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Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation

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Science  30 Aug 2019:
Vol. 365, Issue 6456, pp. 919-922
DOI: 10.1126/science.aav8907
  • Fig. 1 Rhizobial tRFs and their putative target genes in soybean.

    (A) Origins of three rhizobial tRFs. Anticodons in corresponding tRNAs are colored in blue. (B) Abundance of the three tRFs, measured by means of stem-loop quantitative RT-PCR, in free-living B. japonicum (B. j.) USDA110 (1) and 10–day and 20–day post-inoculation (dpi) nodules (2 and 3, respectively). (C) Expression of the putative tRF target genes, measured with quantitative RT-PCR, in the 10-day-old and 20-day-old nodules (2 and 4) and uninoculated soybean roots (1 and 3). Values in (B) and (C), with one set as “1” and the others adjusted accordingly, are shown as means ± SE from three biological replicates. Asterisks indicate the significance level at P < 0.01 (Student’s t test). (D) The three tRFs, their putative target transcripts, and the cleavage sites and frequencies (indicated with arrows and ratios) were detected in the 20-dpi nodules.

  • Fig. 2 Modulation of soybean nodulation by rhizobial tRFs and their putative targets.

    (A) Knockouts of the putative tRF targets by means of CRISPR-Cas9 (CR) resulted in increased nodule numbers. (B) Overexpression (OX) of the putative tRF targets resulted in decreased nodule numbers. (C) Silencing of individual tRFs by means of STTM resulted in decreased nodule numbers. (D) Nodule numbers, with all data points represented by dots, are shown as box and whisker plots displaying 95 to 5% interval from three biological replicates (12 plants per replicate) collected 28 days after inoculation. Controls are transgenic roots of empty vectors used for the CRISPR-Cas9 knockouts, the gene-overexpression roots, and the STTM tRF-silencing roots, respectively. Asterisks indicate the significance level of P < 0.01 (Student’s t test).

  • Fig. 3 Rhizobial tRF-guided gene regulation by hijacking the host RNAi machinery.

    (A) Abundance of artificial miRNAs measured with stem-loop quantitative RT-PCR in aMIR-tRF001 (2) and aMIR-tRF003 transgenic roots (3) and respective empty-vector transgenic roots (1) 28 days after inoculation. (B) Expression of the putative tRF/artificial miRNA targets measured with quantitative RT-PCR in the same samples as described in (A). Values in (A) and (B), with one set as “1” and the others adjusted accordingly, are shown as means ± SE from three biological replicates. Asterisks indicate the significance level at P < 0.01 (Student’s t test). (C) Nodule numbers in the same samples as described in (A), with all data points represented by dots, are shown as box and whisker plots displaying 95 to 5% interval from three biological replicates (12 plants per replicate). (D) GFP activity in transgenic roots of “GFP-tRF target site” fusion genes (W1 to W3) and “GFP-mutated tRF target site” fusion genes (M1 to M3) 24 hours after inoculation with USDA110. Bj– and Bj+ indicate uninoculated and inoculated roots, respectively. (E) Association of the three tRFs with soybean GmAGO1b in nodules 28 days after inoculation detected from three experimental replicates. “+” and “−” indicate the GmAGO1b-Myc fusion protein–associated fraction immunoprecipitated by the Myc antibody and the nodule lysate without Myc antibody incubation, respectively.

  • Fig. 4 Modulation of early-stage rhizobial infection by rhizobial tRFs and their targets in soybean.

    (A and B) Morphological differences between the root hairs overexpressing the rhizobial tRF targets and the negative control and between the STTM root hairs inhibiting the rhizobial tRF function and the negative control. (C) Quantitation of deformed root hairs and curled root hairs with infection foci in samples as exemplified in (A) and (B). The values are shown as means ± SD from three biological replicates (n = 25 hairy roots per replicate). Asterisks indicate the significant level at P < 0.05 (Student’s t test).

Supplementary Materials

  • Rhizobial tRNA-derived small RNAs are signal molecules regulating plant nodulation

    Bo Ren, Xutong Wang, Jingbo Duan, Jianxin Ma

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