NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice

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Abstract

Nitrogen-use efficiency of indica varieties of rice is superior to that of japonica varieties. We apply 16S ribosomal RNA gene profiling to characterize root microbiota of 68 indica and 27 japonica varieties grown in the field. We find that indica and japonica recruit distinct root microbiota. Notably, indica-enriched bacterial taxa are more diverse, and contain more genera with nitrogen metabolism functions, than japonica-enriched taxa. Using genetic approaches, we provide evidence that NRT1.1B, a rice nitrate transporter and sensor, is associated with the recruitment of a large proportion of indica-enriched bacteria. Metagenomic sequencing reveals that the ammonification process is less abundant in the root microbiome of the nrt1.1b mutant. We isolated 1,079 pure bacterial isolates from indica and japonica roots and derived synthetic communities (SynComs). Inoculation of IR24, an indica variety, with an indica-enriched SynCom improved rice growth in organic nitrogen conditions compared with a japonica-enriched SynCom. The links between plant genotype and root microbiota membership established in this study will inform breeding strategies to improve nitrogen use in crops.

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Fig. 1: Root microbiota of indica and japonica.
Fig. 2: Random-forest model detects bacterial taxa that accurately predict indica and japonica subspeciation.
Fig. 3: Taxonomic and functional characteristics of differential bacteria between the indica and japonica root microbiota.
Fig. 4: NRT1.1B contributes to the variation in the root microbiota of indica and japonica.
Fig. 5: Rice root-associated bacterial culture collections capture the majority of bacterial species that are reproducibly detectable by culture-independent sequencing.
Fig. 6: Indica-enriched SynCom have stronger ability to promote rice growth under the supply of organic nitrogen than japonica-enriched SynCom.

Data availability

Raw sequence data reported in this paper have been deposited (PRJCA001214) in the Genome Sequence Archive in the BIG Data Center62, Chinese Academy of Sciences under accession codes CRA001372 for bacterial 16S rRNA gene sequencing data and CRA001362 for metagenomic sequencing data that are publicly accessible at http://bigd.big.ac.cn/gsa. All pure strains (Supplementary Table 11) are deposited in two national culture collection centers, the China Natural Gene Bank and the Agricultural Culture Collection of China. All information about these strains, such as the 16S rRNA gene sequences, taxonomy and isolation details, as well as any further updates are available at http://bailab.genetics.ac.cn/culture_collection/.

Code availability

Scripts employed in the computational analyses are available at https://github.com/microbiota/Zhang2019NBT.

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Acknowledgements

We thank P. Schulze-Lefert and S. Hacquard at the Max Planck Institute for Plant Breeding Research for their suggestions for improving the manuscript. This work was financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (grant nos. XDB11020700 to Y.B. and XDA08010104 to C.C.), the Key Research Program of Frontier Sciences of the Chinese Academy of Science (grant nos. QYZDB-SSW-SMC021 to Y.B. and QYZDJ-SSW-SMC014 to C.C.), the National Natural Science Foundation of China (grant nos. 31772400 to Y.B. and 31801945 to J.Z.), and the Key Research Program of the Chinese Academy of Sciences (grant no. KFZD-SW-219 to Y.B.). J.Z. is supported by the CPSF-CAS Joint Foundation for Excellent Postdoctoral Fellows (grant no. 2016LH00012). Y.B. is supported by the Thousand Youth Talents Plan (grant no. 2060299).

Author information

C.C. and Y.B. conceived the study and supervised the project. J.Z. and N.Z. performed the experiments. Y.-X.L. analyzed the data of 16S rRNA gene profiling. B.H. and L.Y. coordinated field experiments and revised the manuscript. T.J., P.Y. and R.G.-O. analyzed the metagenomic data. X.Z., Y.Q. and G.F. were involved in the informatics analysis. J.H. performed the soil properties analysis. S.C., H.X., X.W., C.W., H.W. and B.Q. participated in growing plants and harvesting samples. X.G. optimized the protocol of library preparation for the 16S rRNA gene profiling. J.Z., C.C. and Y.B. wrote the manuscript.

Correspondence to Chengcai Chu or Yang Bai.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Fig. 1 Coverage of members in the root bacterial microbiota by the representative indica and japonica varieties.

(a) Rarefaction curves of detected bacterial species of the root microbiota reach the saturation stage with increasing numbers of samples, indicating that the root microbiota in our population capture most root bacteria members from each rice subspecies. Indica and japonica varieties in two locations are shown separately. (b) Rarefaction curves of detected bacterial OTUs of the root microbiota from indica and japonica varieties reach saturation stage with increasing sequencing depth. Each vertical bar represents standard error. The numbers of replicated samples in this figure are as follows: in field I, indica (n = 201), japonica (n = 80), soil (n = 12); in field II, indica (n = 201), japonica (n = 81), soil (n = 12).

Supplementary Fig. 2 Comparison of the root microbiota of indica and japonica varieties.

(a,b) Principal coordinate analysis with unweighted (a) and weighted (b) UniFrac distance show that the root microbiota of indica separate from that of japonica in field I in the first two axes, indicating that the root microbiota of indica are distinct from that of japonica (P < 0.001, PERMANOVA by Adonis). Ellipses cover 68% of the data for each rice subspecies. (c,d) Principal coordinate analysis with unweighted (c) and weighted (d) UniFrac distance showing that the root microbiota of indica separate from those of japonica in field II in the first two axes, revealing that root microbiota of indica are distinct from those of japonica (P < 0.001, PERMANOVA by Adonis). The numbers of replicated samples are as follows: in field I, indica (n = 201), japonica (n = 80); in field II, indica (n = 201), japonica (n = 81).

Supplementary Fig. 3 Comparison of the root microbiota in two fields.

(a,b) Unconstrained (a) and constrained (b) principal coordinate analysis of indica in field I, japonica in field I, indica in field II, and japonica in field II with Bray-Curtis distance. (c,d) Principal coordinate analysis of indica in field I, japonica in field I, indica in field II, and japonica in field II with unweighted (c) and weighted UniFrac (d) distance. Ellipses cover 68% of the data for each rice subspecies. The numbers of replicated samples are as follows: in field I, indica (n = 201), japonica (n = 80); in field II, indica (n = 201), japonica (n = 81).

Supplementary Fig. 4 Comparison of within-sample diversity (α-diversity) between indica and japonica.

(a,b) Chao 1 (a) and observed OTUs (b) of the root microbiota of indica, japonica, and corresponding unplanted bulk soils in two fields. The numbers of replicated samples are as follows: in field I, indica (n = 201), japonica (n = 80), soil (n = 12); in field II, indica (n = 201), japonica (n = 81), soil (n = 12). Data in two locations show the consistent trend that the root microbiota of indica show higher alpha diversity than those of japonica. The horizontal bars within boxes represent median. The tops and bottoms of boxes represent 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5 × the interquartile range from the upper edge and lower edge of the box, respectively.

Supplementary Fig. 5 Taxonomic composition of the indica- and japonica-enriched OTUs.

(a,b) The relative abundance of indica-enriched (a) and japonica-enriched (b) OTUs at the phylum level. Proteobacteria are shown at the class level.

Supplementary Fig. 6 Function and time-series shift of the indica- and japonica-enriched OTUs.

(a) Functional annotation of indica-enriched OTUs by FAPROTAX. The presence of functions is shown in red. (b) Shift of relative abundance of indica-enriched OTUs according to time-course data from the rice root microbiota in the field in Changping Farm17. (c) Functional annotation of japonica-enriched OTUs by FAPROTAX. The presence of functions is shown in red. (d) Shift of relative abundance of japonica-enriched OTUs according to time-course data from the rice root microbiota in the field in Changping Farm17.

Supplementary Fig. 7 Correlation between the natural variation of NRT1.1B and nitrogen-related functions in indica and japonica populations.

(ae) The natural variation in NRT1.1B in indica and japonica populations is correlated with nitrogen-related functions in root microbiota from indica and japonica populations, including nitrite ammonification (a) (P = 2.2 × 10–16 in field I; P = 1.8 × 10–12 in field II, two-sided t-test), nitrate reduction (b) (P = 1.1 × 10–13 in field I; P = 2.1 × 10–8 in field II, two-sided t-test), respiration of nitrate (c) (P = 2.2 × 10–16 in field I; P = 6.1 × 10–13 in field II, two-sided t-test), nitrite respiration (d) (P = 2.4 × 10–16 in field I; P = 7.2 × 10–13 in field II, two-sided t-test) and nitrogen respiration (e) (P = 2.2 × 10–16 in field I; P = 6.8 × 10–13 in field II, two-sided t-test). NRT1.1Bindica harbors a “T” at 980 bp downstream of the ATG start codon and NRT1.1Bjaponica harbors a “C” at the same position, resulting in an amino acid substitution (p. Met327Thr). The numbers of replicated samples are as follows: in field I, indica (n = 192), japonica (n = 86); in field II, indica (n = 192), japonica (n = 87).

Supplementary Fig. 8 NRT1.1B and its natural variation modulate the assembly of the rice root microbiota.

(a,b) Principal coordinate analysis with unweighted (a) and weighted (b) UniFrac distance showing that the root microbiota of ZH11 (wild-type), nrt1.1b, Nipponbare NRT1.1Bindica, and Nipponbare NRT1.1Bjaponica separate in the first two axes. Ellipses cover 68% of the data for each genotype. (c) Constrained principal coordinate analysis showing that 57.1% of the root microbiota variations are explained by genotypes (ZH11, nrt1.1b, NRT1.1Bindica, and NRT1.1Bjaponica). The numbers of replicated samples are as follows: ZH11 (n = 16), nrt1.1b (n = 14), NRT1.1Bindica (n = 15), NRT1.1Bjaponica (n = 15). (d,e) A full factorial replication experiment validates the conclusion that NRT1.1B and its natural variation modulate the assembly of the rice root microbiota. Unconstrained (d) and constrained (e) principal coordinate analysis with Bray-Curtis distance showing that the root microbiota of ZH11 (wild-type), nrt1.1b, NRT1.1Bindica, and NRT1.1Bjaponica separate in the first two axes. The plants were grown in different fields and at different time from the samples in Fig. 4. Ellipses cover 68% of the data for each genotype. The numbers of replicated samples are as follows: ZH11 (n = 14), nrt1.1b (n = 10), NRT1.1Bindica (n = 15), NRT1.1Bjaponica (n = 15).

Supplementary Fig. 9 OTUs associated with NRT1.1B in the field condition related to Fig. 4.

(a) Enrichment and depletion of OTUs in the nrt1.1b mutant compared with wild-type ZH11. Each point represents an individual OTU, and the position along the x axis represents the abundance fold change between the nrt1.1b mutant and wild-type. (b) Heat map showing the relative abundance of the differential OTUs between the nrt1.1b mutant and wild-type ZH11. (c) OTUs enriched in NRT1.1Bindica or NRT1.1Bjaponica. Each point represents an individual OTU, and the position along the x axis represents the abundance fold change between NRT1.1Bindica or NRT1.1Bjaponica. (d) Heat map showing the relative abundance of the differential OTUs between NRT1.1Bindica and NRT1.1Bjaponica. The numbers of replicated samples are as follows: ZH11 (n = 16), nrt1.1b (n = 14), NRT1.1Bindica (n = 15), and NRT1.1Bjaponica (n = 15).

Supplementary Fig. 10 Experimental procedure for isolation and identification of rice root-associated bacteria.

Step 1–4 illustrate the isolation procedure; Step 5–11 illustrate the identification of cultivated rice root-associated bacteria by the improved two-step barcoded system.

Supplementary Fig. 11 The scheme for the previous high-throughput barcoding system to determine 16S rRNA gene sequences covering regions V5–V7 of bacterial isolates.

A previously published two-step barcoded pyrosequencing procedure (Bai, Y. et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528, 364–369, 2015). Please note that the second step PCR generates chimera sequences that will contain mislabeled plate and well barcodes for bacterial identification.

Supplementary Fig. 12 Plant growth with or without indica-enriched SynCom under inorganic nitrogen conditions.

(ac) IR24 (indica) rice plants were grown under different ratios of ammonium and nitrate (0:2, 2:0, and 1:1) with or without indica-enriched SynCom. After 2-week bacterial inoculation, rice plants were measured by root length (a), plant height (b), and shoot fresh weight (c). (df) Nipponbare (japonica) rice plants were grown under different ratios of ammonium and nitrate (0:2, 2:0, and 1:1) with or without indica-enriched SynCom. After 2-week bacterial inoculation, rice plants were measured by root length (d), plant height (e), and shoot fresh weight (f). Different letters indicate significantly different groups (P < 0.05, ANOVA, Tukey-HSD). Boxplots show combined data from three independent inoculation experiments with 4–5 technical replicates each (Supplementary Table 13). The horizontal bars within boxes represent medians. The tops and bottoms of boxes represent 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5 × the interquartile range from the upper edge and lower edge of the box, respectively.

Supplementary Fig. 13 Plant growth with indica- or japonica-enriched SynCom under inorganic nitrogen conditions.

IR24 rice plants were grown under the inorganic nitrogen condition with and without SynComs, including indica-enriched SynCom, japonica-enriched SynCom, and corresponding heat-killed bacteria as controls, respectively (Supplementary Table 13). After 2-week bacterial inoculation, rice plants were measured by root length (a), plant height (b), and shoot fresh weight (c). Different letters indicate significantly different groups (P < 0.05, ANOVA, Tukey-HSD). Boxplots show combined data from two independent inoculation experiments with 4–5 technical replicates each. The horizontal bars within boxes represent medians. The tops and bottoms of boxes represent 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5 × the interquartile range from the upper edge and lower edge of the box, respectively.

Supplementary information

Supplementary Figures

Supplementary Figs. 1–13

Reporting Summary

Supplementary Table 1

Soil properties and cultivation practices of field I and field II on Lingshui farm.

Supplementary Table 2

Information on indica and japonica varieties.

Supplementary Table 3

Metadata, OTU representative sequences, taxonomy annotation and OTU table.

Supplementary Table 4

Differential phyla and classes of Proteobacteria between indica and japonica.

Supplementary Table 5

Random-forest: accuracy of the random-forest model at each taxonomy level; feature importance at the family level; outcomes of prediction.

Supplementary Table 6

Differential abundance of OTUs between indica and japonica; details of OTUs in each part of the Venn diagrams in Fig. 3.

Supplementary Table 7

Abundance of OTUs in time-course data and functional annotation by FAPROTAX.

Supplementary Table 8

Differential abundances of OTUs between the nrt1.1b mutant and wild-type ZH11; differential abundances of OTUs between NRT1.1Bindica and NRT1.1Bjaponica; details of OTUs in each part of the Venn diagrams of Fig. 4.

Supplementary Table 9

KEGG orthology of metagenomes in ZH11 and the nrt1.1b mutant.

Supplementary Table 10

Detailed information of all cultivated CFUs and unique bacterial sequences from rice root.

Supplementary Table 11

Taxonomy and sequences of 1,079 bacterial stocks in rice root bacterial culture collection.

Supplementary Table 12

Experimental design of synthetic communities on germ-free plants related to the nitrogen assay.

Supplementary Table 13

Phenotypes of rice plants under inorganic nitrogen and organic nitrogen conditions with and without SynComs.

Supplementary Table 14

Primer sequences and experimental procedures for culture-independent community profiling and bacterial identification.

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