A single bacterial genus maintains root development in a complex microbiome

Plants grow within a complex web of species interacting with each other and with the plant. Many of these interactions are governed by a wide repertoire of chemical signals, and the resulting chemical landscape of the rhizosphere can strongly affect root health and development. To understand how microbe-microbe interactions influence root development in Arabidopsis, we established a model system for plant-microbe-microbe-environment interactions. We inoculated seedlings with a 185-member bacterial synthetic community (SynCom), manipulated the abiotic environment, and measured bacterial colonization of the plant. This enabled classification of the SynCom into four modules of co-occurring strains. We deconstructed the SynCom based on these modules, identifying microbe-microbe interactions that determine root phenotypes. These interactions primarily involve a single bacterial genus, Variovorax, which completely reverts severe root growth inhibition (RGI) induced by a wide diversity of bacterial strains as well as by the entire 185-member community. We demonstrate that Variovorax manipulate plant hormone levels to balance this ecologically realistic root community’s effects on root development. We identify a novel auxin degradation operon in the Variovorax genome that is necessary and sufficient for RGI reversion. Therefore, metabolic signal interference shapes bacteria-plant communication networks and is essential for maintaining the root’s developmental program. Optimizing the feedbacks that shape chemical interaction networks in the rhizosphere provides a promising new ecological strategy towards the development of more resilient and productive crops.

To ascertain the phylogenetic breadth of the Variovorax ability to attenuate RGI, we 153 tested additional Variovorax strains from across the genus' phylogeny (Extended Data  Table 11). 182 Seventeen of these are co-expressed genes with proposed functions related to the root 12 To identify the bacterial mechanisms involved in RGI-attenuation, we compared the 230 genomes of the 10 Variovorax strains in the SynCom to the genomes of the other 175 231 SynCom members. We identified a list of 947 orthogroups that were rare (< 5% 232 prevalence) in the 175 SynCom members and core (100% prevalence) across the 10 233 Variovorax strains. We collapsed these orthogroups into hotspots (regions of physically 234 continuous orthogroups) and focused on the 12 hotspots that contained at least 10  Paraburkholderia growth on IAA 18 (Fig. 5a). To test whether the hotspots we identified  Variovorax CL14 genomic inserts in both of these clones contained portions of hotspot 253 33 (Fig. 5a, Extended Data Fig. 9). The overlap common to these clones contained 9 254 genes, among them the weak homologs to Paraburkholedria iacCDE. To test whether 255 this genomic region is sufficient to revert RGI in the plant, we transformed a relative of 256 Variovorax, Acidovorax Root219 that does not cause or revert RGI (Extended Data Fig.   257 5), with the shorter functional insert containing Vector 2 (V2) or with an empty vector (EV). 258 We inoculated the resulting gain-of-function strain, Acidovorax Root219::V2 or the Tripartite system 45 genes    Primary root elongation (cm)  Fig. 4 Taxonomic composition of the SynCom used in Figure 2D.
Primary root elongation (cm) NS NS *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** *** **    Fig. 7 Root growth inhibition-related genes share gene ontologies. Network of statistically significant gene ontology terms contained in the 18 genes upregulated in Variovorax CL14/Arthrobacter CL28 co-inoculation vs Arthrobacter CL28 alone AND in the full SynCom vs the Variovorax drop-out SynCom (See Figure 4a and 4b). The network was computed using the emapplot function from the package clusterProfiler in R. A p-value for terms across the gene ontology was computed using a hypergeometric test, additionally the size of each point (Gene ontology term) denoted the number of genes mapped in that particular term.  All seeds were surface-sterilized with 70% bleach, 0.2% Tween-20 for 8 min, and rinsed 319 three times with sterile distilled water to eliminate any seed-borne microbes on the seed 320 surface. Seeds were stratified at 4 °C in the dark for two days. Plants were germinated 321 on vertical square 12 X 12 cm agar plates with JM containing 0.5% sucrose, for 7 days.   Roots and shoots were placed in 2 mL Eppendorf tubes with three sterile glass beads.     The resulting abundance tables were processed and analyzed with functions from the 404 ohchibi package (https://github.com/isaisg/ohchibi). An alpha diversity metric (Shannon 405 diversity) was calculated using the diversity function from the vegan package v2.5-3 47 . 406 We used ANOVA to test for differences in alpha diversity between groups. Beta diversity 407 analyses (Principal coordinate analysis, and canonical analysis of principal coordinates) 39 were based on Bray-Curtis dissimilarity calculated from the relative abundance matrices. 409 We used the capscale function from the vegan R package v.2.5-3 47    according to the dendrogram order obtained from the USeqs co-occurrence analysis.

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The heatmap was colored based on the log2FoldChange output by the GLM model. We 448 highlighted in a black shade the comparisons that were significant (q-value < 0.05).

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Finally, for each of the four modules we computed for each family present in that module 450 a hypergeometric test testing if that family was overrepresented (enriched) in that 451 particular module. Families whose FDR p-value < 0.1 were visualized in the figure.   International) to maintain sterility and gas exchange. 47 The entire SynCom, excluding all 10 Variovorax isolates was grown and prepared as  8. Phylogenetic inference of the SynCom and Variovorax isolates (Fig 2A, fig. S1A implemented the same methodology described above.     To present representative root images, we increased contrast and subtracted 857 background in imageJ, then cropped the image to select representative roots.

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Neighboring roots were manually erased from the cropped image.  Variovorax CL14 was grown either alone or in co-culture with Arthrobacter CL28 in 5mL 880 of 1/10 2xYT medium (1.6 g/L tryptone, 1 g/L yeast extract, 0.5 g/L NaCl) in triplicate.

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The mono-culture was inoculated at OD600 of 0.02 and the co-culture was inoculated 882 with OD600 of 0.01 of each strain. Cultures were grown at 28°C to early stationary phase 883 (approximately 22 hours) and cells were harvested by centrifugation at 4100 x g for 15 884 min and frozen at -80°C prior to RNA extraction.