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Horizontally acquired CSP genes contribute to wheat adaptation and improvement

Abstract

Although horizontal gene transfer (HGT) often facilitates environmental adaptation of recipient organisms, whether and how they might affect crop evolution and domestication is unclear. Here we show that three genes encoding cold-shock proteins (CSPs) were transferred from bacteria to Triticeae, a tribe of the grass family that includes several major staple crops such as wheat, barley and rye. The acquired CSP genes in wheat (TaCSPs) are functionally conserved in their bacterial homologues by encoding a nucleic acid-binding protein. Experimental evidence indicates that TaCSP genes positively regulate drought response and improve photosynthetic efficiency under water-deficient conditions by directly targeting a type 1 metallothionein gene to increase reactive oxygen species scavenging, which in turn contributed to the geographic expansion of wheat. We identified an elite CSP haplotype in Aegilops tauschii, introduction of which to wheat significantly increased drought tolerance, photosynthetic efficiency and grain yields. These findings not only provide major insights into the role of HGT in crop adaptation and domestication, but also demonstrate that novel microbial genes introduced through HGT offer a stable and naturally optimized resource for transgenic crop breeding and improvement.

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Fig. 1: Bacterial CSP genes were transferred to Triticeae and encode a nucleic acid-binding protein.
Fig. 2: TaCSP-H1 is involved in abiotic stress responses of wheat.
Fig. 3: TaCSP-H1 improves photosynthetic efficiency of wheat and promotes ROS scavenging via TaMT1 under drought stress.
Fig. 4: AetCSP-H1 natural variations improve wheat drought adaptation and grain yield.
Fig. 5: TaCSP-H1 increases abiotic stress response and grain yield in rice.

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Data availability

The RNA-seq and RIP-seq data generated in this study have been deposited in the National Center for Biotechnology Information (NCBI) under BioProject number PRJNA1114877. The reference genome of wheat (CS v.2.1) was downloaded from IWGSC (http://wheat-urgi.versailles.inra.fr/Seq-Repository/Assemblies). All analyses were conducted using standard software. The settings of software used for analyses are described in Methods. Materials used in this study are available upon request. Source data are provided with this paper.

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Acknowledgements

We thank S. Li for providing the T093-Zhoumai 18 introgression lines; W. Chi for providing the E. coli BX04 strain and PINIII vector; and X. Dong and J. Li for providing the E. coli RL211 strain. This work was supported in part by grants from the National Natural Science Foundation of China (32230079 to C.-P.S.), the National Key Research and Development Program of China (2022YFF1001602 to C.-P.S.), the Key Research and Development Program of Henan Province (231111110200 to Y.Z.), and the Key Research Project of the Shennong Laboratory (SN01-2022-01 to C.-P.S.).

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Authors and Affiliations

Authors

Contributions

C.-P.S., Y.Z. and J.H. conceived and designed the study. K.W., G.G., S.B., J.M. and Z.Z. performed the main experiments. J.H., J.M. and S.B. performed HGT analyses. K.W., G.G., Z.Z., Z.X., J.W., Junrong Li and X.Z. generated the transgenic lines and identified the phenotypes. Z.X., J.W., W.X. and C.Y. performed the field tests and population genetic analyses. W.W. analysed the protein structures. Z.X., W.X., Z.Z., H. Li, Z.L. and Y.L. collected data for the introgression lines. C.-P.S., Y.Z., J.H., K.W. and S.B. analysed the RNA-seq and RIP-seq data. T.B., W.-C.L., H. Liang, X.S., R.-F.S., J. Liang, Q.L., F.Z., X.Q. and T.H. conducted some of the experiments. C.-P.S., Y.Z., J.H. and K.W. wrote the manuscript. C.-P.S., Y.Z., J.H., K.W., G.G. and S.B. revised the manuscript.

Corresponding authors

Correspondence to Jinling Huang, Yun Zhou or Chun-Peng Song.

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Nature Plants thanks Zhong-Hua Chen, Lingrang Kong and Bojian Zhong for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Phylogenetic analyses of CSP proteins sequences.

a. Phylogenetic analyses of CSP proteins. CSP sequences from representative lineages of three domains of life (bacteria, archaea, and eukaryotes) in nr and other related databases were sampled, and the phylogenetic tree was constructed using IQ-TREE with 1,000 replicates from ultrafast bootstrap analyses. Scale bars represent numbers of substitutions per amino acid. b. The distribution of CSP-I and CSP-II proteins in different plant groups. Purple lines indicate the distribution of CSP-I proteins in both bacteria and plants, whereas green lines show the distribution of CSP-II proteins in photosynthetic eukaryotes. The divergence times are estimated by www.timetree.org. The small horizontal line after the CSD domain in Porphyridium purpureum refers to amino acid sequence without a conserved structure. CSD: cold shock domain. RRM: RNA recognition motif; GR: glycine-rich region; Znf: zinc finger. c. Multiple sequence alignment of the TaCSP-H1, TaCSP-H2, and CSP-H3 proteins. d. Phylogenetic analyses of the CSP-H3 proteins. Numbers beside branches represent bootstrap values based on 1,000 replicates from ultrafast bootstrap analyses using IQ-TREE. Scale bars represent numbers of substitutions per amino acid. Also see Supplementary Note 1 on the relationships between CSP-H3 and CSP-H1/H2.

Source data

Extended Data Fig. 2 Nucleic acid melting activity assay of wheat TaCSP-H and TaCSP-V.

a. Nucleic acid unfolding activity assay of TaCSP-H1 from all three sub-genomes of wheat in E. coli BX04 strain. Diluted cultures of BX04 cells expressing bacterial CspA, PINIII (empty vector) or TaCSP-H1 were spotted onto LB-carbenicillin plates and incubated at 15 °C or 37 °C. CspA and PINIII were used as the positive and negative controls, respectively. The mRNA levels indicate the expression of genes used in the assay. b. Transcription anti-termination assay of TaCSP-H2 and TaCSP-H3 in E. coli RL211 strain. Diluted cultures of RL211 cells expressing TaCSP-H2 and TaCSP-H3 were spotted onto LB plates with ( + Cm) or without (-Cm) chloramphenicol. CspA and PINIII were used as the positive and negative controls, respectively. The mRNA levels indicate the expression of genes used in the assay. c-e. Transcriptional anti-termination assay of the TaCSP-V in E. coli RL211 strain. Diluted cultures of RL211 cells expressing TaCSP-V1 (TraesCS1D03G0632700), TaCSP-V2 (TraesCS1D03G0633000), TaCSP-V3 (TraesCS6D03G0140500) and their CSD domain truncations (TaCSP-V-CSD) were spotted onto LB plates with ( + Cm) or without (-Cm) chloramphenicol. CspA and PINIII were used as the positive and negative controls, respectively. The mRNA levels indicate the expression of genes or gene truncations used in the assay.

Source data

Extended Data Fig. 3 Expression patterns of TaCSP-H homoeologs in public transcriptomic data.

a. Expression profiles of TaCSP-H homoeologs in different tissues and developmental stages. The color gradient from yellow to blue indicates expression levels from low to high. Data source: http://www.wheat-expression.com. b-d. Differential expression analysis of TaCSP-H homoeologs in roots (b), spikes (c), and grains (d) in public transcriptome data. All samples used in (d) have no differentially expressed genes. Red and bule rectangles indicate up- and down-regulation compared to the control, respectively, whereas gray rectangles indicate no significant difference. The differential criteria were set as |Fold Change | > 2 and q-value < 0.05.

Extended Data Fig. 4 Expression levels of TaCSP-H1 and TaCSP-H2.

a. Cis-element analyses of TaCSP-H homoeolog promoters. Upstream sequences from the initiation codon were selected for analyses, and the number of each type of elements is shown in the box. b. Expression levels of TaCSP-H1 and TaCSP-H2 homoeologs in WT seedlings at different developmental stages. TaActin was used as the internal control. Each bar represents mean ± SEM of three biological replicates. c-d. Gene expression levels of TaCSP-H1 and TaCSP-H2 homoeologs under normal and drought conditions in transcriptome data. Gene expression levels are indicated by transcripts per kilobase million (TPM). The TaCSP-H1 homoeolog from the D sub-genome of wheat is the most expressed gene and mightily induced by drought stress.

Source data

Extended Data Fig. 5 Identification of TaCSP-H1 CRISPR/Cas9 mutants and overexpression lines in wheat.

a. Editing sites of TaCSP-H1 CRISPR/Cas9 lines in wheat and genomic sequences in two tacsp-h1 mutants. The TaCSP-H1-edited plants were sequenced to detect the targeted mutations by Sanger sequencing. b. Relative expression of TaCSP-H1 in WT and two TaCSP-H1 overexpression lines (TaCSP-H1-OE6 and TaCSP-H1-OE8). TaActin was used as the internal control. Each bar represents mean ± SEM of three biological replicates. c. Protein levels of TaCSP-H1 in WT and two TaCSP-H1 overexpression lines. TaCSP-H1 protein was detected by anti-Flag antibodies. TaActin was used as loading control and detected by anti-Actin antibodies.

Source data

Extended Data Fig. 6 Developmental phenotypes of tacsp-h1 mutants and TaCSP-H1 overexpression plants during the whole life cycle.

a. The WT, tacsp-h1 and TaCSP-H1-OE plants were moved to soil at fifth-leaf stage after germination and photographed every week until maturity. Bars = 20 cm. b. Statistical analyses of plant height, spike length and number of spikes per plant for WT, tacsp-h1 and TaCSP-H1-OE plants. Each bar shows mean ± SEM from 20 biological replicates. Different letters represent significant differences (p < 0.05, one-way ANOVA, Tukey’s HSD test).

Source data

Extended Data Fig. 7 TaCSP-H1 positively regulates abiotic stress responses in wheat.

a-d. Phenotypes of WT, tacsp-h1 and TaCSP-H1-OE plants before (a) and after heat (b), cold (c), and salt (d) treatments. Bars = 10 cm. e. Statistical analyses of shoot fresh weight in WT, tacsp-h1 and TaCSP-H1-OE plants before (a) and after heat (b), cold (c), and salt (d) treatments. Five plants from one pot were measured per replicate, and three different pots were analyzed. Whiskers indicate the minimum and maximum values, central lines indicate medians, and box boundaries indicate the upper (25th percentile) and lower (75th percentile) quartiles. Different letters represent significant differences (p < 0.05, one-way ANOVA, Tukey’s HSD test).

Source data

Extended Data Fig. 8 TaCSP-H1 regulates photosynthesis-related network under drought stress.

a. Numbers of differentially expressed genes (DEGs) in five comparisons. Up- and down-regulated DEGs are shown in blue and green, respectively. WT-ck, WT samples under normal growth condition; WT-dr, WT samples under drought condition; KO-ck, tacsp-h1 samples under normal growth condition; KO-dr, tacsp-h1 samples under drought condition. OE-ck, TaCSP-H1-OE samples under normal growth condition; OE-dr, TaCSP-H1-OE samples under drought condition. b. Venn diagrams of total numbers of DEGs in WT-dr vs WT-ck and KO-dr vs WT-dr comparisons, as well as WT-dr vs WT-ck and OE-ck vs WT-ck comparisons. c. GO enrichment analyses of DEGs in WT vs tacsp-h1 and WT vs TaCSP-H1-OE comparisons. Each circle in the figure represents a GO term, and the number of genes enriched in a pathway corresponds to the size of the circle. The degree of significance for the enrichment of DEGs in a pathway is represented by -log10 (q-value). d. Heatmap of the photosynthesis-related genes in WT vs tacsp-h1 and WT vs TaCSP-H1-OE comparisons. Colors in the heatmap indicate the log2 fold change values (log2 FC).

Extended Data Fig. 9 TaCSP-H1 enhances photosynthetic efficiency under drought stress and promotes wheat spread.

a. Photosystem II (PSII) efficiency (ϕPSII) of flag leaves in WT, tacsp-h1, and TaCSP-H1-OE plants. Each bar shows mean ± SEM of nine biological replicates. Different letters represent significant differences (p < 0.05, one-way ANOVA, Tukey’s HSD test). b. Expression levels of TaCSP-H1 in different geographic regions in (c). CC, Caspian coast; CE, Central Europe; CA, Central Asia; AF, Africa; WE, Western Europe; EA, East Asia. c. Geographic distribution of natural populations of hexaploid wheat and their TaCSP-H1D expression levels. The area where wheat originated is indicated by dotted lines. Black arrows indicate the possible spread routes of wheat speculated by Zhao et al.37. The color of the circle represents the gene expression level (TPM) in public transcriptome data38. The map was drawn using the R ggmap package (http://maps.stamen.com/, map tiles by Stamen Design, under CC BY 4.0. Data by OpenStreetMap, under ODbL).

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Extended Data Fig. 10 TaCSP-H1 expressions and photosynthetic efficiency of the diploid, tetraploid ancestors and hexaploid wheat.

a. Venn diagram of photosynthesis-related DEG numbers in diploid Aegilops (Ae. tauschii, 2n = 14, DD) vs synthetic hexaploid wheat (2n = 6x = 42, AABBDD), tetraploid wheat (T. durum, 2n = 4x = 28, AABB) vs synthetic hexaploid wheat, and WT vs TaCSP-H1-OE comparisons. The synthetic hexaploid wheat was generated by hybridizing diploid Aegilops with tetraploid wheat, followed by spontaneously doubling at F1 generation. b. Heatmap of the photosynthesis-related genes in WT and TaCSP-H1-OE in (a). Cluster I, photosystem related genes; Cluster II, electron transport chain related genes; Cluster III, Rubisco related genes. Numbers in the scale bar stand for z-scores of gene expression. c. Expression levels of CSP-H1 homoeologs in diploid Ae. tauschii (DD1, DD2 and DD3), tetraploid wheat (AABB), and three independent lines of synthetic hexaploid wheat (SHW1, SHW2 and SHW3). Each bar shows mean ± SEM of three biological replicates. Different letters represent significant differences (p < 0.05, one-way ANOVA, Tukey’s HSD test).

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Supplementary Information

Supplementary Figs. 1–8 and Note 1.

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Supplementary Table 1

Supplementary Tables 1–15.

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Source Data

Statistical source data for Figs. 1–5 and Extended Data Figs. 1, 2, 4–7, 9 and 10.

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Wang, K., Guo, G., Bai, S. et al. Horizontally acquired CSP genes contribute to wheat adaptation and improvement. Nat. Plants 11, 761–774 (2025). https://doi.org/10.1038/s41477-025-01952-8

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