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SCIENCE CHINA Life Sciences, Volume 65 , Issue 1 : 1-15(2022) https://doi.org/10.1007/s11427-021-2017-1

A 5′ tRNA-Ala-derived small RNA regulates anti-fungal defense in plants

Hanqing Gu 1,2,†, Bi Lian 1,2,†, Yuxiang Yuan 1,2,†, Ci Kong 1,2, Yan Li 1,2, Chang Liu 1,2, Yijun Qi 1,2,*
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  • ReceivedSep 7, 2021
  • AcceptedOct 18, 2021
  • PublishedOct 22, 2021

Abstract


Funding

the National Natural Science Foundation of China(31801074,31788103)


Acknowledgment

This work was supported by the National Natural Science Foundation of China (31801074, 31788103). Yijun Qi is a visiting investigator of the CAS Center for Excellence in Molecular Plant Sciences.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplementary data

SUPPORTING INFORMATION

The supporting information is available online at https://doi.org/10.1007/s11427-021-2017-1. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


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  • Figure 1

    Profiling of 5′ tsRNAs and 5′ tRNA halves by RtcB sRNA-seq. A, Heatmap showing the relative abundances (RPM) of 5′ tsRNAs and 5′ tRNA halves produced by cleavage in indicated tRNA isodecoder groups as determined by RtcB sRNA-seq. Color intensity represents the fractional density across the row of RPM counts. B, 5′ tsRNAs and 5′ tRNA halves produced from tRNA-AlaAGC and tRNA-GlyUCC and the RPM of 3′-cP/P-terminated and 3′-OH-terminated species. C, Scatterplot showing the RPM of 3′-cP/P-terminated and/or 3′-OH-terminated 5′ tsRNAs, 5′ tRNA halves and miRNAs in RtcB sRNA-seq libraries. Red dots represent 5′ tsRNAs and 5′ tRNA halves that are predominantly terminated with a 3′ cP/P. Blue dots represent 5′ tsRNAs and 5′ tRNA halves that are predominantly terminated with a 3′ OH. Orange dots represent 5′ tsRNAs and 5′ tRNA halves that are bimorphic. Green triangles represent miRNAs that are terminated with a 3′ OH.

  • Figure 2

    RNS1 and RNS3 are required for 5′ tsRNA biogenesis and negatively regulate plant defense to Botrytis cinerea. A, Detection of 5′ tsR-Ala and 5′ tsR-Ser in Col-0 and rns mutants by Northern blot. miR158 and AtRep2 siRNAs were detected and used as controls. B, Venn diagram showing the overlap of downregulated 5′ tsRNAs in rns1-3 and rns3-2 (upper panel). Heatmap showing the relative abundances of 5′ tsRNAs downregulated in rns1-3 or rns3-2 from two biological replicates of cP-sRNA-seq (bottom panel). Colors represent log2-transformed fold changes of 5′ tsRNA abundances in the mutants relative to Col-0. W.T.: wild-type; F.C.: fold change. C, Venn diagrams showing the overlap of upregulated and downregulated genes in rns1-3 and rns3-2 mutants (left panel). Heatmaps showing the relative expression levels of overlapping upregulated and downregulated genes in rns1-3 and rns3-2 mutants (right panel). Colors represent log2-transformed fold changes of gene expression levels in the mutants relative to Col-0. D, Gene ontology analysis of overlapping upregulated and downregulated genes in rns1-3 and rns3-2 mutants. E, Lesion formation on the leaves of Col-0 and the indicated mutants after B. cinerea inoculation (upper panel). The true leaves of 4-week-old plants were inoculated with droplets containing B. cinerea spores. The pictures were taken 48 h post inoculation. Scale bar: 1 cm. Quantification of lesion sizes of B. cinerea on the leaves of Col-0 and the indicated mutants (bottom panel). Each dot represents the size of the lesion on one leaf. Error bars indicate SDs of more than 25 leaves. Significant different groups (P-value≤0.05, Student’s t test) are indicated by different letters.

  • Figure 3

    5′ tsR-Ala negatively regulates CYP71A13 expression and camalexin biosynthesis. A, Base-pairing between 5′ tsR-Ala and its target CYP71A13 mRNA and cleavage in CYP71A13 mRNA as determined by 5′ RACE. The number (out of 15) indicates the proportion of sequences cleaved at the indicated site. B, Relative expression levels of CYP71A13 in Col-0 and the indicated rns mutants as determined by mRNA-seq. CYP71A13 expression levels were FPKM-normalized. Error bars indicate SDs of three biological replicates. Significant different groups (q-value≤0.05, estimated by cuffdiff) are indicated by different letters. C, Detection of CYP71A13 expression levels in Col-0 and the indicated rns mutants by RT-qPCR. CYP71A13 expression levels were normalized against GAPDH. Error bars indicate SDs of three biological replicates. Significant different groups (P-value≤0.05, Student’s t test) are indicated by different letters. D, Detection of 5′ tsR-Ala in Col-0 and ago1 mutants by Northern blot. E, Detection of CYP71A13 expression levels in Col-0 and ago1 mutants by RT-qPCR. Error bars indicate SDs of three biological replicates. Significant different groups (P-value≤0.05, Student’s t test) are indicated by different letters. F, Detection of 5′ tsR-Ala in Col-0 and STTM-Ala lines by Northern blot. G, Detection of CYP71A13 expression levels in Col-0 and STTM-Ala lines by RT-qPCR. H, Detection of camalexin levels in Col-0, STTM-Ala #1 and the indicated rns mutants by Orbitrap Mass Spectrometry. Leaves of 4-week-old seedlings were inoculated with B. cinerea. Camalexin levels were measured 24 h post inoculation. Error bars indicate SDs of three biological replicates. Each replicate consists of at least ten independent seedlings. Significant different groups (P-value≤0.05, Student’s t test) are indicated by different letters.

  • Figure 4

    5′ tsR-AlaAGC negatively regulates plant defense to Botrytis cinerea. A, Lesion formation on the leaves of Col-0 and the indicated plants after B. cinerea inoculation (left panel). Scale bar: 1 cm. Quantification of lesion sizes of B. cinerea on the leaves of Col-0 and the indicated plants (right panel). Each dot represents the size of the lesion on one leaf. Error bars indicate SDs of more than 25 leaves. Significant different groups (P-value≤0.05, Student’s t test) are indicated by different letters. B, Venn diagrams showing the overlap of upregulated and downregulated genes in STTM-Ala and 35S::CYP71A13 plants. F.C.: fold change. C, Heatmaps showing the relative expression levels of overlapping upregulated and downregulated genes in STTM-Ala and 35S::CYP71A13 plants and cyp71A13-1 mutant plants. Colors represent log2-transformed fold changes of gene expression levels in the indicated plants relative to Col-0. D, Detection of 5′ tsR-Ala in mock- and B. cinerea-inoculated Col-0 seedlings by Northern blot. sRNAs were purified 24 h post inoculation for detection. miR158 and AtRep2 siRNAs were used as controls. E, Heatmap showing the relative abundances of differentially expressed 5′ tsRNAs in mock- and B. cinerea-inoculated Col-0 seedlings. Colors represent log2-transformed fold changes of 5′ tsRNA abundances in B. cinerea-inoculated seedlings relative to mock-treated seedlings.

  • Figure 5

    A model for the role of 5′ tsR-Ala in anti-fungal defense. Under normal condition, RNS1/3 cleave tRNA-Ala in the D loop to produce 5′ tsR-Ala. 5′ tsR-Ala is loaded into AGO1 and directs the cleavage of its target CYP71A13, resulting in the repression of anti-fungal defense response. Upon B. cinerea infection, RNS1/3-mediated tRNA cleavage is suppressed. Less 5′ tsR-Ala is produced, which derepresses CYP71A13 and anti-fungal defense response.

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