products. The genome of the small mustard plant Arabidopsis thaliana harbors four root-expressed triterpene biosynthetic gene clusters that encode unknown triterpene biosynthetic pathways. Plant biosynthetic gene clustering is likely to be a result of strong selection pressure during evolution with associated production of small molecules of biological and ecological importance. Several of these clustered Arabidopsis genes have been implicated in defense against root pathogens, further suggesting that metabolites derived from these triterpene biosynthetic gene clusters may modulate the Arabidopsis root microbiota.

RESULTS

We have elucidated a specialized metabolic network expressed in the roots of A. thaliana that consists of functionally divergent triterpene biosynthetic gene clusters connected by scattered genes outside the clusters that encode promiscuous acyltransferases and alcohol dehydrogenases. This metabolic network has a latent capacity for synthesizing more than 50 previously unknown root metabolites. This is a relatively large number considering the total number of nonvolatile root metabolites that we detected (approximately 300). We characterized three divergent pathways for the biosynthesis of root triterpene metabolites: thalianin, thalianyl fatty acid esters, and arabidin. Analysis of the root microbiota of A. thaliana mutants disrupted in the biosynthesis of these compounds revealed shifts in the composition and diversity of their root microbiota compared with those of the wild type. Comparison with the root bacterial profiles of the taxonomically remote species rice and wheat supports a role for this specialized triterpene biosynthetic network in mediating the establishment of an Arabidopsis-specific microbiota. We next tested the activity of purified or synthesized Arabidopsis root triterpenes and representative triterpene cocktails in vitro toward 19 taxonomically diverse bacterial strains isolated from the A. thaliana root microbiota. We found that these compounds could indeed selectively modulate the growth of these bacteria, examples of both positive and negative modulation being evident. The modulation effects of the various triterpenes on the growth of different bacterial strains correlated with the relative differential abundance of the differential bacterial genera in the roots of A. thaliana Col-0 and triterpene mutant lines. Moreover, some root bacteria were found to be able to selectively metabolize certain triterpenes (such as thalianyl fatty acid esters) and use the breakdown products such as palmitic acid as carbon sources for proliferation.

CONCLUSION

We demonstrate that A. thaliana produces a range of specialized triterpenes that direct the assembly and maintenance of an A. thaliana–specific microbiota, enabling it to shape and tailor the microbial community within and around its roots to its own purposes. We speculate that metabolic diversification within the plant kingdom may provide a basis for communication and recognition that enables the sculpting of microbiota tailored to the needs of the host and that this may in part explain the existence of plant-specialized metabolism. Our study opens up opportunities for engineering root microbiota and further paves the way for investigating the functions of root microbiota in plant growth and health.

Dynamic modulation of the Arabidopsis root microbiota by specialized triterpene metabolites derived from biosynthetic gene clusters.

The specialized triterpenes thalianin, thalianyl fatty acid esters, and arabidin selectively modulate A. thaliana root microbiota members by promoting (indicated with the orange and purple bacteria) or inhibiting (indicated with the blue bacteria) the growth of different bacterial taxa and, in some cases, by serving as carbon sources (purple bacteria). These triterpenes are products of pathways encoded by biosynthetic gene clusters and nonclustered genes. Colored arrows indicate genes encoding different types of enzymes: black, triterpene synthase; red, cytochrome P450s; purple, acyltransferases; and blue, alcohol dehydrogenases. The dynamic modulation of root bacteria mediated by these specialized triterpenes contributes to the assembly of an A. thaliana–specific root microbiota.

Abstract

Plant specialized metabolites have ecological functions, yet the presence of numerous uncharacterized biosynthetic genes in plant genomes suggests that many molecules remain unknown. We discovered a triterpene biosynthetic network in the roots of the small mustard plant Arabidopsis thaliana. Collectively, we have elucidated and reconstituted three divergent pathways for the biosynthesis of root triterpenes, namely thalianin (seven steps), thalianyl medium-chain fatty acid esters (three steps), and arabidin (five steps). A. thaliana mutants disrupted in the biosynthesis of these compounds have altered root microbiota. In vitro bioassays with purified compounds reveal selective growth modulation activities of pathway metabolites toward root microbiota members and their biochemical transformation and utilization by bacteria, supporting a role for this biosynthetic network in shaping an Arabidopsis-specific root microbial community.

Plants have evolved the ability to produce a vast array of specialized metabolites. This metabolic diversification is likely to have been driven by the need to adapt to different environmental niches (1). One of the key environmental factors that influences plant health and fitness is the root microbiota (24), yet mechanisms underpinning the establishment of specific root microbiota and how plants direct the microbial communities around their roots remain elusive. Plants are estimated to use ~20% of their photosynthesized carbon to make root-derived organic molecules, stimulating the formation of distinctive root microbiota from surrounding soil (58). However, whether and which specialized metabolites produced by plants can modulate root microbiota is not known. The presence of a substantial number of uncharacterized root-expressed biosynthetic genes in plant genomes also reveals our incomplete understanding of these communication processes.

Triterpenes are specialized plant metabolites that have important functions in plant defense and signaling. They are one of the largest and most structurally diverse families of plant natural products and many have been shown to possess antifungal and antibacterial activities, suggesting potential roles in mediating interactions between the producing plants and microbes (911). Triterpenes are synthesized by means of the mevalonate pathway, the first committed step being carried out by enzymes known as triterpene synthases (TTSs), which collectively are able to make a diverse array of different triterpene scaffolds (12). There are 13 predicted TTS genes in the genome of A. thaliana accession Col-0, five of which (MRN, THAS, ABDS, PEN3, and BARS) are located in a total of four plant biosynthetic gene clusters on chromosomes 4 and 5 (Fig. 1A).

Fig. 1 Identification of a root-expressed triterpene biosynthetic network in A. thaliana.

(A) The four A. thaliana triterpene biosynthetic gene clusters and peripheral genes. Genes encoding previously characterized enzymes are shown in solid color, and uncharacterized ones are dotted. Black, TTSs; red, CYP705 family; orange, CYP708 family; brown, CYP71 family; yellow, CYP716 family; dark yellow, CYP702 family; purple, acyltransferases (ACTs); blue, alcohol dehydrogenase (ALDHs); gray, nonbiosynthetic genes. The scattered/peripheral genes identified as part of this network are also shown. Coexpressed genes have light blue borders. PEN3 was previously shown to be coexpressed with other cluster genes in roots (18). (B) Expression profiles of the triterpene cluster genes and additional scattered (peripheral) biosynthetic genes identified as part of this study in different A. thaliana tissues. The relative expression indicates the log2 ratio of a tissue’s expression level to control signal—typically, the median of different tissues (45). The heatmap was generated by using microarray expression data from the eFP browser (45). For clarity, expression profiles for the same tissue across different developmental stages are labeled with the tissue names only. A more detailed heatmap can be found in fig. S2.

Plant biosynthetic gene clusters represent evolutionary genomic hot spots under strong selection pressure (13), with the potential to encode metabolites of ecological importance (14). Moreover, most genes within these four Arabidopsis biosynthetic gene clusters are root-expressed (Fig. 1B), and the arabidiol gene cluster has been implicated in defense against the root-rot pathogen Pythium irregulare, suggesting that the derived metabolites might play a role in root-microbe interactions (1518). The TTSs of the A. thaliana biosynthetic gene clusters appear to share a common ancestor but have functionally diverged and form a monophyletic clade that is distinct from other A. thaliana TTSs (fig. S1) (15). These clustered TTSs channel primary metabolism into specialized metabolism by converting the ubiquitous precursor 2,3-oxidosqualene into different triterpene scaffolds, specifically marneral/marnerol M1/M2 (19), thalianol T1 (20), arabidiol A1 (21), tirucalladienol Tr1 (22), and baruol B1 (23) through the corresponding bi/tri/tetra-cyclic carbocations, respectively (fig. S1). Four of the 13 cytochrome P450 (CYP) genes in these cluster regions [At5g48000 (THAH), At4g15330 (CYP705A1), At5g62590 (MRO), and At5g36110 (CYP716A1) belonging to the CYP708, CYP705, CYP71, and CYP716 families, respectively] have previously been shown to convert T1, A1, M1/M2, and Tr1 primarily to 3β,7β-thaliandiol T2, 14-apo-arabidiol A2, 23-hydroxy-marner-al/ol M3/M4, and unknown products, respectively (Fig. 2) (18, 24). However, numerous other uncharacterized genes with predicted functions in specialized metabolism are present in these biosynthetic gene clusters and are coexpressed with the characterized TTS and CYP genes in A. thaliana roots (Fig. 1B and fig. S2) (15, 18). Here, we elucidate three biosynthetic pathways derived from the thalianol and arabidiol gene clusters and show that these major root-specialized metabolites play important roles in selectively modulating Arabidopsis root bacteria both in vivo and in vitro and contribute substantially to the establishment of an Arabidopsis-specific root microbial community.

Fig. 2 The triterpene biosynthetic network in A. thaliana roots.

Transformations catalyzed by TTSs are shaded gray, clustered CYPs are shaded red, and other tailoring enzymes are shaded blue. Chemical diversification of pathway scaffolds and intermediates by promiscuous acyltransferases and alcohol dehydrogenases is highlighted in the black box, bottom right. DMNT, (E)-4,8-dimethyl-1,3,7-nonatriene. Black arrows, known transformations; orange and burgundy red arrows, newly characterized steps in the thalianin and arabidin pathways, respectively; cyan arrows, pathway diversification by promiscuous enzymes; dotted black arrow, unknown transformation. The enzymes for each step are color-coded to match the classes of enzyme shown in Fig. 1A, with the exception that all CYPs are in red.

Elucidation of the thalianol gene cluster–derived pathways

The thalianol biosynthetic gene cluster in A. thaliana contains four coexpressed genes, of which the functions of two (the TTS THAS and the CYP THAH) have been characterized (15, 24). A second CYP (THAO, At5g47990) that belongs to the CYP705 family mediates a third step in the pathway, but the precise structure of the resulting product has not been determined (15). A fourth gene (THAA1, At5g47980) that is coexpressed with the other pathway genes is predicted to encode an acyltransferase belonging to the BAHD family, but its function is unknown (15, 25). We used Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves to investigate the functions of the cluster genes THAO and THAA1 (2628). We first coexpressed THAS with the three other cluster genes encoding the hydroxylase (THAH), CYP705A5 (THAO), and acyltransferase (THAA1) in the leaves of N. benthamiana in a combinatorial fashion (table S1). We found that THAO alone could modify thalianol to give two new products—3β,16-thaliandiol T3 and 16-keto-thalianol T4, respectively—when coexpressed with THAS (Fig. 2 and fig. S3). Coexpression of THAO and THAH with THAS gave 16-keto-3β,7β-thaliandiol T5 and 16-keto-3β,7β,15-thaliantriol T6 as the main products, instead of the desaturated thaliandiol proposed previously (Fig. 2 and fig. S3) (15). Furthermore, we found that THAA1 encodes an acyltransferase that was functional only when coexpressed with all three other cluster genes—THAS, THAH, and THAO—yielding a new product identified as 3β,7β-dihydroxy-16-keto-thalian-15-yl acetate T7, along with its C17=C18 cis isomer cis-T7 as the minor product (Fig. 2 and fig. S3). This suggests that THAA1 functions after THAS, THAH, and THAO to add an acetate group on the C15 position of the thalianol scaffold after a hydroxy moiety is installed at this position. We also identified another BAHD acyltransferase gene (THAA2, At5g47950) in close proximity to THAA1 on chromosome five that was coexpressed with the thalianol cluster genes (Fig. 1). In contrast to THAA1, THAA2 is promiscuous and can act on the C3 hydroxy moiety of different thalianol-derived compounds (T1 to T7) to introduce an acetyl group (T11 to T17) when coexpressed with subsets of the thalianol cluster genes (Fig. 2, fig. S4, and table S1). Our results indicate that the four thalianol cluster genes and the nearby coexpressed gene THAA2 are functional and yield 7β-hydroxy-16-keto-thalian-3β-15-yl diacetate T17 when coexpressed in N. benthamiana.

Identification of thalianol-derived root metabolites in Arabidopsis

We next performed targeted metabolomic analysis to identify thalianol-derived metabolites in A. thaliana. Metabolic profiling identified seven major thalianol-derived products (T1, T2, T9, T10, and T18a to T18c) in root extracts from the wild-type Col-0 accession and the THAS overexpression line (thas-oe) that are absent in those from the THAS mutants (thas-ko1 and thas-ko2) (Fig. 3, A to C, and table S2). Mutation of THAH (thah-ko) led to accumulation of 3β,15-thaliandiol T3 and 16-keto-thalianol T4, whereas mutation of THAO (thao-ko) resulted in elevated levels of 3β,7β-thaliandiol T2 and absence of T3 and T4 (Fig. 3, A and B, and fig. S5), indicating that THAO functions as a C16 oxidase of thalianol in A. thaliana. We were unable to detect T5 to T7 or T17 in either Col-0 wild-type or thas-oe root extracts, but we did detect two compounds T9 and T10, which are potential isomers of T7 and T17 (fig. S5). Compound T10 was absent in root extracts from mutants of all thalianol cluster genes and THAA2 (thaa2-ko and thaa2-crispr), whereas T9 was still detected in those of thaa2-ko and thaa2-crispr, suggesting that T9 and T10 are downstream products of the thalianol gene cluster and that THAA2 may act after T9 to form T10 (Fig. 3, A to C, and fig. S5). Targeted metabolomics analysis also revealed the presence of thalianol-derived medium-chain saturated triterpene fatty acid esters (TFAEs) in the root extracts of Col-0 and thas-oe, including thalianyl palmitate (16:0, T18a), myristate (14:0, T18b), and laurate (12:0, T18c) (Fig. 3, A and B). Compounds T18a and T18b were also detected in the roots of other mutant lines thah-ko, thao-ko, thaa1-crispr, thaa2-ko, and thaa2-crispr except thas-ko1 and thas-ko2, suggesting the presence of a branched thalianol-derived biosynthetic pathway. The identities of these TFAEs were confirmed with chemical synthesis.

Fig. 3 Metabolite analysis of roots of different Arabidopsis lines.

(A) Comparative GC-MS total ion chromatograms (TICs) of root extracts of the A. thaliana wild-type Col-0 (black), thas-oe (red), thas-ko1 (dark blue), thas-ko2 (blue), thah-ko (brown), thao-ko (green), thaa1-crispr (dark yellow), thaa2-ko (purple), thaa2-crispr (pink), and an authentic standard mixture containing T1, 3-keto-T1, T2, 3-keto-T2, T4, T9, T10, T18a to T18c, and A5 (orange). (B) Comparative extracted ion chromatograms (EICs) from Fig. 2A for characteristic fragments of the mass spectra of A5 (189), T1 to T4 (229), T9 (362), T10 (344), and T18a to T18c (229). (C) Triterpene pathway metabolites detected in whole-root extracts from wild-type and mutant lines. Col-0, wild type. thas-oe, thas-ko1, thas-ko2, thah-ko, thao-ko, thaa2-ko, and thaa2-crispr are mutants for THAS, THAH, THAO, and THAA2 (supplementary materials, materials and methods). (D) Triterpenoids detected in chloroform (CHCl3) extracts from the surfaces of fresh roots of A. thaliana wild-type Col-0 seedlings and subsequent ethyl acetate extracts of dry and powdered chloroform-extracted roots. All lines used in qualitative and quantitative GC-MS analysis (Fig. 2, A to D) were grown on ¼ Murashige-Skoog (MS) agar plates at 22°C (8 hours dark/16 hours light cycle) for 10 days. (E) Determination of compounds T9 and T10 by LC-MS in root exudates of Col-0 seedlings grown hydroponically in liquid ¼ MS after 10 days. Error bars represent standard deviation of three biological replicates.

Identification of missing genes for the biosynthesis of thalianin, TFAEs, and arabidin

To identify the missing genes required for the biosynthesis of metabolites T9 and T10, we carried out a genome-wide search for coexpressed candidate biosynthetic genes in the ATTED-II plant coexpression database (http://atted.jp) using the four thalianol cluster genes as baits (29) because no biosynthetic genes in or near the gene cluster region appeared to be coexpressed. We selected eight candidates from the top 20 coexpressed genes for functional analysis in N. benthamiana (table S2). We identified two genes, At3g29250 (THAR1) and At1g66800 (THAR2), that encode a pair of promiscuous oxido-reductases capable of epimerizing the C3 hydroxy moiety of T1 to T7 (Figs. 1, A and B, and 2 and fig. S6). In this epimerization sequence, THAR1 converted the C3β hydroxy of T1 to T7 into the C3 ketones [3-keto-(T1-T6) and T8], whereas THAR2 reduced the C3-ketones into 3α alcohols [3-epi-(T1-T6) and T9] sequentially (Fig. 2, fig. S6, and table S9). Coexpression of THAR1 and THAR2 with the thalianol cluster genes and THAA2 completed the biosynthesis of T10 in N. benthamiana (fig. S7). The structure of T10 was established as 7β-hydroxy-16-keto-thalian-3α-15-yl diacetate by means of nuclear magnetic resonance, and T10 is named thalianin hereafter (table S10).

To identify genes responsible for the biosynthesis of TFAEs T18a to T18c, we screened seven O-acyltransferase genes (table S4) from A. thaliana identified based on annotation (30) and found that only one of these [THAA3, At3g51970; previously reported to function in sterol fatty acid ester biosynthesis (31)] could catalyze the formation of thalianyl palmitate T18a when coexpressed with THAS in N. benthamiana (fig. S8, A and B). We also generated a THAA3 overexpression line (thaa3-oe) and found that THAA3 could catalyze the formation of T18a in planta because elevated levels of T18a, but not T18b or T18c, were detected in this line. THAA3 is presumably partially redundant because T18a was still detected in the THAA3 mutant (thaa3-ko) (fig. S8, C and D). We further showed that THAA3 could catalyze the formation of T18b when coexpressed with THAS and a characterized chain-length specific 14:0-acyl carrier protein (ACP) thioesterase gene from Cuphea palustris, CpFatB2 in N. benthamiana (fig. S8A) (32), suggesting that there are as yet unidentified genes encoding chain-length–specific ACP thioesterases involved in TFAE (T18a to T18c) biosynthesis. Besides thalianol, THAA3 could also act on other triterpenes—including arabidiol A1 and its derivative A2, the PEN3 product Tr1, and marnerol M2—to introduce a palmityl or myristyl group depending on the type of fatty acyl CoA available (fig. S9). However, these products were not detected in A. thaliana Col-0 or thaa3-oe roots, possibly because of their very low abundance.

The promiscuity of enzymes encoded by the nonclustered [referred to as peripheral (14)] genes—including THAR1, THAR2, and THAA2 and their strong coexpression with other divergent triterpene gene clusters in roots (Figs. 1B and 2)—prompted us to perform combinatorial biosynthesis experiments in N. benthamiana using these genes and the arabidiol, marneral, and tirucalladienol cluster TTS and CYP genes. Our results showed that THAR1, THAR2, and THAA2 could also act on arabidiol A1, 14-apo-arabidiol A2, and tirucalladienol Tr1 (figs. S10 and S11), but not marnerol M2, to give the corresponding C3 ketones, 3α-alcohols, and acetates, respectively. Arabidiol A1 was fully converted to A5 when THAR1, THAR2, and THAA2 were coexpressed together with ABDS and CYP705A1 (Fig. 2 and fig. S12). A5 is an A. thaliana root metabolite identified through comparative metabolomics analysis of the A. thaliana wild type (Col-0) with ABDS and CYP705A1 mutants (33). Our results show that THAR1, THAR2, and THAA2 encode enzymes that can convert A2 to A3, A3 to A4, and A4 to A5, respectively, reconstituting the complete biosynthesis of A5 in N. benthamiana (Fig. 2, fig. S12, and table S17).

Metabolite analyses of root extracts of A. thaliana T-DNA insertion mutants of THAR1 (thar1-ko), THAR2 (thar2-ko), and THAA2 (thaa2-ko) confirm that these three genes are indeed required for the synthesis of thalianin T10 in A. thaliana. T10 was absent in root extracts from all three mutant genotypes, and the pathway intermediates T7 and T17, T8, and T9, respectively, accumulated instead (fig. S13). Additionally, THAR2 and THAA2 are also responsible for the biosynthesis of 14-apo-arabi-3α-yl acetate A5 (referred hereafter as arabidin) because mutants thar2-ko and thaa2-ko lacked A5 and instead accumulated A3 and A4, respectively (fig. S14). A5 was still detected in the root extracts from the thar1-ko line, indicating that THAR1 is partially redundant in 14-apo-arabi-3α-yl acetate A5 biosynthesis. Thus, these enzymes are involved in multiple pathways.

Triterpene biosynthesis affects Arabidopsis root microbiota assembly

Having discovered and reconstituted the complete biosynthetic pathways of thalianin T10, arabidin A5, and TFAEs T18a and T18b in N. benthamiana, we sought to investigate the potential biological role of this metabolic network. The thalianin T10 and arabidin A5 pathway genes are coexpressed in A. thaliana root epidermis and pericycle/stele, respectively (fig. S15), and thalianol pathway compounds T1, T2, T9, T10, and arabidin A5 could be detected in root surface wax extracts and T9 and T10 also in exudates of hydroponically grown seedlings (Fig. 3, D and E, and fig. S16). Moreover, both the thalianin and arabidin pathway genes are up-regulated upon methyl jasmonate treatment (fig. S17), suggesting that they may have a role in interactions with root microbes (34).

We selected mutants disrupted in the thalianin (T10), TFAE (T18a to T18c), and arabidin (A5) pathways (thas-ko1, thas-ko2, thah-ko, thao-ko, thaa2-ko, and thaa2-crispr) along with the wild type (Col-0) for root microbiota analyses (table S2). Comprehensive unbiased untargeted metabolomics analysis of whole-root extracts, root-surface extracts, and root exudates from these mutant lines versus Col-0 suggests that the thalianin pathway compounds are the most affected of all root metabolites analyzed, showing the largest fold changes across the respective metabolomes of the mutants (figs. S18 to S22). We grew these lines in natural soil from Changping Farm in Beijing for 6 weeks under controlled experimental conditions. The roots were harvested and washed with phosphate-buffered saline buffer to remove soil particles and loosely attached microbes before carrying out deep 16S ribosomal RNA (rRNA) gene sequencing of the root microbiota by using previously established methodology (3, 4, 35). There were no obvious differences in root phenotypes between the wild-type (Col-0) and mutant lines when the plants were harvested. However, we found that mutants affected in these pathways assembled different root microbiota when compared with the wild type (Col-0). Constrained principal coordinate analysis (CPCoA) revealed differences in root microbiota between the wild-type (Col-0) and mutant lines (16.5% of total variance was explained by the plant genotypes, P < 0.001, permutational multivariate analysis of variance) (Fig. 4A and tables S18 to S22). Pairs of independent mutant lines for THAS (thas-ko1 and thas-ko2) and THAA2 (thaa2-ko and thaa2-cripsr) each display similar metabolite defects (figs. S18 to S22) and have similar microbiota profiles and microbial diversity (Fig. 4A, fig. S23, and tables S23 to S26). Furthermore, all the pathway mutants tested showed similar root microbiota modulation patterns for Bacteroidetes (enrichment) and Deltaproteobacteria (depletion) compared with the A. thaliana Col-0 wild type at the phylum and operational taxonomic unit (OTU) levels (Fig. 4B, figs. S24 and S25, and tables S27 to S44), which is consistent with these genes operating in the same pathway for thalianin biosynthesis. Of the OTUs that were coenriched/codepleted in the mutants, 93% (28 out of 30) showed higher abundance in Col-0 roots than bulk soil, suggesting active selection of root bacteria by the A. thaliana plants (fig. S26 and tables S45 and S46). These data indicate that the triterpene biosynthetic network that we have unveiled contributes to root microbiota assembly and establishment.

Fig. 4 Modulation of specific root bacterial taxa in triterpene pathway mutants.

(A) Constrained principal coordinate analysis (CPCoA) of Bray-Curtis dissimilarity showing triterpene mutant effects. Total number of individual plants used for analyses: Col-0 (n = 12), thas-ko1 (n = 12), thas-ko2 (n = 9), thah-ko (n = 14), thao-ko (n = 13), thaa2-ko (n = 9), and thaa2-crispr (n = 12). Biological replicates (individual plants) from two independent experiments (experiment 1 and 2) are indicated with dots and triangles, respectively. Ellipses include 68% of samples from each genotype. (B) Phylum distribution of the root microbiota compositions of the tested A. thaliana genotypes. Because the relative abundance of Proteobacteria is greater than 50%, bacteria in this phylum are shown at the class level. Pound sign (#) indicates Bacteroidetes significantly higher than that in Col-0 roots at P < 0.05; asterisk indicates Deltaproteobacteria significantly lower than that in Col-0 at P < 0.05. (C and D) Venn diagrams showing substantial overlap of OTUs (C) depleted or (D) enriched in the root microbiota of A. thaliana triterpene mutant lines as compared with the wild type (Col-0) (pink circles), compared with those depleted in the root microbiota of rice (blue circles) and wheat (orange circles) versus the A. thaliana Col-0 wild type. The OTU numbers specifically enriched in the root microbiota of A. thaliana Col-0 compared with rice and wheat are highlighted in blue and bold in the Venn diagram overlaps.

To understand whether and how this specialized metabolic network might modulate A. thaliana–specific root bacteria, we also compared the root bacterial profiles of the A. thaliana Col-0 and mutant lines with those from the taxonomically distant species rice (36) and wheat, also previously grown in the soil from Changping Farm on different occasions. Although the A. thaliana, rice, and wheat samples differ from each other in many aspects—including growth conditions, germination periods, and climate conditions (supplementary materials, materials and methods)—the starting inocula (soils) are very similar, sharing substantial overlap in total OTUs (67%, 2377 out of 3531) (fig. S27 and tables S47 to S49). We found that of the 494 OTUs specifically enriched in the root microbiota of A. thaliana Col-0 compared with rice and wheat (represented in the overlap between the blue and orange circles in Fig. 4, C and D), 34% (170 out of 494) (Fig. 4C and tables S50 to S55) were depleted, and 18% (88 out of 494) (Fig. 4D and tables S50 to S55) enriched in the root microbiota of the triterpene mutant lines compared with those of A. thaliana Col-0. Unlike A. thaliana, rice and wheat do not make thalianin, TFAEs, or arabidin (fig. S28). Our results suggest that the specialized triterpene biosynthetic network that we have uncovered may contribute to enrichment of around a third of the A. thaliana–specific root bacteria present in the roots of the wild-type line (Fig. 4C) while deterring another 18% of this bacterial population (Fig. 4D). The relatively larger number of OTUs depleted in the triterpene mutants (versus Col-0) (a total of 380) (Fig. 4C, pink circle) in comparison with the total number of OTUs that are enriched in the mutant lines versus Col-0 (298) (Fig. 4D, pink circle) further suggests that this triterpene biosynthetic network may play a more important role in enriching Arabidopsis–specific root bacteria rather than repelling other bacteria.

Purified triterpenes selectively modulate root bacteria

To test whether triterpene pathway metabolites directly regulate root microbiota members, we isolated and identified bacteria from the roots of A. thaliana Col-0 plants grown in soil from the aforementioned Changping Farm by limiting dilution and barcoded sequencing (35). We selected a total of 19 bacterial strains that belong to 17 genera within three major bacterial phyla (Proteobacteria, Actinobacteria, and Firmicutes). These strains shared >97% 16S rRNA gene similarity to the OTUs that showed differential abundances in the microbiota of A. thaliana wild type (Col-0), mutants, and soil (fig. S29 and table S56). These bacterial strains were grown in liquid culture with a formulated cocktail of purified compounds that reflected the content and composition of the pathway metabolites in the roots of A. thaliana Col-0 (figs. S30 to S34). We found that most of the Proteobacteria strains tested proliferated faster in the presence of the A. thaliana triterpene mixture, whereas all five Actinobacteria strains were inhibited (Fig. 5A and figs. S30 to S34). The OTUs corresponding to these bacterial isolates (16 out of 19) showed consistent enrichment or depletion patterns in plant roots versus soil that corresponded with the growth promotion and inhibitory effects of the triterpene mixture, suggesting that the compounds tested contribute to the active selection of plant root bacteria (figs. S30 to S34). Moreover, the corresponding enrichment or depletion patterns for 10 bacterial genera (59% of the 17 tested) to which the metabolite-sensitive bacterial isolates belong were detected in the microbiota of at least one pathway mutant included in our microbiota analysis; the genera of bacteria for which growth is either promoted or inhibited by the triterpene cocktail mixture are depleted or enriched, respectively, in the pathway mutants compared with the wild type (Col-0) (fig. S35 and tables S57 to S63). Further tests of the sensitive strains with purified individual compounds revealed that pathway metabolites can selectively modulate the growth of bacteria and that small structural differences between compounds can affect activities (fig. S36). For example, we found that Actinobacteria Arthrobacter sp. strain A224 was inhibited by compounds T2, T9, T10, and A5 at 20 μM but not by the other triterpenes tested, whereas all compounds tested showed growth promoting effects on Gammaproteobacteira Arenimonas sp. strain A388 (Fig. 5, B and C). We also found that strain A475-1 (Agromyces sp.) has alcohol dehydrogenase activity and could selectively convert T2 into 3-keto-T2 but not T9/T10 (Fig. 6, A and B, and fig. S37A), whereas strain A215 (Pseudomonas sp.) has lipase activity and is able to cleave the TFAEs T18a to T18c to give T1 and the corresponding fatty acids but not the acetate T11 (Fig. 6, C and D, and fig. S37, B to D). Moreover, strain A215 could use the cleavage product palmitic acid as a carbon source for proliferation (Fig. 6E). Such diverse and substantial interaction patterns of root metabolites with taxonomically distinct root microbiota members suggest that the metabolites from this biosynthetic network alter the assembly of A. thaliana root microbiota. Taking the results of the root microbiota sequencing and in vitro bioassays together, we conclude that this triterpene biosynthetic network tunes the ecological niche for the assembly and maintenance of A. thaliana root microbiota.

Fig. 5 Effects of pathway metabolites on the growth of isolated A. thaliana root–associated bacteria and bacterium-mediated chemical transformations.

(A) Growth modulation activity of compound mixture Mix (10 μM T1, 5 μM T2, 20 μM T9, 20 μM T10, 10 μM T18a, 5 μM T18b, 1 μM T18c, and 10 μM A5) against the 19 strains of A. thaliana root bacteria from different taxa. The heatmap shows log2 fold change of individual strains treated with Mix versus control (ethanol only) over 48 or 72 hours. The corresponding graphical growth curves are depicted in figs. S30 to S34. (B) Heatmap showing the log2 fold change in cell density (OD600) of Arenimonas sp. strain A388 treated with eight different purified compounds (T1, T2, T9, T10, T18a, T18b, T18c, and A5, respectively) at 20 μM and Mix over 72 hours. All compounds were dissolved in ethanol. Control, 0 mM, ethanol. (C) Heatmap showing the log2 fold change in cell density (OD600) of Arthrobacter sp. strain A224 treated with eight different purified compounds (T1, T2, T9, T10, T18a, T18b, T18c, and A5, respectively) at 20 μM and Mix over 48 hours. All compounds were dissolved in ethanol. Control, 0 mM, ethanol.

Fig. 6 Bacterium-mediated transformations of triterpenes.

(A) Conversion of thaliandiol T2 to 3-keto-T2 by Agromyces sp. strain A475-1 as shown by means of (B) comparative GC-MS TICs of EtOAc extracts of bacterial cultures supplemented with T2 on the 2nd, 7th and, 10th day, respectively, with the standard mixture described in Fig. 3A. (C) Selective conversion of TFAEs T18a to T18c but not T11 to T1 by Pseudomonas sp. strain A215 as shown by means of (D) comparative GC-MS EICs [mass/charge ratio (m/z) = 229] of EtOAc extracts of bacterial strain A215 cultures supplemented with T1, T11, and T18a to T18c on the 2nd and 10th day, respectively. (E) Pseudomonas sp. strain A215 could use palmitic acid as carbon source, as shown by the proliferation of A215 in minimal salt media supplemented with 0.5 mM (in ethanol) of T18a or palmitic acid (PA) but not in those with T1, T18b, T18c, T11, myristic acid (MA), lauric acid (LA), and control. The minimal salt media contain 0.3 mg/ml resazurin as indicator of bacterial growth (from purple blue to pink).

Conclusion

The triterpene biosynthetic network described here has a latent capacity for the synthesis of more than 50 root metabolites (Fig. 2). This is a relatively large number considering the total of ~300 nonvolatile root metabolites that we detected in the polar methanolic (~220) and nonpolar ethyl acetate extracts (~85) of A. thaliana Col-0 roots under our experimental conditions (tables S64 and 65). This network originates from evolutionary divergent biosynthetic gene clusters coupled with cross-talk that involves enzymes encoded by peripheral genes that service multiple biosynthetic pathways. These root metabolites can selectively regulate the growth of Arabidopsis root bacteria from different taxa by acting as antibiotics or proliferating agents. Biosynthesis of these specialized root triterpenes dynamically modulate a large portion [52%, 258 (170+88) out of 498 OTUs] (Fig. 4, C and D) of the A. thaliana–specific bacteria, shaping the A. thaliana–specific root microbiota. Triterpenes, a large and diverse groups of plant natural products (>20,000 reported so far) (12), likely also sculpt the microbiota of other plant species, tailoring them to generate plant species–specific microbial communities, and may be useful for engineering root microbiota for sustainable agriculture (3739). It is tempting to speculate that metabolic diversification in the plant kingdom may provide a basis for communication and recognition that enables the shaping of microbial communities tailored to the needs of the host, and that this may in part explain the existence of plant-specialized metabolism.

Materials and methods

Detailed materials and methods can be found in the supplementary materials.

Plant materials

Arabidopsis thaliana accession Columbia (Col-0) was used as wild type in this study. All A. thaliana T-DNA insertion mutants were obtained from Nottingham Arabidopsis Stock Centre (NASC) unless otherwise stated. Homozygous mutants were identified by PCR-based genotyping using the primers listed in the table S5A. A 35S promoter-driven overexpression line of thalianol synthase (thas-oe) was generated previously (15). Overexpression lines for THAA3 were generated by transforming A. thaliana Col-0 plants with Agrobacterium tumefaciens strain LBA4404 harboring the expression vector pMDC32 which contains the coding sequence of THAA3 (At3g51970) under the control of the 35S promoter via floral dipping (40). Homozygous CRISPR knockout lines of THAA1 (At5g47980) and THAA2 (At5g47950) were generated by transforming A. thaliana Col-0 wild type with A. tumefaciens C58C1 harboring CRISPR/Cas9 constructs with specific single guide RNAs as previously described (41).

Cloning and transient expression

The coding sequences for THAS (At5g48010), THAH (At5g48000), THAO (At5g47990), THAA1 (At5g47980) and THAA2 (At5g47950), THAR1 (At3g29250), THAR2 (At1g66800), THAA3 (At3g51970), ABDS (At4g15340), CYP705A1 (At4g15330), At1g14960, At5g23840, At2g16005, At5g38030, At1g50560, At5g38020, MRO (At5g42580), CYP705A12 (At5g42590), MRN (At5g42600), At5g12420, At5g55380, At3g49210, At1g54570, At3g49190, At3g26840 were amplified from a root cDNA library of the A. thaliana Col-0 accession (26). The coding sequences for PEN3 (At5g36150), CYP716A1 (At5g36110) and the truncated HMG CoA reductase (tHMGR) gene from oat had been cloned previously and were included in this study (18, 27). The coding sequence of CpFatB2 from Cuphea palustris (accession no. U38189) was retrieved from NCBI and synthesized by Integrated DNA Technologies. These sequences were cloned into the pEAQ-HT expression vector for transient expression in N. benthamiana leaves as detailed in the supplementary materials.

Metabolite extraction and analysis

N. benthamiana leaves from transient expression experiments were harvested 5 days post infiltration, lyophilized, extracted with EtOAc and analyzed by means of gas chromatography–mass spectrometry (GC-MS). Seedlings of A. thaliana Col-0 and mutants, rice and wheat were grown on Murashige-Skoog (¼ MS) agar plates or liquid media at 22°C under short day conditions (8 hours light/16 hours dark) for 10 days. Roots were harvested from 10-day-old seedlings grown on agar plates, lyophilized, extracted with EtOAc and analyzed by GC-MS or extracted with MeOH and analyzed by means of liquid chromatography–MS (LC-MS). Spent media and whole seedlings of 10-day-old A. thaliana grown in ¼ MS liquid media were harvested separately, lyophilized, extracted with MeOH and analyzed by LC-MS. Targeted and untargeted metabolomics analysis of GC-MS and LC-MS data were performed using Agilent MassHunter and XC-MS, respectively.

Production and isolation of thalianol and arabidiol derived metabolites

Compounds T1-T10, 3-keto-T2, T17, cis-T10, cis-T17, A1, A2, and A4 were obtained by large-scale vacuum infiltration of N. benthamiana leaves with A. tumefaciens LBA4404 strains harboring the corresponding expression constructs followed by extraction and purification. Compounds 3-keto-T1, T11, T18a-c, A3, and A5 were chemically synthesized from the corresponding precursors.

Microbiota analysis

A. thaliana plants (Col-0 and triterpene mutants) were grown under controlled short-day conditions in the natural soil from Changping Farm (40°5′49’’N, 116°24’44’’ E, Beijing, China). Two wheat (Xiaoyan54 and Jing411) and rice (IR24 and Nipponbare) varieties were grown in Changping Farm under field conditions in 2016 and 2017, respectively. Roots from A. thaliana, wheat and rice were harvested six weeks post plantation in natural soil for 16S rRNA gene profiling. Data analysis was performed using QIIME 1.9.1 (42), USEARCH 10.0 (43) and in-house scripts. OTUs with differential abundance were identified with a negative binomial generalized linear model in the edgeR package with a fold change threshold >1.2 (20). Venn diagrams were generated using the Venndiagram package (22).

In vitro bioassays

Root-associated bacteria were isolated and identified from A. thaliana Col-0 plants grown in the aforementioned natural soil from Changping Farm with limiting dilution and barcoded sequencing (35). 19 bacterial isolates from diverse taxa that share greater than 97% 16S rRNA gene similarity with representative OTU sequences were selected for bioassay. The bacteria were cocultured with different formulated cocktails of triterpene mixtures or individual triterpenes in TSB media and their growth (OD600) monitored over 48 to 72 hours. Bacteria-mediated transformation of triterpenes T1, T11, and T18a to T18c was tested against all 19 bacteria, whereas T2, T9, and T10 against sensitive bacterial strains A224, A475-1, and A479.

Supplementary Materials

science.sciencemag.org/content/364/6440/eaau6389/suppl/DC1

Materials and Methods

Figs. S1 to S64

Tables S1 to S65

References (4660)

References and Notes

Acknowledgments: J. Pollier and P. Fernandez-Calvo are acknowledged for their support of Y-C.B.; C. Owen is acknowledged for initial testing of the thalianol cluster genes. Funding: This work has been supported by the National Institutes of Health Genome to Natural Products Network award U101GM110699 (A.O. and A.C.H.); the “Strategic Priority Research Program” of the Chinese Academy of Sciences (XDB11020700) (Y.B.); the Key Research Program of the Chinese Academy of Sciences (grant KFZD-SW-112-02-02, KFZD-SW-219) (Y.B.); the International Cooperation and Exchanges NSFC grant 31761143017 (Y.B.); the Centre of Excellence for Plant and Microbial Sciences (CEPAMS), established between the John Innes Centre and the Chinese Academy of Sciences and funded by the UK Biotechnology and Biological Sciences Research Council (BBSRC) and the Chinese Academy of Sciences (A.O. and Y.B.); the Priority Research Program of the Chinese Academy of Sciences (QYZDB-SSW-SMC021) (Y.B.); the European Community’s Seventh Framework Program (FP7/2007–2013) under grant agreement 613692 (TriForC) (A.O. and A.G.); the joint Engineering and Physical Sciences Research Council/ BBSRC-funded OpenPlant Synthetic Biology Research Centre grant BB/L014130/1 (H.-W.N., A.O.); and the Research Foundation Flanders with a research project grant to A.G. (G008417N). A.C.H. is supported by a European Commission Marie Skłodowska-Curie Individual Fellowship (H2020-MSCA-IF-EF-ST-702478-TRIGEM). H.-W.N. is currently supported by a Royal Society University Research Fellowship (UF160138). A.O.’s laboratory is funded by the UK BBSRC Institute Strategic Programme Grant “Molecules from Nature” (BB/P012523/1) and the John Innes Foundation. Y.-C.B. is supported by a China Scholarship Council (CSC) Ph.D. scholarship. Y.B. is supported by Thousand Youth Talents Plan (grant 2060299). Author contributions. A.C.H., Y. B., and A.O. conceived and designed the project. A.C.H. discovered and characterized the biosynthetic network, performed bacterial growth assay, and coordinated the project; T.J. grew plants in natural soils, harvested roots, prepared the 16S amplicon library for sequencing, isolated A. thaliana root bacteria and performed bacterial growth assays; Y.-X.L. performed bioinformatics analysis on microbiota sequencing results; Y.-C.B. generated the homozygous thaa1-crispr and thaa2-crispr lines; J.R. cloned the thalianol and marneral cluster genes; and B.Q. grew and harvested the wheat samples for microbiota analysis. A.C.H., T.J., Y.-X.L., H.-W.N., A.G., and Y.B. analyzed data; A.C.H., T.J., Y.B., and A.O. wrote the manuscript, with contributions from other authors. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Raw microbiota sequencing data reported in this paper have been deposited in the Genome Sequence Archive in Beijing Institute of Genomics (BIG) Data Center (44), Chinese Academy of Sciences under accession no. PRJCA001296 that are public accessible at http://bigd.big.ac.cn/gsa. Scripts used in the microbiota analyses are available under the following link: https://github.com/microbiota/Huang2019SCIENCE.
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